Mesenchymal stem cell secretome loaded StarPEG-gag hydrogels as a new route to induce spinal cord injury regeneration
Abstract
As lesões medulares têm sido descritas como "uma doença não tratável" desde os tempos do antigo Egipto. Esta é uma condição que afeta milhões de pessoas em todo o mundo, comprometendo tanto as funções motoras como sensoriais do corpo humano, impactando negativamente a qualidade de vida dos pacientes a nível físico, psicológico e económico. Várias terapias foram testadas em ensaios clínicos demonstrando capacidade de melhoria das funções motoras e sensoriais, no entanto não foram eficazes a chegar à clínica por promoverem diversas reações adversas. Nesta tese, o nosso objetivo foi desenvolver um sistema de libertação controlada baseado num hidrogel para libertar o secretoma de células estaminais derivadas do tecido adiposo (ASC) promovendo processos regenerativos, como crescimento axonal, redução da inflamação, aumento da sobrevivência celular e remodelação vascular, levando à recuperação motora. No entanto, embora a via sistémica (por exemplo, i.v. (intravenosa)) possa causar efeitos colaterais em diferentes órgãos, a administração local tem baixa eficiência devido à rápida difusão por fluidos corporais. O sistema de libertação baseia-se num hidrogel formado por poli(etilenoglicol) (starPEG) e o glicosaminoglicano (GAG) heparina (Hep) com cargas aniónicas que promovem uma elevada afinidade para fatores de crescimento, citocinas, e quimiocinas devido a interações eletrostáticas. Demonstramos que o hidrogel é adequado para libertação continua de factores pró-regenerativos, tais como interleucina (IL)-4, IL-6, fator neurotrófico derivado do cérebro (BDNF), fator neurotrófico de células glial (GDNF), e fator de crescimento beta-nervoso (β-NGF) durante dez dias. A rápida libertação após dois dias, está relacionada com a libertação de fatores neuroinflamatórios e angiogénicos, enquanto a libertação contínua e prolongada até dez dias é maioritariamente impulsionada por fatores de crescimento neuroreguladores. O secretoma libertado promoveu a diferenciação de células progenitoras neurais humanas (hNPCs) e o crescimento de neurites em fatias organotípicas da medula espinal. Finalmente, no modelo de transceção T8 em rato, a libertação de secretoma levou a melhorias motoras significativas comparado com animais lesionados ou tratados com secretoma localmente. As melhorias motoras são promovidas essencialmente pela redução da percentagem de microglia ameboide e níveis sistémicos elevados de citocinas anti-inflamatórias, tais como a IL-10. A libertação de secretoma de ASC do hidrogel starPEG-Hep pode oferecer opções sem precedentes para a terapia regenerativa de lesões medulares.
Full text
Universidade do Minho Escola de Medicina Deolinda Isabel Fernandes da Silva Mesenchymal Stem Cell Secretome Loaded StarPEG-GAG Hydrogels as a New Route to Induce Spinal Cord Injury Regeneration abril de 2023 UMinho | 2023 Deolinda Isabel Fernandes da Silva Mesenchymal Stem Cell Secretome Loaded StarPEG-GAG Hy drogels as a New Route to Induce Spinal Cord Injury Regenerationulo
i Universidade do Minho Escola de Medicina Deolinda Isabel Fernandes da Silva Mesenchymal Stem Cell Secretome Loaded StarPEG-GAG Hydrogels as a New Route to Induce Spinal Cord Injury Regeneration Tese de Doutoramento Doutoramento em Ciências da Saúde Trabalho efetuado sobre a orientação de Doutor António José Braga Osório Gomes Salgado e do Doutor Rui Pedro Romero Amandi de Sousa Abril de 2023
ii DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não previstas no licenciamento indicado, deverá contactar o autor, através do RepositóriUM da Universidade do Minho. Licença concedida aos utilizadores deste trabalho Atribuição-NãoComercial-SemDerivações CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/
iii Agradecimentos Life is made up of moments, memories, accomplishments, failures, and people who force you to learn and grow. Um agradecimento, em primeiro lugar, para a orientação científica. Obrigada, “Tó” pela paciência, pelos conselhos e aprendizagens transmitidas ao longo destes anos. Ao Rui Sousa por aceitar esta orientação e por a Stemmatters ser uma parte fundamental neste projeto. To thank Carsten for his warm welcome at the Max Bergmann Institute and for his contributions to my work. Lucas and Passant, thank you for guiding me and being partners in this journey. Às equipas que me acolheram nesta aventura, à “Tó team” que guardo com carinho especial, somos uma equipa como há poucas, e fica a amizade para além do trabalho, à equipa do laboratório do Carsten e à da Stemmatters que me receberam sempre com carinho. Um agradecimento muito especial à minha família. Aos meus pais, pela união que construíram, pelos valores que me transmitiram e pelos esforços que fizeram para me trazer até aqui. Pai, sei que estás orgulhoso desta conquista! Aos meus irmãos (ãs), cunhados (as) e sobrinhos (as). Somos muitos, mas somos incríveis. São a minha “casa”. Os que sempre persistem mesmo quando o mundo parece desmoronar, a eles devo a pessoa que sou. Às pessoas que chegaram e ficam para a vida. A Inês, a Sandra, a Tiffany, a Raquel, a Aline, o João Afonso nunca vou esquecer o gesto que tiveste para comigo, ao Rui. À Catarina e à Cláudia que me acompanham desde a infância. Por fim, agradeço a todas as pessoas, sem exceção, que cruzaram o meu caminho nestes últimos anos mas que não consigo enunciar aqui. De todas levo algum ensinamento, todas me ajudaram a crescer e hoje sou mais feliz porque a cada dia construo uma pessoa melhor em princípios, valores e realização. The work presented in this thesis was performed in the Life and Health Sciences Research Institute (ICVS), at School of Medicine, Minho University, Stemmatters, Biotecnologia e Medicina Regenerativa SA., Barco, Guimarães and at Max Bergmann Center of Biomaterials, Dresden, Germany. This work was supported by Prmios Santa Casa Neurociencias–Prize Melo e Castro for Spinal Cord Injury Research (MC-04/17; MC-18-2021) and the Portuguese Foundation for Science and Technology (Ph.D. Fellowship to D.S (PD/BDE/135567/2018 and COVID/BDE/152051/2022); funded by FEDER, through the Foundation for Science and Technology (FCT), under the scope of the projects UIDB/50026/2020; UIDP/50026/2020; POCI-01-0145-FEDER-029206; POCI-01-0145-FEDER-031392; PTDC/ MEDNEU/31417/2017; NORTE-01-0145-FEDER-029968; POCI-01-0145-FEDER-029751; POCI-01-0145FEDER-032619. This work has been funded by ICVS Scientific Microscopy Platform, a member of the national infrastructure PPBI - Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122. This work has also been developed under the scope of the project NORTE-01-0145FEDER-000013 and NORTE-01-0145-FEDER-000023, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). Work supported by the Portuguese Foundation for Science and Technology (FCT): projects UID/FIS/04650/2020, PTDC/EMD-EMD/28159/2017, and PTDC/BTMMAT/28237/2017.
iv STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho.
v Secretoma de Células Estaminais Mesenquimatosas Encapsulado em Hidrogel StarPEGGAG como uma Nova Terapia para a Regeneração de Lesões Vertebro-medulares. Resumo As lesões medulares têm sido descritas como "uma doença não tratável" desde os tempos do antigo Egipto. Esta é uma condição que afeta milhões de pessoas em todo o mundo, comprometendo tanto as funções motoras como sensoriais do corpo humano, impactando negativamente a qualidade de vida dos pacientes a nível físico, psicológico e económico. Várias terapias foram testadas em ensaios clínicos demonstrando capacidade de melhoria das funções motoras e sensoriais, no entanto não foram eficazes a chegar à clínica por promoverem diversas reações adversas. Nesta tese, o nosso objetivo foi desenvolver um sistema de libertação controlada baseado num hidrogel para libertar o secretoma de células estaminais derivadas do tecido adiposo (ASC) promovendo processos regenerativos, como crescimento axonal, redução da inflamação, aumento da sobrevivência celular e remodelação vascular, levando à recuperação motora. No entanto, embora a via sistémica (por exemplo, i.v. (intravenosa)) possa causar efeitos colaterais em diferentes órgãos, a administração local tem baixa eficiência devido à rápida difusão por fluidos corporais. O sistema de libertação baseia-se num hidrogel formado por poli(etilenoglicol) (starPEG) e o glicosaminoglicano (GAG) heparina (Hep) com cargas aniónicas que promovem uma elevada afinidade para fatores de crescimento, citocinas, e quimiocinas devido a interações eletrostáticas. Demonstramos que o hidrogel é adequado para libertação continua de factores pró-regenerativos, tais como interleucina (IL)-4, IL-6, fator neurotrófico derivado do cérebro (BDNF), fator neurotrófico de células glial (GDNF), e fator de crescimento beta-nervoso (β-NGF) durante dez dias. A rápida libertação após dois dias, está relacionada com a libertação de fatores neuroinflamatórios e angiogénicos, enquanto a libertação contínua e prolongada até dez dias é maioritariamente impulsionada por fatores de crescimento neuroreguladores. O secretoma libertado promoveu a diferenciação de células progenitoras neurais humanas (hNPCs) e o crescimento de neurites em fatias organotípicas da medula espinal. Finalmente, no modelo de transceção T8 em rato, a libertação de secretoma levou a melhorias motoras significativas comparado com animais lesionados ou tratados com secretoma localmente. As melhorias motoras são promovidas essencialmente pela redução da percentagem de microglia ameboide e níveis sistémicos elevados de citocinas anti-inflamatórias, tais como a IL-10. A libertação de secretoma de ASC do hidrogel starPEG-Hep pode oferecer opções sem precedentes para a terapia regenerativa de lesões medulares Palavras-chave: lesões medulares, hidrogel, secretoma, sistemas de libertação, starPEG, heparina
vi Mesenchymal Stem Cell Secretome Loaded StarPEG-GAG Hydrogels as a New Route to Induce Spinal Cord Injury Regeneration Abstract SCI has been described as "an ailment not to be treated" since ancient Egyptian times. Every year, millions of people worldwide are affected by this condition, which affects both motor and sensory functions of the human body with a negative impact on the quality of life of patients who are afflicted not only physically but also psychologically and economically. Several therapies have reached clinical trials and shown to be effective in promoting motor and sensorial improvements in patients. However, severe side effects or problems with route of administration have left those therapies on the way to clinic. We aimed to develop a release system based on a biohybrid hydrogel to deliver the secretome of adipose tissuederived stem cells (ASCs) locally and in a time-dependent manner, which has been shown to promote several regenerative mechanisms, including inducing axonal growth, reducing inflammation, promoting cell survival, and vascular remodeling, ultimately leading to functional recovery. However, while systemic delivery (e.g., i.v. (intravenous)) may cause off-target effects in different organs, the local administration has low efficiency due to fast clearance by body fluids. We started by developing the release system, which is based on a hydrogel formed of star-shaped poly (ethylene glycol) (starPEG) and the glycosaminoglycan (GAG) heparin (Hep) with anionic charges resulting in high affinity for a broad range of growth factors, cytokines, and chemokines due to electrostatic interactions. Due to this particularity, we have shown that the hydrogel is suitable to continuously release pro-regenerative signaling mediators such as interleukin (IL)-4, IL-6, brain-derived neurotrophic factor (BDNF), glial-cell neurotrophic factor (GDNF), and beta-nerve growth factor (β-NGF) over ten days. A burst release was observed after two days, mainly related to the release of neuroinflammatory and angiogenic factors, while the continuous and prolonged release until ten days is driven mainly by neuroregulatory growth factors. The released secretome was shown to significantly induce differentiation of human neural progenitor cells (hNPCs) and neurite outgrowth in organotypic spinal cord slices. Finally, in the in vivo complete transection SCI rat model, the secretome-loaded hydrogel significantly improved motor function in comparison to lesioned or secretome locally treated animals. Motor improvements were mainly supported by reducing the percentage of ameboid microglia and systemically elevated levels of anti-inflammatory cytokines, such as IL-10. Delivery of ASC-derived secretome from starPEG-Hep hydrogels may therefore offer unprecedented options for regenerative therapy of SCI. Keywords: spinal cord injury, hydrogels, secretome, delivery systems, starPEG, heparin
vii Table of contents AGRADECIMENTOS ...................................................................................................................... III STATEMENT OF INTEGRITY ........................................................................................................... IV RESUMO ....................................................................................................................................... V ABSTRACT ................................................................................................................................... VI LIST OF ABBREVIATIONS .............................................................................................................. XI LIST OF FIGURES ........................................................................................................................XIV LIST OF TABLES ..........................................................................................................................XVI THESIS AIMS AND LAYOUT ........................................................................................................ XVIII CHAPTER I ................................................................................................................................. 1 INTRODUCTION ......................................................................................................................... 1 1. NERVOUS SYSTEM AND SPINAL CORD ORGANIZATION ....................................................................... 2 2. SPINAL CORD INJURY HISTORICAL PERSPECTIVE ............................................................................. 3 2.1. Epidemiology......................................................................................................... 4 2.2. Pathophysiology and barriers for regeneration .......................................................... 5 2.3. Current Clinical approaches ................................................................................... 6 2.3.1. Surgical decompression ......................................................................................... 6 2.3.2. Rehabilitation ........................................................................................................ 6 2.4. Clinical trials ......................................................................................................... 7 2.4.1. Neuroprotection..................................................................................................... 8 2.4.1.1. Methylprednisolone ................................................................................................ 8 2.4.1.2. Riluzole ................................................................................................................. 8 2.4.1.3. Minocycline ........................................................................................................... 9 2.4.1.4. Granulocyte Colony-stimulating Factor (G-CSF) ......................................................... 9 2.4.1.5. Hepatocyte growth factor (HGF) .............................................................................. 9 2.4.2. Neuroregeneration ............................................................................................... 10 2.4.2.1. Cethrin ............................................................................................................... 10
xiv List of Figures CHAPTER I Figure I. 1 Organization of Spinal Cord. Figure I. 2 Underlying events that comprise the pathophysiology of SCI. Figure I.3 Clinical trials in SCI, related administration routs and side effects. Figure I. 4 Hydrogel classification, formulation methods and characterization techniques applied in the context of DS. Figure I. 5 Hydrogels have been applied in SCI as delivery systems to improve the therapeutic effect of loading agents. CHAPTER II Figure II. 1 Schematic representation of in vitro cultures to evaluate the bioactivity of hASCs secretome released from starPEG-Hep hydrogels. Figure II. 2 Physical characterization of starPEG-Hep hydrogels. Figure II. 3 Cumulative release of hASCs secretome from starPEG-Hep hydrogels. Figure II. 4 Evaluation of hASCs secretome release profile from starPEG-Hep hydrogels using membrane-based protein arrays. Figure II. 5 Effect of hASCs secretome released from starPEG-Hep hydrogels in hNPCs differentiation in vitro. Figure II. 6 Effect of hASCs secretome released from starPEG-Hep hydrogels in promoting neurite outgrowth in organotypic in vitro cultures. Supplementary Figure II. S1 Characterization of hASCs secretome released from starPEG-Hep hydrogels at tow and twn days using Membrane-based protein array RayBio ® C-Series Human Neuro Discovery array C1 Kit.
xv Supplementary Figure II. S2 Characterization of hASCs secretome released from StarPEG-Hep hydrogels at 2 and 10 days using Membrane-based protein array RayBio ®C-Series Human Cytokine Discovery array C5 Kit. CHAPTER III Figure III. 1 Schematic representation of our biomaterial concept and in vivo model that were used to evaluate the bioactivity of human adipose-tissue derived stem cells (hASCs) secretome released from starPEG-Hep hydrogels injected right before lesion. Figure III. -2 Evaluation of motor performance in SCI rats by BBB test for eight weeks post-injury. Figure III. 3 Evaluation of motor recovery after SCI rat model. Figure III. 4 Representative confocal microscopy images of longitudinal sections of spinal cord tissue for Iba1 staining. Figure III. 5 Representative confocal microscopy images of longitudinal sections of spinal cord tissue for GFAP and NF staining. Figure III.-6 SMI-71 quantification in SCI rat model eight weeks after lesion. Figure III.-7 Molecular analysis of collected sera following SCI using Rat Cytokine Array C2 from RayBiotech. Supplementary Figure III. S1 Molecular analysis of collected sera following SCI using Rat Cytokine Array C2 from RayBiotech. CHAPTER IV Thesis Graphical Abstract
xvi List of tables CHAPTER I Table I. 1 Current hydrogels delivery systems applied in SCI model and it potential to induce regeneration CHAPTER II Supplementary Table II. S1 Characterization of starPEG-Hep hydrogels. Supplementary Table II. S2 Pairwise comparisons between groups using Tukey’s correction for the percentage of hNPCs differentiated in immature (DCX+) and mature (MAP2+) neurons . Supplementary Table II. S3 Multiple comparisons between groups using Tukey’s correction for the percentage of neurofilament are in organotypic spinal cord slices cultures. CHAPTER III Supplementary Table III. S1 Pairwise comparisons between groups using Tukey’s correction for motor improvements evaluation using BBB test for 8 weeks post-injury. Supplementary Table III. S2 Pairwise comparisons between groups using Tukey’s correction for motor improvements evaluation using MST test at eight weeks post-injury. Supplementary Table III. S3 Pairwise comparisons between groups using Tukey’s correction for motor sensorial function using Von Frey test at tow and six weeks post-injury, in left and right hindlimbs. Supplementary Table III. S4 Multiple comparisons between groups using Tukey’s correction for the percentage of Iba1 ameboid area in spinal cord longitudinal sections. Supplementary Table III. S5 Pairwise comparisons between groups using Tukey’s correction for positive area of GFAP and NF.
xvii Supplementary Table III. S6 Pairwise comparisons between groups using Tukey’s correction for SMI71 quantification.
xviii Thesis aims and Layout The primary goal of this thesis was to create a drug delivery system that could load the secretome of human adipose tissue derived stem cells and promote their extended release in order to induce spinal cord injury regeneration. As a result, this thesis is divided into four chapters: The anatomy of the spinal cord and SCI pathophysiology are covered in Chapter I. An overview of current clinical practice and pipeline therapeutic strategies, including drugs used and cell-based therapy approaches, is provided. Furthermore, an overview of current biomaterials approaches used in the field was made, with a focus on the key characteristics to consider when developing efficient drug delivery systems. The development of the hydrogel release system is presented in Chapter II. StarPEG-Hep hydrogels were chosen for the purpose because of their high affinity for growth factors and cytokines, which are the most abundant molecules in the secretome. These hydrogels are mechanically suitable for implantation. In vitro studies also show that they have the ability to release a secretome over a 10-day period that can induce neuronal differentiation in hNPCs and neurite outgrowth in spinal cord slices. In Chapter III, the therapeutic potential of the starPEG-Hep+secretome is assessed in a thoracic transection rat model. Locomotor functional recovery and histological analysis of spinal cord tissue were carried out. Hydrogel loading secretome treatment has been shown to improve motor recovery and reduce inflammation following injury. Chapter IV is a general discussion that incorporates all of the previous chapters' findings. It also goes over the limitations and clinical relevance of these treatments in the clinic. Future work and perspectives were discussed, with the primary goal of improving the findings of this work as a potential combination of different strategies aimed at improving the patients' condition.
CHAPTER I Introduction This chapter is based on the published work in an international peer reviewed journal: Hydrogels as delivery systems for spinal cord injury regeneration Deolinda Silva, Rui A. Sousa, António J. Salgado Article published in Materials Today Bio, January 2021 DOI: 10.1016/j.mtbio.2021.100093 Link: https://doi.org/10.1016/j.mtbio.2021.100093
2 1. Nervous system and spinal cord organization Central Nervous System (CNS) is the structure that controls all functions in the human body. It is responsible for controlling all voluntary and involuntary movements caused by internal or external stimuli. The CNS is divided into two parts: the Brain and the Spinal Cord (SC). The general anatomy of the SC is divided into five categories of segments: cervical (eight segments), thoracic (twelve segments), lumbar (five segments), sacral (five segments) and coccygeal (one segment). This organization can differ between species. The fact that each segment is composed of a pair of roots, dorsal corresponding to sensorial fibers and ventral associated to motor fibers, is shared by all. The dural sleeve and neural foramina allow these roots to enter the SC [1]. Cervical and lumbar regions have higher concentration of motor neuros, which are responsible for finely tuned movements, which explains the enlargement in these regions [1]. Furthermore, SC is made up of the central canal, which is filled with cerebrospinal fluid (CSF) and surrounded by glial cells. The grey matter (nuclei) encloses this structure, and the ascending and descending tracts, white matter, enfold both structures, as shown in Figure I. 1A [2]. All structures are protected by the Blood Spinal Cord Barrier (BSCB), which is made up dura, arachnoid and pia, responsible to maintain SC homeostasis by mediating molecular exchange between the blood and SC [3]. SC has been described as a relay between brain and the peripheral organs, acting as a conduit for neural information to be transmitted from brain to all peripheral nervous system and vice-versa. The SC is a center for some reflexes and has the ability to maintain homeostasis. For example, it oversees coordinating sympathetic and parasympathetic reflexes (Figure I. 1B) [4]. The sympathetic division’s cells bodies are located at T1-L2/L3, while for parasympathetic division cell bodies are located at S2-S4 region [5]. Both divisions have the ability to act antagonistically, independently or synergistically. While the sympathetic nervous system is in charge of responses in times of danger, such as controlling arterial pressure, the parasympathetic system controls functions during rest, such as digestion and energy conservation. Both systems, which perform opposing functions, innervate the heart, bronchi, stomach and bladder. Blood vessels, and brown adipose tissue, found in newborn humans and animals, are only innervated by sympathetic nervous system or ciliary muscle, and the nasopharyngeal glands are only controlled by parasympathetic system [6]. Furthermore, neuroscientist have demonstrated in recent years that the SC is a key structure in controlling peripheral functions due to the presence of central pattern generators (CPGs). These are neuronal networks that can be instructed to move. CPGs controlling ejaculation, defection and micturition, for
3 example, are found in the lumbar and sacral segments of animal models. CPGs involved in locomotion have also been identified in animals and humans [4,7]. Based on these facts, any injury to the SC could jeopardize the body’s normal function. The impact of a mechanical insult in SC will be described in the following sections, as well as the consequences and potential therapies to restore functions. Figure I. 1 Organization of Spinal Cord. A – neural pathway to transmit neuronal information, and organization of spinal cord section. B – Sympathetic and parasympathetic enervation of different organs. 2. Spinal Cord Injury historical perspective Spinal cord injury (SCI) was firstly described by Edwin Smith in an ancient Egyptian papyrus [8]. At that time, it was possible to study injuries in the central nervous system (CNS) due to the high number of accidents caused by construction of Egyptian pyramids. In this document they describe SCI as a loss of function and sensitivity below the level of injury. Moreover, they described it as “an ailment not to be treated” [9]. Later on, Hippocrates associated other complications to this condition, like constipation, dysuria, skin problems and edema, which could lead to patient’s death [9]. The first big improvement in SCI patient care came as consequence of the drastically high numbers of deaths caused by SCI during World War I, due to low level of palliative cares provided to patients. As a consequence, a significant effort was made to establish multidisciplinary care centers and recruiting specialist to treat and follow civilians and military in the World War II [3, 4]. As a result, an improvement in life expectancy of SCI patients in the last decades was achieved, disclosing levels slightly lower than
4 the average rate for non-SCI individuals. However, there are some facts than can influence life expectancy, namely severity of injury, age, gender and also the fact that low income countries have higher mortality rates due to the lack of economic resources available for medical care [12,13]. 2.1. Epidemiology In a study performed by the Global Burden of Diseases (GBD) in 2016 the incidence of new SCI cases was 0.93 millions with a prevalence of 27 millions, being higher in high income countries (0.29 millions) as USA or Canada, compared with low income (0.08 millions) like Zimbabwe, and Mozambique [14]. Curiously, the incidence of SCI is also different among genders in these regions. In high income men are slightly more affected than women, specially between 20-40 years old, while in low income there is a high incidence of men affected, mainly because most of women stay at home to take care of family [13,14]. This is also related with the direct causes of SCI, which mostly are falls, traffic accidents, sport activities. In some regions of North Africa and Middle East terrorism and violence are the main cause of traumatic injuries [14,15]. Regarding non-traumatic injuries, the principal causes are associated with cord infarction, transverse myelitis, spinal abscess, or spinal canal stenosis [16]. SCI can also affect different functions of the body, depending on the region it occurs. Among all, around 50% of injuries occur at cervical level with an impact in respiratory functions, movement of arms and all functions below the neck. Overall, these are the most severe and also the worst regarding survival and life expectancy. Injuries at thoracic, lumbar or sacral regions are less frequent with a better prognosis of survival. Normally, these kind of injuries compromise the control of abdominal muscles, loss of bladder and bowel control as well as sexual function, hips and legs movement [17]. Both can be complete, with a total loss of function below the injury, or incomplete, where only one part of the spinal cord is affected, in which some function bellow the injury level can happen [18]. The visible image of a patient with a SCI is a wheelchair-dependent person, however, there are far more than motor and physiological consequences. For instance, SCI patients are prone to have depression, anxiety, sleep disturbances and autonomic dysreflexia [19], which frequently lead to an increase in suicide among SCI people [20].
5 2.2. Pathophysiology and barriers for regeneration Pathophysiology of SCI comprises three phases, the primary injury caused by a mechanical insult of the bone, followed by the secondary injury and then the onset of the chronic phase (Figure I. 2). Primary injury starts when Spinal Cord (SC) is compressed, contused, lacerated or transected. Immediately upon injury occurs the disruption of ascending and descending pathways, beginning secondary phase, which leads to permanent neuronal damage. Additionally, with the disruption of the blood spinal cord barrier (BSCB) there is the massive infiltration of inflammatory cells [21], that lead to a release of proinflammatory cytokines like TNF-α, IL-1β and IL-1α [22] to the extracellular milieu. Further, the damage of spinal neurons, axons and astrocytes leads to a massive release of glutamate that will bind to NMDA receptors promoting their overactivation allowing higher flow of calcium that triggers cell death and consequently death of healthy neurons [23]. Additionally, T cells may play a role in activation of NADPH oxidase, a protein complex that is involved in the production of reactive oxygen species (ROS), enhancing the inflammatory response and impacting in the clearance of myelin debris [24]. On top of this, the death of oligodendrocyte precursors cells (OPC), responsible for myelination of axons, will not allow the myelination of sprouting axons [25]. Thenceforth a chronic phase is established, with demyelination of white matter and dissolution of grey matter, formation of cystic cavity due to enhancement of astrogliosis and surrounded by reactive astrocytes, microglia/macrophages and components of the ECM, particularly chondroitin sulphate proteoglycans (CSPG) [26]. Figure I. 2 Underlying events that comprise the pathophysiology of SCI. The mechanical insult triggers a cascade of events mainly occurring at secondary phase with a massive damage of neuronal tissue.
