In Vitro Comparison of 2 Clinically Applied Biomaterials for Autologous Chondrocyte Implantation: Injectable Hydrogel Versus Collagen Scaffold

https://doi.org/10.1177/19476035231154507 Cartilage 2023, Vol. 14(2) 220 –234 © the author(s) 2023 DOi: 10.1177/19476035231154507 journals.sagepub.com/home/Car Creative Commons Non Commercial CC BY-NC: this article is distributed under the terms of the Creative Commons attributionNonCommercial 4.0 license (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the Sage and Open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). Cartilage Repair Scaffolds 1154507CarXXX10.1177/19476035231154507CARTILAGEWeitkamp et al. research-article20232023 1Department of Oral and Maxillofacial Surgery, University Medical Center Schleswig-Holstein, Campus Kiel, Kiel, germany 2Department of anatomy, Kiel University, Kiel, germany 3teteC tissue engineering technologies ag, reutlingen, germany 4g.e.r.N. research Center for tissue replacement, regeneration & Neogenesis, Department of Orthopedics and trauma Surgery, Medical Center albert-ludwigs-University of Freiburg, Faculty of Medicine, albert-ludwigs-University of Freiburg, Freiburg, germany 5Clinic for trauma Surgery, Diako Hospital Flensburg, Flensburg, germany 6Department of trauma and Orthopedic Surgery, University Medical Center Schleswig-Holstein, Campus Kiel, Kiel, germany 7Department of trauma Surgery, Orthopedics and Sportsorthopedics, asklepios Klinik St. georg, Hamburg, germany Corresponding Author: Jan-tobias Weitkamp, Department of Oral and Maxillofacial Surgery, University Medical Center Schleswig-Holstein, Campus Kiel, arnold-Heller-Str. 3, Kiel 24105, germany. email: [email protected] In Vitro Comparison of 2 Clinically Applied Biomaterials for Autologous Chondrocyte Implantation: Injectable Hydrogel Versus Collagen Scaffold Jan-Tobias Weitkamp1,2 , Karin Benz3, Bernd Rolauffs4, Andreas Bayer2, Matthias Weuster5, Ralph Lucius2, Aydin Gülses1, Hendrik Naujokat1, Jörg Wiltfang1, Sebastian Lippross6, Michael Hoffmann7, Bodo Kurz2, and Peter Behrendt2,7 Abstract Objective. in autologous chondrocyte implantation (aCi), there is no consensus about used bioscaffolds. the aim of this study was to perform an in vitro comparative analysis of 2 clinically applied biomaterials for cartilage lesion treatment. Design. Monolayer expanded human chondrocytes (n = 6) were embedded in a collagen scaffold (CS) and a hyaluronic acid–based hydrogel (Ha). Cells were cultured in chondropermissive medium supplemented with and without interleukin-10 (il-10) and bone morphogenetic protein–2 (BMP-2). gene expression of chondrogenic markers (COl1a1, COl2a1, COl10a1, aCaN, SOX9) was detected via quantitative real-time–polymerase chain reaction (rt-qPCr). Biosynthesis of matrix compounds, cell viability, morphology as well as migration from surrounding native bovine cartilage into cell-free scaffolds were analyzed histologically. adhesion of the material to adjacent cartilage was investigated by a custom-made push-out test. Results. the shift of COl1/2 ratio toward COl2a1 was more pronounced in Ha, and cells displayed a more spherical morphology compared with CS. BMP-2 and il-10 significantly increased COl2a1, SOX9, and aCaN expression, which was paralleled by enhanced staining of glycosaminoglycans (gags) and type 2 collagen in histological sections of CS and Ha. COl10a1 was not significantly expressed in Ha and CS. Better interfacial integration and enhanced cell invasion was observed in CS. Push-out tests using CS showed higher bonding strength to native cartilage. Conclusion. Ha-based hydrogel revealed a more chondrocyte-like phenotype but only allowed limited cell invasion, whereas CS were advantageous in terms of cellular invasion and interfacial adhesion. these differences may be clinically relevant when treating cartilaginous or osteochondral defects. Keywords autologous chondrocyte implantation, collagen scaffold, hydrogel, hyaluronic acid, human articular chondrocytes, in vitro comparison Weitkamp et al. 221 Introduction Articular cartilage repair remains a challenging task in the field of orthopedic surgery aiming to prevent joint dysfunction and the