Effect of Tetracyclines on Osteopenic - and Osteoporotic-Derived Bone-Marrow Osteoblastic Cell Cultures

EFFECT OF TETRACYCLINES ON OSTEOPENICAND OSTEOPOROTIC-DERIVED BONE-MARROW OSTEOBLASTIC CELL CULTURES Joana Pereira de Pinho Dissertação para obtenção do grau de Mestre em Engenharia Biomédica Faculdade de Engenharia, Universidade do Porto Porto, julho de 2013 EFFECT OF TETRACYCLINES ON OSTEOPENICAND OSTEOPOROTIC-DERIVED BONE-MARROW OSTEOBLASTIC CELL CULTURES Orientadores Professor Doutor Pedro Sousa Gomes Faculdade de Medicina Dentária da Universidade do Porto Professora Doutora Maria Helena Raposo Fernandes Faculdade de Medicina Dentária da Universidade do Porto Faculdade de Engenharia, Universidade do Porto Porto, julho de 2013 i INDEX AGRADECIMENTOS........................................................................................................................ v ABSTRACT ..................................................................................................................................... vii RESUMO ........................................................................................................................................ ix FIGURE INDEX ................................................................................................................................ xi GRAPHIC INDEX ........................................................................................................................... xiii ABREVIATION LIST ........................................................................................................................ xv I. LITERATURE OVERVIEW ........................................................................................................ 1 BONE TISSUE ................................................................................................................................. 2 BONE STRUCTURE ..................................................................................................................... 2 CLASSIFICATION ........................................................................................................................ 3 Macroscopic Classification .................................................................................................... 3 Microscopic Classification ..................................................................................................... 3 BONE TISSUE FORMATION ........................................................................................................ 6 BONE CELLS ............................................................................................................................... 8 Osteobastic Lineage Cells ...................................................................................................... 9 Osteoclastic Lineage Cells ................................................................................................... 11 BONE MATRIX .......................................................................................................................... 12 BONE REMODELING ................................................................................................................ 13 BONE METABOLISM ................................................................................................................ 16 BONE REGENERATION ............................................................................................................. 19 OSTEOPOROSIS AND OSTEOPENIA.............................................................................................. 21 EPIDEMIOLOGY ....................................................................................................................... 23 PATHOPHYSIOLOGY................................................................................................................. 24 DIAGNOSIS............................................................................................................................... 28 Radiography ........................................................................................................................ 28 BMD Measurement ............................................................................................................. 28 PREVENTION AND TREATMENT .............................................................................................. 31 TETRACYCLINES ........................................................................................................................... 32 II. RESEARCH HYPOTHESIS AND OBJECTIVES .......................................................................... 35 RESEARCH HYPOTHESIS ............................................................................................................... 36 OBJECTIVES .................................................................................................................................. 36 III. MATERIALS AND METHODS ............................................................................................ 37 ii MATERIALS .................................................................................................................................. 38 METHODS .................................................................................................................................... 38 ANIMALS .................................................................................................................................. 38 Animals and Experimental Groups ...................................................................................... 38 Ovariectomy and Sham Surgical Procedure ........................................................................ 40 Assessment of the Bone Tissue Structure ........................................................................... 40 ESTABLISHMENT OF THE BONE-MARROW DERIVED OSTEOBLASTIC CULTURES.................... 41 CHARACTERIZATION OF CELL CULTURES ................................................................................ 42 Optical Microscopy .............................................................................................................. 42 Cell Proliferation Assay (DNA Content) ............................................................................... 42 Metabolic Activity Assay (MTT Assay) ................................................................................. 42 Apoptosis Assay ................................................................................................................... 43 Assessment of Cell Morphology .......................................................................................... 43 Total Protein Content .......................................................................................................... 44 Alkaline Phosphatase Activity ............................................................................................. 44 Histochemical Methods ...................................................................................................... 45 Gene Expression Analysis .................................................................................................... 46 STATISTICAL ANALYSIS ............................................................................................................ 46 IV. RESULTS ........................................................................................................................... 47 ASSESSMENT OF THE BONE TISSUE STRUCTURE ........................................................................ 48 ESTABLISHMENT OF BONE MARROW-DERIVED OSTEOBLASTIC CULTURES FROM SHAM (CONTROL) ANIMALS................................................................................................................... 48 CELL PROLIFERATION .............................................................................................................. 49 METABOLIC ACTIVITY .............................................................................................................. 49 APOPTOSIS .............................................................................................................................. 50 CELL MORPHOLOGY ................................................................................................................ 51 ALKALINE PHOSPHATASE ACTIVITY ......................................................................................... 52 ALKALINE PHOSPHATASE STAINIG .......................................................................................... 52 COLLAGEN STAINING ............................................................................................................... 53 EXPRESSION OF OSTEOGENIC-RELATED MARKERS ................................................................. 54 EXPRESSION OF ADIPOGENIC-RELATED MARKERS ................................................................. 55 ESTABLISHMENT OF BONE MARROW-DERIVED OSTEOBLASTIC CULTURES FROM OSTEOPENIC ANIMALS ...................................................................................................................................... 56 CELL PROLIFERATION .............................................................................................................. 57 METABOLIC ACTIVITY .............................................................................................................. 57 iii APOPTOSIS .............................................................................................................................. 58 CELL MORPHOLOGY ................................................................................................................ 59 ALKALINE PHOSPHATASE ACTIVITY ......................................................................................... 60 ALKALINE PHOSPHATASE STAINIG .......................................................................................... 61 COLLAGEN STAINING ............................................................................................................... 62 EXPRESSION OF OSTEOGENIC-RELATED MARKERS ................................................................. 63 EXPRESSION OF ADIPOGENIC-RELATED MARKERS ................................................................. 64 ESTABLISHMENT OF BONE MARROW-DERIVED OSTEOBLASTIC CULTURES FROM OSTEOPOROTIC ANIMALS ........................................................................................................... 65 CELL PROLIFERATION .............................................................................................................. 66 METABOLIC ACTIVITY .............................................................................................................. 66 APOPTOSIS .............................................................................................................................. 67 CELL MORPHOLOGY ................................................................................................................ 68 ALKALINE PHOSPHATASE ACTIVITY ......................................................................................... 69 ALKALINE PHOSPHATASE STAINIG .......................................................................................... 