12 months after which improvements in sensorimotor functions and reduction of neuropathic pain was achieved. Additionally, this study showed the safety and feasibility of scaffold implantation in SCI patients [74]. This study is now in process to recruit patients for clinical trials (INSPIRE 2 - NCT03762655) Figure I. 3 Clinical trials in SCI, related administration routs and side effects. 3. Hydrogels as delivery systems in Spinal Cord Injury Over the last decades there was an increased number of clinical trials conducted in SCI. However, a considerable number failed to promote an effective recovery. Despite some promising results in promoting gain function, most therapies presented upwards, such MPSS or MH, are not already in clinics due to the high risk to trigger of severe side effects (Figure I. 3). For researchers it is quite a challenge to reduce these effects because they are mainly due to the administration route that requires the use of high doses to have a local effect, which became toxic for other organs, particularly liver [75]. On the other side, local administration is also not an option due to fast clearance by fluids. In some cases, the administration is done by mini pumps intrathecally, which is still an invasive method with some risk of infection [76,77].
13 In an attempt to overcome these problems, biomaterials have flourished as a promising tool in SCI therapeutic strategies. Particularly hydrogels, which are high water content crosslinking structures, with a particularity of being similar to nervous tissue. They are known of being biocompatible, have the capacity to fill the cystic cavity and support axonal growth or cell differentiation. Specifically they can be implanted or injected at lesion site without promoting a further immune response [78]. These characteristics made hydrogels very attractive to be used as DS of drugs, growth factors, to be injected locally in a minimally invasive way for site oriented effect avoiding the use of high doses of therapeutic agents and consequent adverse side effects [79]. Moreover, several preclinical trials have shown the accomplishment of using hydrogels as DS (Table I. 1). Although, cell transplantation is considered as a very promising approach, there is the concern of cell migration for other regions of CNS, forming ectopic colonies or triggering abnormal tissue formation [80,81]. Once more, hydrogels can be used as matrixes to retain cells locally. Furthermore, hydrogels can be functionalized with peptides, such as fibronectin (GRGDS) or a laminin motifs (IKVAV), that support cell adhesion and growth and have shown to improve recovery after lesion [78,82]. As previously referred, hydrogels appear as excellent and versatile platforms to be used in SCI therapies. More than being used as depots for drugs, growth factors or increasing the potential effect of transplanted cells they protect molecules from being degraded by enzymes or an adverse immune response as they can support axonal growth, and promote tissue regeneration while they are degraded [83]. Another advantage is that their formulation characteristics can be modulated in order to improve their performance in vivo , reducing further inflammatory responses as well as control drug delivery. An ideal DS will promote a burst release in the first days, and a slow release for the maximum time possible. This implies that a therapeutic agent has an immediate relief upon injection, normally in the first 2-3 days, but with a prolonged effect with a continuous release, which will allow a continuous therapeutic effect without needing to perform several administrations in time [84]. Below some important characteristic of hydrogels will be highlighted in order to improve the hydrogel designing and consequently the deliver efficacy. 3.1. Hydrogels formulation methods and characterization techniques Hydrogels can be obtained from natural, synthetic sources, or formed by both natural and synthetic polymers forming a hybrid hydrogel [85]. While natural sources have the advantage of higher biocompatibility and biodegradability, synthetic biomaterials have high water absorption, long life and higher variety in chemistry which confers them strength and resistance [78,82]. Formulation of hydrogels
14 is of extreme importance when considering their use in context of SCI, they require outstanding improvements in order to enhance therapeutic effect and avoid additional surgical interventions. As an alternative injection of in situ forming hydrogels is a more reliable strategy. Upon injection the fast transition from liquid to gel allows a better adaptation to the tissue at injury site, eliminating free spaces and forming a template for tissue regeneration. Another advantage of this method is the easy formulation of hydrogels with cells, growth factors or drugs in liquid state formerly injection [86]. In this sense, several methods can be used to synthetize them, herein a brief introduction of the main processes used will be presented (Figure I. 4). Hydrogels can be physically or chemicaly crosslinked, depending if it is a non-covalent bond promoted by ionic interations, hydrogen bounds or hydrophobic interations, or if the covalent bond is formed by irradiation or a chemical crosslinker, respectively. Comparing both methods physical hydrogels have a reversible sol-gel transition and are formed by non-covalent cross-links, while chemical formulations have the disadvantage of using a chemical crosslinker, which can be toxic and interfere in the integrity of loading molecules [87]. However they do offer the advantage of an easy control over mechanical properties and form irreversible ligations [88,89]. As an example of chemical crosslinking EDC/NHS reaction is conducted to activate carboxyl groups of heparin for posterior formation of an amide bond between them and the amine groups of Poloxamer. These hydrogels were formulated to deliver GDNF orthotopically in a compression injury rat model [90]. A wide range of chemical coupling reactions are used to sinthetise hydrogels namely click reactions such as Michael type addition, thiol-maleimide reaction and Diels-Alder chemistry. The term “click chemistry” is used to characterize quick and versatile reactions, with high yield and highly selective resulting low toxic bioproducts with well-defined spatiotemporally controlled chemical network structures [91,92]. Mentioned chemical reactions allow the formation of hydrogels in water solution at physiological pH and formation of in situ hydrogels, which in context of SCI will help the hydrogels to better accommodate to the lesion site. Briefly, a maleimide-KAFAK was covalently bonded to methylcellulose by thiol-maleimide click chemistry for the delivery of BDNF improving neural behavior in a SCI rat model [93]. Among several other methods to formulate hydrogels, photopolymerization is used with intention to induce in situ polymerization controlled by photo-induced gelation by ultraviolet or visible light [86]. In this process the photo-initiator react to form a covalent bond via reacting groups. The main concern with this approach is cell viability that can be compromised by the radiation applied, mainly those with high proliferation rate. In this sense it has to be done a compromise to have a fast light exposure that will promote fast polymerization [94]. Piantino et al. developed a PLA-PEG-PLA triblock copolymer that are
15 assembled by photopolymerization of methacrylated groups of the macromers for delivery of NT-3. Upon injection hydrogel was exposed to light for 60s, an adequate time to induce polymerization and prevent dispersion of the hydrogel from the injection site [95]. Another interesting strategy is the use of self-assembly peptides. This strategy allows the formation of micro-building blocks rationally and coherently into a tissue-like assembly, more likely synthetic aminoacids based molecules that have a fast sol-gel transition at neutral pH. Moreover, self-assembly is a natural phenomenon used to construct complex structures from simple building blocks. For instance microgels can be induced by magnetic, acoustic, mechanical, capillary forces, surface tension or polarity [94,96]. Considering hydrogel characterization, it is crucial to study their structure and functionality. For that purpose, several microscopical techniques are the gold standard to explore hydrogel structure. Scanning electron microscopy (SEM) is by far the most popular technique used to perform morphological characterization of hydrogels. In this context SEM allows the identification of pore formation and size, crosslinking status of a hydrogel and evaluate the effect of loading molecules in general structure of matrices [97]. Although SEM is widely used it has a limitation capacity of generation two-dimensional projections. To overcome this limitation Laser Scanning Confocal Microscopy (LSCM) has been introduced in the field. Additionally, LSCM can be combined with fluorescent dyes, like fluorescent recovery after bleaching (FRAP) to study the diffusivity from hydrogels, which will contribute to predict the release profile in DS [97,98]. Spectroscopic methods include Fourier transforms infrared (FTIR) spectroscopy is an analytical method used to study the bonding structure of atoms based on the interaction between infrared (IR) irradiation with matter [99]. It can be used to characterize reactions between specific chemical groups such as chitosan and β-glycerophosphate [100].
16 Figure I. 4 Hydrogel classification, formulation methods and characterization techniques applied in the context of DS. 3.2. Rheology Mechanical properties of hydrogels are of the utmost importance when they are considered for implantation. Rheology is a process where the hydrogel is subject to small deformation, normally small amplitude oscillatory shear measurements, in a rheometer where is measured the deformation energy stored during the shear process (G’) also mentioned as elastic modulus or stiffness, and the energy dissipated during the process (G”) also referred as viscosity. Hydrogels are more elastic if G’> G’’ or are more viscous if G” > G’[101]. In 2010 Lampe and co-workers investigated the effect of different macroscopic properties of polyethylene glycol (PEG) hydrogels in the growth and differentiation of neural progenitor cells (NPCs), in which by increasing the amount of polymer in solution, hydrogels ranging from of 1kPa to 20kPa elastic modulus were obtained. In 3D models the increased stiffness hindered the metabolic activity of cells leading to a higher apoptotic rate. Noteworthy, with increased polymer concentration the access to nutrient could be hampered by the reduced mesh size, which could explain cell apoptosis [102]. In line with this finding, hyaluronic acid (HA) hydrogels were modified with increased methacrylate (MA) groups ratio in order to increase the hydrogel stiffness. Elastic modulus varied between 3kPa to 5kPa with increased MA substitution. Notably, neuronal differentiation was better supported by soft hydrogels, in which more mature neurons and increased axonal length were detected. Surprisingly no effect of mechanical properties was noticed in the morphology of astrocytes isolated from SC [103]. Later on, Schultz et al . developed a DS using a very soft agarose hydrogel with approximately 1.5kPa. In this work, they aimed the release of thyroid hormone 3,3′,5-triiodothy-ronine (T3) in order to tackle the enhancement of oligodendrocytes (OL) at injury site favoring the myelination of sprouting axons [104].
17 The release of T3 in a unilateral cervical contusion injury rat model increased the number of newly formed mature OLs and consequent axonal myelination rostral to lesion site [105]. In a different approach, hydrogels can be used in a strategy to decrease the mechanical properties of scaffolds. The combination of collagen in a scaffold of poly (propylene fumarate) (PPF), allow the formation of a porous structure filled with collagen for the development of a DS. Furthermore, collagen ameliorated the release of neurotrophin-3 (NT3), previously combined with collagen binding domain (CBD) which binds to biomaterials and can retain high content of neurotrophic factors, for 7 days. In a complete SC transection at thoracic level the scaffold surpasses organized axonal growth throughout the porous structure. The incorporation of collagen improved the production of mature neurons and NT3 enhanced survival of endogenous NPCs [106]. Beyond elastic modulus, viscosity of hydrogels must also be considered. It is related with the capacity of hydrogels to be injected through a needle and can diffuse prior to gelation [107]. Heparin poloxamer (HP) was used to deliver bFGF at a thoracic SCI hemisection. The hydrogel viscosity ranged between 8 Pa·s to 10 Pa·s as the temperature change from 32 ºC to 37 ºC [108]. Imidazole-poly(organophophazenes) polymer hydrogel (I-5), was implanted in a thoracic contusion injury rat model. This hydrogel has the capacity to rapidly form a gel-like material with a viscosity of 50 Pa·s and in just 150s achieving a viscosity of 600 Pa·s at body temperature. In vivo , its implantation almost eliminated cystic cavity and promote neuron repair and motor recovery [109]. Accordingly, hydrogels to be used in SCI therapies should mimic the mechanical properties of host tissue to allow the regenerative processes to take place. In agreement with literature the stiffness of SC could range between 3kPa and 300kPa [110,111] which means that hydrogels have to be stiff enough to assemble itself and soft enough to create an appropriate environment for cells to growth, adhere and differentiate. Moreover, it was shown that soft hydrogels (< 1kPa) with low viscosity are suitable for SCI implantation to favor tissue regeneration [112]. 3.3. Mesh size Hydrogels are polymer networks that form a gel when exposed to a polymerizing agent promoting the crosslinking between polymer chains. The free space between two points is named as mesh or pore of a hydrogel. Depending on the distance between the entangled points, hydrogels could be classified as macro-porous, micro-porous or non-porous [113]. Mesh size can be modulated by polymer and crosslinker concentration or external stimulus as pH or temperature. The porosity of hydrogels is crucial
18 for delivery of therapeutic agents in injury because in most cases this process occurs by diffusion or by interactions between polymer and loading agent. If the pores are smaller than the size of therapeutic agent, it will be entrapped into the hydrogel and will not be released. On the other hand, if the hydrogel pores and molecule size are similar the result will be a slow release. Contrarily if pores are higher than the agent, small molecules will move freely in the network and release will occur by diffusion, release is not directly correlated with mesh size [114]. Recent developments have been made to determine mesh size. Among them FRAP bleaching is used to study the diffusivity of fluorescent molecules from hydrogels. This technique has the advantage of being performed in a common confocal laser scanning microscope with a fluorescent probe capable of photobleaching [98]. Fluorescence correlation spectroscopy (FCS) is a complement to FRAP, once it correlates diffusivity coefficients from resident times of fluorescent particles moving through a small, static illuminated volume, being ideal for microscale heterogeneities in the hydrogel structure. This method has the advantage of using less powerful laser than FRAP as well as low fluorophore concentrations [115]. Currently, the most effective technique to determine mesh size (ξ), is correlating with swelling data, a theory of Canal and Pepas, equation 1 [116,117] 𝜉=𝜑−1 3 ((1 − 2𝔣)𝑙−2𝐶∞𝜆𝑀𝑐 𝑀𝑟)1 2 (Equation 1) where, 𝜑 is polymer volume fraction, 𝑙 polymeric carbon-carbon bond length, C∞ is the polymer-specific characteristic ratio for a chain of ∞ repeating units, 𝜆 polymer backbone bond factor, Mc number of average molecular weight between crosslinks in a polymer network and Mr molecular weight of the polymer repeating unit. Porosity in network structure is also imperative for diffusion of nutrients within the lesion site or even to allow the migration of cells or axons to growth through the implanted hydrogel. Chen et al. developed a macro-porous hydrogel based on 2-hydroxyethil methacrylate (HEMA) with MOETACL to deliver bFGF at injury site. This network was favored by communicating pores with an average size of 80µm. In a complete thoracic transection, the implantation of this DS allowed the recovery or motor performance evidenced by the increase ingrowth of axons and blood vessel in the hydrogel only 5 days after injury [118]. In the field of DS the possibility to modulate pore size is of extreme importance, considering also the injectability of the matrix. In this sense the formulation of matrices as DS using an oil-in-water emulsion at different ratios and surfactant concentrations allows the control over porous structure. Briefly
19 conjugation of both methods allows in situ pore formation by incorporation of oil droplets within crosslinkable precursor solution and polymerization induced by UV light, upon immersion in water the diffusion of oil droplets results in pore formation. Pore size was of the oil droplet size, but with increasing concentration of surfactant pore size diminish [119]. In a more simple and easy way, pore size can be tunable by controlling crosslinking degree, namely increased crosslinking degree or hydrogel precursor content decrease mesh size in a matrix hydrogel [120]. 3.4. Swelling Swelling behavior of the hydrogels is the process in which water molecules will enter in the structure of the polymer promoting the expansion of mesh size which will allow more water molecules to enter. In this process elasticity offset the extreme expansion of the network, avoiding it destruction. Thus, the equilibrium is reached when there is a balance of these two forces, also known as swelling pressure (Psw), is equal to zero. Generally speaking, swelling takes in account the increase in weight, volume or length of a hydrogel, and can be measured as degree of swelling (Dsw), equation 2 [121] 𝐷𝑠𝑤 =𝑚ℎ𝑤 𝑚ℎ𝑑 ⁄ (Equation 2) where mhw and mhd is the weight of wet and dry hydrogels, respectively. It could also be measured by the diameter of swollen hydrogels, equation 3 [122] 𝐷𝑠𝑤 =(𝑑ℎ𝑤 𝑑ℎ𝑑 ⁄ )3 (Equation 3) where dhw and dhd are, respectively, diameter of hydrogels after and before swelling, which always assume values Dsw ≥1 [121]. After SCI there is an increased pressure in surrounding tissues responsible by the edema formation, which causes cell death and tissue loss [107]. Considering this, hydrogels to be used in therapeutic strategies should not hold a high degree of swelling in order to avoid more pressure in the site of implantation [107]. Moreover, it is also important to control the swelling behavior in order to control the
20 release of molecules. Hydrogel swelling is directly correlated with porous structure, so all strategies to modulate mesh size mentioned ahead influence swelling behavior. Hydrogels with small pores will swell very slowly while macroporous structures swell fast [123]. For instance the increase of crosslinking density of HA-tyramine conjugate by increasing the concentration of H2O2 will decrease the swelling ratio and therefore decrease the rate of protein release [124]. Besides, swelling can also be controlled by the thiolation process of chitosan, that when modified with Traut’s reagent immediately swell after chitosan and PEG precursors are mixed and form a gel, slightly shrinking. Such approaches can reduce the likeliness of hydrogels to swell in the injury site, avoiding adverse reactions such as inflammation [125]. Likewise, hydrogel swelling can be modulated by incorporation of thermoresponsive segments in a hydrogel matrix. They have the particularity of being sensitive to thermal stimuli. Basically, these segments are water soluble and at low temperatures they are hydrated due to interactions between water molecules and hydrophilic domains extending polymer chains. On the other hand, high temperatures promote their dehydration and polymer chains aggregate due to hydrophobic interactions. Briefly at low temperature hydrogels swell while at high temperatures the swelling is slow, so hydrogel swelling can be controlled by combining hydrophilic and hydrophobic components [126]. Such polymers are poly(ethylene glycol)/ poly(propylene glycol), poly (glycidyl ethers) cellulose derivates, poly (N-substituted acrylamide) [127–129]. The main advantage of this strategy for injectable hydrogels is that hydrogels will polymerize upon injection normally at body temperature. 3.5. Degradation Introduction of biomaterials in regenerative strategies for CNS imply that progressively after implantation they will degrade as axons regenerate. This is extremely important to avoid inflammation or nerve compression. Accordingly, despite some promising results with synthetic nondegradable materials, such as silicone, polyacrylonitrile/polyvinylchloride (PAN/PVC), poly(tetrafluoro-ethylene) (PTEE), and poly (2hydroxyethyl methacrylate) (PHEMA), their use is not frequent due to their non-biodegradability. On the other hand, most of degradable materials used are provided by natural sources or synthetic materials, such as PLGA, PLA, PGA or PEG also used in medical devices [130]. Network degradation plays an important role in controlling molecular release, this is, controlling hydrogel degradation alter release of therapeutic agent. Piantino et al . controlled the release of NT3 by modulating the number of degradable units and macromer concentration in an acrylated PLA-b-PEG-b-PLA polymer.
21 This hydrogel is formed by the addition of degradable lactic units of hydroxyl groups. NT3 had a burst release in the first 24h, being slowly released thereafter for periods up to 2 weeks. This sustained release in a thoracic SCI transection promoted recovery of motor function as plasticity of raphespinal tracts [95]. Hydrogel degradation can also be modulated by crosslinking with specific sequences that will be recognized for endogenous or implanted enzymes. For instance, a HA hydrogel crosslinked with MMPsensitive peptide was tested in a thoracic compression injury rat model for BDNF delivery. When implanted in vivo , the endogenous cells secrete MMPs that degrade the sensitive peptides and consequently promote the release of BDNF, which induce motor neuron regeneration and motor function recovery [131]. Delivery of BDNF was also performed using a peptide amphiphilic (PA) hydrogel. In this work, a hydrogel was functionalized with IKVAV. The particularity of this hydrogel is that when implanted in vivo , it will assemble in nanofibers and BDNF release is controlled by electrostatic interactions. However, ultimately the release is controlled by hydrogel degradation once nanofibers will get shorter and mesh size will increase. This explains the release profile with a burst release in the first 3 days, followed by a slow release until 21 days post-implantation. In a thoracic compression injury rat model, such approach preserved axonal degeneration and diminish astrogliosis [132]. 3.6. Gelation temperature The gelation timing of a hydrogel to be used in DS is crucial to determine the therapeutic effect of loading agent. The gelation temperature (GT) is determined when elastic modulus is the halfway for gel formation [133]. Hydrogels can also be responsive to stimulus, in this case can gelate in response to temperature. The ideal behavior is that hydrogels are liquid at room temperature, and gel at body temperature, in situ gelation. This will ensure that hydrogel could be injected in the lesion site using a needle and, once there, a fast gelation process will allow prolonged therapeutic effect avoiding wash away by CSF [134]. One example of thermoresponsive hydrogels is poloxamers, that are composed of a triblock with a central hydrophobic block of polyoxypropylene (PPO) flanked by two hydrophilic blocks of polyoxyethylene (PEO) [135]. Liu and co-workers investigated the impact of polymer concentration on GT. They formulated hydrogels with different ratios of PPO:PEO, Pol-1(PEO101-PPO60-PEO101) and Pol-2 (PEO88-PPO27-PEO80), which form a gel at 15ºC and 50ºC, respectively. Interactions between hydrophilic PEO and hydrophobic units will determine the GT and nature of hydrogel. None of the created hydrogels were suited for implantation, so they combine both and investigated the GT by varying their concentrations and found out that forming a hydrogel with 5% (w/w) Pol-2 and 17,8% (w/w) Pol-1 the GT was around 39ºC. GT was determined by
28 demonstrating that their secretory profile differ [193]. Only 134 of the 451 proteins found were shared by all cell types. Some proteins involved in CNS processes were found to be expressed only in certain cell types. Indeed, ß1-4-galoctosyltransferase (ß4Gal-T) was only found in ASCS, whereas Stromal-derived factor-1α (SDF-1α) and Gelsolin were found in BMSCs, and Cyr61 and colony-stimulating factor (CSF-1) were found in HUCPVs. Moreover, anti-oxidative and anti-apoptotic molecules CYPA and CYPB were found to be upregulated in BMSCs. These results suggest that BMSCs may have higher anti-oxidative profile. In addition, molecules involved in axonal growth, such as SEM7A and GDN, were found to be upregulated in BMSCs and ASCs, indicating that these cells may play an important role in promoting neurite outgrowth. When compared to the secretome of BMSCs, the secretome of ASCs was able to induce significantly higher neurite outgrowth of dorsal root ganglia (DRGs) cultures [194]. Thus, ASCs-derived secretome has been shown to protect pheochromocytoma (PC12) cells from glutamate excitotoxicity and apoptosis by reducing the levels of cleaved-caspase-3 [184]. Regarding inflammation, the ASCs secretome is able to reduce the release of pro-inflammatory tumor necrosis factor-α (TNF-α) after exposure to an inflammatory stimulus, as well as to induce actively macrophages (M2) polarization and the secretion of antiinflammatory cytokines IL-10 and transforming growth factor (TGFβ1) [195]. Furthermore, the ASCs secretome was shown to be capable of promoting neuronal survival and differentiation [196,197], axonal outgrowth in dorsal root ganglia (DRG) explants [188,194] and also tissue vascularization [198]. Moreover, after exposure to spinal cord injury conditioned medium, the secretome of ASCs can increase MAP2 positive neurons, while decreasing GFAP cells, acting primarily as a protective factor in cortical neurons [199]. Exosomes or extracellular vesicles (EVs), in addition to proteins, make up the secretome. These structures can be important in cell-to-cell communication as well as transporting other proteins, mRNA, miRNA and organelles [200,201]. In vitro , EVs can protect neurons from glutamate excitotoxicity. Moreover, in a contusion SCI rat model they induce autophagy to reduce neural cell apoptosis, inflammation and contribute to improved motor performance [202]. Because it can address a wide range of pathways involved in trauma or disease, the secretome has emerged as a promising approach in SCI. The positive effects on neuroregeneration after SCI was demonstrated for the first time by our group. The multiple systemic injections of ASCs secretome after a contusion SCI in a mice model promoted functional recovery [203]. The beneficial effects have only been observed by systemic administration in this study, whereas the action of local injection was greatly diminished due to its quick clearance of the secretome from the target site. Furthermore, the authors emphasized the importance of administering the secretome as a whole, including both vesicular and protein fractions, after single administration did not significantly promote motor recovery.