development of post-traumatic osteoarthritis. Autologous chondrocyte implantation (ACI) has become the gold standard for the regenerative treatment of large size cartilage defects.1,2 Within the last 3 decades, the originally introduced technique was further developed by utilizing additional biomaterials as cell-carrier systems.3,4 Different generations of ACI development have aimed to overcome the issue of chondrocyte de-differentiation, which impairs the chondrocyte function and results in inferior quality of the regenerative tissue.5 Third-generation ACI consists of a cell-biomaterial product that has improved both the clinical outcome and the histological quality of the regenerated tissue, which emphasizes the constant optimization process of cell-based tissue regeneration techniques.3,6 Numerous biomaterials have been extensively studied in pre-clinical investigations, but comparative studies are rare. Therefore, identification of the ideal biomaterial candidate regarding its cell-matrix interactions, cytocompatibility, biomechanical properties, and surgical application techniques is insufficient. In addition, due to regulatory restrictions and financial burdens, it is not feasible to examine the whole spectrum of biomaterials clinically. A deepened knowledge about biomaterial advantages in clinically established biomaterials may help to focus current efforts in further ACI development. In this regard, biomaterials based on collagen and hyaluronic acid, which are inspired by the natural architecture of articular cartilage, are of high interest for investigators in this field.7-9 Fiber-based matrices have shown to support chondrocyte differentiation and formation of cartilaginous matrix in several in vitro and in vivo studies.10,11 Hydrogels, on the contrary, have also been investigated intensively due to their water-binding capacity that mimics cartilage tissue.12 Especially, gels based on naturally derived biopolymers such as hyaluronic acid are interesting for cartilage tissue engineering due to its chondroprotective effects.13 Another improvement in ACI treatment may be achieved by a pre-conditioning using soluble adjuvants that improve cell differentiation prior to ACI implantation. In this context, interleukin-10 (IL-10) and bone morphogenetic protein–2 (BMP-2) have been described,14,15 which were shown to have potential chondrogenic effects, even in the post-traumatic micromilieu after ACI graft transplantation.16,17 Our study group previously demonstrated that IL-10 significantly enhances chondrogenic differentiation of cells embedded in collagen matrices and stabilized the chondrogenic phenotype with less extracellular matrix loss after mechanical cartilage injury.14,18 BMP-2, a member of the transforming growth factor–β (TGF-β) super-family, plays an essential role in the regulation of chondrocyte proliferation.15 Given the abundant spectrum of eligible biomaterials, deepened understanding of clinically applied scaffolds may help to focus further ACI developments. This study aims to identify advantageous biomaterial properties of 2 clinically used ACI grafts (macroporous type I/III collagen matrix vs. injectable hyaluronic acid-based hydrogel) in a comparative in vitro study with respect to (1) cell viability, (2) chondrogenic potential, (3) bio-adhesion, (4) lateral integration, and (5) additional adjuvants such as IL-10 and BMP-2. It was hypothesized that both biomaterials have distinct biological differences regarding cell differentiation and graft integration. Method Articular Chondrocyte Isolation and Culture Human articular chondrocytes (hCh; n = 6) were isolated from femoral heads of patients (mean = 70.3 ± 5.3 years) undergoing hip replacement surgery (ethical approval was obtained of the University Kiel D572/17). The cartilage tissue was dissected into small pieces and digested with 0.1% pronase (Roche, Mannheim, Germany) followed by digestion with type 2 collagenase 600 U/ml (Worthington, Lakewood, USA) in Dulbecco’s Modified Eagle Medium (DMEM). Isolated hCh were cultured at a density of 10,000 cells/cm2 in chondropermissive medium (CPM) consisting of high-glucose DMEM (HG-DMEM) supplemented with 10% Sera