69 COLLAGEN STAINING ............................................................................................................... 70 EXPRESSION OF OSTEOGENIC-RELATED MARKERS ................................................................. 71 EXPRESSION OF ADIPOGENIC-RELATED MARKERS ................................................................. 72 V. DISCUSSION ......................................................................................................................... 73 VI. CONCLUSION ................................................................................................................... 83 VII. REFERENCES .................................................................................................................... 85 iv v AGRADECIMENTOS Ao Professor Doutor Pedro Sousa Gomes e à Professora Doutora Maria Helena Fernandes, agradeço o apoio e incentivo constantes, a disponibilidade, os ensinamentos transmitidos no último ano e meio, e a supervisão do trabalho de investigação que levou à realização desta dissertação. À Dra. Mónica Garcia e ao José Carlos do Laboratório de Farmacologia e Biocompatibilidade Celular da Faculdade de Medicina Dentária, Universidade do Porto, agradeço a motivação, paciência e ajuda nos procedimentos laboratoriais. Aos meus amigos, em especial à Sofia Ribeiro, agradeço a apoio e amizade durante a elaboração desta dissertação. Aos meus pais e irmão, agradeço a motivação e amor incondicionais, não só durante esta fase, como ao longo de todos os momentos da minha vida. xii Figure 19 – Collagen staining of osteopenic rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. ............................................... 63 Figure 20 – CLSM imaging of osteoporotic rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. ............................................... 68 Figure 21 – ALP staining of osteoporotic rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. ..................................................... 70 Figure 22 – Collagen staining of osteoporotic rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. ............................ 71 xiii GRAPHIC INDEX Graphic 1 – Cell proliferation of sham rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. .......................................................... 49 Graphic 2 – Metabolic activity of sham rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. ..................................................... 50 Graphic 3 – Apoptosis of sham rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. ................................................................... 50 Graphic 4 – ALP activity of sham rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. .......................................................... 52 Graphic 5 – RT-PCR gene expression of ALP, BMP-2, Col I, OPG,OPN and OC, in sham rat bone marrow-derived cell cultures established at days 7 and 14, in the absence and in the presence of doxycycline. ............................................................................................................................. 55 Graphic 6 – RT-PCR gene expression of PPARγ, CFD and AP-2, in sham rat bone marrow-derived cell cultures established at days 7 and 14, in the absence and in the presence of doxycycline 56 Graphic 7 – Cell proliferation of osteopenic rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. ............................................... 57 Graphic 8 – Metabolic activity of osteopenic rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. ............................................... 58 Graphic 9 – Apoptosis of osteopenic rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. .......................................................... 59 Graphic 10 – ALP activity of osteopenic rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. ..................................................... 61 Graphic 11 – RT-PCR gene expression of ALP, BMP-2, Col I, OPG, OPN and OC, in osteopenic rat bone marrow-derived cell cultures established at days 7 and 14, in the absence and in the presence of doxycycline. ............................................................................................................. 64 Graphic 12 – RT-PCR gene expression of PPARγ, CFD and AP-2, in osteopenic rat bone marrowderived cell cultures established at days 7 and 14, in the absence and in the presence of doxycycline. ................................................................................................................................. 65 Graphic 13 – Cell proliferation of osteoporotic rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. ............................ 66 Graphic 14 – Metabolic activity of osteoporotic rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. ............................ 67 xiv Graphic 15 – Apoptosis of osteoporotic rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. ..................................................... 67 Graphic 16 – ALP activity of osteoporotic rat bone marrow-derived cell cultures, .................... 69 Graphic 17 – RT-PCR gene expression of ALP, BMP-2, Col I, OPG, OPN and OC, in osteoporotic rat bone marrow-derived cell cultures established at days 7 and 14, in the absence and in the presence of doxycycline. ............................................................................................................. 72 Graphic 18 – RT-PCR gene expression of PPARγ, CFD and AP-2, in osteoporotic rat bone marrow-derived cell cultures established at days 7 and 14, in the absence and in the presence of doxycycline. ............................................................................................................................. 72 xv ABREVIATION LIST AA – Ascorbic Acid ALP – Alkaline Phosphatase AMV-RT – Avian Myeloblastosis Virus RT BMD – Bone Mineral Density BMP – Bone Morphogenetic Protein BMU – Bone Multicellular Unit BV/TV – Bone Volume/Total Volume Cbfa1 – Core-binding Factor α1 CLSM – Confocal Laser Scanning Microscopy CMT – Chemically Modified Tetracyclines CNS – Central Nervous System CO2 – Carbon Dioxid CT – Computed Tomography DNA – Deoxyribonucleic Acid dNTP – Deoxynucleoside Triphosphate DTT – Dithiothreitol DXA – Dual-Energy X-ray Absorptiometry D1 – Doxycycline ECM – Extracellular Matrix FBS – Fetal Bovine Serum FUNGI – Fungizone GH – Growth Hormone HA – Hydroxyapatite IGF-I – Insulin-like Growth Factor-I IGF-II – Insulin-like Growth Factor-II IL-1 – Interleukin-1 IP – Intraperitoneal MMP – Matrix Metalloproteinase MSC – Mesenchymal Stem Cell NaOH – Sodium Hydroxid OC – Osteocalcin OPG – Osteoprotegerin xvi OPN – Osteopontin Osx – Osterix PDGF – Platelet-derived Growth Factor PEST – Penicillin-streptomycin pNPP – p-nitrophenylphosphate disodium PTH – Parathyroid Hormone QUS – Quantitative Ultrasound RANK – Receptor Activator of Nuclear Factor κB RANKL – RANK Ligand Tb N – Trabecular Number Tb Sp – Trabecular Separation Tb Th – Trabecular Thickness TGF-β – Transforming Growth Factor-β TNF – Tumor Necrosis Factor TRAPase – Tartrate-resistant Acid Phosphatase VOI – Volume-Of-Interest WHO – World Health Organization α-MEM – α-Minimal Essential Medium JOANA PINHO 1 I. LITERATURE OVERVIEW JOANA PINHO 2 BONE TISSUE BONE STRUCTURE Bone is a connective tissue considerable complex and important since it is responsible for strength and stiffness of the skeleton, ensuring the protection of soft tissues and organs (1). Bone is constantly remodeling (bone resorption and bone formation) (2) and has two main components: the extracellular bone matrix and bone cells (3). Due to its unique characteristics, its material composition and structural properties (4), bone tissue has various functions of extreme importance in the human body such as: o Movement and locomotion (3); o Support – shape the skeleton that serves to support muscle insertion (3); o Protection – protects internal tissues and organs (e.g.: the central nervous system (CNS), the brain and spinal canal, and bone marrow) (3); o Plasticity – must be flexible enough to be able to remodel in response to external forces, but should also be rigid and resilient to forces (4); o Maintenance of mineral homeostasis, serving as a reservoir for storing minerals – calcium and phosphate ions (3,5-7). The bone contains 99% of the body’s calcium and acts as a reserve of this ion; o Provides the proper environment for hematopoiesis within the bone marrow spaces (6,7). Figure 1 – Structure of bone tissue in a long bone (8). LITERATURE OVERVIEW 3 CLASSIFICATION Macroscopic Classification Bones can be classified regarding the form, being characterized in long, short and flattened bones (3,7). The first ones have greater length than width, and most of the bones of the upper and lower members are of this type; the second ones exhibit the length similar to the width; the last ones have a thin and flattened shape and are, as a rule, rounded (3,9). The long bone comprises three components: the diaphysis that corresponds to the bone’s body; the epiphysis are the edges of the bone; and the epiphyseal plate is composed of cartilage that ossified and becomes the epiphyseal line (3). Microscopic Classification At the microscopic level, the bone tissue can be classified as cortical or compact bone, with high density and rich in mineralized bone matrix, and trabecular or cancellous bone, constituted by small cavities surrounded by bone matrix (Figure 2) (3,10). These two types of bone are formed by a lamellar pattern, in which the collagen fibrils are placed in alternating directions (7). Figure 2 – Schematic representation of the bone tissue – cortical and trabecular bone (11) JOANA PINHO 4 The cortical bone is approximately 80% of the bone tissue existing in the body and is located in the diaphysis of long bones (1,10) and on the surface of the majority of bones (Figure 3). It is a dense bone due to the compaction of the collagen fibers (3). It’s organized in a Haversian system (Figure 4) (7) that consists of a central canal, the Haversian canal, which is surrounded by concentric bone, lamellae. Inside of these lamellae are the osteocytes within small spaces, lacunae (3,7). Inside are the blood vessels, which are responsible for osteocytes, nerves and connective tissue nutrition (7). The cortical bone has two surfaces, the endosteum and the periosteum (7). The periosteum is a membrane of connective tissue that covers the outer surface of the bone and has a high osteogenic potential (1,3). The endosteum has a similar structure and it’s located in the inner surface of long bones. It consists of a single cell layer, including osteoblasts, osteoclasts and osteochondral progenitor cells (3). These two membranes play an important role in the nutrition of bone tissue and contain the major cells of bone metabolism. Thereby, have a significant contribution to bone remodeling and regeneration processes. Figure 3 – Typical structure of a long bone. (a) Long bone components – epiphysis and diaphysis; (b) Epiphysis constitution – trabecular bone; (c) Diaphysis constitution – cortical bone (12). LITERATURE OVERVIEW 5 The trabecular bone is located inside the cortical bone, in the epiphyses of long bones (Figure 3) (10). It forms a network of bone plates connected to each other, the trabeculae, and the space between these is filled with bone marrow and