29 Overall, these studies demonstrated the feasibility of using ASCs secretome in SCI regenerative therapies. These cells have the ability to release bioactive molecules that are involved in neuroprotective, neuroregenerative and immunomodulatory pathways, which help to restore lost functions. Moreover, using ASCs secretome overcomes concerns associated with cell transplantation, such as host rejection or incorrect differentiation. Figure I. 5 Hydrogels have been applied in SCI as delivery systems to improve the therapeutic effect of loading agents. Characteristics as rheology, mesh size, swelling, degradation rate, gelation temperature or surface charge, illustrated in the center of figure, can be modulated in order to improve their performance when injected at injury site. Additionally, hydrogels can be combined with other molecules to deliver agents at and extended time. Among all, growth factors, drugs, antibodies, hormones or
30 enzymes are the most frequent therapeutic agents loaded in hydrogels in SCI. In preclinical models of SCI these systems have shown promising impact in promoting tissue regeneration. 7. Conclusions and future perspective Spinal cord injury affects both motor and sensorial functions of millions of people worldwide being a serious social, economic and emotional problem. Despite the growing research in the field with multidisciplinary approaches raising to promote regeneration there is still a lack of an effective therapy in the clinic that could improve life quality of patients. Biomaterials can represent a good alternative to apply as DS, overcoming side effects reported in several clinical trials. As discussed, it is possible to modulate their designing characteristics to improve its performance at injury site and support regenerative events promoted by delivered molecular therapies. In preclinical trials, the use of these systems has allowed preservation of respiratory functions reduction of inflammation, enhanced axonal regeneration, reducing cellular apoptosis leading to recovery of motor function (Figure I. 5). While all reported strategies are relevant when considering the design of effective DS some can be considered most favorable to further explore, mainly due to easy chemistry used or simplest modulation of important characteristics. For instance, it is clear that the modulation of characteristics such as stiffness, mesh size, swelling and gelation temperature have in common alterations of crosslinking degree achieved by altering hydrogel precursor content. In this context poloxamer hydrogels appear to easily modulate these characteristics. Moreover, they have the advantage of being thermoresponsive hydrogels undergoing sol-gel transition at body temperature. Additionally, the possibility of incorporating molecules for affinity-based release, like heparin, is advantageous once is based on natural affinity for proteins or growth factors, prolonging the release profile and taking also advantage of heparin being a glycosaminoglycan present in ECM. Notwithstanding the promising results achieved in preclinical trials there is still a big question related to biomaterials, that why so few of them have reached clinical trials. The long path from bench to clinics comprehends the regulatory approval, which often tends to be quite challenging for strategies dealing with scaffolds [204]. Moreover, production of biomaterials for human implantation has to ensure Good Manufacturing Practices (GMP), as well as low batch to batch variability of natural or synthetic materials, a fact that is also challenging when scaling up lab run processes to an industrial scale [84]. Upon overcoming all mentioned problems, another concern is the injection method, which needs to be fast and
31 low invasive, do not promote further damage as well as protecting all therapeutic agents within the formulation. As mentioned, most of the strategies to test hydrogels are conducted in transection models, because of the gap created by the lesion that will allow the hydrogel to fill the cavity without fostering pressure to surrounding tissue. However, most human SCI are contusions/compressions, so another facing problem for clinical translation is the injection of biomaterials in these conditions. Upon several developments in SCI therapies, there is a strong know-how about its pathophysiology, which is crucial when developing effective therapies. Taking this into consideration, the development of a therapy will benefit from a multidisciplinary strategy of integrated knowledge of several areas, namely biology, physics, medicine, engineering and bioengineering and material science to try to tackle all degenerative events. Hydrogels can help with the adhesion of cells, their differentiation in neurons or uphold axonal growth, while at the same time have the ability to release growth factors or drugs that will reduce inflammation, induce axonal growth or decrease tissue damage. Likewise, materials with electrical properties could be embedded in hydrogels as well as the combination with stimulation will favor tissue regeneration and improve patient’s rehabilitation. Nonetheless, stem cell-based therapy has been shown in numerous studies to be a promising option to tackle SCI condition. Their therapeutic potential has been attributed primarily to secreted bioactive molecules that modulate neuroprotective, neuroregenerative, and immunomodulatory processes, as well as promoting motor recovery following SCI. As a result, a new era in cell-based therapy has begun with a better understanding of the key mechanisms modulated by these factors, as well as the development of an efficient and feasible method to inject secretome. This option may overcome the issues raised by cell transplantation. As a result, hydrogels could be a promising vehicle for delivering the secretome to the site of injury. Altogether, overcoming these problems will contribute to improve patient’s life quality. Acknowledgements This work was supported by Prmios Santa Casa Neurociencias–Prize Melo e Castro for Spinal Cord Injury Research (MC-04/17; MC-18-2021) and the Portuguese Foundation for Science and Technology (Ph.D. Fellowship to D.S (PD/BDE/135567/2018 and COVID/BDE/152051/2022). This work was funded by national funds and FEDER, through the Foundation for Science and Technology (FCT), under the scope of the projects UIDB/50026/2020; UIDP/50026/2020; POCI-01-0145-FEDER-029206; POCI01-0145-FEDER-031392; PTDC/ MED-NEU/31417/2017; NORTE-01-0145-FEDER-029968; POCI-010145-FEDER-029751; POCI-01-0145-FEDER-032619. This work has been funded by ICVS Scientific
32 Microscopy Platform, a member of the national infrastructure PPBI - Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122. This work has also been developed under the scope of the project NORTE-01-0145FEDER-000013 and NORTE-01-0145-FEDER-000023, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). Work supported by the Portuguese Foundation for Science and Technology (FCT): projects UID/FIS/04650/2020, PTDC/EMDEMD/28159/2017, and PTDC/BTM-MAT/28237/2017.
33 Table I. 2 Current hydrogels delivery systems applied in SCI model and it potential to induce regeneration Hydrogel type Functionaliz ation Therapeuti c agent Characteristics Injury model Delivery approach Therapeutic effects Reference Natural HAMC KAFKA BDNF • Low swelling • Release for 4 days Rat T10 clip compression Local administration after injury • Locomotor recovery • Inhibition of proinflammatory cytokines expression • Upregulation of anti-inflammatory cytokines [93] Alginate PLGA NPs MH and PTX in NPs • Irregular microporous structure • Low viscosity allowing syringe injection • MH and PTX released for 63 days Rat T8 lateral hemisection Local administration after injury • Axonal regrowth • Neuroprotection mediated by reducing proinflammatory cytokines • Locomotor recovery [205]
34 Gelatin CBD HGF • Photocrosslinkable hydrogel • Furfurylamine enhanced retention of CBD-HGF into hydrogel • Release for 7 days Mice compression injury Intrathecal injection after injury • Reduction of scar formation • Reduced inflammatory response • Motor recovery [206] Agarose DS-CH BDNF • Released over 17 days Rat unilateral C4/C5 contusion Local administration after injury • Preservation of respiratory function • Enhanced diaphragm innervation • Increase 5-HT positive fibers [207] Chitosan Collagen Serp-1 Rat T10 compression Local administration after injury • Improved motor function • Reduced SC damage • Reduced CD3positive cell at injury site [208]
35 β-glycerophosphate/h ydroxyethylcell ulose lentiviral mediated NGFoverexpressin g ADSCs Rat T8-T9 moderate contusion Local injection one week postinjury • Higher survival and proliferation cells at injury site • Improved motor function • Reduced cavity formation [100] Synthetic Laponite Heparin FGF4 • Elastic modulus of 0.1 kPa • 3D porous structure • Easily injected using syringe • FGF4 released for 35 days Rat T9 clip compression Orthotopic injection • Reduced cystic cavity • Axonal regeneration and remyelination • Suppression of astrocyte migration and polarization • Reduction of M1 phenotype macrophages [209] Fmoc RGD S-220 (Epac2 agonist that could enhance axonal growth) • Stiffness ~100 Pa • Release for 28 days Rat T10 contusion Local injection 3 weeks after injury • Improve in control limb movements • Enhanced neurite growth • Suppression of astrocyte activation [210]
36 Poloxamer Heparin GDNF • Gelation temperature at 37ºC • Porous structure • 80% GDNF released after 24h Rat T9 compression Orthotopic injection after injury • Neuroprotection of injured SC • Enhanced axonal repair • Inhibited expression of caspase-3 [90] NGF • Porous sponge like structure • Interconnected inner porous Rat T9 contusion Orthotopic injection after injury • Motor recovery • Decreased astrocytes at injury site • Increased number of CD31 positive cells • Reduced apoptosis [211]
37 References [1] A. Tracey, C. Minneap, M. February, S.C. Disorders, Spinal_Cord_Functional_Anatomy___Erratum.3, 21 (2015) 13–35. [2] O. Bican, A. Minagar, A.A. Pruitt, The Spinal Cord. A Review of Functional Neuroanatomy, Neurol. Clin. 31 (2013) 1–18. https://doi.org/10.1016/j.ncl.2012.09.009. [3] L.Y. Jin, J. Li, K.F. Wang, W.W. Xia, Z.Q. Zhu, C.R. Wang, X.F. Li, H.Y. Liu, Blood-Spinal Cord Barrier in Spinal Cord Injury: A Review, J. Neurotrauma. 38 (2021) 1203–1224. https://doi.org/10.1089/neu.2020.7413. [4] I. Steuer, P.A. Guertin, Central pattern generators in the brainstem and spinal cord: An overview of basic principles, similarities and differences, Rev. Neurosci. 30 (2018) 107–164. https://doi.org/10.1515/revneuro-2017-0102. [5] A. Farley, E. Mclafferty, C. Johnstone, C. Hendry, Nervous system: part 3 Farley, Nurs. Stand. 28 (2013) 46–50. [6] E.A. Wehrwein, H.S. Orer, S.M. Barman, Overview of the Anatomy, Physiology, and Pharmacology of the Autonomic Nervous System, Compr. Physiol. 6 (2016) 1239–1278. https://doi.org/10.1002/cphy.c150037. [7] K. Minassian, U.S. Hofstoetter, F. Dzeladini, P.A. Guertin, A. Ijspeert, The Human Central Pattern Generator for Locomotion: Does It Exist and Contribute to Walking?, Neuroscientist. 23 (2017) 649–663. https://doi.org/10.1177/1073858417699790. [8] R. Porter, The Cambridge Illustrated History of Medicine, Ann. Intern. Med. 128 (1996) 415. https://doi.org/10.7326/0003-4819-128-5-199803010-00020. [9] L. Anderberg, H. Aldskogius, A. Holtz, Spinal cord injury - Scientific challenges for the unknown future, Ups. J. Med. Sci. 112 (2007) 259–288. https://doi.org/10.3109/2000-1967-200. [10] J.F. Ditunno, Linking spinal cord injury rehabilitation between the World Wars: The R. Tait McKenzie legacy, J. Spinal Cord Med. 40 (2017) 641–648. https://doi.org/10.1080/10790268.2017.1370522. [11] D.J. Lanska, The influence of the two world wars on the development of rehabilitation for spinal cord injuries in the United States and Great Britain, Front. Neurol. Neurosci. 38 (2016) 56–67. https://doi.org/10.1159/000442569. [12] G. Savic, M.J. Devivo, H.L. Frankel, M.A. Jamous, B.M. Soni, S. Charlifue, Long-term survival after traumatic spinal cord injury: A 70-year British study, Spinal Cord. 55 (2017) 651–658. https://doi.org/10.1038/sc.2017.23. [13] T. Øderud, Surviving spinal cord injury in low income countries, African J. Disabil. 3 (2014) 1–9. https://doi.org/10.4102/ajod.v3i2.80. [14] S.L. James, A. Theadom, et.al, Global, regional, and national burden of traumatic brain injury and spinal cord injury, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016, Lancet Neurol. 18 (2019) 56–87. https://doi.org/10.1016/S1474-4422(18)30415-0. [15] N. Level, Facts and Figures at a Glance, J. Spinal Cord Med. 30 (2018) 304–305. https://doi.org/10.1080/10790268.2007.11753944. [16] P.W. New, R. Marshall, International Spinal Cord Injury Data Sets for non-traumatic spinal cord injury, Spinal Cord. 52 (2014) 123–132. https://doi.org/10.1038/sc.2012.160. [17] L.H.S. Sekhon, M.G. Fehlings, Epidemiology, demographics, and pathophysiology of acute spinal cord injury, Spine (Phila. Pa. 1976). 26 (2001) 2–12. https://doi.org/10.1097/00007632200112151-00002. [18] M.J. Eckert, M.J. Martin, Trauma: Spinal Cord Injury, Surg. Clin. North Am. 97 (2017) 1031– 1045. https://doi.org/10.1016/j.suc.2017.06.008. [19] N.T. North, The psychological effects of spinal cord injury: a review, J. Spinal Cord Med. (1999) 671–679.
44 https://doi.org/10.1038/srep38332. [109] L.T.A. Hong, Y.M. Kim, H.H. Park, D.H. Hwang, Y. Cui, E.M. Lee, S. Yahn, J.K. Lee, S.C. Song, B.G. Kim, An injectable hydrogel enhances tissue repair after spinal cord injury by promoting extracellular matrix remodeling, Nat. Commun. 8 (2017) 1–14. https://doi.org/10.1038/s41467-017-00583-8. [110] H. Ozawa, T. Matsumoto, T. Ohashi, M. Sato, S. Kokubun, Comparison of spinal cord gray matter and white matter softness: Measurement by pipette aspiration method, J. Neurosurg. 95 (2001) 221–224. https://doi.org/10.3171/spi.2001.95.2.0221. [111] R.J. Oakland, R.M. Hall, R.K. Wilcox, D.C. Barton, The biomechanical response of spinal cord tissue to uniaxial loading, Proc. Inst. Mech. Eng. Part H J. Eng. Med. 220 (2006) 489–492. https://doi.org/10.1243/09544119JEIM135. [112] M.C. Mosley, H.J. Lim, J. Chen, Y.H. Yang, S. Li, Y. Liu, L.A. Smith Callahan, Neurite extension and neuronal differentiation of human induced pluripotent stem cell derived neural stem cells on polyethylene glycol hydrogels containing a continuous Young’s Modulus gradient, J. Biomed. Mater. Res. - Part A. 105 (2017) 824–833. https://doi.org/10.1002/jbm.a.35955. [113] Q. Chai, Y. Jiao, X. Yu, Hydrogels for Biomedical Applications: Their Characteristics and the Mechanisms behind Them, Gels. 3 (2017) 6. https://doi.org/10.3390/gels3010006. [114] J. Li, D.J. Mooney, Designing hydrogels for controlled drug delivery, Nat. Rev. Mater. 1 (2016). https://doi.org/10.1038/natrevmats.2016.71. [115] S.T. Hess, S. Huang, A.A. Heikal, W.W. Webb, Biological and chemical applications of fluorescence correlation spectroscopy: A review, Biochemistry. 41 (2002) 697–705. https://doi.org/10.1021/bi0118512. [116] T. Canal, N.A. Peppas, Correlation between mesh size and equilibrium degree of swelling of polymeric networks, J. Biomed. Mater. Res. 23 (1989) 1183–1193. https://doi.org/10.1002/jbm.820231007. [117] N.R. Richbourg, N.A. Peppas, The swollen polymer network hypothesis: Quantitative models of hydrogel swelling, stiffness, and solute transport, Prog. Polym. Sci. 105 (2020) 101243. https://doi.org/10.1016/j.progpolymsci.2020.101243. [118] B. Chen, J. He, H. Yang, Q. Zhang, L. Zhang, X. Zhang, E. Xie, C. Liu, R. Zhang, Y. Wang, L. Huang, D. Hao, Repair of spinal cord injury by implantation of bFGF-incorporated HEMA-MOETACL hydrogel in rats, Sci. Rep. 5 (2015) 1–10. https://doi.org/10.1038/srep09017. [119] O. Yom-Tov, L. Neufeld, D. Seliktar, H. Bianco-Peled, A novel design of injectable porous hydrogels with in situ pore formation, Acta Biomater. 10 (2014) 4236–4246. https://doi.org/10.1016/j.actbio.2014.07.006. [120] U. Freudenberg, A. Hermann, P.B. Welzel, K. Stirl, S.C. Schwarz, M. Grimmer, A. Zieris, W. Panyanuwat, S. Zschoche, D. Meinhold, A. Storch, C. Werner, A star-PEG-heparin hydrogel platform to aid cell replacement therapies for neurodegenerative diseases, Biomaterials. 30 (2009) 5049–5060. https://doi.org/10.1016/j.biomaterials.2009.06.002. [121] M. Sefidgaran, Hydrogels in Controlled Drug Delivery Systems, 6 (2010) 125–131. [122] P. Atallah, L. Schirmer, M. Tsurkan, Y.D. Putra Limasale, R. Zimmermann, C. Werner, U. Freudenberg, In situ-forming, cell-instructive hydrogels based on glycosaminoglycans with varied sulfation patterns, Biomaterials. 181 (2018) 227–239. https://doi.org/10.1016/j.biomaterials.2018.07.056. [123] F. Ganji, S. Vasheghani-Farahani, E. Vasheghani-Farahani, Theoretical Description of Hydrogel Swelling: AReview Fariba, Iran. Polym. J. 7 (2010) 41–48. [124] F. Lee, J.E. Chung, M. Kurisawa, An injectable hyaluronic acid-tyramine hydrogel system for protein delivery, J. Control. Release. 134 (2009) 186–193. https://doi.org/10.1016/j.jconrel.2008.11.028.
45 [125] A.E. Mohrman, M. Farrag, R.K. Grimm, N.D. Leipzig, Evaluation of in situ gelling chitosan-PEG copolymer for use in the spinal cord, J. Biomater. Appl. 33 (2018) 435–446. https://doi.org/10.1177/0885328218792824. [126] H. Kamata, X. Li, U. Il Chung, T. Sakai, Design of Hydrogels for Biomedical Applications, Adv. Healthc. Mater. 4 (2015) 2360–2374. https://doi.org/10.1002/adhm.201500076. [127] S. Reinicke, J. Schmelz, A. Lapp, M. Karg, T. Hellweg, H. Schmalz, Smart hydrogels based on double responsive triblock terpolymers, Soft Matter. 5 (2009) 2648–2657. https://doi.org/10.1039/b900539k. [128] D.C. Harsh, S.H. Gehrke, Controlling the swelling characteristics of temperature-sensitive cellulose ether hydrogels, J. Control. Release. 17 (1991) 175–185. https://doi.org/10.1016/01683659(91)90057-K. [129] M.A. Ward, T.K. Georgiou, Thermoresponsive polymers for biomedical applications, Polymers (Basel). 3 (2011) 1215–1242. https://doi.org/10.3390/polym3031215. [130] K.S. Straley, C.W.P. Foo, S.C. Heilshorn, Biomaterial design strategies for the treatment of spinal cord injuries, J. Neurotrauma. 27 (2010) 1–19. https://doi.org/10.1089/neu.2009.0948. [131] J. Park, E. Lim, S. Back, H. Na, Y. Park, K. Sun, Nerve regeneration following spinal cord injury using matrix metalloproteinase-sensitive, hyaluronic acid-based biomimetic hydrogel scaffold containing brain-derived neurotrophic factor, J. Biomed. Mater. Res. - Part A. 93 (2010) 1091– 1099. https://doi.org/10.1002/jbm.a.32519. [132] Z. Hassannejad, S.A. Zadegan, A.R. Vaccaro, V. Rahimi-Movaghar, O. Sabzevari, Biofunctionalized peptide-based hydrogel as an injectable scaffold for BDNF delivery can improve regeneration after spinal cord injury, Injury. 50 (2019) 278–285. https://doi.org/10.1016/j.injury.2018.12.027. [133] H.E. Park, N. Gasek, J. Hwang, D.J. Weiss, P.C. Lee, Effect of temperature on gelation and crosslinking of gelatin methacryloyl for biomedical applications, Phys. Fluids. 32 (2020). https://doi.org/10.1063/1.5144896. [134] C.A. McKay, R.D. Pomrenke, J.S. McLane, N.J. Schaub, E.K. Desimone, L.A. Ligon, R.J. Gilbert, An injectable, calcium responsive composite hydrogel for the treatment of acute spinal cord injury, ACS Appl. Mater. Interfaces. 6 (2014) 1424–1438. https://doi.org/10.1021/am4027423. [135] S.M. Moghimi, A.C. Hunter, Poloxamers and poloxamines in nanoparticle engineering and experimental medicine, Trends Biotechnol. 18 (2000) 412–420. https://doi.org/10.1016/S0167-7799(00)01485-2. [136] D. Liu, T. Jiang, W. Cai, J. Chen, H. Zhang, S. Hietala, H.A. Santos, G. Yin, J. Fan, An In Situ Gelling Drug Delivery System for Improved Recovery after Spinal Cord Injury, Adv. Healthc. Mater. 5 (2016) 1513–1521. https://doi.org/10.1002/adhm.201600055. [137] S. Lakard, G. Herlem, A. Propper, A. Kastner, G. Michel, N. Vallès-Villarreal, T. Gharbi, B. Fahys, Adhesion and proliferation of cells on new polymers modified biomaterials, Bioelectrochemistry. 62 (2004) 19–27. https://doi.org/10.1016/j.bioelechem.2003.09.009. [138] A. Hejčl, P. Lesný, M. Přádný, J. Šedý, J. Zámečník, P. Jendelová, J. Michálek, E. Syková, Macroporous hydrogels based on 2-hydroxyethyl methacrylate. Part 6: 3D hydrogels with positive and negative surface charges and polyelectrolyte complexes in spinal cord injury repair, J. Mater. Sci. Mater. Med. 20 (2009) 1571–1577. https://doi.org/10.1007/s10856-009-3714-4. [139] T. Schackel, P. Kumar, M. Günther, S. Liu, M. Brunner, B. Sandner, R. Puttagunta, R. Müller, N. Weidner, A. Blesch, Peptides and Astroglia Improve the Regenerative Capacity of Alginate Gels in the Injured Spinal Cord, Tissue Eng. - Part A. 25 (2019) 522–537. https://doi.org/10.1089/ten.tea.2018.0082. [140] N.J. Tester, A.H. Plaas, D.R. Howland, Effect of Body Temperature on Chondroitinase ABC’s Ability To Cleave Chondroitin Sulfate Glycosaminoglycans Nicole, J. Neurosci. Res. 85 (2007) 1110– 1118. https://doi.org/10.1002/jnr.
46 [141] M.M. Pakulska, K. Vulic, M.S. Shoichet, Affinity-based release of chondroitinase ABC from a modified methylcellulose hydrogel, J. Control. Release. 171 (2013) 11–16. https://doi.org/10.1016/j.jconrel.2013.06.029. [142] M.M. Pakulska, C.H. Tator, M.S. Shoichet, Local delivery of chondroitinase ABC with or without stromal cell-derived factor 1α promotes functional repair in the injured rat spinal cord, Biomaterials. 134 (2017) 13–21. https://doi.org/10.1016/j.biomaterials.2017.04.016. [143] Q. Wang, Y. He, Y. Zhao, H. Xie, Q. Lin, Z. He, X. Wang, J. Li, H. Zhang, C. Wang, F. Gong, X. Li, H. Xu, Q. Ye, J. Xiao, A Thermosensitive Heparin-Poloxamer Hydrogel Bridges aFGF to Treat Spinal Cord Injury, ACS Appl. Mater. Interfaces. 9 (2017) 6725–6745. https://doi.org/10.1021/acsami.6b13155. [144] P.M. Medberry, Christopher J. Crapo, B.F. Siu, C.A. Carruthers, M.T. Wolf, S.P. Nagarkar, V. Agrawal, K.E. Jones, J. Kelly, S.A. Johnson, S.S. Velankar, S.C. Watkins, M. Modo, S.F. Badylak, Hydrogels Derived from Central Nervous System Extracellular Matrix, Biomaterials. 34 (2013) 1033–1040. https://doi.org/10.1007/s11103-011-9767-z.Plastid. [145] H.L. Xu, F.R. Tian, J. Xiao, P.P. Chen, J. Xu, Z.L. Fan, J.J. Yang, C.T. Lu, Y.Z. Zhao, Sustainedrelease of FGF-2 from a hybrid hydrogel of heparin-poloxamer and decellular matrix promotes the neuroprotective effects of proteins after spinal injury, Int. J. Nanomedicine. 13 (2018) 681–694. https://doi.org/10.2147/IJN.S152246. [146] R.C. Robinson, C. Radziejewski, G. Spraggon, J. Greenwald, M.R. Kostura, L.D. Burtnick, D.I. Stuart, S. Choe, E.Y. Jones, The structures of the neurotrophin 4 homodimer and the brain-derived neurotrophic factor/neurotrophin 4 heterodimer reveal a common Trk-binding site., Protein Sci. 8 (1999) 2589–2597. [147] I. Elliott Donaghue, C.H. Tator, M.S. Shoichet, Sustained delivery of bioactive neurotrophin-3 to the injured spinal cord, Biomater. Sci. 3 (2015) 65–72. https://doi.org/10.1039/c4bm00311j. [148] A.H. Sheikh, B. Raghuram, L. Eschen-lippold, D. Scheel, Local delivery of neurotrophin-3 and antiNogoA promotes repair after spinal cord injury, v (2017) 1–36. https://doi.org/10.2174/138161211796197016. [149] Y. Cho, R. Shi, R.B. Borgens, Chitosan produces potent neuroprotection and physiological recovery following traumatic spinal cord injury, J. Exp. Biol. 213 (2010) 1513–1520. https://doi.org/10.1242/jeb.035162. [150] M. Boido, M. Ghibaudi, P. Gentile, E. Favaro, R. Fusaro, C. Tonda-Turo, Chitosan-based hydrogel to support the paracrine activity of mesenchymal stem cells in spinal cord injury treatment, Sci. Rep. 9 (2019) 1–16. https://doi.org/10.1038/s41598-019-42848-w. [151] J.S. Rao, C. Zhao, A. Zhang, H. Duan, P. Hao, R.H. Wei, J. Shang, W. Zhao, Z. Liu, J. Yu, K.S. Fan, Z. Tian, Q. He, W. Song, Z. Yang, Y.E. Sun, X. Li, NT3-chitosan enables de novo regeneration and functional recovery in monkeys after spinal cord injury, Proc. Natl. Acad. Sci. U. S. A. 115 (2018) E5595–E5604. https://doi.org/10.1073/pnas.1804735115. [152] B. Ghosh, Z. Wang, J. Nong, M.W. Urban, Z. Zhang, V.A. Trovillion, M.C. Wright, Y. Zhong, A.C. Lepore, Local BDNF delivery to the injured cervical spinal cord using an engineered hydrogel enhances diaphragmatic respiratory function, J. Neurosci. 38 (2018) 5982–5995. https://doi.org/10.1523/JNEUROSCI.3084-17.2018. [153] E. Vecino, J.C.F. Kwok, The Extracellular Matrix in the Nervous System: The Good and the Bad Aspects, Compos. Funct. Extracell. Matrix Hum. Body. (2016). https://doi.org/10.5772/62527. [154] C. Tonda-Turo, F. Ruini, M. Ramella, F. Boccafoschi, P. Gentile, E. Gioffredi, G. Falvo D’Urso Labate, G. Ciardelli, Non-covalently crosslinked chitosan nanofibrous mats prepared by electrospinning as substrates for soft tissue regeneration, Carbohydr. Polym. 162 (2017) 82–92. https://doi.org/10.1016/j.carbpol.2017.01.050. [155] P.G. Popovich, C.A. Tovar, S. Lemesshoe, Q. Yin, L.B. Jakeman, Independent evaluation of the
47 anatomical and behavioral effects of Taxol in rat models of spinal cord injury, Exp. Neurol. (2015) 97–108. https://doi.org/10.1016/j.expneurol.2014.06.020.Independent. [156] Q. Wang, H. Zhang, H. Xu, Y. Zhao, Z. Li, J. Li, H. Wang, D. Zhuge, X. Guo, H. Xu, S. Jones, X. Li, X. Jia, J. Xiao, Novel multi-drug delivery hydrogel using scar-homing liposomes improves spinal cord injury repair, Theranostics. 8 (2018) 4429–4446. https://doi.org/10.7150/thno.26717. [157] G.M. Soliman, A.O. Choi, D. Maysinger, F.M. Winnik, Minocycline block copolymer micelles and their anti-inflammatory effects on microglia, Macromol. Biosci. 10 (2010) 278–288. https://doi.org/10.1002/mabi.200900259. [158] Z. Zhang, Z. Wang, J. Nong, C.A. Nix, H.-F. Ji, Y. Zhong, Metal ion-assisted self-assembly of complexes for controlled and sustained release of minocycline for biomedical applications, Biofabrication. 7 (2016) 1–25. https://doi.org/10.1088/1758-5090/7/1/015006.Metal. [159] Z. Wang, J. Nong, R.B. Shultz, Z. Zhang, V.J. Tom, R.K. Ponnappan, Y. Zhong, Local delivery of minocycline from metal ion-assisted self-assembled complexes promotes neuroprotection and functional recovery after spinal cord injury, Biomaterials. 112 (2017) 62–71. https://doi.org/10.1016/j.biomaterials.2016.10.002. [160] B. Ghosh, J. Nong, Z. Wang, M.W. Urban, N.M. Heinsinger, V.A. Trovillion, M.C. Wright, A.C. Lepore, Y. Zhong, A hydrogel engineered to deliver minocycline locally to the injured cervical spinal cord protects respiratory neural circuitry and preserves diaphragm function, Neurobiol. Dis. 127 (2019) 591–604. https://doi.org/10.1016/j.nbd.2019.04.014. [161] X. Lu, T.H. Perera, A.B. Aria, L.A.S. Callahan, Polyethylene glycol in spinal cord injury repair: A critical review, J. Exp. Pharmacol. 10 (2018) 37–49. https://doi.org/10.2147/JEP.S148944. [162] R. Censi, P. Di Martino, T. Vermonden, W.E. Hennink, Hydrogels for protein delivery in tissue engineering, J. Control. Release. 161 (2012) 680–692. https://doi.org/10.1016/j.jconrel.2012.03.002. [163] S. Ren, Z.H. Liu, Q. Wu, K. Fu, J. Wu, L.T. Hou, M. Li, X. Zhao, Q. Miao, Y.L. Zhao, S.Y. Wang, Y. Xue, Z. Xue, Y.S. Guo, S. Canavero, X.P. Ren, Polyethylene glycol-induced motor recovery after total spinal transection in rats, CNS Neurosci. Ther. 23 (2017) 680–685. https://doi.org/10.1111/cns.12713. [164] X. Bin Kong, Q.Y. Tang, X.Y. Chen, Y. Tu, S.Z. Sun, Z.L. Sun, Polyethylene glycol as a promising synthetic material for repair of spinal cord injury, Neural Regen. Res. 12 (2017) 1003–1008. https://doi.org/10.4103/1673-5374.208597. [165] J. Luo, R. Shi, Polyethylene glycol inhibits apoptotic cell death following traumatic spinal cord injury, Brain Res. 1155 (2007) 10–16. https://doi.org/10.1016/j.brainres.2007.03.091. [166] I. Elliott Donaghue, M.S. Shoichet, Controlled release of bioactive PDGF-AA from a hydrogel/nanoparticle composite, Acta Biomater. 25 (2015) 35–42. https://doi.org/10.1016/j.actbio.2015.08.002. [167] R.M. Namba, A.A. Cole, K.B. Bjugstad, M.J. Mahoney, Development of porous PEG hydrogels that enable efficient, uniform cell-seeding and permit early neural process extension, Acta Biomater. 5 (2009) 1884–1897. https://doi.org/10.1016/j.actbio.2009.01.036. [168] S.-Q.K.S.O.D.T. Hagop Kantarjian Guillermo Garcia-Manero Hui Yang, Three-dimensional scaffolding to investigate neuronal derivatives of human embryonic stem cells, Bone. 23 (2005) 1–7. https://doi.org/10.1007/s10544-012-9662-7.Three-dimensional. [169] X.Q. Zheng, J.F. Huang, J.L. Lin, Y.X. Zhu, M.Q. Wang, M.L. Guo, X.J. Zan, A.M. Wu, Controlled release of baricitinib from a thermos-responsive hydrogel system inhibits inflammation by suppressing JAK2/STAT3 pathway in acute spinal cord injury, Colloids Surfaces B Biointerfaces. 199 (2021) 111532. https://doi.org/10.1016/j.colsurfb.2020.111532. [170] I. Capila, R.J. Linhardt, Heparin-protein interactions, Angew. Chemie - Int. Ed. 41 (2002) 390– 412. https://doi.org/10.1021/j150493a017.