Plus (PAN-Biotech, Aidenbach, Germany), 10 mg/ml Penicillin G, 10 mg/ml of streptomycin (PAA Laboratories, Pasching, Germany),1% L-ascorbic acid (Sigma-Aldrich, St. Louis, USA) and 2 ng/ml fibroblastic growth factor–2 (FGF-2; R&D Systems, Minneapolis, USA). Medium change was twice a week. Cells were harvested at cell passage 3 (P3) by trypsin–ethylenediaminetetraacetic acid (EDTA; Lonza, Cologne, Germany) treatment and used for further in vitro cultivation. Embedding of hCh in HA and CS Monolayer expanded chondrocytes (P3) (n = 6) were seeded either onto the macroporous part of a biphasic type 1/3 collagen scaffold (CS) as used in Novocart 3D® (TETEC Tissue Engineering Technologies AG, Reutlingen, Germany) or were embedded in an albumin-hyaluronic acid–based hydrogel (HA) as used in Novocart Inject® (TETEC Tissue Engineering Technologies AG, Reutlingen, Germany). Therefore, 2.5 × 106 hCh/ml/cm2 were re-suspended in HG-DMEM and seeded onto the CS and HA, respectively, and cultivated in 24-well plates coated with 2% agarose (Sigma-Aldrich, St. Louis, USA). The hydrogel is based on chemically activated maleolyl-albumin supplemented with HA and was cross-linked by a specific thio-polyethylene glycol (PEG).13 222 CARtIlAgE 14(2) Cellularized constructs were cultured in CPM (without Sera Plus) that was supplemented with 1% insulin-tranferrinsodium selinite liquid media (Sigma-Aldrich, St. Louis, USA) and 0.1 mM nonessential amino acids (Sigma-Aldrich, St. Louis, USA) and renewed every 3 days. The samples were divided into 4 different treatment groups: non-supplemented, additional human IL-10 (100 pg/ml; Kingfisher Biotech, Saint-Paul, USA), additional human bone morphogenetic protein–2 (hBMP-2; 250 ng/ml; R&D Systems, Minneapolis, USA), and co-treatment with IL-10 and BMP-2. Cell Viability, Morphology, and Expression of Microfiber Assessment To determine the morphology and viability of cells within the biomaterial live/dead (L/D), staining was performed after 7 and 28 days of culture. Therefore, HA and CS samples were stained with 10 μM calcein-AM and 5 μM ethidium homodimer-1 (both Sigma-Aldrich, Buchs, Switzerland). After 1-hour incubation, the samples were imaged using confocal laser scanning microscopy (CLSM 510; Carl Zeiss, Germany). To quantify the number of L/D cells, 3 images were taken from 3 different fields of view, and a minimum of 100 cells were counted using image J (Wayne Rasband, NIH, USA). To visualize the expression of F-actin, additional staining with phalloidin and nuclear staining (4′,6-diamidino-2-phenylindole [DAPI]) was performed followed by CLSM. gene Expression Analyses by Quantitative Realtime PCR Gene expression was analyzed after 1 and 28 days. HA samples were pre-digested with proteinase K (3 mg/ml in HG-DMEM; Roche, Germany). Total RNA of the digest of HA and CS samples was extracted using the RNeasy Mini Kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany). Complementary DNA was obtained by reverse transcription using Qiagen RT-PCR Kit (Qiagen, Hilden, Germany). Quantitative real-time–polymerase chain reaction (RT-qPCR) was performed using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene and Qiagen QuantiTect SYBR® Green RT-PCR Kit according to manufacturer’s instructions with a 7500 Fast Real-Time PCR System (Applied Biosystems, Darmstadt, Germany). Human aggrecan (ACAN), type 2 collagen (COL2A1), transcription factor SOX-9 (SOX9), type 1 collagen (COL1A1), type 10 collagen (COL10A1), and GAPDH (all Biomers, Ulm, Germany) primers were used at a concentration of 0.3 µM (Table 1). Data analysis was performed using a comparative quantification (ΔΔCTmethod). Untreated monolayer (P4) served as control group. DNA Quantification and DMMB-Assay After 28 days, CS and HA samples (n = 3) digested with 0.5 mg/ml proteinase K (Roche, Mannheim, Germany). Sulphated glycosaminoglycans (sGAG) content within the digest and the cumulative supernatant was detected with modified 1,9-dimethylmethylene blue (DMMB) assay (Sigma-Aldrich, Darmstadt, Germany) according to Zheng and Levenston.19 The DNA content of the digest was quantified using a bisbenzimide-based DNA quantification assay according to manufacturer’s