blood vessels (Figure 4) (3). Trabeculae ensure the skeleton elasticity and stability. This type of bone is continuously remodeling, while cortical bone has a much slower renewal (13). Figure 4 – Structure of cortical and trabecular bone tissue (14). JOANA PINHO 12 BONE MATRIX The bone matrix is composed of two different components, an inorganic component (5070%) and an organic component (30-50%). The rest are lipids and water. The organic material consists mainly of collagen which confers resistance to the matrix – types I, III and V, where the most abundant is type I – and proteoglycans (1-3,17). There are also non-structural proteins, such as growth factors, blood proteins, osteonectin and OC (7). The inorganic material is responsible for the hardness of the bone matrix and is composed mostly by calcium (Ca2+) and phosphate (PO43-) ions that form crystals, also called HA crystals – Ca10(PO4)6(OH)2, when deposited in the matrix (1,3). These crystals are arranged along the collagen fibrils and are surrounded by proteoglycans and glycoproteins. Magnesium, sodium, potassium, bicarbonate and citrate ions also exist in the bone matrix but in lowest amount. The combination of HA with collagen fibrils gives the hardness and strength to the bone tissue that characterize it. Calcium is the main mineral in the skeleton formation, acting in its structure, internal organs protection and locomotion. Moreover, muscle contraction, blood clotting and neuronal excitation mechanisms require calcium (7,29). Thereby, the calcium concentration must be kept within specific limits in the different physiological compartments. The phosphate anion also exists in high concentration in bone tissue, being the major constituent of their crystalline structure. It is also an essential component of the membrane lipids and DNA, so it’s crucial for cell signaling (7). The balance and homeostasis of plasmatic phosphate are the reflection of food diet, renal excretion and internal exchanges between the extracellular fluid, soft tissues content and skeletal reservoirs (30). LITERATURE OVERVIEW 13 BONE REMODELING The bone tissue is in constant remodeling, i.e. there is a continuous balance between resorption and formation of new bone (25,31,32), regulated by the action of osteoclasts and osteoblasts, respectively (1,2,5,15,33). Bone remodeling is a physiological process that maintains the skeleton integrity (34). Through this process, about 5% of cortical bone and 20% of trabecular bone is renewed per year (35). It can be said that it is the cellular basis of bone metabolism, thus a change in the process may lead into a metabolic disorder. Figure 10 – Bone remodeling process (36) At the microscopic level, the bone remodeling process takes place in small areas of the cortical and trabecular surface called basic multicellular units (BMUs), characterized by the coordinated action of osteoclasts and osteoblasts (2,27,35). BMUs are a unique temporary structure with about 6-9 months of lifespan. During this time numerous generations of osteoclasts (lifespan of about 2 weeks) and osteoblasts (lifespan of about 3 months) are formed. The BMU comprises a group of osteoclasts in the front, a group of osteoblasts in the rear, a central vascular capillary, a nerve supply and associated connective tissue (2). A bone remodeling cycle consists of four different and sequential phases – activation, resorption, reversal and formation. JOANA PINHO 14 Bone remodeling begins with the activation of resting cells to the resorption place, including osteoclastic precursors and bone-marrow mononuclear monocyte-macrophages, which differentiate into multinucleated cells and active resorbing osteoclasts that begin the resorption process (27). In these phase the osteoclasts begin to dissolve the mineral matrix and decompose the osteoid matrix. There is also the release of growth factors contained within the matrix, essentially TGF-β, PDGF, IGF-I and IGF-II (35). After the bone resorption, osteoclasts migrate from the resorption site that is then invaded by a heterogeneous population of mononuclear cells with macrophage and pre-osteoblastic cells. When resorption is completed, it starts the reversal phase where the osteoclasts die through apoptosis and osteoblast precursors locally proliferate, differentiate into mature osteoblasts and migrate into the resorption lacuna made by osteoclasts (27). The following stage is the formation phase, in which osteoblasts synthesize new osteoid material that fills the resorption lacuna and becomes mineralized in the resting phase (27,35). As the organic matrix is mineralized, it surrounds the osteoblasts, which lose their synthetic activity and are therefore called osteocytes. The activation and regulation of bone resorption requires an interaction between osteoblasts and osteoclasts. The molecular mechanism that supports this relationship is explained by the RANK/RANKL/OPG system (Figure 11). RANK is a protein member of the tumor necrosis factor (TNF)-receptor superfamily and is expressed by mature osteoclasts, dendritic cells and some cancer cells (27). RANKL is a TNF superfamily member expressed by osteoblasts and is essential for the recruitment, differentiation, activation and survival of osteoclastic cells through binding to its specific receptor RANK (27). OPG is a soluble receptor of RANK and is synthesized by osteoblasts, stromal cells, vascular smooth muscle cells, B lymphocytes and articular chondrocytes (27). By modulating RANKL and OPG, osteoblasts can control osteoclast differentiation and activity and consequently bone remodeling: RANKL binds to RANK that is present on the surface of osteoclast precursors leading to the activation of Nuclear Factor κB and transcription of genes involved in osteoclastogenesis. OPG has the ability to act as a decoy by binding to RANKL and blocking the RANKL/RANK interaction (2,27). This way, OPG inhibits osteoclastogenesis, osteoclast activity and bone resorption. LITERATURE OVERVIEW 15 Figure 11 – Molecular mechanism of the RANK/RANKL/OPG system. Bone remodeling is a key process in the normal body behavior because it gives the characteristics that bone needs to maintain physiological functions (15). Furthermore, bone remodeling replaces old bone with inferior biomechanical properties and helps maintaining the normal calcium and phosphate concentrations (37). In young adults, the rate of bone resorption and formation are ideally equal, and the total bone mass remains constant, which do not occur in children, where bone formation rate is higher than bone resorption, since the bones are growing (7). Bone remodeling increases in premenopausal and postmenopausal women being then attenuated with age in both genders, continuing to be faster than in pre-menopausal periods (7,38). In pathological situations, it is common to see a change in the normal functioning of bone remodeling, i.e., an imbalance between the processes of bone formation and bone resorption, which leads to bone loss (39). Thus, there is an increase in the osteoclasts number and activity, the cells responsible for the bone resorption process, and/or a decreased differentiation of osteoblasts, which are responsible for bone formation (39). Osteoporosis is an example of a bone disease where this occurs. JOANA PINHO 16 BONE METABOLISM Bone is a mineral that releases calcium and phosphate in response to hormones secreted by several organs. These elements are very significant in the bone composition, and the levels monitoring are essential to the performance of many vital physiological functions. Therefore, normal levels of calcium, phosphate and related minerals are maintained by a complex regulatory system. The major hormones that regulate these values are the parathyroid hormone (PTH) and vitamin D (38). The calcitonin, growth hormone (GH) and estrogens also play an important role in the regulation of bone metabolism. The parathyroid hormone (PTH) is released by the parathyroid glands and its secretion is regulated by plasmatic concentration of calcium (13,29,40). The primary effect of PTH is to increase plasmatic calcium levels and reduce plasma phosphate concentration, acting in three main organs – the bone and the kidney, directly, and the gastrointestinal tract, indirectly (13,29,41). In the bone, PTH receptors are present in osteoblasts as well as in osteoclasts, and its effect in this tissue involves two mechanisms: o An immediate stimulation consists in the activation of the bone cells present in the bone tissue. PTH binds to specific receptors in osteoblasts and osteocytes and activates a calcium pump, where calcium is transferred from the bone fluid to the extracellular medium. Calcium levels in the bone fluid are rapidly replenished by removing calcium phosphate salts from amorphous deposits existent along the osteoblastic cells; o A slower effect, that can take several days or weeks, consists in the activation of the osteoclast system, with an increase in the osteoclasts recruitment and activity. This process will lead to an increased resorption of mineralized bone tissue. LITERATURE OVERVIEW 17 Vitamin D in its active form – 1.25-dihydroxyvitamin D3 (calcitriol) – is a steroid hormone (35) and one of the major regulators of calcium metabolism. Its main functions are to stimulate intestinal absorption of ingested calcium and to promote the calcium resorption by the bones (13,40). In the bone, only osteoblasts have calcitriol receptors, despite vitamin D stimulate bone resorption. As a result, recruitment, differentiation, and fusion of precursors into active osteoclast increase. Vitamin D is also involved in the process of new bone formation by inducing the synthesis of osteocalcin in osteoblasts. Moreover, it has an action of negative feedback suppressing, in a direct way, the gene responsible for the PTH synthesis (13,40). Calcitonin is produced in the parafollicular cells of the thyroid and its greater effect on bone metabolism is the reduction of plasma calcium concentration caused by the inhibition of bone resorption (28,35,41). By this way, it reduces the number and activity of osteoclasts (35). Hence, calcitonin is a physiological antagonist of PTH hormone in relation to calcium levels. However, their effect on phosphate concentration is similar, i.e. reduces plasma phosphate levels. The greatest stimulus for calcitonin secretion is the increase in plasma calcium (13,30), but vitamin D may also promote calcitonin levels by food intake, without increasing calcium concentration. Growth hormone (GH) is produced and stored in the acidophilic cells of the anterior lobe of pituitary and promotes bone growth. GH increases both bone resorption and bone formation processes (13,30). Its action on cell growth requires the formation of peptides known as somatomedins, also called insulin growth factors (IGFs). These mediate the responses of GH on cartilage, bone tissue, muscle, fat tissue and fibroblasts (13). To highlight the effect of IGF-I on bone metabolism as it increases chondrocytes and osteogenic cells proliferation and proteins deposition by these two cell types. It also stimulates chondrocytes conversion into osteogenic cells leading to bone tissue formation (13). JOANA PINHO 18 Estrogens are hormones synthesized by the