48 [171] D.R. Coombe, Biological implications of glycosaminoglycan interactions with haemopoietic cytokines, Immunol. Cell Biol. 86 (2008) 598–607. https://doi.org/10.1038/icb.2008.49. [172] S. Roy, H. Lai, R. Zouaoui, J. Duffner, H. Zhou, L. P Jayaraman, G. Zhao, T. Ganguly, T.K. Kishimoto, G. Venkataraman, Bioactivity screening of partially desulfated low-molecular-weight heparins: A structure/activity relationship study, Glycobiology. 21 (2011) 1194–1205. https://doi.org/10.1093/glycob/cwr053. [173] O. Ostrovsky, B. Berman, J. Gallagher, B. Mulloy, D.G. Fernig, M. Delehedde, D. Ron, Differential effects of heparin saccharides on the formation of specific fibroblast growth factor (FGF) and FGF receptor complexes, J. Biol. Chem. 277 (2002) 2444–2453. https://doi.org/10.1074/jbc.M108540200. [174] U. Freudenberg, A. Hermann, P.B. Welzel, K. Stirl, S.C. Schwarz, M. Grimmer, A. Zieris, W. Panyanuwat, S. Zschoche, D. Meinhold, A. Storch, C. Werner, A star-PEG-heparin hydrogel platform to aid cell replacement therapies for neurodegenerative diseases, Biomaterials. 30 (2009) 5049–5060. https://doi.org/10.1016/j.biomaterials.2009.06.002. [175] M. V. Tsurkan, K. Chwalek, S. Prokoph, A. Zieris, K.R. Levental, U. Freudenberg, C. Werner, Defined polymer-peptide conjugates to form cell-instructive starpeg-heparin matrices in situ, Adv. Mater. 25 (2013) 2606–2610. https://doi.org/10.1002/adma.201300691. [176] M. V. Tsurkan, K. Chwalek, K.R. Levental, U. Freudenberg, C. Werner, Modular StarPEG-heparin gels with bifunctional peptide linkers, Macromol. Rapid Commun. 31 (2010) 1529–1533. https://doi.org/10.1002/marc.201000155. [177] A. Watarai, L. Schirmer, S. Thönes, U. Freudenberg, C. Werner, J.C. Simon, U. Anderegg, TGFβ functionalized starPEG-heparin hydrogels modulate human dermal fibroblast growth and differentiation, Acta Biomater. 25 (2015) 65–75. https://doi.org/10.1016/j.actbio.2015.07.036. [178] L. Schirmer, P. Atallah, C. Werner, U. Freudenberg, StarPEG-Heparin Hydrogels to Protect and Sustainably Deliver IL-4, Adv. Healthc. Mater. 5 (2016) 3157–3164. https://doi.org/10.1002/adhm.201600797. [179] M. Kassem, M. Kristiansen, B.M. Abdallah, Mesenchymal stem cells: Cell biology and potential use in therapy, Basic Clin. Pharmacol. Toxicol. 95 (2004) 209–214. https://doi.org/10.1111/j.1742-7843.2004.pto950502.x. [180] S. Wang, X. Qu, R.C. Zhao, Mesenchymal stem cells hold promise for regenerative medicine, Front. Med. China. 5 (2011) 372–378. https://doi.org/10.1007/s11684-011-0164-4. [181] M. Dominici, K. Le Blanc, I. Mueller, I. Slaper-Cortenbach, F.C. Marini, D.S. Krause, R.J. Deans, A. Keating, D.J. Prockop, E.M. Horwitz, Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement, Cytotherapy. 8 (2006) 315–317. https://doi.org/10.1080/14653240600855905. [182] R.M. Samsonraj, M. Raghunath, V. Nurcombe, J.H. Hui, A.J. van Wijnen, S.M. Cool, Concise Review: Multifaceted Characterization of Human Mesenchymal Stem Cells for Use in Regenerative Medicine, Stem Cells Transl. Med. 6 (2017) 2173–2185. https://doi.org/10.1002/sctm.170129. [183] A. J. Braga Osorio Gomes Salgado, R. L. Goncalves Reis, N. Jorge Carvalho Sousa, J. M. Gimble, A. J. Salgado, R. L. Reis, N. Sousa, Adipose Tissue Derived Stem Cells Secretome: Soluble Factors and Their Roles in Regenerative Medicine, Curr. Stem Cell Res. Ther. 5 (2010) 103–110. https://doi.org/10.2174/157488810791268564. [184] S. Lu, C. Lu, Q. Han, J. Li, Z. Du, L. Liao, R.C. Zhao, Adipose-derived mesenchymal stem cells protect PC12 cells from glutamate excitotoxicity-induced apoptosis by upregulation of XIAP through PI3-K/Akt activation, Toxicology. 279 (2011) 189–195. https://doi.org/10.1016/j.tox.2010.10.011.
49 [185] S.K. Kang, E.S. Jun, Y.C. Bae, J.S. Jung, Interactions between human adipose stromal cells and mouse neural stem cells in vitro, Dev. Brain Res. 145 (2003) 141–149. https://doi.org/10.1016/S0165-3806(03)00224-4. [186] S.N. Rafiei Alavi, A. Madani Neishaboori, H. Hossein, A. Sarveazad, M. Yousefifard, Efficacy of adipose tissue-derived stem cells in locomotion recovery after spinal cord injury: a systematic review and meta-analysis on animal studies, Syst. Rev. 10 (2021). https://doi.org/10.1186/s13643-021-01771-w. [187] E.D. Gomes, S.S. Mendes, H. Leite-Almeida, J.M. Gimble, R.Y. Tam, M.S. Shoichet, N. Sousa, N.A. Silva, A.J. Salgado, Combination of a peptide-modified gellan gum hydrogel with cell therapy in a lumbar spinal cord injury animal model, Biomaterials. 105 (2016) 38–51. https://doi.org/10.1016/j.biomaterials.2016.07.019. [188] E.D. Gomes, S.S. Mendes, R.C. Assunção-Silva, F.G. Teixeira, A.O. Pires, S.I. Anjo, B. Manadas, H. Leite-Almeida, J.M. Gimble, N. Sousa, A.C. Lepore, N.A. Silva, A.J. Salgado, Co-Transplantation of Adipose Tissue-Derived Stromal Cells and Olfactory Ensheathing Cells for Spinal Cord Injury Repair, Stem Cells. 36 (2018) 696–708. https://doi.org/10.1002/stem.2785. [189] E.D. Gomes, B. Ghosh, R. Lima, M. Goulão, T. Moreira-Gomes, J. Martins-Macedo, M.W. Urban, M.C. Wright, J.M. Gimble, N. Sousa, N.A. Silva, A.C. Lepore, A.J. Salgado, Combination of a Gellan Gum-Based Hydrogel With Cell Therapy for the Treatment of Cervical Spinal Cord Injury, Front. Bioeng. Biotechnol. 8 (2020) 1–14. https://doi.org/10.3389/fbioe.2020.00984. [190] C.C. Chen, S.F. Yang, I.K. Wang, S.Y. Hsieh, J.X. Yu, T.L. Wu, W.J. Huang, M.H. Su, H.L. Yang, P.C. Chang, A.C. Teng, C. Chia-Yi, S.L. Liang, The Long-Term Efficacy Study of Multiple Allogeneic Canine Adipose Tissue-Derived Mesenchymal Stem Cells Transplantations Combined With Surgery in Four Dogs With Lumbosacral Spinal Cord Injury, Cell Transplant. 31 (2022). https://doi.org/10.1177/09636897221081487. [191] F. Mussano, T. Genova, M. Corsalini, G. Schierano, F. Pettini, D. Di Venere, S. Carossa, Cytokine, Chemokine, and Growth Factor Profile Characterization of Undifferentiated and Osteoinduced Human Adipose-Derived Stem Cells, Stem Cells Int. 2017 (2017). https://doi.org/10.1155/2017/6202783. [192] D.W. Greening, R.J. Simpson, Understanding extracellular vesicle diversity–current status, Expert Rev. Proteomics. 15 (2018) 887–910. https://doi.org/10.1080/14789450.2018.1537788. [193] A.O. Pires, B. Mendes-Pinheiro, F.G. Teixeira, S.I. Anjo, S. Ribeiro-samy, E.D. Gomes, S.C. Serra, N.A. Silva, B. Manadas, A.J. Salgado, Unveiling the Differences of Secretome of Human Bone Marrow Mesenchymal Stem Cells, Adipose Tissue derived Stem Cells and Human Umbilical Cord Perivascular Cells: A Proteomic Analysis, Stem Cells Dev. 25 (2016) 1073–1083. https://doi.org/10.1089/scd.2016.0048. [194] R.C. Assunção-Silva, B. Mendes-Pinheiro, P. Patrício, L.A. Behie, F.G. Teixeira, L. Pinto, A.J. Salgado, Exploiting the impact of the secretome of MSCs isolated from different tissue sources on neuronal differentiation and axonal growth, Biochimie. 155 (2018) 83–91. https://doi.org/10.1016/j.biochi.2018.07.026. [195] M.I. Guillén, J. Platas, M.D. Pérez del Caz, V. Mirabet, M.J. Alcaraz, Paracrine anti-inflammatory effects of adipose tissue-derived mesenchymal stem cells in human monocytes, Front. Physiol. 9 (2018) 1–10. https://doi.org/10.3389/fphys.2018.00661. [196] C.A. Ribeiro, J.S. Fraga, M. Grãos, N.M. Neves, R.L. Reis, J.M. Gimble, N. Sousa, A.J. Salgado, The secretome of stem cells isolated from the adipose tissue and Wharton jelly acts differently on central nervous system derived cell populations, Stem Cell Res. Ther. 3 (2012) 18. https://doi.org/10.1186/scrt109. [197] S.C. Serra, J.C. Costa, R.C. Assunção-Silva, F.G. Teixeira, N.A. Silva, S.I. Anjo, B. Manadas, J.M. Gimble, L.A. Behie, A.J. Salgado, Influence of passage number on the impact of the secretome of
50 adipose tissue stem cells on neural survival, neurodifferentiation and axonal growth, Biochimie. 155 (2018) 119–128. https://doi.org/10.1016/j.biochi.2018.09.012. [198] L.A. Rocha, E.D. Gomes, J.L. Afonso, S. Granja, F. Baltazar, N.A. Silva, M.S. Shoichet, R.A. Sousa, D.A. Learmonth, A.J. Salgado, In vitro Evaluation of ASCs and HUVECs Co-cultures in 3D Biodegradable Hydrogels on Neurite Outgrowth and Vascular Organization, Front. Cell Dev. Biol. 8 (2020) 1–14. https://doi.org/10.3389/fcell.2020.00489. [199] E. Szekiova, L. Slovinska, J. Blasko, J. Plsikova, D. Cizkova, The neuroprotective effect of rat adipose tissue-derived mesenchymal stem cell-conditioned medium on cortical neurons using an in vitro model of SCI inflammation, Neurol. Res. 40 (2018) 258–267. https://doi.org/10.1080/01616412.2018.1432266. [200] H. Valadi, K. Ekström, A. Bossios, M. Sjöstrand, J.J. Lee, J.O. Lötvall, Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells, Nat. Cell Biol. 9 (2007) 654–659. https://doi.org/10.1038/ncb1596. [201] B. Mead, S. Tomarev, Bone Marrow-Derived Mesenchymal Stem Cells-Derived Exosomes Promote Survival of Retinal Ganglion Cells Through miRNA-Dependent Mechanisms, Stem Cells Transl. Med. (2017) 1273–1285. https://doi.org/10.1002/sctm.12056. [202] Y. Rong, W. Liu, J. Wang, J. Fan, Y. Luo, L. Li, F. Kong, J. Chen, P. Tang, W. Cai, Neural stem cell-derived small extracellular vesicles attenuate apoptosis and neuroinflammation after traumatic spinal cord injury by activating autophagy, Cell Death Dis. 10 (2019). https://doi.org/10.1038/s41419-019-1571-8. [203] A.G. Pinho, J.R. Cibrão, R. Lima, E.D. Gomes, S.C. Serra, J. Lentilhas-Graça, C. Ribeiro, S. Lanceros-Mendez, S.F.G. Teixeira, S. Monteiro, N.A. Silva, A.J. Salgado, Immunomodulatory and regenerative effects of the full and fractioned adipose tissue derived stem cells secretome in spinal cord injury, Exp. Neurol. 351 (2022) 113989. https://doi.org/10.1016/j.expneurol.2022.113989. [204] E. Thomas Pashuck, M.M. Stevens, Designing regenerative biomaterial therapies for the clinic, Sci. Transl. Med. 4 (2012). https://doi.org/10.1126/scitranslmed.3002717. [205] Z. Nazemi, M.S. Nourbakhsh, S. Kiani, Y. Heydari, M.K. Ashtiani, H. Daemi, H. Baharvand, Codelivery of minocycline and paclitaxel from injectable hydrogel for treatment of spinal cord injury, J. Control. Release. 321 (2020) 145–158. https://doi.org/10.1016/j.jconrel.2020.02.009. [206] K. Yamane, T. Mazaki, Y. Shiozaki, A. Yoshida, K. Shinohara, M. Nakamura, Y. Yoshida, D. Zhou, T. Kitajima, M. Tanaka, Y. Ito, T. Ozaki, A. Matsukawa, Collagen-Binding Hepatocyte Growth Factor (HGF) alone or with a Gelatin-furfurylamine Hydrogel Enhances Functional Recovery in Mice after Spinal Cord Injury, Sci. Rep. 8 (2018) 1–12. https://doi.org/10.1038/s41598-018-19316-y. [207] B. Ghosh, Z. Wang, J. Nong, M.W. Urban, Z. Zhang, V.A. Trovillion, M.C. Wright, Y. Zhong, A.C. Lepore, Local BDNF delivery to the injured cervical spinal cord using an engineered hydrogel enhances diaphragmatic respiratory function, J. Neurosci. 38 (2018) 5982–5995. https://doi.org/10.1523/JNEUROSCI.3084-17.2018. [208] J.M. Kwiecien, L. Zhang, J.R. Yaron, L.N. Schutz, C.J. Kwiecien-Delaney, E.A. Awo, M. Burgin, W. Dabrowski, A.R. Lucas, Local Serpin Treatment via Chitosan-Collagen Hydrogel after Spinal Cord Injury Reduces Tissue Damage and Improves Neurologic Function, J. Clin. Med. 9 (2020) 1221. https://doi.org/10.3390/jcm9041221. [209] C. Wang, Z. Gong, X. Huang, J. Wang, K. Xia, L. Ying, J. Shu, C. Yu, X. Zhou, F. Li, C. Liang, Q. Chen, An injectable heparin-Laponite hydrogel bridge FGF4 for spinal cord injury by stabilizing microtubule and improving mitochondrial function, Theranostics. 9 (2019) 7016–7032. https://doi.org/10.7150/thno.37601. [210] A. Guijarro-Belmar, M. Viskontas, Y. Wei, X. Bo, D. Shewan, W. Huang, Epac2 Elevation Reverses Inhibition by Chondroitin Sulfate Proteoglycans In Vitro and Transforms Postlesion Inhibitory
51 Environment to Promote Axonal Outgrowth in an Ex Vivo Model of Spinal Cord Injury, J. Neurosci. 39 (2019) 8330–8346. https://doi.org/10.1523/JNEUROSCI.0374-19.2019. [211] Y.Z. Zhao, X. Jiang, J. Xiao, Q. Lin, W.Z. Yu, F.R. Tian, K.L. Mao, W. Yang, H.L. Wong, C.T. Lu, Using NGF heparin-poloxamer thermosensitive hydrogels to enhance the nerve regeneration for spinal cord injury, Acta Biomater. 29 (2016) 71–80. https://doi.org/10.1016/j.actbio.2015.10.014.
CHAPTER II Sustained Release of hASCs Secretome from StarPEG-GAG Hydrogels promotes neuronal differentiation of hNPCS and neurite outgrowth in organotypic spinal cord slices This chapter is part of published work in an international peer reviewed journal: Sustained Release of Human Adipose Tissue Stem Cell Secretome from Star-Shaped Poly (ethylene glycol) Glycosaminoglycan Hydrogels Promotes Motor Improvements after Complete Transection in Spinal Cord Injury Rat Model Deolinda Silva; Lucas Schirmer; Tiffany S. Pinho; Passant Atallah; Jorge R. Cibrão; Rui Lima; João Afonso; Sandra B-Antunes; Cláudia R. Marques; João Dourado; Uwe Freudenberg; Rui A. Sousa; Carsten Werner; António J. Salgado Advanced Healthcare Materials 2023
53 Sustained Release of hASCs Secretome from StarPEG-GAG Hydrogels promotes neuronal differentiation of hNPCS and neurite outgrowth in organotypic spinal cord slices Deolinda Silva 1,2,3; Lucas Schirmer 4; Tiffany S. Pinho1,2,3; Passant Atallah 4; Jorge R. Cibrão1,2; Rui Lima1,2; João Afonso1,2; Sandra B-Antunes1,2,3; Cláudia R. Marques1,2; João Dourado5; Uwe Freudenberg4; Rui A. Sousa 3 ; Carsten Werner 4,6*; António J. Salgado1,2* 1 Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal; 2 ICVS/3B’s – PT Government Associated Laboratory, Braga/Guimarães, Portugal; 3Stemmatters, Biotecnologia e Medicina Regenerativa SA, Guimarães, Portugal; 4Leibniz Institute of Polymer Research Dresden (IPF), Max Bergmann Center of Biomaterials Dresden (MBC), Dresden, Germany; 5School of Medicine, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal 6Technische Universitat Dresden, Center for Regenerative Therapies Dresden (CRTD), Dresden, Germany; *These authors shared senior authorship Corresponding author António Salgado – [email protected]o.pt
60 To obtain samples for these experiments, secretome (15 µL) was mixed with heparin (7.5 µL) and then loaded into starPEG (7.5 µL) into 24-well plate. Secretome was allowed to release by adding Neurobasal medium (1 mL, ThermoFisher, USA) supplemented with 1% penicillin/streptomycin (ThermoFisher, USA). Samples were collected at defined time points (two and ten days) and replaced by an equal volume of fresh medium. The released samples were stored at -80ºC until analyzed by Human Neuro Discovery Array C1 (C-Series RayBiotech, USA) and Human Cytokine Antibody Array C5 (C-Series RayBiotech, USA). For secretome analysis, after blocking the membrane for 30 min at RT, released samples (1 mL) were incubated in each well overnight at 4ºC. After washing five times, the membranes were incubated with a biotinylated antibody cocktail for 2 h at RT. Then membranes were incubated with HRP-Streptavidin for 2 h at RT and analyzed by chemiluminescence detection in Sapphire Biomolecular Imager (Azure Biosystems, USA). Analysis of the membranes was performed in AzureSpot Analysis software (Azure Biosystems, USA) where the relative intensity of each spot was measured. Afterward, quantification was done by subtracting the background in each spot and normalizing it to positive control in each membrane. 2.5. Bioactivity of released secretome in in vitro cultures 2.5.1. Neural progenitor cells (hNPCs) growth and incubation with released secretome To study the potential of secretome released from starPEG-Hep hydrogels in promoting neuronal differentiation hNPCs were used. hNPCs were kindly gifted from Prof. Leo A. Behie (University of Calgary, Canada). Cells were isolated from the telencephalon region of a 10 week post-conception fetus, as described previously [41]. Ethical guidelines were previously established and approved by the Conjoint Health Research Ethics Board (CHREB, University of Calgary, Canada; ID: E-18786). Cells were thawed and cultured in suspension as neurospheres in Complete NeuroCult™ Proliferation Medium (Stem Cell Technologies, California) for 3 days. After that, neurospheres were mechanically triturated using a 200 µL micropipette by pipetting up and down 50-100 times in a firm consistent rhythm until obtaining a single cell suspension. At the end, viable cells were counted and cells seeded (density of 1x104 viable cells/cm2) in fresh medium. hNPCs were maintained in culture for 10-14 days until obtain neurospheres with approximately 100 µm diameter, adding medium every two days as suggested in reference protocol (StemCells Technologies, California). For differentiation assays, neurospheres were mechanically dissociated as previously described and plated in 24-well plates in coverslips coated with poly-D-Lysine (100 µg/mL; Merck, USA) and laminin (10 µg/mL; Merck, USA) using 60,000 cells per well. After seeding
61 hNPCs on the bottom, hydrogels were prepared and pipetted onto the insert membrane as illustrated in Figure II.1A. Briefly, concentrated secretome (15µL) collected in Neurobasal A was mixed with Heparin (7.5µL) and starPEG (7.5µL) and pipetted per well. NbA supplemented with 1% kanamycin and 1 % GlutaMAX (Gibco, USA) (basal medium) was added as negative control, and the same amount of loaded secretome diluted in this medium added as a free condition. Additionally, NbA supplemented with B27 (2%, ThermoFisher, USA), FGF-2 (0.05%; R&D Systems, USA), kanamycin (1%) and GlutaMAX (1%) was used as positive control. Cultures were maintained for 5 days. 2.5.2. Spinal cord slices isolation and incubation with released secretome Organotypic spinal cord slice cultures were prepared from Wistar Han rats of postnatal day 7 (P7). Briefly, pups were decapitated, the spinal cord exposed, and the thoracic segment removed and placed in icecold high glucose (6mg/mL) Hanks’ Balanced Salt Solution (HBSS) without Ca2+ and Mg2+(ThermoFisher, USA) with 1 % pen/strep for some time. Under sterile conditions, meninges and roots were removed to ensure the cleaning of the tissue. Then spinal cord was sectioned in a Mcllwain Tissue Chopper (Gomshall, UK), transversal segments with 350 µm thickness and placed in cold HBSS. Rat tail type I collagen (3.4 mg/mL, BD Biosciences, USA) was prepared by adding Basal Eagle’s medium (10x, Gibco, USA) in a proportion of collagen (450µL) to basal medium (50µL) and sodium bicarbonate solution (7.5%, 2µL) [42]. Single drops (30µL) were placed on 24-well plate and incubated at 37ºC and 5% CO2 for approximately 1h to promote collagen gel formation. Then, viable slices were deposited on top of collagen drops. Additionally, drops of starPEG hydrogel loading secretome (30µL) were allowed to polymerized and release secretome in the same well as illustrated in Figure II.1B. Afterwards, Neurobasal medium supplemented with B27 (2%, ThermoFisher, USA), glucose (2%, 300mg, Merck, USA), LGlutamine (1%, ThermoFisher, USA) and 1% pen/strep. A total of 200µL was added to each well and medium changed after 2 days and the cultures maintained at 37ºC and 5% CO2. 2.5.3. Immunocytochemistry hNPCs and Spinal Cord slices were fixed with 4% paraformaldehyde (PFA; Merck, Portugal) for 20 and 45 min at RT, respectively, to retain antigenicity of the target molecules and preserve cell morphology. For hNPCs, permeabilization was performed using 0.3% Triton X-100 in PBS (PBS-T; Merck, USA) for five min, followed by blocking of non-specific binding sites using PBS with newborn calf serum (10 %, NBCS, Biochrom, Germany) or 1h. In case of spinal cord slices permeabilization and blocking were performed
62 using phosphate-buffered saline (PBS) with Triton X-100 (0.5%, PBS-T, Merck, USA) NBCS (10%) for 1 h at RT. hNPCs were incubated with the following primary antibodies: rabbit anti-doublecortin (DCX, , 1:300, Abcam, UK) for immature neurons and anti-microtubule associated protein-2 (MAP-2, 1:500, Merck, USA) for more mature neurons, diluted in PBS with NBCS (10%) for 1h at RT, while slices incubated with antineurofilament (NF, 1:200; Merck, USA) in PBS-T 0.5% +10% NBCS for 48h at 4 ºC. After washing 3x with PBS with NBCS (0.5%), cells were incubated with secondary antibodies Alexa Fluor 488 goat anti-rabbit (1:1000; Thermo) and Alexa Fluor 594 goat anti-mouse (1:1000; ThermoFisher, USA) for DCX and MAP2 detection, respectively, for 1 h 30 at RT. Spinal cord slices were incubated with Alexa Fluor 488 goat anti-mouse (1:1000; ThermoFisher, USA) for 24h at 4 ºC. Cell nuclei were stained with 4-6-diamidino-2phenylindoledhydrochloride (DAPI, 1:1000; ThermoFisher, California) for five and 30 min at RT, hNPCs and spinal cord slices, respectively. Imaging was performed with a fluorescence microscope (BX61, Olympus, Germany) for hNPCS and with a confocal point-scanning microscope Olympus FV1000 for spinal cord slices. 2.5.4. Neuronal differentiation analysis Cell counts were performed under blinded conditions using Image J (NIH) software for quantification. Ten representative fields per condition were selected and analyzed. The number of positive cells for DCX and MAP-2 were counted per field and normalized to a total number of cells in each field stained with DAPI. Results are presented as a percentage of differentiated cells. 2.5.5. Neurite extension analysis To quantify the area occupied by neurites, Fiji software was used. Firstly, the scale was defined, and the total slice area was measured, using the proper drawing tools. Then applying the threshold contrast was possible to emphasize the neurite identification. Using the function "Analyze particles," the total area occupied by neurite was calculated. The results were then normalized to the total area of the slice and presented as a percentage of neurite area.