ovaries and have an important role in maintaining bone mass in women. They promote bone tissue formation (13). When estrogen levels are reduced there is an increase in the intensity of bone remodeling (4), which causes an imbalance between osteoblastic and osteoclastic activities. So there is an increase in osteoclasts activity and a decrease in osteoblasts activity, which leads to the formation of resorption spaces that are not filled by immature bone. Estrogens bind to specific receptors in the cytoplasm of bone cells, which results in formation and release of soluble factors with autocrine and paracrine actions in both osteoblasts and osteoclasts. This will influence their recruitment, proliferation, differentiation and metabolic activity (4). LITERATURE OVERVIEW 19 BONE REGENERATION Bone is a highly specialized and dynamic tissue that has the capacity of self-regeneration. During development and growth bone undergoes a modeling process in which it is removed from one site to a different one (2). The bone regeneration is a process that takes place throughout life to replace the old bone with new and it has special importance when a fracture occurs. This is more common in bone pathologies, or by trauma, osteonecrosis and tumors. A fracture is a break in a bone that is always accompanied with damage in the adjacent tissues (42). In cases of bone tissue loss it is necessary the immediate stimulation of new bone production. There are a number of events that follow a bone fracture. They usually start with the formation of a wound, followed by the healing and finally the repair process. The injury process begins with the stimulation of growth and differentiation factors which then activate localized pluripotent osteoprogenitor cells. These cells produce bone morphogenetic proteins (BMPs) that induce the migration of mesenchymal cells, which in turn proliferate and differentiate into bone-forming cells (42). Afterward, it starts a rapid inflammatory response resulting in tissue edema and chemotaxis through the release of cytokines and growth factors. Then follows the first phase of collagen repair that involves the deposition and formation of granulation tissue, which becomes a new and temporary weak tissue. This process ends with the second stage of collagen repair that results in extracellular matrix remodeling, localized angiogenesis and reproduction of full-strength tissue. Bone remodeling is initiated with both endochondral and intramembranous ossification which are stimulated by hypoxia and vascular disruption at the fracture site (17,42). The above coordinated processes result in the reconstruction of normal bone and restoration of a structural unit. However, these normal bone regenerative mechanisms breakdown during failure of optimal bone remodeling and repair, or in large bone fractures and defects. JOANA PINHO 20 In these cases, it is necessary to resort to Tissue Engineering: an interdisciplinary field that applies the principles of engineering and the life sciences to the development of biological substitutes that restore, maintain, or improve tissue function (43). This is a new field of research that suggests the regeneration of the tissue instead of its substitution (44) and it has gained special attention due to the lack of clinical treatments capable of restoring full functionality. For that the use of grafts for bone repair is a good option. The autologous graft (graft from the individuals themselves) is the clinical gold standard, but although effective, with no immunogenicity, superior grafting and minimal risk of disease transmission, it has a limited supply of viable donor tissue, there is the need of additional surgeries, increased risk of infection and donor site morbidity (45,46). Allograft (graft from another individual) bone is an alternative to autografts but it also has some disadvantages related to limited donor supply, disease transmission and inadequate physiologic and biomechanical responses (46). The biologic mechanisms that provide a basis for bone grafting are osteoconduction, osteoinduction and osteogenesis. This way, the bone graft material must be osteoconductive serving as a structure (scaffold) that allows cells to migrate and function within its limits (46); osteoinductive providing factors that stimulate the proliferation and differentiation of osteoprogenitor cells into osteoblasts that then begin the new bone formation (46); and osseointegrative being capable of integrate into the surrounding tissue (46). Thus, the superposition of two or more materials in order to completely achieve these characteristics is a logical strategy. In effect, the creation of composites is a biomimetic approach, as bone can be viewed as a composite of collagen, the principal organic component; HA, the inorganic mineral component; water; and small amounts of other organic phases. LITERATURE OVERVIEW 21 OSTEOPOROSIS AND OSTEOPENIA Osteoporosis is a pathological condition characterized by reduced bone mineral density (BMD) and microarchitectural changes in bone tissue that result in bone fragility and increased risk of fracture (2,31,32,47). A pathological imbalance between bone resorption and bone formation during the remodeling process may be the cause of this disease (31,39,48,49). Its most obvious morphological characteristic is bone loss, which tends to be more evident in skeleton areas with a high content in trabecular bone (Figure 12). Figure 12 – Morphological difference between a bone in physiological conditions and an osteoporotic bone (50). There are two classic forms of osteoporosis, the primary or physiological osteoporosis and the secondary osteoporosis (32), that is caused by other associated conditions. The first one can be divided into two forms: o Postmenopausal osteoporosis – often identified in women aged over 50 years (47,51) and characterized by high bone resorption rate resulting in an accelerated and increased osteoclastic activity caused by estrogen deficiency (52); o Senile osteoporosis – affects individuals of both genders with ages over 70 and has a normal or slightly increased bone resorption, associated with a decrease in the osteoblast activity that leads to low bone formation (52,53). JOANA PINHO 28 DIAGNOSIS Osteoporosis is difficult to diagnose because it remains asymptomatic until the occurrence of a fracture (38). In the past, it was often diagnosed in postmenopausal women and older men, based on radiological findings and in the presence of small fractures. Recently, it was established an inverse relationship between BMD and fracture risk, which allowed making the diagnosis based on BMD measurements that enable disease identification even before a fracture occurs (62,64). Radiography In old times, radiographs were used to evaluate the peripheral skeleton BMD, usually at metacarpals level. The metacarpal cortical thickness was thus used for many years for diagnosing and predicting the risk for osteoporosis. However, the sensitivity of this radiographic technique is poor (65), and metacarpal measurement results do not reflect the BMD at most important sites such as the hip and spine (61). Radiographic analysis reveals bone loss recognizable only when 25-30% of bone density is lost, when osteoporosis is considered being in a more developed state. Although there is a correlation between BMD in central and peripheral skeleton, the association is not strong enough to predict central BMD measurements from a given peripheral bone (61). Nowadays, the main role of radiography is the diagnosis of secondary fractures to osteoporosis. BMD Measurement Quantitative analysis of bone density can be performed by several methods, among which we highlight the Dual-Energy X-Ray Absorptiometry (DXA), Quantitative Computed Tomography (qCT) and Quantitative Ultrasound (QUS). LITERATURE OVERVIEW 29 Dual-Energy X-Ray Absorptiometry (DXA) Dual-energy X-ray absorptiometry (DXA) is a very precise and accurate technique for assessing bone density at several skeletal sites (66), that determine the absorption of two beams of photons at two different energies (67). With this method it is possible to measure BMD (mass/area) in proximal femur and lumbar spine, as well as the mineral density throughout the body. However, it fails to differentiate between measurements of cortical and trabecular bone. This method allows the BMD measurement in the hip or in the spine, with greater accuracy than other methods (accuracy error: 0.5-2%). The scanning time is about 5 minutes at each spot and the radiation dose is low (68,69). Besides the evaluation of normal anatomical sites, it may be used to provide side views of vertebral morphometric evaluations to determine vertebral fractures and deformities. The results are reported as a density measurement in g/cm2 and T and Z scores. The T scores represent the number of standard deviations below the mean BMD value for young adults (20-30 years old) (66). This is used to make diagnosis of normal bone density and with osteoporosis, in postmenopausal women and in men from the age of 50. The Z scores represent the number of standard deviations above or below the mean BMD value for subjects of the same age (66). These values are rather used to assess bone loss in premenopausal women and men younger than 50 years old. The World Health Organization (WHO) (70) defines osteoporosis as a result of BMD evaluation of standard deviation of 2.5 or higher. The WHO continues to seek a new algorithm for defining the limits of osteoporosis treatment that includes other factors such as age. Osteopenia is an intermediate category of bone loss defined as a T-score between 1 and 2.5 (71). It’s a physiological condition characterized by the decrease of BMD. JOANA PINHO 30 Quantitative Computed Tomography (qCT) Quantitative Computed Tomography (qCT), with appropriate software, allows determining the absorption of different calcified tissues so that areas of interest as the vertebral body can be studied. This technique has the capability of evaluating bone density in three dimensions (g/cm3) (66). qCT has the advantage of distinguish trabecular from cortical bone. In addition, it can provide details related to the bone structure, including width and density in specific sub regions of cortical bone (66). The high resolution CT enables the measurement of trabecular diameter, inter-trabecular spaces and can identify abnormal trabecular architecture. Recent developments of tridimensional CT (3D) allowed the evaluation of the 3D trabecular structure and could improve the ability to estimate biomechanical properties of the bone. Dual energy scanning (with twice the dose of radiation) can improve accuracy but the precision worsens. The radiation dose and the price are considerably higher than those of conventional qCT. Quantitative Ultrasound (QUS) Quantitative ultrasound (QUS) provides information regarding bone mass and structural organization of the bone. It assesses the speed of sound or the absorption pattern of different sound wavelengths designated attenuation of ultrasound bandwidth (66). The attenuation of ultrasonic signals during the passage through the bone may be measured by determining the reduction in signal amplitude of ultrasounds. The ultrasound instruments have theoretical advantages in relation to DXA because they do not need radiation, are portable and cheaper. However, currently, it is difficult to apply the ultrasound to the clinic due to the lack of specific diagnosis criteria and the need to use manifold instruments. Moreover, as there are technical differences between devices, the results cannot be extrapolated from one device to another (66). LITERATURE OVERVIEW 31 PREVENTION AND TREATMENT Osteoporosis is a bone disease that affects a large number of people at ever earlier ages. Thereby, the main goal is to identify individuals at risk of developing osteoporosis and provide them a safe, effective and economical intervention to prevent appearance of this disease. However, the factors that contribute to the occurrence of osteoporosis are very heterogeneous and there are a large number of genetic parameters that are unknown, making it difficult to create a complete plan for its prevention and treatment (52). Peak bone mass and resulting BMD are important throughout the growth and aging, which means that must be paid special attention to procedures that can optimize the level of bone mass (52). Thus, the approaches used for treatment and prevention of osteoporosis focus on prevention of bone loss. This can be done by several mechanisms: o Increase of calcium levels to reduce bone loss – calcium and vitamin D supplements (29,37); o Decrease in osteoclasts action to suppress bone resorption through pharmacological treatments – calcium, biophosphates, estrogens or analogs and calcitonin (29,37); o Increase in osteoblasts action to promote bone formation – low doses of PTH and testosterone (29,37). Prevention of this disease is also related to lifestyle, so it is encouraged the practice of regular physical exercise that is beneficial to maintain bone mass and reduce fracture risk (29,37,52); the reduction in alcohol, salt, caffeine and tobacco consumption, and nutritional care, such as increased consumption of animal protein and adequate calcium intake that maintains bone mass (37,52). With use of continuous treatments, probably indefinitely, it is possible to decrease the risk of fracture or at least keep it down. This strategy can be changed with the emergence of agents that substantially increase bone mass and restore microscopic architecture of the bone (37). However, the treatment of osteoporosis is difficult, both theoretical and practical, and the efforts that imposed directly on prevention measures are continuous. JOANA PINHO 32 TETRACYCLINES Tetracyclines are broad-spectrum antibiotics which act at the ribosomal level and interfere with protein synthesis (72). They were first used in the treatment of acne in the early 1950s and more recently have been considered for the biologic management of distinct conditions ranging from inflammation, proteolysis, angiogenesis, apoptosis, metal chelation, ionophoresis and bone metabolism. At the present, are available three groups of tetracyclines – naturalderived products, semisynthetic compounds, and chemically modified tetracyclines (CMTs) (72). Tetracyclines can act in several clinical conditions that include periodontitis, osteoporosis/osteopenia, rheumatoid arthritis, cancer invasion and metastasis, corneal ulceration, abdominal aortic aneurysms, inflammatory skin diseases and other immuneinflammatory conditions (73). Upon administration, tetracyclines are widely distributed into the tissues and can also achieve the cerebrospinal fluid. Besides that, as they chelate with the calcium ions, they are concentrated at mineralized tissues, namely bone and teeth. Tetracyclines and their derivatives are responsible for several actions that are independent from their antibiotic activity. Many of the non-antimicrobial properties may be related with their capacity for divalent cation quelation (74). These drugs proved to be effective against a range of mediators of the inflammatory cascade. Several direct or indirect mechanisms have been proposed – suppression of neutrophilic migration and chemotaxis, inhibition of T cell activation and consequent inhibition of their proliferation, inhibition and increased degradation of nitric oxide syntheses and pro-inflammatory cytokines inhibition. Tetracyclines can modify the expression of these mediators which makes them attractive for therapeutic action. LITERATURE OVERVIEW 33 The anti-inflammatory actions include the inhibition of matrix metalloproteinases (MMPs) (75). MMPs are extracellular enzymes that rely on the availability of two cations per molecule to fulfill their enzymatic activity (76). Some MMPs (MMP-1, MMP-8 and MMP-13) are known as collagenases due to their capacity to break down fibrillar collagens. Others (MMP-2 and MMP-9) are known as gelatinases and are responsible for the degradation of collagen type IV, which can be found in the basement membrane. The MMPs family plays a role in angiogenesis, the process of formation of new blood vessels from pre-existing ones. Recent studies have shown that collagenases and other MMPs may participate in the degradation of type I collagen, the main component of the bone organic matrix and in the destruction of other connective tissue constituents. Thus, preliminary in vivo research has shown that tetracyclines were able to inhibit bone loss through inhibition of osteoclast-mediated bone resorption, but also by enhancing osteoblast activity, up regulation of type I collagen expression and increased bone formation (77,78). Other important non-antimicrobial property of tetracyclines and related molecules is associated with their eventual antiapoptotic effect. Furthermore, the interaction between tetracyclines and the bone system is known for a long time. These agents have been used as diagnostic markers for a long time (79). Overall, tetracyclines have been shown to inhibit MMPs, retard proliferation, induce apoptosis, and impair mitochondrial function in various experimental settings (80). Since the mid-twentieth century, they have found application beyond their anti-microbial activity in both the clinic and biomedical research; therefore, several translational studies were performed to demonstrate the clinical validity of it. After some research approaches, it was developed a low dose therapeutic regimen of doxycycline that showed to be non-antimicrobial, effective and safe. In this therapeutic regimen, doxycycline was found to achieve peak plasma levels of around 1 μg/mL. Doxycycline is a semi-synthetic tetracycline and remains the preferred tetracycline agent for most indications (81). JOANA PINHO 34 35 II. RESEARCH HYPOTHESIS AND OBJECTIVES JOANA PINHO 36 RESEARCH HYPOTHESIS It was hypothesized that due to the known effects of osteopenia/osteoporosis in the bone tissue structure and metabolism, doxycycline, known to enhance osteoblast functionality in physiological conditions, could further assist on the improvement of the osteogenic activity in the established impaired conditions. OBJECTIVES 1) To establish rat models representative of the human condition of osteopenia and osteoporosis; 2) To establish and characterize bone marrow-derived osteoblastic cultures from sham (control), osteopenic and osteoporotic animals, in the absence of exogenous osteogenic stimuli; 3) To address the effects of doxycycline in the proliferation and functional activity of established sham, osteopenic and osteoporotic-derived osteoblastic cultures. 37 III. MATERIALS AND METHODS JOANA PINHO 44 Total Protein Content The total protein content was determined using the Lowry method (88,89). This is based on the reaction of Folin-Ciocalteau reagent with aromatic amino acids, and there’s the formation of a colored product, which is measured by spectrophotometry. After removing the culture medium, cells were washed twice with PBS buffer ("Dulbeco's Phosphate Buffered Saline," Sigma D-1480), pH 7.4. It was added 100µL of Triton X-100 (0.1%) to each well and plates were placed in an incubator at 37°C for one hour. Once this time, samples were collected into eppendorf tubes, make up to volume of 300μl with NaOH 0.1 M and added to each sample 1.5 ml of an alkaline solution of copper. Allowed to act for 10 minutes, phenol reagent was added and the plate was placed in the dark for one hour. Once this time, absorbance was read in a spectrophotometer (Jenway, model 6300) at 750nm. Absorbance was compared with values obtained for a serie of standards of bovine serum albumin, made from a solution of 0.5 mg/mL in NaOH 0.1. Results were expressed in µg of total protein in the sample per square centimeter (µg/cm2). Alkaline Phosphatase Activity The alkaline phosphatase (ALP) activity was assessed by spectrophotometric determinination of the product obtained by hydrolysis of p-nitrophenylphosphate disodium (pNPP), upon reaction with ALP (p-nitrophenol). The culture medium was removed and cells were washed twice with PBS buffer, pH 7.4. It was added 100µL of Triton X-100 (0.1%) to each well to induce cell lysis. Thereafter, the substrate was prepared, 80μl were placed in each well and plate was incubated at 37°C for 30 minutes. Results were expressed as nanomoles of p-nitrophenol produced per minute per microgram of protein (nmol/min.μg protein) as compared with respective calibration straight line. MATERIALS AND METHODS 45 Histochemical Methods Cell cultures were fixed at specific time points for further performance of histochemical techniques. The culture medium was removed and cells were washed with PBS at 37°C, pH 7.4. Cells were fixed with glutaraldehyde 1.5% for ten minutes. Thereafter, was added sodium cacodylate buffer at 0.14 M and samples were preserved at 4°C. Alkaline Phosphatase Staining Cell cultures were stained immunohistochemically to identify ALP activity by a method based on the hydrolysis of sodium naphthyl phosphate by ALP, and precipitation of phosphate liberated by reaction with a diazonium salt (Fast Blue RR). Hence, originates a colored product that, depending on amount of enzyme, may be yellow, brown or black. Cultures were placed in the dark for one hour, after fixation with buffer Tris 0.1 M, pH 10, with 2 mg/ml of sodium naphthyl phosphate and 2 mg/ml of Fast Blue RR. After the incubation, samples were washed with distilled water and allowed to dry under ambient conditions. Cultures were photographed in a Nikon TMS Inverted Phase Contrast microscope with magnifications of 40x and 100x. Collagen Staining Collagen assay is based on the binding of a dye Sirius red F3BA (BDH, UK) – with the triple helical collagen fibril. The staining of the fixed cells was performed with 0.1% siriusred F3BA in saturated picric acid at room temperature, during 1 hour, under mild shaking. Afterward, the dye solution was removed by suction and the stained cell layers intensively washed with 0.01 N hydrochloric acid to remove all non-bound dye. JOANA PINHO 46 Gene Expression Analysis RT-PCR analysis was done in the three established cultures at specific time points. These were evaluated for the expression of ALP, bone morphogenic protein-2 (BMP-2), Col I, OPN, OC, OPG, PPARγ, CFD and AP-2. Total RNA was extracted using the NucleoSpin® RNA II Kit (Macherey-Nagel) according to the manufacturer’s instructions. The concentration and purity of total RNA in each sample were assessed by UV spectrophotometry at 260 nm and by calculating the A260nm/A280nm ratio, respectively. RT-PCR was done using the Titan One Tube RT-PCR system (Roche® Applied Science), according to the manufacturer’s instructions, for 28 cycles. RT reaction mixtures consisted of extracted RNA, Titan RT-PCR buffer, dithiothreitol (DTT), deoxynucleoside triphosphate (dNTP), primers for each tested gene, avian myeloblastosis virus RT (AMV-RT) and water, in a total volume of 25 μl. Total RNA was reverse transcribed with cDNA (30 minutes at 50ºC), which was then amplified with recombinant TaqDNA polymerase at different annealing temperatures. For all the genes, the annealing temperature was 60ºC. The PCR products were electrophoresed in a 1% agarose gel, stained with ethidium bromide and semi-quantitatively assessed by densitometry with Image J® software. STATISTICAL ANALYSIS Data presented in this work are the result of three separate experiments performed in cell cultures established from different animals. For biochemical data (MTT assay, total protein content and ALP activity) each point represents the mean ± standard error of 6 independent assays. Statistical analysis was done by one-way analysis of variance (ANOVA). P values ≤ 0.05 were considered significant. 