63 Figure II. 1 Schematic representation of in vitro cultures to evaluate the bioactivity of hASCs secretome released from starPEG-Hep hydrogels. Capacity to promote differentiation of human Neural Progenitor Cells (hNPCs) (A) and neurite outgrowth in organotypic spinal cord slices (B) were performed under described conditions. 2.6. Statistical Analysis Data regarding neurodifferentiation of hNPCs was analyzed using Mixed ANOVA to compare the mean values of five groups. When evaluating the neurite outgrowth in spinal cord slices one-way ANOVA was performed. A pairwise comparison between groups based on estimated marginal means using Turkey’s correction was performed. The significance value was set as p ≤ 0.05 for all statistical tests and graphs are presented as mean ± SEM. For all data normality was assessed using Shapiro-Wilk statistical tests and taking into account the measures of skewness and kurtosis. Moreover, is important to highlight that for repeated measures ANOVA distribution can be approximately normal distributed [43]. IBM SPSS® Statistics version 27 for IOS (IBM Co., USA) was used to perform all statistical analysis. For graphical representations GraphPad Prism ver.8.4.3 (GraphPad Software, La Jolla, USA) was used. 3. Results 3.1. Physical characterization of in-situ forming hydrogels Hydrogels were formed by desulfated heparin derivatives bearing six maleimide groups and thiolterminated starPEG. Hydrogels were characterized with respect to their physicochemical network properties, such as stiffness, mesh size and swelling (Figure II. 2 and Supplementary Table II. S1). The physical properties of hydrogels can be modulated by the number of starPEG molecules reacting with
64 heparin maleimide group, changing the degree of crosslinking, and by the solid content [22]. Increasing the crosslinking degree from 0.75 to 1 and 1.5, hydrogels increase their storage modulus from 1.01 ± 0.13 to 2.69 ± 0.30 to 7.14 ± 0.97 kPa, respectively (Figure II.2A), which range from soft to stiff materials. On the other hand, mesh size and swelling degree decrease with increased solid content, or a higher number of starPEG molecules in solution (Figure II. 2B and C). To notice, the modulation of physical properties is extremely important for further implantation in vivo. Figure II. 2 Physical characterization of starPEG-Hep hydrogels. At different crosslinking degrees (starPEG/Hep molar ratio) hydrogels characteristics such as stiffness (A), mesh size (B) and swelling degree (C) were measured. Hydrogels can vary from soft to stiff materials, decrease mesh size and swelling degree with increased crosslinking degree. Measurements are plotted as mean ± SEM, corresponding to 9 replicates 3.2. Characterization of secretome released from starPEG-Hep hydrogels In the context of SCI, administering a therapy is still a concern, considering the administration route and associated problems, such as toxic side effects promoted by high doses or the rapid clearance by the fluids at injury site. Moreover, the enzymatic degradation at the injury site can compromise the bioactivity of drugs and molecules [44].
65 To minimize these problems, starPEG-GAG hydrogels can be easily integrated at the lesion site and bind, protect, and sustainably deliver the secretome of ASCs. As the secretome of ASCs has already been characterized to be composed of a wide range of signaling factors [29,30,45], our approach capitalize from the high number of binding sites within the starPEG-Hep hydrogels that allow for independent release of multiple factors [46]. Several release assays were conducted to evaluate the release profile of the hASCs secretome from starPEG-Hep hydrogels. In a first approach, the secretome was labeled with a FITC dye that binds to amine groups of proteins, enabling their detection by fluorescence intensity. Then, the labeled secretome was loaded into starPEG-Hep hydrogels with an efficiency of immobilization of about 95% and incubated in phosphate buffered saline (PBS) with 1% bovine serum albumin (BSA). To characterize the release of the secretome, samples from the supernatant that have been in contact with the hydrogel were collected over ten days, revealing a controlled and prolonged release profile (Figure II. 3A). Briefly, after a burst release during the first day (approximately 45%) the hydrogel showed a cumulative release of the secretome until ten days, reaching approximately 70% of release at that time point of the experiment. Over the following 9 days, a sustained release of the secretome was detected. Regarding multiplex results, Gal-1 was detected to be continuously released for the entire time of the experiment (Figure II. 3B). Figure II. 3 Cumulative release of hASCs secretome from starPEG-Hep hydrogels. A, Prior of loading in starPEG-Hep hydrogels, secretome was labeled with a FITC dye and samples collected at 0 and 3h, 1, 2, 3, 4, 5, 6, 7 and 10 days. Secretome was detected by fluorescence absorbance and percentage of cumulative release calculated over time. B, For Multiplex assay, samples were collected at 0, 3h, 1,3,5,7,10 days and Gal-1 was detected to be controlled and prolonged released over that time. Values
66 are plotted as mean ± SEM from 2 independent experiment white four replicates each (A), and one independent experiment (B). Though these results have shown the potential of the hydrogel as a good release system, secretome is composed of a wide range of proteins, cytokines growth factors and other molecules [29]. In order to decipher more closely which components of the secretome were being released from starPEG-Hep hydrogels, membrane-based protein arrays were used (Figure II 4A and B). For this purpose, samples of released secretome were collected only at 2 and 10 days and evaluated separately using Human Neuro Discovery Array C1 and Human Cytokine Antibody Array C5 (Figure II. 4 A-D), allowing the detection of known ASCs released neurotrophic factors and cytokines after two and ten days, respectively. Each dot was quantified and normalized to the positive controls to determine the relative amount released at different time points. The full array of molecules detected, and their relative expression can be found in Supplementary Figures II. S1 and S2. Membrane arrays revealed a different release profile of neuroinflammatory and angiogenic factors (Figure II. 4C) from neuroregulatory molecules (Figure II. 4D). In fact, cytokines that play a role in modulating the immune response such as IL-1α, IL-1ß, IL-2, IL-3 and IL-4, or factors involved in angiogenesis like angiogenin or VEGF had a burst release after two days. Despite that, the cumulative release observed after ten days evidenced their continuous and extended release. In contrast, growth factors that promote neuronal growth and survival like BDNF, ß-NGF, HB-EGF are mainly released from two to ten days, indicating overall an instant release of immune modulating factors and slightly delayed but therefore more prolonged release of neuro-regulating factors. Moreover, it was possible to detect a wide range of molecules present in the secretome of hASCs that were continuously released from hydrogels such as FGF, TGF-ß, HGF, NT-3, NT-4, IL-16 (Supplementary Figure II. S1 and S2). Altogether, these results support and explain the cumulative release profile presented in Figure II. 3A. While Figure II. 3A shows the total release of the secretome, membrane-based protein arrays show which of these factors are released over time. The burst release results from high amounts of neuroinflammatory and angiogenic factors released in the first days, while the continuous and prolonged release until ten days is driven mainly by neuroregulatory growth factors.
67 Figure II. 4 Evaluation of hASCs secretome release profile from starPEG-Hep hydrogels using membrane-based protein arrays. A and B show the membranes of Cytokines Antibody arrays and Neuro Discovery arrays, respectively. C, Relative intensity of factors involved in neuroinflammatory and angiogenesis, such as IL-4, tissue inhibitor of metalloproteinase (TIMP)-1, and VEGF, present a higher release in the first two days. On the opposite neuroregulatory molecules (C) like BDNF, β-NGF, or TGF-β are mainly released within two to ten days of release. Values are presented as relative intensity, in percentage, for the positive control in each membrane. 3.3. hASCs secretome released from starPEG-Hep hydrogels promote differentiation of hNPCs Testing of the bioactivity of the developed hydrogel system then followed through the evaluation of the differentiation of hNPCs. For this goal, hNPCs were seeded as a single monolayer of adherent cells on precoated coverslips with poly-D-Lysin and laminin, after which secretome-loaded starPEG-Hep (starPEGHep+sec) hydrogels were placed on an insert, above the cell layer. After 5 days in culture, differentiation of hNPCs was assessed by immunocytochemistry analysis for doublecortin positive cells (DCX+) and microtubule-associated protein positive cells (MAP2+), staining immature and mature neurons, respectively. Fluorescence microscopy imagens showed hNPCs differentiation (Figure II. 5A). Statistical analysis showed that there was an effect of factor treatment (F (4, 474) = 57.87, p<0.0001, η2partial = 0.33),
68 and the differentiation condition (F (1, 474) = 153.66, p<0.0001, η2partial = 0.25), and the interaction between this two factors (F(4, 474) = 40.15, p<0.0001, η2partial = 0.25). As shown in Figure II. 5B, hASCs secretome released from starPEG-Hep hydrogels present significative higher percentage of DCX+ cells when compared with hydrogel loading the vehicle in which secretome was collected (Neurobasal A) (starPEG-Hep+NbA) (n = 120; 25.47±17.90 vs n = 99; 9.08± 12.66; p<0.0001) or the vehicle alone (n = 80; 17.57±15.37; p<0.0001). No statistical differences were observed when comparing with control (n = 100; 24.77±13.87; p = 0.72) or free secretome (n = 80;28.78±9.97; p = 0.11), which was in contact with cells for the entire experiment. The same tendency is observed regarding the staining for mature neurons (MAP2*). Cells treated with secretome released from starPEG-Hep hydrogels promoted a higher differentiation for MAP2+ cells (Figure II. 5C) compared to cells treated with the vehicle released from the hydrogel (n = 120;30.75±19.80 vs n = 99;10.43±13.23; p<0.0001) and cells treated only with vehicle (n = 80; 17.95±15.01; p<0.0001). Significance was also observed when comparing with control (n = 100; 44.78±13.34; p<0.0001) or free secretome (n = 80; 36.72±11.74; p = 0.007). All statistical values are presented in Supplementary Table II. S2.
69 Figure II. 5 Effect of hASCs secretome released from starPEG-Hep hydrogels in hNPCs differentiation in vitro . A, Representative micrographs of hNPCs differentiation in immature (DCX) and mature (MAP2) neurons when exposed to control, secretome or NbA released from starPEG-Hep hydrogels, free secretome or NbA conditions. Nuclei are stained with DAPI and neurons with MAP2 and DCX. B-C, Quantification of the percentage of DCX+, MAP2+ cells from total cells, respectively. Results represented three independent experiments with 8/12 replicates in a total of ten representative fields per replicate. Repeated measures ANOVA; *** p < 0.0001. Error bars represented mean ± SEM. Scale bar: 50 μm. MAP2 microtubule associated protein 2, DCX doublecortin, DAPI 4’,6’-diamino-2-fenil-indol
76 Acknowledgments This work was supported by Prmios Santa Casa Neurociencias–Prize Melo e Castro for Spinal Cord Injury Research (MC-04/17; MC-18-2021) and the Portuguese Foundation for Science and Technology (Ph.D. Fellowship to D.S (PD/BDE/135567/2018 and COVID/BDE/152051/2022); T. S. P. (PD/BDE/143150/2019); J.R.C (SFRH/BD/145860/2019); J.A (2021.08337.BD); S.B.-A ((PD/BDE/135568/2018). This work was funded by national funds and FEDER, through the Foundation for Science and Technology (FCT), under the scope of the projects UIDB/50026/2020; UIDP/50026/2020; POCI-01-0145-FEDER-029206; POCI-01-0145-FEDER-031392; PTDC/ MEDNEU/31417/2017; NORTE-01-0145-FEDER-029968; POCI-01-0145-FEDER-029751; POCI-01-0145FEDER-032619. This work has been funded by ICVS Scientific Microscopy Platform, a member of the national infrastructure PPBI - Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122. This work has also been developed under the scope of the project NORTE-01-0145FEDER-000013 and NORTE-01-0145-FEDER-000023, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). Work supported by the Portuguese Foundation for Science and Technology (FCT): projects UID/FIS/04650/2020, PTDC/EMD-EMD/28159/2017, and PTDC/BTMMAT/28237/2017. Dr. Jeff Gimble and LaCell, Inc. kindly provided the adipose tissue-derived stem cells used in this study. Nelly Rein for her technical advice and support in working with the hydrogel material. Author contributions D.S designed and performed most of the experiments, collected and analyzed the data, and drafted the manuscript. T.S.P, J.C.R, R.L, J.A, S.B-A, C.R.M, JD helped in vitro and animal experiments. P.A, L.S helped in designing experiments. U. F. and C.W provided hydrogel materials. A.J.S conceived and financially support the study, participated in its design and coordination. L.S, U. F., C.W., A.J. S. critically read the manuscript. Supervision: R.A.S, U. F., C.W., A.J.S. All authors read and approved the final manuscript. Conflict of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
77 Data availability The data that supports this study is available from the authors upon reasonable request.
78 References [1] S. David, A.J. Aguayo, Axonal elongation into peripheral nervous system “bridges” after central nervous system injury in adult rats, Science (80-. ). 214 (1981) 931–933. https://doi.org/10.1126/science.6171034. [2] J. Silver, Jerry; Miller, Regeneration beyond the glial scar, Sci. Rep. 9 (2018) 1–10. https://doi.org/10.1016/j.physbeh.2017.03.040. [3] G. Yiu, Z. He, Glial inhibition of CNS axon regeneration Glenn, Net Rev Neurosci. 7 (2006) 617– 627. https://doi.org/10.1038/nrn1956.Glial. [4] C.C. Stichel, H.W. Müller, Experimental strategies to promote axonal regeneration after traumatic central nervous system injury, Prog. Neurobiol. 56 (1998) 119–148. https://doi.org/10.1016/S0301-0082(98)00033-1. [5] L.F. Martins, R.O. Costa, J.R. Pedro, P. Aguiar, S.C. Serra, F.G. Teixeira, N. Sousa, A.J. Salgado, R.D. Almeida, Mesenchymal stem cells secretome-induced axonal outgrowth is mediated by BDNF, Sci. Rep. 7 (2017) 4153. https://doi.org/10.1038/s41598-017-03592-1. [6] P. Lu, L.L. Jones, M.H. Tuszynski, BDNF-expressing marrow stromal cells support extensive axonal growth at sites of spinal cord injury, Exp. Neurol. 191 (2005) 344–360. https://doi.org/10.1016/j.expneurol.2004.09.018. [7] M.M. Migliore, R. Ortiz, S. Dye, R.B. Campbell, M.M. Amiji, B.L. Waszczak, Neurotrophic and neuroprotective efficacy of intranasal GDNF in a rat model of Parkinson’s disease, Neuroscience. 274 (2014) 11–23. https://doi.org/10.1016/j.neuroscience.2014.05.019. [8] M.B. Bracken, M.J. Shepard, T.R. Holford, L. Leo-Summers, E.F. Aldrich, M. Fazl, M. Fehlings, D.L. Herr, P.W. Hitchon, L.F. Marshall, R.P. Nockels, V. Pascale, P.L. Perot, J. Piepmeier, V.K.H. Sonntag, F. Wagner, J.E. Wilberger, H.R. Winn, W. Young, Administration of methylprednisolone for 24 or 48 hours or tirilazad mesylate for 48 hours in the treatment of acute spinal cord injury: Results of the Third National Acute Spinal Cord Injury randomized controlled trial, J. Am. Med. Assoc. 277 (1997) 1597–1604. https://doi.org/10.1097/00132586-199808000-00011. [9] V.W. Yong, J. Wells, F. Giuliani, S. Casha, C. Power, L.M. Metz, The promise of minocycline in neurology, Lancet Neurol. 3 (2004) 744–751. https://doi.org/10.1016/S1474-4422(04)009378. [10] T. Hachiya, Y. Ochi, M. Yoshimura, T. Miyazaki, Serum T3 Level in the Patients with Hyperthyroidism After Therapy, Endocrinol. Jpn. 22 (1975) 255–260. https://doi.org/10.1507/endocrj1954.22.255. [11] Z. Liu, Y. Yang, L. He, M. Pang, C. Luo, B. Liu, L. Rong, High-dose methylprednisolone for acute traumatic spinal cord injury: A meta-analysis, Neurology. 93 (2019) e841–e850. https://doi.org/10.1212/WNL.0000000000007998. [12] F.G. Teixeira, M.M. Carvalho, K.M. Panchalingam, A.J. Rodrigues, B. Mendes-pinheiro, S.I. Anjo, B. Manadas, L.A. Behie, N. Sousa, A.J. Salgado, Impact of the Secretome of Human Mesenchymal Stem Cells on Brain Structure and Animal Behavior in a Rat Model of Parkinson’s Disease, Stem Cells Transl. Med. (2017) 634–646. https://doi.org/10.5966/sctm.2016-0071. [13] K.A. Follett, R.L. Boortz-Marx, J.M. Drake, S. DuPen, S.J. Schneider, M.S. Turner, R.J. Coffey, Prevention and management of intrathecal drug delivery and spinal cord stimulation system infections, Anesthesiology. 100 (2004) 1582–1594. https://doi.org/10.1097/00000542200406000-00034. [14] L.L. Jones, M.H. Tuszynski, Chronic intrathecal infusions after spinal cord injury cause scarring and compression, Microsc. Res. Tech. 54 (2001) 317–324. https://doi.org/10.1002/jemt.1144. [15] B. Ghosh, Z. Wang, J. Nong, M.W. Urban, Z. Zhang, V.A. Trovillion, M.C. Wright, Y. Zhong, A.C. Lepore, Local BDNF delivery to the injured cervical spinal cord using an engineered hydrogel
79 enhances diaphragmatic respiratory function, J. Neurosci. 38 (2018) 5982–5995. https://doi.org/10.1523/JNEUROSCI.3084-17.2018. [16] E.D. Gomes, S.S. Mendes, H. Leite-Almeida, J.M. Gimble, R.Y. Tam, M.S. Shoichet, N. Sousa, N.A. Silva, A.J. Salgado, Combination of a peptide-modified gellan gum hydrogel with cell therapy in a lumbar spinal cord injury animal model, Biomaterials. 105 (2016) 38–51. https://doi.org/10.1016/j.biomaterials.2016.07.019. [17] Z. Nazemi, M.S. Nourbakhsh, S. Kiani, Y. Heydari, M.K. Ashtiani, H. Daemi, H. Baharvand, Codelivery of minocycline and paclitaxel from injectable hydrogel for treatment of spinal cord injury, J. Control. Release. 321 (2020) 145–158. https://doi.org/10.1016/j.jconrel.2020.02.009. [18] G.E. Rooney, A.M. Knight, N.N. Madigan, L. Gross, B. Chen, C.V. Giraldo, S. Seo, J.J. Nesbitt, M. Dadsetan, M.J. Yaszemski, A.J. Windebank, Sustained delivery of dibutyryl cyclic adenosine monophosphate to the transected spinal cord via oligo [(polyethylene glycol) fumarate] hydrogels, Tissue Eng. - Part A. 17 (2011) 1287–1302. https://doi.org/10.1089/ten.tea.2010.0396. [19] A.S. Samaddar, P. Chatterjee, A. Roy, Thermosensitive heparin-poloxamer hydrogels enhance the effects of GDNF on neuronal circuit remodelling and neuroprotection after spinal cord injury, Microbiol. Res. 2 (2018) 1–38. https://doi.org/10.1016/j.micres.2018.11.005. [20] B. Chen, J. He, H. Yang, Q. Zhang, L. Zhang, X. Zhang, E. Xie, C. Liu, R. Zhang, Y. Wang, L. Huang, D. Hao, Repair of spinal cord injury by implantation of bFGF-incorporated HEMA-MOETACL hydrogel in rats, Sci. Rep. 5 (2015) 1–10. https://doi.org/10.1038/srep09017. [21] U. Freudenberg, A. Hermann, P.B. Welzel, K. Stirl, S.C. Schwarz, M. Grimmer, A. Zieris, W. Panyanuwat, S. Zschoche, D. Meinhold, A. Storch, C. Werner, A star-PEG-heparin hydrogel platform to aid cell replacement therapies for neurodegenerative diseases, Biomaterials. 30 (2009) 5049–5060. https://doi.org/10.1016/j.biomaterials.2009.06.002. [22] M. V. Tsurkan, K. Chwalek, S. Prokoph, A. Zieris, K.R. Levental, U. Freudenberg, C. Werner, Defined polymer-peptide conjugates to form cell-instructive starpeg-heparin matrices in situ, Adv. Mater. 25 (2013) 2606–2610. https://doi.org/10.1002/adma.201300691. [23] O. Ostrovsky, B. Berman, J. Gallagher, B. Mulloy, D.G. Fernig, M. Delehedde, D. Ron, Differential effects of heparin saccharides on the formation of specific fibroblast growth factor (FGF) and FGF receptor complexes, J. Biol. Chem. 277 (2002) 2444–2453. https://doi.org/10.1074/jbc.M108540200. [24] F. Peysselon, S. Ricard-Blum, Heparin-protein interactions: From affinity and kinetics to biological roles. Application to an interaction network regulating angiogenesis, Matrix Biol. 35 (2014) 73– 81. https://doi.org/10.1016/j.matbio.2013.11.001. [25] A. Watarai, L. Schirmer, S. Thönes, U. Freudenberg, C. Werner, J.C. Simon, U. Anderegg, TGFβ functionalized starPEG-heparin hydrogels modulate human dermal fibroblast growth and differentiation, Acta Biomater. 25 (2015) 65–75. https://doi.org/10.1016/j.actbio.2015.07.036. [26] S. Prokoph, E. Chavakis, K.R. Levental, A. Zieris, U. Freudenberg, S. Dimmeler, C. Werner, Sustained delivery of SDF-1α from heparin-based hydrogels to attract circulating pro-angiogenic cells, Biomaterials. 33 (2012) 4792–4800. https://doi.org/10.1016/j.biomaterials.2012.03.039. [27] K. Schurig, A. Zieris, A. Hermann, U. Freudenberg, S. Heidel, M. Grimmer, A. Storch, C. Werner, Neurotropic growth factors and glycosaminoglycan based matrices to induce dopaminergic tissue formation, Biomaterials. 67 (2015) 205–213. https://doi.org/10.1016/j.biomaterials.2015.07.029. [28] L. Schirmer, P. Atallah, C. Werner, U. Freudenberg, StarPEG-Heparin Hydrogels to Protect and Sustainably Deliver IL-4, Adv. Healthc. Mater. 5 (2016) 3157–3164. https://doi.org/10.1002/adhm.201600797.
80 [29] A.O. Pires, B. Mendes-Pinheiro, F.G. Teixeira, S.I. Anjo, S. Ribeiro-samy, E.D. Gomes, S.C. Serra, N.A. Silva, B. Manadas, A.J. Salgado, Unveiling the Differences of Secretome of Human Bone Marrow Mesenchymal Stem Cells, Adipose Tissue derived Stem Cells and Human Umbilical Cord Perivascular Cells: A Proteomic Analysis, Stem Cells Dev. 25 (2016) 1073–1083. https://doi.org/10.1089/scd.2016.0048. [30] S.C. Serra, J.C. Costa, R.C. Assunção-Silva, F.G. Teixeira, N.A. Silva, S.I. Anjo, B. Manadas, J.M. Gimble, L.A. Behie, A.J. Salgado, Influence of passage number on the impact of the secretome of adipose tissue stem cells on neural survival, neurodifferentiation and axonal growth, Biochimie. 155 (2018) 119–128. https://doi.org/10.1016/j.biochi.2018.09.012. [31] C.A. Ribeiro, J.S. Fraga, M. Grãos, N.M. Neves, R.L. Reis, J.M. Gimble, N. Sousa, A.J. Salgado, The secretome of stem cells isolated from the adipose tissue and Wharton jelly acts differently on central nervous system derived cell populations, Stem Cell Res. Ther. 3 (2012) 18. https://doi.org/10.1186/scrt109. [32] R.C. Assunção-Silva, B. Mendes-Pinheiro, P. Patrício, L.A. Behie, F.G. Teixeira, L. Pinto, A.J. Salgado, Exploiting the impact of the secretome of MSCs isolated from different tissue sources on neuronal differentiation and axonal growth, Biochimie. 155 (2018) 83–91. https://doi.org/10.1016/j.biochi.2018.07.026. [33] E.D. Gomes, S.S. Mendes, R.C. Assunção-Silva, F.G. Teixeira, A.O. Pires, S.I. Anjo, B. Manadas, H. Leite-Almeida, J.M. Gimble, N. Sousa, A.C. Lepore, N.A. Silva, A.J. Salgado, Co-Transplantation of Adipose Tissue-Derived Stromal Cells and Olfactory Ensheathing Cells for Spinal Cord Injury Repair, Stem Cells. 36 (2018) 696–708. https://doi.org/10.1002/stem.2785. [34] S. Lu, C. Lu, Q. Han, J. Li, Z. Du, L. Liao, R.C. Zhao, Adipose-derived mesenchymal stem cells protect PC12 cells from glutamate excitotoxicity-induced apoptosis by upregulation of XIAP through PI3-K/Akt activation, Toxicology. 279 (2011) 189–195. https://doi.org/10.1016/j.tox.2010.10.011. [35] A.G. Pinho, J.R. Cibrão, R. Lima, E.D. Gomes, S.C. Serra, J. Lentilhas-Graça, C. Ribeiro, S. Lanceros-Mendez, S.F.G. Teixeira, S. Monteiro, N.A. Silva, A.J. Salgado, Immunomodulatory and regenerative effects of the full and fractioned adipose tissue derived stem cells secretome in spinal cord injury, Exp. Neurol. 351 (2022) 113989. https://doi.org/10.1016/j.expneurol.2022.113989. [36] A. J. Braga Osorio Gomes Salgado, R. L. Goncalves Reis, N. Jorge Carvalho Sousa, J. M. Gimble, A. J. Salgado, R. L. Reis, N. Sousa, Adipose Tissue Derived Stem Cells Secretome: Soluble Factors and Their Roles in Regenerative Medicine, Curr. Stem Cell Res. Ther. 5 (2010) 103–110. https://doi.org/10.2174/157488810791268564. [37] J. Rehman, D. Traktuev, J. Li, S. Merfeld-Clauss, C.J. Temm-Grove, J.E. Bovenkerk, C.L. Pell, B.H. Johnstone, R. V. Considine, K.L. March, Secretion of Angiogenic and Antiapoptotic Factors by Human Adipose Stromal Cells, Circulation. 109 (2004) 1292–1298. https://doi.org/10.1161/01.CIR.0000121425.42966.F1. [38] Q. Chai, Y. Jiao, X. Yu, Hydrogels for Biomedical Applications: Their Characteristics and the Mechanisms behind Them, Gels. 3 (2017) 6. https://doi.org/10.3390/gels3010006. [39] M. Rubinstein, R.H. Colby, Polymer physics, 2003. [40] S.G. Dubois, E.Z. Floyd, S. Zvonic, G. Kilroy, X. Wu, S. Carling, Y.D.C. Halvorsen, E. Ravussin, J.M. Gimble, Isolation of human adipose-derived stem cells from biopsies and liposuction specimens, Methods Mol. Biol. 449 (2008) 69–79. https://doi.org/10.1007/978-1-60327-169-1_5. [41] F.G. Teixeira, K.M. Panchalingam, S.I. Anjo, B. Manadas, R. Pereira, N. Sousa, A.J. Salgado, L.A. Behie, Do hypoxia/normoxia culturing conditions change the neuroregulatory profile of Wharton Jelly mesenchymal stem cell secretome?, Stem Cell Res. Ther. 6 (2015) 1–14. https://doi.org/10.1186/s13287-015-0124-z.
81 [42] A. Tucker, Cranial motor axons respond differently to the floor plate and sensory ganglia in collagen gel co-cultures, Eur. J. Neurosci. 8 (1996) 906–916. https://doi.org/10.1111/j.14609568.1996.tb01577.x. [43] LUND ADAM, L. MARK, Laerd Statistics, (n.d.). https://statistics.laerd.com/spss-tutorials/oneway-anova-repeated-measures-using-spss-statistics.php (accessed January 6, 2022). [44] J.E. Springer, R.D. Azbill, S.E. Kennedy, J. George, J.W. Geddes, Rapid calpain I activation and cytoskeletal protein degradation following traumatic spinal cord injury: Attenuation with riluzole pretreatment, J. Neurochem. 69 (1997) 1592–1600. https://doi.org/10.1046/j.14714159.1997.69041592.x. [45] R. Vawda, A. Badner, J. Hong, M. Mikhail, R. Dragas, K. Xhima, A. Jose, M.G. Fehlings, Harnessing the Secretome of Mesenchymal Stromal Cells for Traumatic Spinal Cord Injury: Multicell Comparison and Assessment of in Vivo Efficacy, Stem Cells Dev. 29 (2020) 1429–1443. https://doi.org/10.1089/scd.2020.0079. [46] A. Zieris, K. Chwalek, S. Prokoph, K.R. Levental, P.B. Welzel, U. Freudenberg, C. Werner, Dual independent delivery of pro-angiogenic growth factors from starPEG-heparin hydrogels, J. Control. Release. 156 (2011) 28–36. https://doi.org/10.1016/j.jconrel.2011.06.042. [47] D. Dooley, E. Lemmens, P. Ponsaerts, S. Hendrix, Interleukin-25 is detrimental for recovery after spinal cord injury in mice, J. Neuroinflammation. 13 (2016) 1–6. https://doi.org/10.1186/s12974-016-0566-y. [48] H.J. Lee, J.S. Ryu, P.J. Vig, Current strategies for therapeutic drug delivery after traumatic CNS injury, Ther. Deliv. 10 (2019) 251–263. https://doi.org/10.4155/tde-2019-0006. [49] G. Perale, F. Rossi, E. Sundstrom, S. Bacchiega, M. Masi, G. Forloni, P. Veglianese, Hydrogels in spinal cord injury repair strategies, ACS Chem. Neurosci. 2 (2011) 336–345. https://doi.org/10.1021/cn200030w. [50] D. Silva, R.A. Sousa, A.J. Salgado, Hydrogels as delivery systems for spinal cord injury regeneration, Mater. Today Bio. 9 (2021) 100093. https://doi.org/10.1016/j.mtbio.2021.100093. [51] P. Atallah, L. Schirmer, M. Tsurkan, Y.D. Putra Limasale, R. Zimmermann, C. Werner, U. Freudenberg, In situ-forming, cell-instructive hydrogels based on glycosaminoglycans with varied sulfation patterns, Biomaterials. 181 (2018) 227–239. https://doi.org/10.1016/j.biomaterials.2018.07.056. [52] A.G. Pinho, J.R. Cibrão, N.A. Silva, S. Monteiro, A.J. Salgado, Cell secretome: Basic insights and therapeutic opportunities for CNS disorders, Pharmaceuticals. 13 (2020) 1–18. https://doi.org/10.3390/ph13020031. [53] U. Freudenberg, Y. Liang, K.L. Kiick, C. Werner, Glycosaminoglycan-based biohybrid hydrogels: a sweet and smart choice for multifunctional biomaterials, Adv. Mater. 28 (2016) 8861–8891. https://doi.org/10.1016/j.physbeh.2017.03.040. [54] U. Freudenberg, A. Zieris, K. Chwalek, M. V. Tsurkan, M.F. Maitz, P. Atallah, K.R. Levental, S.A. Eming, C. Werner, Heparin desulfation modulates VEGF release and angiogenesis in diabetic wounds, J. Control. Release. 220 (2015) 79–88. https://doi.org/10.1016/j.jconrel.2015.10.028. [55] R.D. Bartlett, D. Eleftheriadou, R. Evans, D. Choi, J.B. Phillips, Mechanical properties of the spinal cord and brain: Comparison with clinical-grade biomaterials for tissue engineering and regenerative medicine, Biomaterials. 258 (2020) 120303. https://doi.org/10.1016/j.biomaterials.2020.120303. [56] A.P. Balgude, X. Yu, A. Szymanski, R. V. Bellamkonda, Agarose gel stiffness determines rate of DRG neurite extension in 3D cultures, Biomaterials. 22 (2001) 1077–1084. https://doi.org/10.1016/S0142-9612(00)00350-1.
82 [57] Z.Z. Khaing, A. Ehsanipour, C.P. Hofstetter, S.K. Seidlits, Injectable Hydrogels for Spinal Cord Repair: A Focus on Swelling and Intraspinal Pressure, Cells Tissues Organs. 202 (2016) 67–84. https://doi.org/10.1159/000446697. [58] M.C. Jimenez Hamann, E.C. Tsai, C.H. Tator, M.S. Shoichet, Novel intrathecal delivery system for treatment of spinal cord injury, Exp. Neurol. 182 (2003) 300–309. https://doi.org/10.1016/S0014-4886(03)00040-2. [59] P. V. Turner, T. Brabb, C. Pekow, M.A. Vasbinder, Administration of substances to laboratory animals: Routes of administration and factors to consider, J. Am. Assoc. Lab. Anim. Sci. 50 (2011) 600–613. [60] A. Zieris, S. Prokoph, K.R. Levental, P.B. Welzel, M. Grimmer, U. Freudenberg, C. Werner, FGF-2 and VEGF functionalization of starPEG-heparin hydrogels to modulate biomolecular and physical cues of angiogenesis, Biomaterials. 31 (2010) 7985–7994. https://doi.org/10.1016/j.biomaterials.2010.07.021. [61] H.R. Quintá, J.M. Pasquini, G.A. Rabinovich, L.A. Pasquini, Glycan-dependent binding of galectin1 to neuropilin-1 promotes axonal regeneration after spinal cord injury, Cell Death Differ. 21 (2014) 941–955. https://doi.org/10.1038/cdd.2014.14. [62] R.P. Huang, W. Yang, D. Yang, L. Flowers, I.R. Horowitz, X. Cao, R. Huang, The promise of cytokine antibody arrays in the drug discovery process, Expert Opin. Ther. Targets. 9 (2005) 601–615. https://doi.org/10.1517/14728222.9.3.601. [63] U. Freudenberg, P. Atallah, Y.D.P. Limasale, C. Werner, Charge-tuning of glycosaminoglycanbased hydrogels to program cytokine sequestration., Faraday Discuss. 219 (2019) 244–251. https://doi.org/10.1039/c9fd00016j. [64] H.L. Xu, F.R. Tian, J. Xiao, P.P. Chen, J. Xu, Z.L. Fan, J.J. Yang, C.T. Lu, Y.Z. Zhao, Sustainedrelease of FGF-2 from a hybrid hydrogel of heparin-poloxamer and decellular matrix promotes the neuroprotective effects of proteins after spinal injury, Int. J. Nanomedicine. 13 (2018) 681–694. https://doi.org/10.2147/IJN.S152246. [65] H.L. Xu, F.R. Tian, C.T. Lu, J. Xu, Z.L. Fan, J.J. Yang, P.P. Chen, Y.D. Huang, J. Xiao, Y.Z. Zhao, Thermo-sensitive hydrogels combined with decellularised matrix deliver bFGF for the functional recovery of rats after a spinal cord injury, Sci. Rep. 6 (2016) 1–15. https://doi.org/10.1038/srep38332. [66] J. Langhnoja, L. Buch, P. Pillai, Potential role of NGF, BDNF, and their receptors in oligodendrocytes differentiation from neural stem cell: An in vitro study, Cell Biol. Int. 45 (2021) 432–446. https://doi.org/10.1002/cbin.11500. [67] S.Q. Chen, Q. Cai, Y.Y. Shen, X.Y. Cai, H.Y. Lei, Combined use of NGF/BDNF/bFGF promotes proliferation and differentiation of neural stem cells in vitro, Int. J. Dev. Neurosci. 38 (2014) 74– 78. https://doi.org/10.1016/j.ijdevneu.2014.08.002. [68] A.M. Nicaise, K.M. Johnson, C.M. Willis, R.M. Guzzo, S.J. Crocker, TIMP-1 Promotes Oligodendrocyte Differentiation Through Receptor Mediated Signaling, Mol Neurobiol. 56 (2019) 3380–3392. https://doi.org/10.1007/s12035-018-1310-7.TIMP-1. [69] C.A. Ribeiro, A.J. Salgado, J.S. Fraga, N.A. Silva, R.L. Reis, N. Sousa, The secretome of bone marrow mesenchymal stem cells-conditioned media varies with time and drives a distinct effect on mature neurons and glial cells (primary cultures), J. Tissue Eng. Regen. Med. 5 (2011) 668– 672. https://doi.org/10.1002/term.365. [70] S.A. Eming, T.A. Wynn, P. Martin, Inflammation and metabolism in tissue repair and regeneration, Science (80-. ). 1356 (2017) 1026–1030. https://doi.org/10.1126/science.aam7928. [71] N.G. Frangogiannis, The Inflammatory Response in Tissue Repair, in: J.-M. Cavaillon, M. Singer (Eds.), Inflamm. From Mol. Cell. Mech. to Clin., Weinheim,G, Weinheim,Germany: John Wiley and Sons, 2017: pp. 1517–1537.
83 [72] L.I. Benowitz, P.G. Popovich, Inflammation and axon regeneration, Curr. Opin. Neurol. 24 (2011) 577–583. https://doi.org/10.1097/WCO.0b013e32834c208d. [73] L. Crigler, R.C. Robey, A. Asawachaicharn, D. Gaupp, D.G. Phinney, Human mesenchymal stem cell subpopulations express a variety of neuro-regulatory molecules and promote neuronal cell survival and neuritogenesis, Exp. Neurol. 198 (2006) 54–64. https://doi.org/10.1016/j.expneurol.2005.10.029. [74] K.M. Keefe, I.S. Sheikh, G.M. Smith, Targeting neurotrophins to specific populations of neurons: NGF, BDNF, and NT-3 and their relevance for treatment of spinal cord injury, Int. J. Mol. Sci. 18 (2017) 1–17. https://doi.org/10.3390/ijms18030548. [75] H.J. Lee, J.S. Ryu, P.J. Vig, Current strategies for therapeutic drug delivery after traumatic CNS injury, Ther. Deliv. 10 (2019) 251–263. https://doi.org/10.4155/tde-2019-0006. [76] D. Hakkoum, L. Stoppini, D. Muller, Interleukin-6 promotes sprouting and functional recovery in lesioned organotypic hippocampal slice cultures, J. Neurochem. 100 (2007) 747–757. https://doi.org/10.1111/j.1471-4159.2006.04257.x. [77] F.Y.H. Teng, B.L. Tang, Axonal regeneration in adult CNS neurons - Signaling molecules and pathways, J. Neurochem. 96 (2006) 1501–1508. https://doi.org/10.1111/j.14714159.2006.03663.x. [78] M. Zhang, N. Mal, M. Kiedrowski, M. Chacko, A.T. Askari, Z.B. Popovic, O.N. Koc, M.S. Penn, SDF‐1 expression by mesenchymal stem cells results in trophic support of cardiac myocytes after myocardial infarction, FASEB J. 21 (2007) 3197–3207. https://doi.org/10.1096/fj.066558com.
84 Supplementary Information Supplementary Table II. S1 Characterization of starPEG-Hep hydrogels. Hydrogels with crosslinking degree of 0.75, 1 and 1.5 were characterized in relation to their stiffness, mesh size and swelling degree. Characteristics Crosslinking degree 0.75 1 1.5 Stiffness (kPa) 1.01 ± 0.13 2.69 ± 0.30 7.14 ± 0.97 Mesh size (nm) 16.02 ± 0.72 11.55 ± 0.42 8.36 ± 0.38 Swelling degree (Qv) 1.54 ± 0.01 1.32 ± 0.01 1.21 ± 0.02 Supplementary Figure II. S1 Characterization of hASCs secretome released from starPEG-Hep hydrogels at tow and twn days using Membrane-based protein array RayBio ® C-Series Human Neuro Discovery array C1 Kit. BD NF be ta-N G F HB-EG F IFNg IGF-1 IL-6 I L-8 MCP-1 M IP -1 al ph a MMP -2 MM P -3 TG F b e ta TN F al p h a 0 5 10 40 50 60 70 80 Relative Intensity (%) PEG-Hep_2D PEG-Hep_10D starPEG-Hep_2D starPEG-Hep_10D
85 Supplementary Figure II. S2 Characterization of hASCs secretome released from StarPEG-Hep hydrogels at 2 and 10 days using Membrane-based protein array RayBio ®C-Series Human Cytokine Discovery array C5 Kit. Supplementary Table II. S2 Pairwise comparisons between groups using Tukey’s correction for the percentage of hNPCs differentiated in immature (DCX+) and mature (MAP2+) neurons. NPCs differentiation DCX Comparison between groups Mean Difference SEM p -Value Positive Control vs starPEG-Hep+sec -0.70 1.96 0.72 Positive Control vs starPEG-Hep+NbA 15.68 2.05 <0.0001 Positive Control vs Secretome -4.00 2.17 0.066 Positive Control vs NbA 7.2 2.17 <0.001 starPEG-Hep+sec vs starPEG-Hep+NbA 16.38 1.97 <0.0001 starPEG-Hep+Sec vs Secretome -3.30 2.18 0.11 starPEG-Hep+Sec vs NbA 7.90 2.09 <0.0001 EN A -78 GRO GRO-al ph a I -309 I L-1al p ha I L-1b e ta I L-2 IL-3 I L-4 IL12-p 40 IL-15 M CP-2 MC P -3 M -C SF MDC MIG M I P -1 b eta MIP-1-d elta RAN TES SCF S D F -1 TA RC TN F-b e ta EG F I G F -1 Angi oge n in O ncos tati n M TP O 0 10 20 20 40 60 80 Relative Intensity (%) PEG-Hep_2D PEG-Hep_10D VEG F P D G F-BB Le p ti n BLC C K b e ta 8-1 Eotaxi n Eotaxi n -2 F G F-4 FG F -6 F G F -7 FG F -9 G C P-2 GDNF HGF IG F BP -1 IGFBP -2 I G F BP -3 I G F BP -4 IL-16 I P -10 LI F LI G H T M C P -4 MIF MI P -3-al p h a N AP -2 N T -3 NT-4 PA R C PIGF TG F - b 2 TG Fb 3 TI M P-1 TI M P -2 0 10 20 40 60 80 Relative Intensity (%) PEG-Hep_2D PEG-Hep_10D starPEG-Hep_2D starPEG-Hep_10D
92 interactions with proteins, are favorable to create a permissive environment to drive the diffusion of molecules which confers an advantage of using them as encapsulating platforms [39]. For instance the controlled delivery of brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF) from PEG-based hydrogels into the brain have been beneficial to reduce microglial response [40], as the local delivery of neurotrophin-3 (NT-3) in SCI induced axonal growth and functional recovery [32]. However, hydrogels lacking affinity sites for cytokines and growth factors can hardly provide long term release. To overcome this limitation, biohybrid hydrogels made of starPEG (star-shaped PEG) and the glycosaminoglycan (GAG) heparin (Hep)have been produced and demonstrated to enable the sustained release of various growth factors [41–43]. In this system the anionic charge arising from the high density of sulfate moieties on heparin results in a high affinity for a broad range of growth factors, cytokines, and chemokines mainly due to electrostatic interactions [44]. This effect has been previously utilized to modulate the release of multiple growth factors such as FGF-2 and vascular endothelial growth factor (VEGF) [45] or cytokines, such as IL-4 [46] in a controlled and sustained manner over weeks. Additionally, these matrices can be applied as in situ forming hydrogels, which allow for the injection in a liquid state at the injury site, and posterior fast polymerization due to a Michael type reaction [47]. We have demonstrated in chapter II that starPEG-Hep hydrogel is efficient in promoting controlled release of hASCs secretome and its effective in modulate regenerative processes. Herein, we accessed the potential of this release system in promoting regeneration after a complete transection in an SCI rat model (Figure III.1). Histological analysis was conducted in an attempt to decipher which molecular mechanisms were underlying motor function. Moreover, the systemic inflammation was evaluated through a defined panel of cytokines in three different stages of lesion (acute, intermediate and chronic).
93 Figure III. 1 Schematic representation of our biomaterial concept and in vivo model that were used to evaluate the bioactivity of human adipose-tissue derived stem cells (hASCs) secretome released from starPEG-Hep hydrogels injected right before lesion. Recovery of thoracic (T8) level transected rats by secretome-loaded starPEG-Hep hydrogels was analyzed.
94 2. Materials and Methods 2.1. Secretome collection 2.1.1. Cell isolation and culture The hASCs were obtained from lipoaspirates from consenting donors under a protocol approved and reviewed by an institutional board of LaCell LLC. Cells were isolated according to the protocol described by Dubois et al. [48] and maintained in culture, at 37 ºC and 5% CO2, in α-MEM (Invitrogen, USA), with 10% Fetal Bovine Serum (FBS, Biochrom AG, Germany) and 1% antibiotic/antimycotic solution – penicillin/streptomycin (pen/strep; Invitrogen, USA). Medium was changed every 2/3 days until reach 85% confluence, afterwards cells were enzymatically detached and seeded onto new cell culture flasks. 2.1.2. Secretome collection For secretome collection, hASCs were seeded at a density of 4000 cells/cm2 in cell culture flasks with alpha minimum essential medium (α-MEM), as described above. The medium was harvested 72 hours after culture and the cells were washed four times with PBS without Ca2+/Mg2+ (Merk, Germany). The cells were then placed 24 hours in Neurobasal A medium (ThermoFisher, USA) with 1% kanamycin, being this medium harvested at the end of that time (conditioned medium). It was further centrifuged (249g, Megafuge 1.0R, Heraeus, Germany) for 5 minutes to remove any cell debris. Then, hASCs secretome was concentrated (100x) by centrifugation (3000g) using 5kDa cut-off concentrator (Vivaspin, GE Healthcare, UK) and frozen at -80ºC until used. 2.2. In vivo proof of concept 2.2.1. Study Design The goal of this study was to evaluate the capacity of a secretome release system, based on the use of starPEG-Hep hydrogel in promoting regeneration in an SCI animal model. Thus, regenerative processes were assessed by functional recovery and histological alterations. Animals were randomly treated and all data collection (behavior and histology) was obtained in blinded conditions. All procedures were carried out in accordance with EU directive 2010/63/EU and were approved by the ethical committee in life and health sciences (ID: SECVS116/2016, University of Minho, Braga, Portugal).
95 2.2.2. Animals and groups In this in vivo study, Wistar Han female rats (8-11 weeks old, weighing 170g-190g) were used. Animals were kept in light and temperature-controlled cages and fed ad libitum with a standard diet. The handling of the animals was carried out five days before the surgery. The animals were divided into five different groups according to the treatment/procedure instituted: 1) Animals subjected to SCI that were injected with starPEG-Hep loading secretome (starPEG-Hep+sec) (n = 7); 2) SCI animals treated with starPEGHep hydrogel and vehicle (NbA) (starPEG-Hep+NbA)(n = 4); or 3) SCI animals treated with secretome locally (n = 7); 4)SCI animals treated only with vehicle (NbA) (n = 6); and 5) Animals with laminectomy only (SHAM) (n = 7). 2.2.3. Spinal Cord Injury Surgery For surgery, animals were previously anesthetized with an intraperitoneal injection of a mixture (1:5:1) of ketamine (100 mg/mL, Ketamidor/Richter Pharma, Austria) and medetomidine hydrochloride (1 mg/mL, Seedorm/ProdivetZN, Portugal). After anesthesia, the animals' fur was shaved and the skin disinfected with 70% ethanol and chlorhexidine. The incision was made in the dorsal midline, between T7 and T13, with subsequent retraction of the paravertebral muscles. A laminectomy (removal of the spinous processes to expose the spinal cord) was performed at T8 level and a total spinal cord transection was performed at this level. After administering the respective treatment, the paravertebral muscles and the skin were sutured with Vicryl sutures (Johnson and Johnson, USA). After surgery, all rats were kept under heat lamps and received daily post-operative care, during the first week, of vitamins (10 mL/Kg, Duphalyte,Pfizer, USA), 0.9% NaCl, the analgesic butorphanol (Butomidor, Richter Pharma AG, Austria), the antibiotic enrofloxacin (5mg/mL, Baytril, Bayer, Germany) and atipamezole (5mg/mL, Tipafar, ProdivetZN, Portugal) to reverse anesthesia (given on the first day only). In addition, manual bladder emptying was performed twice a day. The animals were examined for symptoms of disease and urinary infections detected during the eight weeks of study were treated with antibiotic enrofloxacin oral solution (Baytril, Bayer, Germany) and evaluated potential adverse reactions to treatment. 2.2.4. Hydrogel preparation Amine end-functionalized 4-arm starPEG (MW 10,000, USA), Heparin (MW 15,000, Merck, Germany) and RGD (990 g/mol, Peptides International) were synthesized as previously described [41,47]. Hydrogel
96 formation was obtained by dissolving the maleimide functionalized heparin and thiol end-functionalized 4-arm starPEG in PBS on ice with an appropriate molar ratio. Briefly, 1.5mM heparin dissolved in PBS was mixed with an equal volume of starPEG solution in the concentration of 1.1mM to produce hydrogels in the crosslinking of 0.75 (molar ratio of starPEG to heparin). For in vivo application heparin, starPEG and RGD were dissolved as previously described and filtered using low protein binding 0.2 µm filter (Acrodisc, PALL, USA) to ensure sterile conditions of the materials prior to injecting. Briefly, hASCs secretome (5 µL collected in NbA) were mixed with an equal amount of heparin/RGD and starPEG (2.5 µL). 2.2.5. Behavioral Analysis 2.2.5.1 Locomotor rating The Basso, Beattie, Bresnahan Locomotor Rating Scale (BBB) [49] was employed to evaluate motor behavior and recovery. The test was performed for four minutes for two blinded researchers starting three days after surgery and performed 1, 2, 4, 6 weeks up to a total of eight weeks. Locomotion of the affected hindlimbs was rated in a score of 0 if no movement was observed, 1 to 8 indicates some movement of joints without weight support. From 9 to 20 animals are capable of weight support, have coordinated steps and trunk stability and finally, a 21 score corresponds to a normal animal with perfect movements. To attribute the final score to each animal the average score of both hindlimbs was made and plotted over eight weeks of behavior analysis. 2.2.5.2. Motor Swimming test (MST) Animals have a natural capability to swim and the buoyancy provided by the water enables rats to perform locomotor movements without having to totally support their body weight. MST was performed once at 8 weeks post-injury where animals were placed in a quadrangular pool (water temperature 24-25ºC) and had to reach the platform to get out of the water. After a short training trial, all trials were recorded by a video camera. To access locomotor activity, the velocity of the animals from one side of the pool to reach the platform was measured by using 3 trials per animal. Values were extracted using Ethovision XT 13 software (Noldus, Wageningen, Netherlands) and values of 3 trials averaged for each animal. Animals that weren’t capable to perform the test, either if they don’t have motivation to swim or needed more than 3 min to perform all trials were excluded.