47 IV. RESULTS JOANA PINHO 48 ASSESSMENT OF THE BONE TISSUE STRUCTURE The bone morphometric parameters were assessed by μCT analysis and the results are in Table 1. The BV/TV and Tb N values decreased in the osteopenic group and were even lower in osteoporotic group. The Tb Th and Tb Sp were found not to differ significantly between groups despite that Mean Density was significant reduced in osteopenic and osteoporotic groups, as comparing to sham. Table 1 – μCT values from Sham, Osteopenic and Osteoporotic groups. * – significantly different from control (p< 0.05). ESTABLISHMENT OF BONE MARROW-DERIVED OSTEOBLASTIC CULTURES FROM SHAM (CONTROL) ANIMALS In the first part of this study, the principal aim was to establish a bone marrow-derived osteoblastic culture from sham animals in two different conditions – in the absence and in the presence of doxycycline at 1 μg/ml. Cultures established in the absence of doxycycline were referred as ‘Control’; and cultures grown with doxycycline were termed as ‘D1’. Cells were cultured in control medium and the first subcultures were evaluated for cell morphology, cell viability/proliferation and osteoblastic differentiation throughout the 14 days of culture time. Sham Osteopenic Osteoporotic BV/TV (%) 54.33 ± 6.3 42 ± 4.78 * 17.78 ± 3.6 * Tb N (1/mm) 6.073 ± 0.54 5.329 ± 0.68 * 1.892 ± 0.41 * Tb Th (mm) 0.0897 ± 0.012 0.1065 ± 0.009 0.0917 ± 0.008 Tb Sp (mm) 0.0998 ± 0.018 0.1278 ± 0.012 0.5452 + 0.014 Mean Density (mg HA/ccm) 883.5004 ± 57.302 846.3288 ± 32.795 * 836.1752 ± 44.784 * RESULTS 49 CELL PROLIFERATION Graphic 1 shows the results of cell proliferation that was assessed by the DNA content. Control cell culture established from sham animals increased the proliferation rate during the time of culture. The culture with doxycycline had a similar behavior – increase in cell proliferation from day 5 to day 14 of culture. The addition of doxycycline revealed higher values in the proliferation rate. METABOLIC ACTIVITY Metabolic activity was evaluated by the MTT assay (Graphic 2). Cultures established from sham animals presented an increase in MTT reduction values till day 10 and suffered a decrease of almost 50% at day 14. The cultures with doxycycline showed a similar behavior – MTT values increased from day 5 to day 10 and decreased at day 14. The metabolic activity in the culture with doxycycline is slightly higher than the one in the control culture in the first days of culture, but this pattern is reverted in the last days of culture. 0 20 40 60 80 100 120 140 160 180 5 8 10 14 DNA (ng/ml) Days Control D1 * Graphic 1 – Cell proliferation of sham rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. * – significantly different from control (p< 0.05). JOANA PINHO 50 APOPTOSIS Apoptosis was assessed by the Caspase-3 assay and is represented in Graphic 3. The apoptosis is broadly constant during the time of culture. The values between the control culture and the one with doxycycline are very similar with no significant differences between experimental conditions. Graphic 2 – Metabolic activity of sham rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. 0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700 0.800 5 8 10 14 Abs (λ=550 nm) Days Control D1 0 50 100 150 200 250 300 5 8 10 14 Fluorescence Days Control D1 Graphic 3 – Apoptosis of sham rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. RESULTS 51 CELL MORPHOLOGY CLSM was the method used for the assessment of cell morphology and the acquired images are shown in Figure 14. At day 8, cells of the control culture exhibited expanded cytoplasm and an adequate nuclear organization. Besides that, several cell to cell contact were established. The cells proliferated adequately and, at this time point, a large area of the culture surface was already covered by cells. The cellular behavior of cultures with doxyxycline was similar. 3 days 8 days 14 days Figure 14 – CLSM imaging of sham rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. Cytoskeleton was stained in green and nucleus counterstained in red. A to C corresponds to control cultures, and D to F corresponds to cultures with doxycycline. Magnification 400x. A B C F E D JOANA PINHO 52 ALKALINE PHOSPHATASE ACTIVITY Results of ALP activity were normalized by total protein content and are show in Graphic 4. In the culture established from sham animals, alkaline phosphatase activity increased from day 5 to day 10 and suffered a decrease at day 14. The culture established in the presence of doxycycline showed a similar behavior – increased in the first days of culture and decreased at day 14. Comparing the two cultures, the ALP activity is higher in doxycycline culture at eighth and tenth days of culture. ALKALINE PHOSPHATASE STAINIG Representative figures of ALP staining are shown in Figure 15. Both control and doxycycline cultures presented a similar behavior concerning the production and distribution of ALP. Grown cells show a homogeneous ALP staining at day 5, in which the first cell clusters are detectable. This organization is easier identified at days 8 and 10, in which aggregates of cells have a more intensive staining – dark brown/black staining. This demonstrates an increase in ALP activity over time culture. Graphic 4 – ALP activity of sham rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. * – significantly different from control (p< 0.05). 0 1 2 3 4 5 6 7 8 9 5 8 10 14 ALP activity (nmol/min/μg prot) Days Control D1 * RESULTS 53 5 days 8 days 10 days 14 days Figure 15 – ALP staining of sham rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. A to D corresponds to control cultures, and E to H corresponds to cultures with doxycycline. Magnification 100x. COLLAGEN STAINING Figure 16 shows representative images of collagen staining in cultures established from sham animals. In control culture is evident the formation of nodular structures since day 5 of culture which increased till day 14. Both cultures revealed an increase in the color staining in later time points, days 11 and 14. A B C D H G F E JOANA PINHO 60 3 days 8 days 14 days Figure 17 – CLSM imaging of osteopenic rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. Cytoskeleton was stained in green and nucleus counterstained in red. A to C corresponds to control cultures, and D to F corresponds to cultures with doxycycline. Magnification 400x. ALKALINE PHOSPHATASE ACTIVITY Results of ALP activity were normalized by total protein content and are show in Graphic 10. In the control culture established from osteopenic animals, ALP activity showed an increase over the time of culture. The culture established in the presence of doxycycline demonstrates a similar behavior, increasing the ALP activity from the first day till the last day of culture. Comparing the two experimental conditions, the ALP activity in the culture with doxycycline was significantly higher since the eighth day of culture. A B C F E D RESULTS 61 Graphic 10 – ALP activity of osteopenic rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. * – significantly different from control (p< 0.05). ALKALINE PHOSPHATASE STAINIG Representative images of ALP staining are shown in Figure 18. It was verified an increased intensity for the two experimental conditions throughout the culture time. In both conditions the nodular aggregates were already visible at day 5 and increased over time, covering the vast majority of the culture. In later time points, the staining increased acquiring a dark brown/black staining, which is detectable in both conditions. This is in agreement with the ALP activity values showed above. 0 1 2 3 4 5 6 7 8 5 8 10 14 ALP activity (nmol/min/μg prot) Days C osteopenic D1 osteopenic * * JOANA PINHO 62 5 days 8 days 10 days 14 days Figure 18 – ALP staining of osteopenic rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. A to D corresponds to control cultures, and E to H corresponds to cultures with doxycycline. Magnification 100x. COLLAGEN STAINING Representative images of collagen staining in osteopenic cultures with and without doxycycline are shown in Figure 19. In both conditions it’s verified the formation of nodular structures that increased over the time culture. The intensity of the staining also increases and in later time points, days 11 and 14, the pink is more accentuated. A B C D E F G H RESULTS 63 5 days 8 days 11 days 14 days Figure 19 – Collagen staining of osteopenic rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. A to D corresponds to control cultures, and E to H corresponds to cultures with doxycycline. Magnification 100x. EXPRESSION OF OSTEOGENIC-RELATED MARKERS RT-PCR analysis showed that both experimental conditions – osteopenic control cultures and with doxycycline – expressed high levels of significant osteogenic markers. Results are shown in Graphic 11. At day 7, cultures with doxycycline expressed higher levels of ALP, Col I and OPN, comparing with the control culture. At day 14, control cultures expressed reduced levels of OPN and OC, while the expression of ALP, BMP-2, Col I and OPG was elevated. At the same time point, cultures with doxycycline expressed higher levels of these genes. A B C D E F G H JOANA PINHO 64 EXPRESSION OF ADIPOGENIC-RELATED MARKERS RT-PCR analysis of adipogenic markers is shown in Graphic 12. At day 7, the expression levels of CFD decreased in cultures with doxycycline. On the other hand, at day 14, in cultures with doxycycline the expression of CFD increased in comparison with the control culture. Graphic 11 – RT-PCR gene expression of ALP, BMP-2, Col I, OPG, OPN and OC, in osteopenic rat bone marrowderived cell cultures established at days 7 and 14, in the absence and in the presence of doxycycline. * – significantly different from control (p< 0.05). * * * * 0 5 10 15 20 25 30 35 ALP BMP-2 COL I OPG OPN OC Genes C osteopenic Day 7 D1 osteopenic Day 7 C osteopenic Day 14 D1 osteopenic Day 14 * * * * RESULTS 65 ESTABLISHMENT OF BONE MARROW-DERIVED OSTEOBLASTIC CULTURES FROM OSTEOPOROTIC ANIMALS In the third part of this study, the principal aim was to establish a bone marrow-derived osteoblastic culture from osteoporotic animals in two different conditions – in the absence and in the presence of doxycycline at 1 μg/ml. Cultures established in the absence of doxycycline were referred as ‘C osteoporotic’; and cultures with doxycycline were termed as ‘D1 osteoporotic’. Cells were cultured in control medium and the first subcultures were evaluated for cell morphology, cell viability/proliferation and osteoblastic differentiation throughout the 14 days of culture time. Graphic 12 – RT-PCR gene expression of PPARγ, CFD and AP-2, in osteopenic rat bone marrow-derived cell cultures established at days 7 and 14, in the absence and in the presence of doxycycline. * – significantly different from control (p< 0.05). 