97 2.2.5.3. Von Frey Loss or gain of sensitivity in the hindlimbs of the lesioned animals were assessed through mechanical allodynia that measures the evoked pain using Von Frey test. In this test monofilaments are applied in the center of the hindlimbs by the up-down method [50]. Once at a time, animals are placed in the Von Frey apparatus and left for some time undisturbed to be acquainted, then a monofilament (2.0g) is applied to the hindlimb center, once at a time, for no more than 4s. If the animals reacted, withdrew the paw, was considered a positive response and a monofilament of a lower force was applied; if no reaction was observed negative response was considered and consequently a monofilament of a high force is applied [51]. The test is finished if the extreme monofilament was reached or 4 measurements around the turning point were obtained. The range of VF monofilaments used was the following: 0.4, 0.6, 1.0, 2.0, 4.0, 6.0, 8.0, 15.0g. The assessment was performed twice at 2and 6-weeks post-injury. The measurement used was the 50% threshold, that is an approximation of the monofilament that causes pain or sensitivity, for that the following equation 1 was used: 50%𝑔 _𝑡𝑟𝑒𝑠ℎ𝑜𝑙𝑑=(10𝑋𝑓+𝐾.𝛿) 10000 Equation 1 where Xf=value (in log units) of the final VF monofilament; K=tabular value corresponding to pattern of positive and negative responses; 𝛿=mean difference (in log units) between stimuli (0.224). 2.2.6. Immunohistochemistry 2.2.6.1. Histological Analysis Eight-weeks post-injury, rats were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital (200 mg/mL, Euthasol, Ecuphar, Spain) and perfused through the ascending aorta with NaCl (0.9 %; 100mL) followed by PFA (4 %; 100mL). A rough dissection of the spine and spinal cord was performed, centered on the site of transection and the tissues were fixed in 4 % PFA overnight at 4ºC. A more detailed dissection of the spinal cord was then done, and the tissues were carefully placed on a solution of sucrose (30 % (w/v)) on the next day. Afterwards, 1.5 cm length of spinal cord tissues, centered on the lesion, were involved in optimal cutting temperature compound (OCT, ThermoFisher, USA), frozen
98 with liquid nitrogen and stored at -20 °C. Later, longitudinal sections of 20 µm thickness were performed using a Leica CM1900 cryostat. 2.2.6.2. Immunohistochemistry Spinal cord longitudinal sections were initially permeabilized with PBS-T (0.2 % ) for 10 min. Then, the slides were blocked with a solution of NBCS (5 %) in PBS-T (0,2% ) for 30 min. After that, the samples were incubated overnight with the following primary antibodies: rabbit anti-rat GFAP for astrocytes (1:200, Dako Denmark, Glostrup, Denmark), mouse anti-neurofilament (NF, 1:200, Merck, USA), rabbit anti-Iba1 (1:1000, Wako, Japan), mouse anti-SMI-71 (1:200, Biolegend, USA). On the next day, samples were incubated for 2 h with the respective secondary antibodies: Alexa Fluor 594 goat anti-rabbit for GFAP, Alexa Fluor 488 goat anti-mouse for NF, Alexa Fluor 488 goat anti-rabbit for Iba-1, Alexa Fluor 594 goat anti-mouse for SMI-71 (all from Invitrogen, USA). All samples were counterstained with DAPI (1:1000, Life Technologies) for 10 min. Between steps, three washes with PBS were performed. Finally, the slides were mounted in Immu-Mount® (ThermoFisher, USA) and observed at a fluorescence microscope, Olympus Widefield Inverted Microscope IX81. All images were treated using Fiji software. 2.2.7. Immunofluorescence Analysis For immunofluorescence analysis, images of the longitudinal section were obtained for each animal as a mosaic of all tissue, 4-6 micrographs per animal. Then images were open with Fiji software and scale was determined first. For GFAP and NF analysis the positive area for each marker was calculated. For that a section of 2000µm was delineated for rostral, epicenter and caudal part of the spinal cord. Following, imagens were converted to 8 bits and processed in the command “make binary”. Finally, after applying the threshold command, using the menu “analyze particles”, the program automatically calculated the positive area occupied for each marker, using the dark background as contrast. The positive area of each marker was normalized to the area of the tissue for each region. For Iba-1 the immunofluorescence quantification was performed by measuring the total ameboid area and normalized to the total area of the spinal cord segment. For SMI-71 analysis a 1500µm region rostral and caudal to the lesion were selected and vessels analyzed using Angio Tool software as previously described [52] three parameters where selected, vessel area, vessel length, average vessel length.
99 2.2.8. Serum collection and analysis by Neuro array membrane Rat blood was collected directly from the tail vein at 48h and four weeks post-injury and from the heart at sacrifice. Blood was allowed to coagulate for approximately 15min and centrifuged at 15330 g for 15min. Serum was collected and stored at -80ºC until use. For each group a pool of serum was made and analyzed using Rat Cytokine Array C2 (C-Series RayBiotech, USA) according to the manufacture’s instructions. Analysis of the membranes was performed in AzureSpot Analysis software (Azure Biosystems, USA) where the relative intensity of each spot was measured. Afterwards, to quantify the relative intensity of each spot corresponding to a different protein, the background was subtracted, and intensity normalized to positive controls. 2.3. Statistical Analysis Data regarding BBB and Von Frey test were analyzed using Mixed ANOVA to compare the mean values of five groups. When evaluating the velocity in MST, the ameboid area of Iba-1 and positive area of NF, GFAP and SMI-71 one-way ANOVA was performed. A pairwise comparison between groups based on estimated marginal means using Turkey’s correction was performed. The significance value was set as p ≤ 0.05 for all statistical tests and graphs are presented as mean ± SEM. For all data normality was assessed using Shapiro-Wilk statistical tests and taking into account the measures of skewness and kurtosis. Moreover, it is important to highlight that ANOVA distribution can be approximately normal distributed [53]. IBM SPSS® Statistics version 27 for IOS (IBM Co., USA) was used to perform all statistical analysis. For graphical representations GraphPad Prism ver.8.4.3 (GraphPad Software, La Jolla, USA) was used. 3. Results 3.1. hASCs secretome released from starPEG-Hep hydrogels promotes motor recovery in an SCI animal model. The secretome of hASCs has previously been shown to induce regeneration after SCI in animal models [24,54]. Herein we aimed to develop a hydrogel system capable of controlled and prolonged release of secretome at the injury site in order to potentiate its effect. Considering the pro-regenerative capacity
100 shown in in vitro studies (Chapter II) we explored the potential of the released secretome in promoting motor improvements after a complete transection in a rat model. Animals were divided into five groups: SCI treated with starPEG-Hep hydrogel loading secretome (starPEG-Hep+sec; n = 7); SCI treated with starPEG-Hep loading Neurobasal A (starPEG-Hep+NbA; n = 4); SCI treated with secretome locally (Secretome; n = 7); SCI treated with Neurobasal A (NbA; n = 6); and non-injured animals (laminectomy – SHAM; n = 7). Complete transection was performed at T8 level and animals were immediately treated after lesion. Motor recovery was evaluated every two weeks until eight weeks post-injury by BBB test. Animal treated with starPEG-Hep+sec displayed improved motor outcomes compared with other groups, Figure III.2. In addition, SHAM animals did not present motor deficits during that time. Statistical analysis revealed an effect of factor time (weeks F (5, 130) = 33.54 p<0.0001, η2partial = 0.56), and treatment F (4, 26) = 948.54 p<0.0001, η2partial = 0.99) and the interaction between these two factors (F (20, 130) = 3.90 p<0.0001, η2partial = 0.38). In particular, the mean BBB score was significantly improved by starPEG-Hep+sec treatment at two, sixand eight weeks post-injury (wpi). Moreover, at two and six wpi, animals treated with starPEGHep+sec showed improved motor function when compared with animals treated with only secretome (2wpi: 2 ± 2 vs 0.71 ±0.57; p = 0.032; 6wpi: 3.5 ± 2.25 vs 1.29 ± 0.95; p = 0.004). Notably, at eight wpi animals treated with starPEG-Hep+sec showed statistically significant improvements compared with starPEG-Hep+NbA (4.07 ± 2.24 vs 2.00 ± 0.82; p = 0.012), animals treated with only secretome (1.86 ± 0.85, p = 0.002) and animals lesioned treated with vehicle (2.25 ± 0.69; p = 0.013). Interestingly, comparing the group treated with starPEG-Hep+sec with free secretome, a difference of approximately two points in BBB scale was reported clearly showing a beneficial effect of the hydrogel-based release. All statistical data is presented in Supplementary Table III.S1.
101 Figure III. 2 Evaluation of motor performance in SCI rats by BBB test for eight weeks post-injury. Animals treated with secretome presented improved motor recovery eight weeks after treatment. Mixed ANOVA; (*) represented differences between starPEG-Hep+sec and Secretome, (#) differences between starPEGHep+sec vs starPEG-Hep+NbA and NbA, and (&) differences between SHAM group and all other; * and # p < 0.05; **p < 0.001 and &&& p < 0.0001. Error bars represented mean ± SEM. The BBB score of animals treated with starPEG-Hep+sec indicates that animals could perform extensive movements of two joints (such as ankle, knee or hip), while animals treated with starPEG-Hep+NbA or NbA were only able to perform extensive movement of one joint and slight movement of the third. Finally, animals treated with secretome only performed slight movements of hindlimbs joints. Other motor tests were performed for motor recovery or sensorial function after injury. Regarding the MST, animals treated with starPEG-Hep+sec were able to perform the task at a higher velocity when compared with animals treated with starPEG-Hep+NbA (9.68±3.85 vs 5.34±1.08), Secretome (8.88±1.97) or NbA (8.24±3.65), Figure III.3A. While animals treated with starPEG-Hep+NbA had the lowest velocity. Nevertheless, no statistical significance was observed among groups and all were slower than SHAM animals, as shown in Supplementary Table III.S2. Sensorial function was evaluated by performing Von Frey test, in which different force filaments were applied to the hindlimbs. Then, results were evaluated as a 50% chance of being the final response filament. For the left hindlimb animals treated with starPEG-Hep+sec showed slightly less sensitivity when compared with starPEG-Hep+NbA (4.53±2.68 vs 3.75±2.26), secretome (4.45±4.86) or NbA (3.37± 1.49) at two wpi. However, a slight increase in sensitivity was observed at six wpi (4.08±4.43) when compared with other groups (starPEGHep+NbA 6.90± 5.87; Secretome 4.84±4.55, NbA 4.33±3.91) as
108 Figure III. 6 SMI-71 quantification in SCI rat model eight weeks after lesion. A, Representative confocal microscopy images of selected areas at rostral and caudal regions in animals treated with starPEGHep+sec, starPEG-Hep+NbA, Secretome, NbA and SHAM eight weeks after lesion. B, Quantification of SMI-71 vessel area, C, Quantification of SMI-71 vessel length, D, Quantification of SMI-71 average vessel length. One-Way ANOVA, Error bars represented mean ± SEM. Scale bar: 100 μm.SMI-71 – Anti Blood Brain Barrier, DAPI 4’,6’-diamino-2-fenil-indol SCI is characterized by an exacerbation of inflammatory response, which could be one of the causes of the massive damage to the tissue after lesion. After we observed an attenuation of inflammation characterized by the reduction of ameboid microglia in the damage tissue of animals treated with hydrogel and secretome, we evaluated if the same could be observed systemically. For this purpose, animal serum was collected at three different time points (48h, four and eight wpi) and the expression of cytokines was evaluated by a cytokine array. Molecular analyses of sera at 48h post-lesion are presented in Figure III.7B, where the expression of a heatmap (Figure III. 7A). Animals treated with starPEG-Hep+sec have an increase in IL-10 compared with animals treated with hydrogel only, secretome or NbA, and a decrease of inflammatory cytokine monocyte chemoattractant protein (MCP-1) compared with other groups. Moreover, cluster of differentiation 89 (CD86), a classically (M1) marker, presented values similar to SHAM animals at this time point.
109 Figure III. 7 Molecular analysis of collected sera following SCI using Rat Cytokine Array C2 from RayBiotech. A, Representative heat map at 48 h pi of the panel for the different groups was generated using the BROAD Institute's R implementation of Morpheus with Euclidean distance hierarchical clustering. B, Relative expression of selected pro-inflammatory and anti-inflammatory cytokines at 48 h pi, C, 4 wpi and D, 8 wpi. Data are shown as mean Log2 (fold change - FC) relative to the normalization of each cytokine to the positive controls of each membrane. Error bars represented mean ± SEM.
110 After four weeks (Figure III. 7C and Supplementary Figure III.S1), animals treated with starPEG-Hep+sec continue to have a higher expression of IL-10 when compared with animals treated with starPEGHep+NbA, secretome or NbA, Figure III.7C. Interestingly, at this time point, a decrease in expression of CD86 is observed compared with NbA treated animals. On the other hand, values of MCP-1 remained similar to values obtained at 48h, with a decrease in groups treated with starPEG-Hep+NbA, secretome or NbA. Right before sacrifice (eight weeks, Figure III. 7D and Supplementary Figure III.S1), sera analysis revealed an increase in IL-10 levels in animals treated with starPEG-Hep+sec and secretome compared to other groups, Figure III. 7D. CD86, a marker of M1 phenotype [55], demonstrated a slight decrease from four weeks post-injury and compared with animals treated with hydrogel and NbA at this time point. It is also worth noting that animals treated with starPEG-Hep+sec have higher levels of VEGF expression than all other groups at all time points. 4. Discussion Cell-based therapy, particularly transplantation of ASCs has upraised therapeutical approaches to tackle devastating conditions caused by SCI. Furthermore, several studies pointed that their beneficial effects are mediated by their secretome, rather than its presence at injury site [9,17]. Along these lines, stem cell secretome has been raised as a cell free-based therapy in the field of regenerative medicine [56,57]. However, administration methodology is an obstacle to foster their application. Local injection is characterized by the rapid clearance at SCI, while multiple systemic administrations promote their fast diffusion in the body, meaning higher dosage to have effect. Biomaterials, particularly hydrogels, flourished as suitable platforms, not only by their singular characteristics but also their potential in controlling drug delivery at injury site [32–34]. In accordance, in this work we aimed to develop a combinatorial therapy in which hASCs secretome is loaded in starPEG-Hep hydrogels to promote a controlled and prolonged release at SCI injury site. With this strategy, secretome effect can be locally potentiated and regeneration favored. From data presented in Chapter II, we have shown that starPEG-Hep hydrogels were effective in promoting controlled and prolonged release of hASCs secretome, which was capable of inducing neural differentiation and axonal growth. However, validation of this therapeutic approach in an in vivo injury mode was still lacking. Likewise, a complete transection at T8 level was performed in a rat model. These results in a very severe lesion, when compared with hemisection or contusion models [58]. Animals were immediately treated and motor recovery evaluated for eight weeks.
111 Animals treated with starPEG-Hep+sec presented significant improved motor recovery at two-, sixand eight-weeks post injury in BBB test. Interestingly, comparing the group treated with starPEG-Hep+sec with free secretome, a difference in approximately two points in BBB scale was reported clearly showing a beneficial effect of the hydrogel-based release. Despite these motor improvements, no differences were observed on the swimming velocity in MST. Lesioned animals had no ability to move hindlimbs, but they swim using forelimbs (videos not shown). This may explain the similarities among groups. On the other hand, this behavioral test evaluates gross motor function where animals have no sensory input from water similar to the stimulus, they experienced with direct contact with BBB table. Sensorial inputs are critical to demand locomotion through activation of central pattern generators (CPGs). These circuitries receive inputs from afferent neurons such as muscles or joints and shape locomotor outputs [59,60]. Additionally, proprioceptive feedback and mechanoreceptive circuits are closely interconnected and may contribute for motor recovery. They are known to be important as an alternative for transmission of afferent information after lesion [61,62]. However, this is not the most likely pathway governing motor recovery as evidenced by low sensorial recovery on Von Frey test. Histological analysis has shown a reduced area occupied by ameboid microglia, a phenotype associated with higher inflammation, in animals treated with starPEG-Hep+sec compared with local secretome or NbA. Previous studies have shown that the starPEG-Hep hydrogels can reduce inflammation by scavenging inflammatory mediators [63,64]. In line with those findings, a slight attenuation of Iba-1 expression was observed in animals treated with starPEG-Hep+NbA, which point to the anti-inflammatory characteristics of the pure hydrogels with only the starPEG-Hep+sec effectively attenuating inflammation in the SCI model. In addition, no major differences were found in GFAP or NF quantification positive area. The integrity of BBB was also evaluated by SMI-71 staining, but no statistical differences were observed regarding vessel area or length. Altogether, these data indicate that motor recovery seems to be favored by a reduced inflammatory response to treatment in local tissue. Furthermore, it has been previously demonstrated that hASCs secretome promoted a reduced inflammatory response in vivo , either by treatment with secretome systemically in mice model [24], or the local cell transplantation [9,10]. Moreover, ASCs secretome can induce M2 polarization, through mammalian target of rapamycin complex 1 (mTORC1) and mTORC2 pathway [65]. Other authors also hypothesized that hASCs transplantation in a contusion injury mice model could reduce neuroinflammation by reducing microglia/macrophages at lesion site, as well as inhibiting Jagged1/Notch pathway [66]. This pathway has been implicated in modulating the immune response after lesion in central nervous system (CNS) by impacting cell fate decisions and
112 endogenous neurogenesis [67]. On the other hand, interferon gamma (IFN-γ), present in secretome and released by hydrogels, may determine microglia's anti or pro-inflammatory state. Indeed a lower concentration can induce a neuroprotective function [68,69]. Moving to sera analysis, animals treated with starPEG-Hep+sec show an increase in IL-10, an antiinflammatory cytokine, in all time points after lesion (48h, four and eight wpi). The release of IL-4 or IFNγ by secretome-loaded hydrogels has been previously shown to induce IL-10 production by microglia/macrophages, leading to increased circulation levels [70,71]. Thus administration of IL-10 has been shown to decrease the levels of pro-inflammatory cytokines and contributed to motor recovery after lesion [72,73]. At the same time, as MCP-1 has been shown to play a critical role in mediating neuronmacrophage interactions that contribute to axonal growth and M2 phenotype polarization, the moderate levels of MCP-1 observed over time may down regulate the inflammatory response and endure the regenerative process [74]. On the other hand, CD86, an M1 marker, is decreased at four and eight weeks after lesion, while MCP1 was low expressed in the acute phase (48h). Together, the reported data demonstrate for the first time that a biohybrid hydrogel-based sustained hASCs secretome release comprising pro-regenerative cytokines (IL-4, IL-2) and growth factors (BDNF, GDNF). Significant motor recovery in a complete SCI rat model further confirmed the robustness of the delivery system, accompanied by a reduced ameboid microglia area, as well as increased levels of the anti-inflammatory cytokine IL-10 in animal sera. 5. Conclusions A starPEG-Hep hydrogel was successfully tuned for sustained hASCs secretome release over weeks as well as for mechanical tissue stabilization to promote neural regeneration [75,76]. The system was demonstrated to be effective in an SCI animal model in reducing inflammation management, reducing ameboid microglia area, as well as increasing anti-inflammatory cytokine IL-10 in animal serum, and initiating pro-regenerative processes. In perspective, the approach may offer unprecedented clinical treatment modalities for SCI.
113 Acknowledgments This work was supported by Prmios Santa Casa Neurociencias–Prize Melo e Castro for Spinal Cord Injury Research (MC-04/17; MC-18-2021) and the Portuguese Foundation for Science and Technology (Ph.D. Fellowship to D.S (PD/BDE/135567/2018 and COVID/BDE/152051/2022); T. S. P. (PD/BDE/143150/2019); J.R.C (SFRH/BD/145860/2019); J.A (2021.08337.BD); S.B.-A ((PD/BDE/135568/2018). This work was funded by national funds and FEDER, through the Foundation for Science and Technology (FCT), under the scope of the projects UIDB/50026/2020; UIDP/50026/2020; POCI-01-0145-FEDER-029206; POCI-01-0145-FEDER-031392; PTDC/ MEDNEU/31417/2017; NORTE-01-0145-FEDER-029968; POCI-01-0145-FEDER-029751; POCI-01-0145FEDER-032619. This work has been funded by ICVS Scientific Microscopy Platform, a member of the national infrastructure PPBI - Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122. This work has also been developed under the scope of the project NORTE-01-0145FEDER-000013 and NORTE-01-0145-FEDER-000023, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). Work supported by the Portuguese Foundation for Science and Technology (FCT): projects UID/FIS/04650/2020, PTDC/EMD-EMD/28159/2017, and PTDC/BTMMAT/28237/2017. Dr. Jeff Gimble and LaCell, Inc. kindly provided the adipose tissue-derived stem cells used in this study. Nelly Rein for her technical advice and support in working with the hydrogel material. Author contributions D.S designed and performed most of the experiments, collected and analyzed the data, and drafted the manuscript. T.S.P, J.C.R, R.L, J.A, S.B-A, C.R.M, JD helped in vitro and animal experiments. P.A, L.S helped in designing experiments. U. F. and C.W provided hydrogel materials. A.J.S conceived and financially support the study, participated in its design and coordination. L.S, U. F., C.W., A.J. S. critically read the manuscript. Supervision: R.A.S, U. F., C.W., A.J.S. All authors read and approved the final manuscript.
114 Conflict of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability The data that supports this study is available from the authors upon reasonable request.