0 5 10 15 20 25 30 PPAR CFD AP-2 Genes C osteopenic Day 7 D1 osteopenic Day 7 C osteopenic Day 14 D1 osteopenic Day 14 * * JOANA PINHO 66 CELL PROLIFERATION The DNA content was the method used to assess the cell proliferation of the established cultures (Graphic 13). In the control culture from osteoporotic animals, cell proliferation increased till the tenth day of culture, being the increase from day 8 to day 10 very accentuated. Then cell proliferation decreased greatly at day 14. The culture with doxycycline didn’t have a similar behavior, increasing in a more regular way over the time of culture. The DNA values are lower in the doxycycline culture than in the control culture, except for the fourteenth day of culture. METABOLIC ACTIVITY Metabolic activity was evaluated by the MTT assay (Graphic 14). Cultures established from osteoporotic animals without introduction of any osteogenic inducers presented an increasing from the first day till day 10 and then showed a slight decrease in the last day of culture. The time-course growth pattern of cultures with doxycycline was a little bit different – MTT reduction values increased over the 14 days of culture. Graphic 13 – Cell proliferation of osteoporotic rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. * – significantly different from control (p< 0.05). * 0 20 40 60 80 100 120 140 160 180 5 8 10 14 DNA (ng/ml) Days C osteoporotic D1 osteoporotic * * RESULTS 67 APOPTOSIS Apoptosis was assessed by the Caspase-3 assay and is represented in Graphic 15. The apoptosis is constant throughout the culture time and there are no significant differences between the two experimental conditions. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 5 8 10 14 Abs (λ=550 nm) Days C osteoporotic D1 osteoporotic Graphic 14 – Metabolic activity of osteoporotic rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. Graphic 15 – Apoptosis of osteoporotic rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. 0 50 100 150 200 250 300 4 8 10 14 Fluorescence Days C osteoporotic D1 osteoporotic JOANA PINHO 68 CELL MORPHOLOGY CLSM was the method used for the assessment of cell morphology and the acquired images are shown in Figure 20. In control cultures, cells were elongated and the actin fibers were with an intense staining. Cell to cell contacts were established and, at day 8, an organized flattened sheet of continuous cell layers were verified. The cultures with doxycycline presented a similar cellular behavior. 3 days 8 days 14 days Figure 20 – CLSM imaging of osteoporotic rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. Cytoskeleton was stained in green and nucleus counterstained in red. A to C corresponds to control cultures, and D to F corresponds to cultures with doxycycline. Magnification 400x. A B C F E D RESULTS 69 ALKALINE PHOSPHATASE ACTIVITY Results of ALP activity were normalized by total protein content and are show in Graphic 16. In the control osteoporotic culture, ALP activity increased over the time of culture. The cultures established in the presence of doxycycline demonstrated a similar time-course growth pattern – increased ALP activity values from the first day till the last day of culture. Doxycycline culture showed higher ALP activity than the control culture during the entire culture time. ALKALINE PHOSPHATASE STAINIG Images shown in Figure 21 are representative of ALP staining. At day 5 clusters of cells can be acknowledged, which start to grow and cover a vast majority of the culture plate in later time points, days 10 and 14. The staining intensity also increased over the time of culture, passing through a light brown in day 5 to a dark brown/black in days 10 and 14. There are no significant differences between the two experimental conditions. Graphic 16 – ALP activity of osteoporotic rat bone marrow-derived cell cultures, established for 14 days, in the absence and in the presence of doxycycline. * – significantly different from control (p< 0.05). 0 1 2 3 4 5 6 7 8 9 5 8 10 14 ALP activity (nmol/min/μg prot) Days C osteoporotic D1 osteoporotic * JOANA PINHO 76 lower proliferation and defective function compared with normal osteoblasts. Related mechanisms might involve abnormalities of the IGF-I signaling system (97), a different production pattern of cytokines involved in the regulation of bone metabolism (98), which converge to occurrence of a different metabolic phenotype in osteoporotic osteoblasts and indicate the presence of reduced anabolic function (99). Furthermore, animal models and in vitro studies have demonstrated a disequilibrium in the RANK/RANKL/OPG system in osteoporotic conditions. A human in vitro study has further suggested that the up-regulation of RANKL on bone marrow cells is an important determinant of increased bone resorption induced by estrogen deficiency (55). Overall, within the established in vitro model of osteoblastic cultures derived from three distinct metabolic conditions, it was verified that the aging-related osteopenic condition was found to greater affect the functionality of cultured cells, sustaining a preponderant effect of the senescence process over the ovariectomy – osteoporotic condition. Despite this realization, microtomographic indexes were found to be more significantly affected in osteoporotic animals sustaining that the metabolic function/functional activity of bone marrow-derived osteoblasts might not be directly related to morphometric indexes of the bone tissue. Besides that, we aimed to assess the doxycycline effect in these three conditions. Tetracyclines are a well-characterized family of antibiotics that may have specific therapeutic value in the treatment of bone diseases, since they have great affinity for mineralized bone matrix (100). Besides that, they have been found to exert an extensive variety of catabolic actions over the metabolic balance of the bone tissue by modulating both osteoblastic and osteoclastic functions (101). Doxycycline is a representative of broad-spectrum tetracyclines antibiotics that is extensively used in medical and dental practice. These antimicrobial drugs revealed an overall positive effect on bone, being considered potential inductive agents for the management of the osteopenic and osteoporotic conditions (102). Knowing the ability of tetracyclines to improve the osteogenic function in a wide range of models, both in physiological and pathological conditions (like osteoporosis), the choice of using it was based on the possibility of enhancing the osteogenic potential of osteoblast precursor cells in the metabolic conditions above mentioned – sham, osteopenia and osteoporosis. Therefore, when accomplished the subculture, doxycycline (1 μg/ml) was added to the established cultures, in the absence of any osteogenic inducer. DISCUSSION 77 In the first part of this study we established and characterized bone marrow derived osteoblastic cultures from sham animals. Were settled two conditions – control and with doxycycline at 1 μg/ml. The cells were then characterized assessing the cell proliferation, metabolic and functional activities. Cell proliferation was evaluated by the DNA content and the results showed an increased over the time of culture. The addition of doxycycline showed to induce the proliferation of cells. Metabolic activity, assessed by the MTT assay, increased in the first 10 days of culture and decreased at day 14, in the control cell culture. Culture with doxycycline presented a similar time-course growth pattern – metabolic activity increased till day 10, day where the confluence was reached, and decreased at fourteenth day. Comparing the two cultures, it’s visible that doxycycline stimulated metabolic activity in the first 8 days of culture, but from day 10 to day 14 the control culture showed higher MTT reduction values. A similar behavior is found in functional activity – ALP activity increased till day 10 and diminished at day 14. This activity is stimulated by doxycycline at days 8 and 10. Histochemical staining for ALP correlated the biochemical determination, revealing a high and increased stain intensity and nodular aggregation throughout the time of culture. The histochemical for collagen also showed an increase in staining in both cultures. The apoptosis, assessed by the Caspase-3 activity, was constant during the time of culture and it was not altered with the introduction of doxycycline. The RT-PCR analysis of osteogenic markers revealed that the expression level of ALP, BMP2, Col I, OPG and OPN was similar at day 7 and the addition of doxycycline stimulated the expression of these markers. At day 14, the expression level of ALP, BMP-2, Col I and OPG was almost the same, but OPN and OC were expressed in reduced levels. As expected, doxycycline addition enhanced the expression of ALP, such as the expression of OPN and OC. However, the expression levels of BMP-2, Col I and OPG slightly decreased. In terms of expression of adipogenic markers, at day 7, PPARγ and AP-2 were expressed at a similar level and the addition of doxycycline decreased their expression. CFD was expressed in higher levels at day 7 and its expression was enhanced with introduction of doxycycline. At day 14, PPARγ and AP-2 were the markers with higher levels of expression. However, at this time point, the addition of doxycycline reduced the expression of all the adipogenic markers. JOANA PINHO 78 In the second part of this study, the aim was to establish and characterize bone marrowderived osteoblastic cultures from osteopenic animals. Once again, were stated two conditions – control and with doxycycline to enhance osteogenic function – and cell proliferation, metabolic and functional activities were then assessed. Cell proliferation increased throughout the time of culture and it’s evident that the presence of doxycycline stimulated cell proliferation, especially in the last days of culture. In metabolic activity the cultures presented a different behavior, in both cultures the metabolic activity increased till day 10, day where confluence was reached, and decreased at day 14. However, the culture with doxycycline showed lower MTT values than the control culture. In terms of Caspase-3 activity, the apoptosis had a decline at day 10 but returned to normal values at day 14. There were no significant differences between the two experimental conditions. The ALP activity showed a similar behavior in both conditions, increasing over the time of culture. The addition of doxycycline enhanced the ALP activity in the eighth and tenth days of culture. These results were corroborated with histochemical data, in which it’s possible to see the increasing in nodular aggregates and stain intensity. Being ALP an important osteoblastic marker, the osteoblastic phenotype was maintained in the two conditions. Collagen staining revealed an increasing in the coloration with no evident differences between the two cultures. ALP is one of the most important genetic markers for osteoblasts being expressed during osteoblastic differentiation. The expression of ALP showed a slight increase from day 7 to day 14 and, in both time points, it was intensified with the addiction of doxycycline. OPN, other marker for osteoblast differentiation, showed the higher levels of expression at day 7 in both conditions; however, this marker showed the lower expression levels in the control culture at day 14 and was enhanced with doxycycline. The expression of BMP-2, Col I and OPG didn’t show significant differences in the two conditions at day 5 but the expression of these markers increased at day 14 and it was stimulate with introduction of doxycycline. The expression of OC, often used as a marker for the bone formation process, didn’t show significant differences at day 5 in both conditions, but the addiction of doxycycline stimulated greatly its expression at day 14. In general, the expression of osteogenic markers increased with addition of doxycycline, being this other parameter that confirms that this antibiotic stimulates the differentiation of osteoblasts. DISCUSSION 79 In terms of the expression of adipogenic markers, PPARγ, a marker expressed in adipocytes, showed reduced levels of expression at day 7 that increased at day 14 and, in both time points, its expression levels were enhanced by addition of doxycycline. CFD was the adipogenic marker that revealed a higher expression at day 5 being reduced with doxycycline. At day 14, the expression of CFD in control cultures was lower than at day 14 but it was stimulated when doxycycline was introduced. AP-2 expression increased with introduction of doxycycline in both experimental conditions. Despite the different levels of adipogenic expression, doxycycline stimulated the expression of all of the genes used. In terms of values, the levels of expression of adipogenic markers are similar to those obtained in osteogenic markers, so the established osteopenic culture had a propensity to the adipogenic differentiation alike the osteogenic one. In the third part of this work, we had the goal of establish and characterize bone marrowderived osteoblastic cultures from osteoporotic animals. The osteoblastic cells were cultured with and without doxycycline and cell proliferation, metabolic and functional activities were evaluated. Cell proliferation of the control culture increased from day 5 to day 10 and decreased at day 14. On tenth day of culture, control cells cultures reached confluence, and cell proliferation is inhibited by cell to cell contact what justify the decrease at the fourteenth day. Culture with doxycycline didn’t present this type of behavior, the proliferation rate increased throughout the whole time of culture, so doxycycline presented a slower cell proliferation rate and its peak was reached later, at day 14. Metabolic activity of control culture increased till day 10 of culture and decreased at day 14, while culture with doxycycline showed an increase over the 14 days of culture. Accordingly, doxycycline delays the metabolic activity and the cells achieve confluence later. There were no substantial differences between the two cultures in apoptosis, being the apoptosis values low in both experimental conditions, so doxycycline doesn’t interfere in the controlled cell death. In terms of functional activity, control culture revealed a regular increase in ALP throughout the time culture. The addition of doxycycline showed a stimulatory effect, increasing the activity of ALP since the first day of culture, being this outcome more marked at day 10. Histochemical results for ALP underpinned the biochemical data being visible the increase in staining intensity and number of clusters in both conditions. At day 10 it was also detectable a darker coloration in the doxycycline culture when compared with the control. The histochemical for collagen revealed an increasing in staining intensity for both cultures and no significant differences were visible. JOANA PINHO 80 In terms of the gene expression of osteogenic markers, ALP and BMP-2 had a similar level of expression at day 7 and then suffered a slight decrease when doxycycline was added. At day 14, the expression of both markers increased but, while ALP revealed a decrease in the culture with doxycycline, the expression of BMP-2 was stimulated by doxycycline. Weren’t found significant differences in Col I expression at days 7 and 14, and it’s evident that doxycycline enhanced its expression in both time points. The expression level of OPG at day 7 was similar in both experimental conditions. At day 14, the OPG expression increased which was stimulated with addition of doxycycline. This pattern of expression wasn’t observable in OPN and OC expression; these two genes showed the highest level of expression at day 7 that was stimulated when doxycycline was introduced. However, at day 14 their expression was much reduced, but doxycycline revealed a stimulatory effect once again. The expression of adipogenic markers was really irregular and showed different results. At day 7 the expression levels of PPARγ and AP-2 were low, while CFD was expressed in elevated levels. The doxycycline had different effects in these markers, stimulating the expression of PPARγ and AP-2 and inhibiting the CFD expression. At day 14 the osteoporotic cells from the control culture expressed PPARγ, CFD and AP-2 at a similar level; however, the doxycycline enhanced the expression of the first one and decreased the expression of the other two markers. With these results we can see that the established osteoporotic culture didn’t have great propensity to differentiate into an adipogenic phonotype at day 7, but at day 14 the levels of expression were higher, so it’s possible that the adipogenic differentiation happens later in the culture time. The results obtained in this experimental work are in line with the evidences found in the literature about bone metabolism and pathology in animal models. In a mouse model used to simulate diabetic-induced osteopenia was verified that the pathological situation (characterized by decreased bone formation and not augmented bone resorption) was significantly ameliorated with tetracycline uptake (103). Bone regeneration in a dog model revealed that the administration of tetracycline induced a more regenerative healing and minimized crestal resorption, in comparison to control (104). Also in a dog model, the administration of doxycycline reduced the severity degree of osteoarthritis, with reduced levels of total collagenase activity and inhibition of the proliferation and hypertrophy of chondrocytes (105). These results were later confirmed in a double-blinded, randomized, placebo-controlled trial that reported a doxycycline-dependent reduction in the rate of joint space narrowing, in knees of obese women with established osteoarthritis (106). Studies with ovariectomized rats revealed that low dose of tetracycline administration increased bone DISCUSSION 81 formation and mass levels (107). Tetracycline administration to squirrel monkeys, in a model of normal bone metabolism, increased the deposition of osteoid in the alveolar process by increasing the number of active osteoblasts (108). In human bone marrow-derived osteoblastic cells, doxycycline has increased significantly the number of active osteoblastic cells that provided a proportional amount of a normal mineralized ECM (109). This behavior was further confirmed when the human osteoblastic cultures were grown on the surface of two biomaterials for bone regeneration – HA and a glass-ceramic composite (110). A similar effect was found in human osteoblasts, in which doxycycline enhanced cell maturation and differentiation rather the process of proliferation itself (111). Recently, studies revealed that doxycycline at 1 μg/ml increase the OC, osteonectin and ALP expression in human periodontal ligament cells. Besides that, the increase in ALP expression was found to be more significant than the one induced by BMP-2 (112). Regarding to bone metabolism relevance, tetracyclines showed to have some effect in the modulation of the osteoclastogenic response. Recent studies found that doxycycline and minocycline (is also a broad-spectrum tetracyclines antibiotics) have the ability to inhibit the RANKL-induced osteoclastogenesis of precursor cells, but they didn’t show any effects on cell growth and phagocytic activity. In addition, the authors also reported the absence of effect on cell proliferation or differentiation in bone-forming osteoblast. Similarly, were perfomed some in vivo studies which revealed that the injection of tetracyclines into RANKL-injected mice and RANKL-transgenic mice suppressed the RANKL-mediated osteoclastogenesis process and promoted the concomitant appearance of CD11c+ cells (113). JOANA PINHO 82 83 VI. CONCLUSION JOANA PINHO 84 Osteoporosis is a prevalent disease characterized by a low bone mass and changes in the microarchitecture of the bone tissue, which result in increased bone fragility and fracture risk. In this pathological condition, the new bone formation decreases and the bone regeneration process can be affected. This way, bone tissue engineering strategies require biomodulators to achieve a successful regeneration of the bone tissue and tetracyclines are a good example of this type of biomodulators. In this work, there were established bone marrow-derived cultures from sham, osteopenic and osteoporotic animals and it was assessed the effect of tetracycline in these three conditions. Cell cultures established from the sham group shown an increased cell proliferation in the presence of doxycycline. Doxycycline was also found to enhance metabolic activity and ALP expression of the established cultures. Doxycycline stimulated cell proliferation and ALP activity in cultures established from osteopenic animals. This stimulatory effect of doxycycline was corroborated by the high expression of osteogenic markers such as ALP, BMP-2, Col I, OPG, OPN and OC. In cultures established from osteoporotic animals, doxycycline showed a delay in cell proliferation and in metabolic activity; however, the expression of ALP was stimulated, what confirmed the maintenance of an osteoblastic cell-like phenotype. RT-PCR evaluation of significant osteogenic markers also revealed an enhancement with addition of doxycycline. The results presented in this study showed that doxycycline can be a suitable candidate to improve the osteogenic potential of osteoblast precursor cells in osteopenic and osteoporotic conditions. Therefore, these antimicrobial drugs can be useful in tissue engineering and have a potential biomedical application, being used in the treatment of osteoporosis 85 VII. 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