115 References [1] L. Anderberg, H. Aldskogius, A. Holtz, Spinal cord injury - Scientific challenges for the unknown future, Ups. J. Med. Sci. 112 (2007) 259–288. https://doi.org/10.3109/2000-1967-200. [2] N. Level, Facts and Figures at a Glance, J. Spinal Cord Med. 30 (2018) 304–305. https://doi.org/10.1080/10790268.2007.11753944. [3] S. Huh, H.Y. Ko, Recovery target priorities of people with spinal cord injuries in Korea compared with other countries: a survey, Spinal Cord. 58 (2020) 998–1003. https://doi.org/10.1038/s41393-020-0457-z. [4] K.D. Anderson, Targeting recovery: Priorities of the spinal cord-injured population, J. Neurotrauma. 21 (2004) 1371–1383. https://doi.org/10.1089/neu.2004.21.1371. [5] M.D. Norenberg, J. Smith, A. Marcillo, The Pathology of Human Spinal Cord Injury: Defining the Problems, J. Neurotrauma. 21 (2004) 429–440. https://doi.org/10.1089/089771504323004575. [6] J.W. Rowland, G.W.J. Hawryluk, B. Kwon, M.G. Fehlings, Current status of acute spinal cord injury pathophysiology and emerging therapies: Promise on the horizon, Neurosurg. Focus. 25 (2008) 1–3. https://doi.org/10.3171/FOC.2008.25.11.E2. [7] A. Anjum, M.D. Yazid, M.F. Daud, J. Idris, A.M. Hwei Ng, A.S. Naicker, O.H. Rashidah Ismail, R.K.A. Kumar, Y. Lokanathan, Spinal cord injury: Pathophysiology, multimolecular interactions, and underlying recovery mechanisms, Int. J. Mol. Sci. 21 (2020) 1–35. https://doi.org/10.3390/ijms21207533. [8] A.P. Tran, P.M. Warren, J. Silver, The biology of regeneration failure and success after spinal cord injury, Physiol. Rev. 98 (2018) 881–917. https://doi.org/10.1152/physrev.00017.2017. [9] E.D. Gomes, S.S. Mendes, R.C. Assunção-Silva, F.G. Teixeira, A.O. Pires, S.I. Anjo, B. Manadas, H. Leite-Almeida, J.M. Gimble, N. Sousa, A.C. Lepore, N.A. Silva, A.J. Salgado, Co-Transplantation of Adipose Tissue-Derived Stromal Cells and Olfactory Ensheathing Cells for Spinal Cord Injury Repair, Stem Cells. 36 (2018) 696–708. https://doi.org/10.1002/stem.2785. [10] Z. Zhou, X. Tian, X. Tian, B. Mo, H. Xu, L. Zhang, L. Huang, S. Yao, Z. Huang, Y. Wang, H. Xie, L. Xu, L. Xu, H. Zhang, H. Zhang, Adipose mesenchymal stem cell transplantation alleviates spinal cord injury-induced neuroinflammation partly by suppressing the Jagged1/Notch pathway, Stem Cell Res. Ther. 11 (2020) 1–17. https://doi.org/10.1186/s13287-020-01724-5. [11] M. Bydon, A.B. Dietz, S. Goncalves, F.M. Moinuddin, M.A. Alvi, A. Goyal, Y. Yolcu, C.L. Hunt, K.L. Garlanger, A.S. Del Fabro, R.K. Reeves, A. Terzic, A.J. Windebank, W. Qu, CELLTOP Clinical Trial: First Report From a Phase 1 Trial of Autologous Adipose Tissue–Derived Mesenchymal Stem Cells in the Treatment of Paralysis Due to Traumatic Spinal Cord Injury, Mayo Clin. Proc. 95 (2020) 406–414. https://doi.org/10.1016/j.mayocp.2019.10.008. [12] J. Vaquero, M. Zurita, M.A. Rico, C. Bonilla, C. Aguayo, J. Montilla, S. Bustamante, J. Carballido, E. Marin, F. Martinez, A. Parajon, C. Fernandez, L. De Reina, An approach to personalized cell therapy in chronic complete paraplegia: The Puerta de Hierro phase I/II clinical trial, Cytotherapy. 18 (2016) 1025–1036. https://doi.org/10.1016/j.jcyt.2016.05.003. [13] A. J. Braga Osorio Gomes Salgado, R. L. Goncalves Reis, N. Jorge Carvalho Sousa, J. M. Gimble, A. J. Salgado, R. L. Reis, N. Sousa, Adipose Tissue Derived Stem Cells Secretome: Soluble Factors and Their Roles in Regenerative Medicine, Curr. Stem Cell Res. Ther. 5 (2010) 103–110. https://doi.org/10.2174/157488810791268564. [14] S. Lu, C. Lu, Q. Han, J. Li, Z. Du, L. Liao, R.C. Zhao, Adipose-derived mesenchymal stem cells protect PC12 cells from glutamate excitotoxicity-induced apoptosis by upregulation of XIAP through PI3-K/Akt activation, Toxicology. 279 (2011) 189–195. https://doi.org/10.1016/j.tox.2010.10.011. [15] S.K. Kang, E.S. Jun, Y.C. Bae, J.S. Jung, Interactions between human adipose stromal cells and
116 mouse neural stem cells in vitro, Dev. Brain Res. 145 (2003) 141–149. https://doi.org/10.1016/S0165-3806(03)00224-4. [16] E.D. Gomes, S.S. Mendes, H. Leite-Almeida, J.M. Gimble, R.Y. Tam, M.S. Shoichet, N. Sousa, N.A. Silva, A.J. Salgado, Combination of a peptide-modified gellan gum hydrogel with cell therapy in a lumbar spinal cord injury animal model, Biomaterials. 105 (2016) 38–51. https://doi.org/10.1016/j.biomaterials.2016.07.019. [17] E.D. Gomes, B. Ghosh, R. Lima, M. Goulão, T. Moreira-Gomes, J. Martins-Macedo, M.W. Urban, M.C. Wright, J.M. Gimble, N. Sousa, N.A. Silva, A.C. Lepore, A.J. Salgado, Combination of a Gellan Gum-Based Hydrogel With Cell Therapy for the Treatment of Cervical Spinal Cord Injury, Front. Bioeng. Biotechnol. 8 (2020) 1–14. https://doi.org/10.3389/fbioe.2020.00984. [18] F. Cofano, M. Boido, M. Monticelli, F. Zenga, A. Ducati, A. Vercelli, D. Garbossa, Mesenchymal stem cells for spinal cord injury: Current options limitations, and future of cell therapy, Int. J. Mol. Sci. 20 (2019) 2698. https://doi.org/10.3390/ijms20112698. [19] M.I. Guillén, J. Platas, M.D. Pérez del Caz, V. Mirabet, M.J. Alcaraz, Paracrine anti-inflammatory effects of adipose tissue-derived mesenchymal stem cells in human monocytes, Front. Physiol. 9 (2018) 1–10. https://doi.org/10.3389/fphys.2018.00661. [20] C.A. Ribeiro, J.S. Fraga, M. Grãos, N.M. Neves, R.L. Reis, J.M. Gimble, N. Sousa, A.J. Salgado, The secretome of stem cells isolated from the adipose tissue and Wharton jelly acts differently on central nervous system derived cell populations, Stem Cell Res. Ther. 3 (2012) 18. https://doi.org/10.1186/scrt109. [21] S.C. Serra, J.C. Costa, R.C. Assunção-Silva, F.G. Teixeira, N.A. Silva, S.I. Anjo, B. Manadas, J.M. Gimble, L.A. Behie, A.J. Salgado, Influence of passage number on the impact of the secretome of adipose tissue stem cells on neural survival, neurodifferentiation and axonal growth, Biochimie. 155 (2018) 119–128. https://doi.org/10.1016/j.biochi.2018.09.012. [22] R.C. Assunção-Silva, B. Mendes-Pinheiro, P. Patrício, L.A. Behie, F.G. Teixeira, L. Pinto, A.J. Salgado, Exploiting the impact of the secretome of MSCs isolated from different tissue sources on neuronal differentiation and axonal growth, Biochimie. 155 (2018) 83–91. https://doi.org/10.1016/j.biochi.2018.07.026. [23] L.A. Rocha, E.D. Gomes, J.L. Afonso, S. Granja, F. Baltazar, N.A. Silva, M.S. Shoichet, R.A. Sousa, D.A. Learmonth, A.J. Salgado, In vitro Evaluation of ASCs and HUVECs Co-cultures in 3D Biodegradable Hydrogels on Neurite Outgrowth and Vascular Organization, Front. Cell Dev. Biol. 8 (2020) 1–14. https://doi.org/10.3389/fcell.2020.00489. [24] A.G. Pinho, J.R. Cibrão, R. Lima, E.D. Gomes, S.C. Serra, J. Lentilhas-Graça, C. Ribeiro, S. Lanceros-Mendez, S.F.G. Teixeira, S. Monteiro, N.A. Silva, A.J. Salgado, Immunomodulatory and regenerative effects of the full and fractioned adipose tissue derived stem cells secretome in spinal cord injury, Exp. Neurol. 351 (2022) 113989. https://doi.org/10.1016/j.expneurol.2022.113989. [25] C.A. Bowers, B. Kundu, G.W.J. Hawryluk, Methylprednisolone for acute spinal cord injury: An increasingly philosophical debate, Neural Regen. Res. 11 (2016) 882–885. https://doi.org/10.4103/1673-5374.184450. [26] K.A. Follett, R.L. Boortz-Marx, J.M. Drake, S. DuPen, S.J. Schneider, M.S. Turner, R.J. Coffey, Prevention and management of intrathecal drug delivery and spinal cord stimulation system infections, Anesthesiology. 100 (2004) 1582–1594. https://doi.org/10.1097/00000542200406000-00034. [27] J.R.W. Kestle, H.J. Hoffman, D. Soloniuk, R.P. Humphreys, J.M. Drake, E.B. Hendrick, A concerted effort to prevent shunt infection, Child’s Nerv. Syst. 9 (1993) 163–165. https://doi.org/10.1007/BF00272269. [28] Z. Gong, D. Lei, C. Wang, C. Yu, K. Xia, J. Shu, L. Ying, J. Du, J. Wang, X. Huang, L. Ni, C. Wang,
117 J. Lin, F. Li, Z. You, C. Liang, Bioactive Elastic Scaffolds Loaded with Neural Stem Cells Promote Rapid Spinal Cord Regeneration, ACS Biomater. Sci. Eng. 6 (2020) 6331–6343. https://doi.org/10.1021/acsbiomaterials.0c01057. [29] J. Pan, J. Deng, Y. Luo, L. Yu, W. Zhang, X. Han, Z. You, Y. Liu, Thermosensitive Hydrogel Delivery of Human Periodontal Stem Cells Overexpressing Platelet-Derived Growth Factor-BB Enhances Alveolar Bone Defect Repair, Stem Cells Dev. 28 (2019) 1620–1631. https://doi.org/10.1089/scd.2019.0184. [30] L. Zeng, J. He, Y. Cao, J. Wang, Z. Qiao, X. Jiang, L. Hou, J. Zhang, Tissue-adhesive and highly mechanical double-network hydrogel for cryopreservation and sustained release of anti-cancer drugs, Smart Mater. Med. 2 (2021) 229–236. https://doi.org/10.1016/j.smaim.2021.07.005. [31] R. Shultz, Y. Zhong, Hydrogel-based local drug delivery strategies for spinal cord repair, Neural Regen. Res. 16 (2021) 247–253. https://doi.org/10.4103/1673-5374.290882. [32] J. Piantino, J.A. Burdick, D. Goldberg, R. Langer, L.I. Benowitz, An injectable, biodegradable hydrogel for trophic factor delivery enhances axonal rewiring and improves performance after spinal cord injury, Exp. Neurol. 201 (2006) 359–367. https://doi.org/10.1016/j.expneurol.2006.04.020. [33] B. Ghosh, J. Nong, Z. Wang, M.W. Urban, N.M. Heinsinger, V.A. Trovillion, M.C. Wright, A.C. Lepore, Y. Zhong, A hydrogel engineered to deliver minocycline locally to the injured cervical spinal cord protects respiratory neural circuitry and preserves diaphragm function, Neurobiol. Dis. 127 (2019) 591–604. https://doi.org/10.1016/j.nbd.2019.04.014. [34] H.L. Xu, F.R. Tian, J. Xiao, P.P. Chen, J. Xu, Z.L. Fan, J.J. Yang, C.T. Lu, Y.Z. Zhao, Sustainedrelease of FGF-2 from a hybrid hydrogel of heparin-poloxamer and decellular matrix promotes the neuroprotective effects of proteins after spinal injury, Int. J. Nanomedicine. 13 (2018) 681–694. https://doi.org/10.2147/IJN.S152246. [35] D. Silva, R.A. Sousa, A.J. Salgado, Hydrogels as delivery systems for spinal cord injury regeneration, Mater. Today Bio. 9 (2021) 100093. https://doi.org/10.1016/j.mtbio.2021.100093. [36] V. Estrada, N. Brazda, C. Schmitz, S. Heller, H. Blazyca, R. Martini, H.W. Müller, Long-lasting significant functional improvement in chronic severe spinal cord injury following scar resection and polyethylene glycol implantation, Neurobiol. Dis. 67 (2014) 165–179. https://doi.org/10.1016/j.nbd.2014.03.018. [37] L.T.A. Hong, Y.M. Kim, H.H. Park, D.H. Hwang, Y. Cui, E.M. Lee, S. Yahn, J.K. Lee, S.C. Song, B.G. Kim, An injectable hydrogel enhances tissue repair after spinal cord injury by promoting extracellular matrix remodeling, Nat. Commun. 8 (2017) 1–14. https://doi.org/10.1038/s41467-017-00583-8. [38] J.A. Burdick, K.S. Anseth, Photoencapsulation of osteoblasts in injectable RGD-modified PEG hydrogels for bone tissue engineering, Biomaterials. 23 (2002) 4315–4323. https://doi.org/10.1016/S0142-9612(02)00176-X. [39] C.C. Lin, K.S. Anseth, PEG hydrogels for the controlled release of biomolecules in regenerative medicine, Pharm. Res. 26 (2009) 631–643. https://doi.org/10.1007/s11095-008-9801-2. [40] K.J. Lampe, D.S. Kern, M.J. Mahoney, K.B. Bjugstad, The administration of BDNF and GDNF to the brain via PLGA microparticles patterned within a degradable PEG-based hydrogel: Protein distribution and the glial response, J. Biomed. Mater. Res. - Part A. 96 A (2011) 595–607. https://doi.org/10.1002/jbm.a.33011. [41] U. Freudenberg, A. Hermann, P.B. Welzel, K. Stirl, S.C. Schwarz, M. Grimmer, A. Zieris, W. Panyanuwat, S. Zschoche, D. Meinhold, A. Storch, C. Werner, A star-PEG-heparin hydrogel platform to aid cell replacement therapies for neurodegenerative diseases, Biomaterials. 30 (2009) 5049–5060. https://doi.org/10.1016/j.biomaterials.2009.06.002.
124 starPEG-Hep+sec vs SHAM -8.49 2.11 <0.0001 starPEG-Hep+NbA vs Secretome 0.26 2.47 0.92 starPEG-Hep+NbA vs NbA 1.57 2.55 0.54 starPEG-Hep+NbA vs SHAM -8.38 2.47 0.002 Secretome vs NbA 1.31 2.20 0.56 Secretome vs SHAM -8.64 2.11 <0.0001 NbA vs SHAM -9.95 2.20 <0.0001 Supplementary Table III. S4 Multiple comparisons between groups using Tukey’s correction for the percentage of Iba1 ameboid area in spinal cord longitudinal sections. Iba1 ameboid area Comparison between groups Mean Difference SEM p -Value starPEG-Hep+sec vs starPEG-Hep+NbA -5.55 3.39 0.49 starPEG-Hep+sec vs Secretome -9.71 2.89 0.019 starPEG-Hep+sec vs NbA -10.40 3.01 0.015 starPEG-Hep+sec vs SHAM 38.49 2.89 <0.0001 starPEG-Hep+NbA vs Secretome -4.16 3.39 0.74 starPEG-Hep+NbA vs NbA -4.86 3.49 0.64 starPEG-Hep+NbA vs SHAM 44.04 3.39 <0.0001 Secretome vs NbA -0.70 3.01 0.99 Secretome vs SHAM 48.20 2.89 <0.0001 NbA vs SHAM 48.89 3.01 <0.0001 Supplementary Table III. S5 Pairwise comparisons between groups using Tukey’s correction for positive area of GFAP and NF. GFAP Rostral Comparison between groups Mean Difference SEM p -Value starPEG-Hep+secr vs starPEG-Hep+NbA -1.19 2.49 0.99 starPEG-Hep+sec vs Secretome -0.35 2.12 1 starPEG-Hep+sec vs NbA -1.36 2.21 0.97 starPEG-Hep+sec vs SHAM 4.37 2.12 0.27 starPEG-Hep+NbA vs secretome 0.83 2.49 0.99 starPEG-Hep+NbA vs NbA -0.18 2.56 1 starPEG-Hep+NbA vs SHAM 5.55 2.49 0.2 Secretome vs NbA -1.00 2.21 0.99 Secretome vs SHAM 4.72 2.12 0.20 NbA vs SHAM 5.73 2.21 0.10 Epicenter starPEG-Hep+sec vs starPEG-Hep+NbA 0.03 1.79 1 starPEG-Hep+secvs Secretome -1.60 1.53 0.83 starPEG-Hep+sec vs NbA -1.2 1.59 0.94
125 starPEG-Hep+sec vs SHAM -3.75 1.53 0.13 starPEG-Hep+NbA vs secretome -1.62 1.79 0.25 starPEG-Hep+NbA vs NbA -1.23 1.85 0.96 starPEG-Hep+NbA vs SHAM -3.77 1.79 0.25 Secretome vs NbA 0.39 1.59 0,99 Secretome vs SHAM -2.15 1.53 0.63 NbA vs SHAM -2.54 1.59 0.51 Caudal starPEG-Hep+secr vs starPEG-Hep+Nba 0.83 2.27 0.99 starPEG-Hep+sec vs Secretome -2.20 1.94 0.79 starPEG-Hep+sec vs NbA -0.41 2.02 1 starPEG-Hep+sec vs SHAM 2.96 1.94 0.56 starPEG-Hep+NbA vs secretome -3.02 2.27 0.99 starPEG-Hep+NbA vs NbA -1.24 2.34 0.98 starPEG-Hep+NbA vs SHAM 2.14 2.27 0.88 Secretome vs NbA 1.79 2.02 0.89 Secretome vs SHAM 5.16 1.94 0.08 NbA vs SHAM 3.37 2.02 0.46 NF Rostral starPEG-Hep+sec vs starPEG-Hep+Nba -2.14 1.55 0.64 starPEG-Hep+sec vs Secretome -0.88 1.32 0.96 starPEG-Hep+sec vs NbA -2.49 1.38 0.39 starPEG-Hep+sec vs SHAM -0.80 1.32 0.97 starPEG-Hep+NbA vs secretome 1.26 1.55 0.92 starPEG-Hep+NbA vs NbA -0.34 1.60 0.99 starPEG-Hep+NbA vs SHAM 1.35 1.55 0.91 Secretome vs NbA -1.61 1.38 0.77 Secretome vs SHAM 0.085 1.32 1 NbA vs SHAM 1.69 1.38 0.74 Epicenter starPEG-Hep+sec vs starPEG-Hep+NbA 1.50 1.48 0.85 starPEG-Hep+sec vs Secretome 0.68 1.26 0.98 starPEG-Hep+sec vs NbA 0.64 1.31 0.99 starPEG-Hep+sec vs SHAM 0.46 1.26 0.99 starPEG-Hep+NbA vs secretome -0.82 1.48 0.98 starPEG-Hep+NbA vs NbA -0.86 1.53 0.97 starPEG-Hep+NbA vs SHAM -1.04 1.48 0.95 Secretome vs NbA -0.04 1.31 1 Secretome vs SHAM -0.22 1.26 1 NbA vs SHAM -0.18 1.26 1 Caudal starPEG-Hep+sec vs starPEG-Hep+NbA -1.63 1.30 0.72 starPEG-Hep+sec vs Secretome -1.22 1.11 0.80 starPEG-Hep+sec vs NbA -1.81 1.15 0.53 starPEG-Hep+sec vs SHAM -0.04 1.11 1
126 starPEG-Hep+NbA vs secretome 0.40 1.30 0.99 starPEG-Hep+NbA vs NbA -0.18 1.34 1 starPEG-Hep+NbA vs SHAM 1.59 1.30 0.74 Secretome vs NbA -0.58 1.15 0.99 Secretome vs SHAM 1.18 1.11 0.82 NbA vs SHAM 1.77 1.15 0.55 Supplementary Table III. S6 Pairwise comparisons between groups using Tukey’s correction for SMI71 quantification. Parameters such as Vessel Area, Vessel Length and Average Vessel Length in Angio Tool software. SMI Vessel Area Comparison between groups Mean Difference SEM p -Value starPEG-Hep+sec vs StarPEG-Hep+NbA 0.02 0.02 0.83 starPEG-Hep+sec vs Secretome 0.00 0.02 1 starPEG-Hep+sec vs NbA 0.01 0.02 0.96 starPEG-Hep+sec vs SHAM -0.02 0.02 0.83 starPEG-Hep+NbA vs Secretome -0.02 0.02 0.85 starPEG-Hep+NbA vs NbA -0.01 0.02 0.99 starPEG-Hep+NbA vs SHAM -0.04 0.02 0.31 Secretome vs NbA 0.01 0.02 0,97 Secretome vs SHAM -0.02 0.02 0.79 NbA vs SHAM -0.03 0.02 0.46 Vessel Length starPEG-Hep+sec vs starPEG-Hep+NbA 1.03 1.01 0.84 starPEG-Hep+sec vs Secretome 0.47 0.86 0.98 starPEG-Hep+sec vs NbA 0.32 0.89 1 starPEG-Hep+sec vs SHAM -0.81 0.86 0.88 starPEG-Hep+NbA vs Secretome -0.56 1.01 0.98 starPEG-Hep+NbA vs NbA -0.71 1.04 0.96 starPEG-Hep+NbA vs SHAM -1.84 1.01 0.38 Secretome vs NbA -0.15 0.89 1 Secretome vs SHAM -1.28 0.86 0.58 NbA vs SHAM -1.13 0.89 0.71 Average vessel length starPEG-Hep+sec vs starPEG-Hep+NbA -0.01 0.01 0.74 starPEG-Hep+sec vs Secretome 0.00 0.01 0.99 starPEG-Hep+sec vs NbA -0.00 0.01 0.94 starPEG-Hep+sec vs SHAM 0.01 0.01 0.88 starPEG-Hep+NbA vs Secretome 0.01 0.01 0.53 starPEG-Hep+NbA vs NbA 0.00 0.01 0.98 starPEG-Hep+NbA vs SHAM 0.01 0.01 0.29
127 Secretome vs NbA -0.00 0.01 0.78 Secretome vs SHAM 0.00 0.01 0.98 NbA vs SHAM 0.01 0.01 0.48 Supplementary Figure III. S1 Molecular analysis of collected sera following SCI using Rat Cytokine Array C2 from RayBiotech. Representative heat map of the panel for the different groups was generated using the BROAD Institute's R implementation of Morpheus with Euclidean distance hierarchical clustering at four wpi (A) and eight wpi (B).
CHAPTER IV Conclusions and Future Perspectives
129 General Discussion Central Nervous System reveals as a fascinating structure that encompasses all functions in the body. Injuries in each region totally disturb the overall normal function. Particularly, spinal cord injury (SCI) is responsible for total or partial loss of both motor and sensorial functions below the level of injury. Both have a huge impact on patient’s life quality at physical and psychological burdens. For scientists, SCI remains a challenge either by its pathophysiological complexity or unknown processes that are responsible for degeneration or regeneration. A cascade of events goes from acute to chronic phase within seconds to months. After a mechanical insult, a myriad of events comprehends I) vascular disruption; II) inflammation; III) release of inflammatory cytokines; IV) excitotoxicity; V) apoptosis and VI) axonal degeneration [1]. Ultimately, the establishment of the chronic phase is characterized by the glial scar formation, which is composed of lesion core and border. In the lesion core, stromal-derived fibroblasts and inflammatory immune cells fill all space, while the lesion border is formed by reactive astrocytes. The scar acts as a barrier for axonal growth or tissue regeneration and at the same time circumvents the injury locally, preventing its spread [2,3]. In the last years, we have witnessed a growth of multivariate fields in developing therapies to tackle SCI regeneration. Drugs such as methylprednisolone (MPSS) were vastly used in an attempt to reduce inflammation and promote recovery [4,5]. Although upon the report of severe side effects, such as liver toxicity or hemorrhages, it was highly unadvised it use [6,7]. Other approaches in clinical trials try to promote regeneration by administering growth factors [8,9] or even anti-bodies [10], promising approaches to improve neurological outcomes. However, side effects or administration routes are still a concern in their translation to the clinic. Other interesting approaches rely on the use of cell-based therapy. The purpose is the repopulation of spinal cord tissue with new cells, that will migrate to injured tissue and replace dead neuronal cells. In clinical trials, they were able to promote improvements in sensory function [11] or bladder control [12] after transplantation. Our group has shown that adipose tissue-derived stem cells (ASCs) were able to promote recovery after transplantation at injury site [13– 15]. However, their effects are mainly attributed to intense paracrine effects, their secretome. Secreted molecules have been shown to play an important role in regenerative processes, such as axonal growth, suporte cell survival, and differentiation [16–18] and ultimately lead to motor recovery after systemic administration [19]. Considering all mentioned facts, in this work, we envisioned designing a more effective way to deliver hASCs secretome at SCI injury site. The advantages include a more target and specific therapy, reduce the dosage needed, protecting molecules from enzymatic degradation, and minimizing the risk of off-
130 target effects. For that, hydrogels have been revealed as a suitable platform to use. They are similar to the extracelular matrix (ECM), have high water content and per se improve motor function in SCI animal models as in the case of use poly(ethilene glycol) (PEG) hydrogels [20]. Freudenberg et al developed a biohybrid hydrogel made of star-shaped PEG hydrogel starPEG) and the glycosaminoglycan (GAG) Heparin (Hep) [21]. The rationale is based on affinity-based systems where, anionic charge arising from the high density of sulfate moieties on heparin results in a high affinity for a broad range of growth factors, cytokines, and chemokines mainly due to electrostatic interactions [22]. Remarkably, controlling their release in a time-dependent manner. Importantly is the fact that these matrices can be injected in a liquid state and by Michael type reaction form in situ polymerization hydrogels [23]. In chapter II, we performed the mechanical characterization of starPEG-Hep hydrogels showing modulation of stiffness, mesh size, and swelling degree. Those are important features not only for in vivo implantation but also for their use as delivery systems. Upon hASCS secretome loading in starPEG-Hep hydrogels, a controlled and prolonged release was achieved for over 10 days. At that time point approximately 70% of cumulative release, was demonstrated in fluorescence experiments. A similar profile to other release systems of growth factors and cytokines [24,25]. Moreover, Gal-1 was found to be released in a continuous and extended way in the multiplex assay. The sensitivity of the multiplex assay was revealed to be very low, most probably by the low concentration of analytes in released samples, which did not allow the identification of more molecules. To pursue this, membrane-based protein arrays were used and released samples were collected at only two and ten days. The results revealed a release profile in which cytokines or factors involved in angiogenesis processes were mainly released after two days. The burst release in the first days, shown in fluorescence assays, was mainly due to the high release of these molecules. On the other side, extended release is supported by neuroregulatory growth factors that had a higher cumulative release from two to ten days. In studies in which these molecules are independently loaded in these hydrogels, continuous release profile was reported [26–28]. Despite the known role of heparin in affinity-based systems, is interesting to highlight this release profile. The results suggest that molecules have a different interaction with heparin as well as dissociation rate which impacts their release [29]. Another alternative is that the high amount of diverse proteins was impacting individual interactions with heparin. It would be interesting to perform electrophoretic techniques such as surface plasmon resonance [30], or in silico calculations [31] to decode affinity and kinetics of heparin-protein interactions. Additionally, starPEG-Hep loading secretome could promote significant higher neuronal differentiation of immature (DCX) and mature (MAP2) neurons, when compared with hydrogel loading vehicle. Also, higher
131 neurite outgrowth was observed in organotypic spinal cord slices. In addition, we hypothesized that these processes were mediated by molecules identified in membrane-based protein arrays, such as BDNF, GDNF, ß-NGF, previously shown to modulate those processes [32,33]. Subsequently, these results corroborate the fact that starPEG-Hep hydrogels are continuously releasing secretome, once the percentage of differentiated cells was similar to the levels of free secretome, which was in higher contact with cells during the experiment. In the case of neurite outgrowth, starPEG-Hep+sec promoted a significantly higher percentage of neurofilament area, in contrast to free secretome that was removed after 48h. The last question of this work (Chapter III) was to evaluate the capacity of the developed release system in promoting motor recovery in T8 transection SCI rat model. This results in a very severe lesion, when compared with hemisection or contusion models [34]. This happens due to complete interruption of neuronal transmission when cutting the spinal cord, resulting in total paralysis of the hindlimbs. Besides this, the total transection model does not reproduce the most frequent lesions in human [35]. However, in tissue engineering field, particularly with biomaterials, the advantage of this model is related with the depot created upon lesion that is easily filled by the hydrogel (combined or not with a loading agent) [34,36]. Motor recovery indicated that starPEG-Hep+sec led to a more robust motor recovery, traducing in significance BBB score at two, and six and eight weeks in comparison treated groups. Interestingly, lower motor recovery was observed in animals treated with local secretome, evidencing that local treatment has a low potential to promote regeneration, probably due to fast clearance from injury site. These results are in accordance with neurite outgrowth in spinal cord slices cultures (Chapter II) where the free secretome condition had a lower neurite extension. Gross motor function or sensorial function were evaluated, but no significant recovery was observed. Histological analysis revealed a significant reduction of ameboid microglia, associated with a more active state, in animals treated with starPEG-Hep+sec in comparison to free secretome or vehicle. Importantly, animals treated with only starPEG-Hep+NbA, did not show further inflammation at injury site, which has disclosed the safety of the hydrogel. Other markers for astrocytes (GFAP), axonal regeneration (NF), or blood brain barrier integrity (SMI-71) did not present significant results. Regarding the SMI-71 results, it would be interesting to evaluate the vascular integrity in early time points, once BSCB integrity is gradually restored until 14 days post-injury [37]. Coupling with an assessment of BBB permeability with occludin, would clarify the revascularization process after lesion [38]. Restoration of vasculature in SCI is crucial to restore nutrient diffusion and cellular maintenance [39]. Altogether, these results reinforce the fact that hASCs secretome was mainly impacting as
132 immunomodulatory agent locally. Systemic characterization of inflammatory levels was also performed during three distinct phases of the condition (acute, intermediate and chronic). From acute to chronic stage, starPEG-Hep+sec treated group promoted increased levels of IL-10, an anti-inflammatory cytokine. At the same time, CD86 and MCP-1, have different expressions. CD86, a M1 marker, is highly expressed at 48h and decrease in intermediate and chronic phase, compared with animals treated with vehicle. In case of MCP-1, a decreased expression in acute phase is counteracted with increased expression in chronic phase. All inflammatory processes could be mediated by proand anti-inflammatory cytokines highly released in membrane-based protein arrays (Chapter II). This includes IL-4 and IFN-γ demonstrated to be able of inducing IL-10 increase systemically [40,41] or VEGF that promote motor recovery by reducing autophagy [42]. This could also point out that inflammatory stimulus could in some extent contribute to regenerative favoring [43,44]. Remarkably, even though the treatment was applied locally, it also has the capacity to modulate the systemic inflammatory response. Despite some improvements that could be done in order to refine the use of starPEG-Hep+sec as delivery systems, in this work we demonstrated for the first time that a biohybrid hydrogel was able to sustainably release hASCs secretome. Several factors released were found to play a critical role in modulating essential processes to foster regenerative processes, demonstrated by increase neuronal differentiation and neurite outgrowth in in vitro assays. SCI lesioned animals were capable of a significant motor improvement when treated with starPEG-Hep+sec compared to other treated groups. We have shown that modulating the inflammatory response after lesion is primarily responsible for this recovery. Altogether, this data highly supports the use of starPEG-Hep+sec to trigger and modulate pro-regenerative processes, which may offer an effective clinical approach to tackle SCI.
133 Thesis Graphical Abstract Previous studies have highlighted the robust beneficial effects of ASCs secretome in modulating crucial processes in SCI regenerative medicine. Herein, using a hydrogel as a release system, we showed the controlled and prolonged release of ASCs secretome that is positively associated with regenerative events in vitro and in vivo models.