protocol (Promega, Mannheim, Germany). Histology Studies After 28 days, samples were mounted in tissue compound and cryo-sectioned (thickness: 10 μm) using Microm HM 56Cryostat-Microtome (Carl Zeiss AG, Zürich, Switzerland). CS and HA samples were stained with toluidine blue according to standard protocol.20 Immunohistochemistry (IHC) of type 2 collagen (mouse anti-type-II-collagen antibody; Clone CIIC1, DSHB, Iowa, USA; as described in) and type 1 collagen (mouse anti type-I-collagen antibody; C-2456 SigmaAldrich) was performed as described in Gille et al.21 and Kurz et al.22 Preparation of Bovine Cartilage Plugs and Invasion Assay Cartilage cylinders of bovine stifle joints were harvested as described before.23 The defect was filled with cell-free HA and CS (n = 6). Agarose 2% (Lonza, Basel, Switzerland) served as negative control. Chondrocyte invasion from a bovine cartilage into the embedded biomaterials was analyzed using CLSM (as described above) after 28 Table 1. Human Primer Sequences (5'-3'). Human target Sequence (5'-3') ACAN sense gaggCCagCagagaagattCtg ACAN antisense gaCgCCtCgCCttCttgaa COl2A1 sense CaaCaCtgCCaaCgtCCagat COl2A1 antisense CtgCttCgtCCagataggCaat SOX9 sense CtCggagaCttCtgaaCgagag SOX9 antisense CgttCttCaCCgaCttCCtCC COl1A1 sense aattCCaaggCCaagaagCatg COl1A1 antisense ggtagCCatttCCttggtggtt COl10A1 sense CCCtttttgCtgCtagtatCCttga COl10A1 antisense aaCtgtgtCttggtgttgggtagtg gAPDH sense gCCtCaagatCatCagCaatgC gAPDH antisense tggtCatgagtCCttCCaCgat ACAN = human aggrecan; COl2A1 = type 2 collagen; SOX9 = transcription factor SOX-9; COl1A1 = type 1 collagen, COl10A1 = type 10 collagen; gAPDH = glyceraldehyde 3-phosphate dehydrogenase. Weitkamp et al. 223 days cultivation time. The plugs were cultivated in an 2% agarose coated 24-well plate with CPM. For quantification of cell invasion in %, the total area of embedded biomaterials was divided into 100 equal areas. Using image J, areas colonized with cells were counted. Images of n = 6 were used to quantify cell invasion in CS, HA, and agarose. Investigations of the Biomaterial/tissue Interface To determine the bonding strength of HA and CS to native cartilage tissue, push-out tests were performed as described before.23 For the push-out test, the defect was filled with HA and CS (without and with 2.5 × 106 hCh/ml/cm2) or 2% agarose (Lonza, Basel, Switzerland) as negative control. Tissue/scaffold constructs were cultured in CPM for up to 28 days. The mechanical test was performed with 6 cartilage rings for each condition and repeated using the cartilage rings isolated from 3 different animals. The bonding strength was calculated by dividing the loading peak (N) by the bonding area (m2). Biomechanical testing: Young’s Modulus Biomechanical tests were carried out in a standard materialtesting machine (Instron 5866 electromechanical test device) equipped with a 10 N load cell. The initial sample height h0 was measured with a caliper and samples placed in a cell culture dish filled with chondrogenic medium. A 1-step unconfined compression test was performed by loading the sample through a flat-ended indenter (0.02 N preload) at a strain rate of 0.5 mm/min until 50% h0 strain was reached. The Young’s modulus was then calculated at the initial linear part of the stress-strain-curve (n = 6). Statistics All data were tested for normality using the Kolmogorov– Smirnov test. Statistical analysis was performed using Graph Pad prism 5 program (San Diego, CA, USA). Oneway analysis of variance (ANOVA) with Bonferroni’s multiple comparison was used to compare means among the independent experimental groups. Differences were considered significant if P ≤ 0.05. Quantitative data in the text are presented as mean and standard deviation (SD). Results Both Biomaterials Support High Cell Viability Detection of metabolic cell activity at day 1 and day 28 showed similar results for hCh seeded in both biomaterials. Relative fluorescence units increased significantly from day 1 to day 28 in non-supplemented and IL-10 supplemented HA and CS (HA day 1 vs. day 28: P = 0.0004; HA + IL-10 day 1 vs. day 28: P = 0.0176; CS day 1 vs. day 28: P = 0.0005; CS + IL-10 day 1 vs. day 28: P < 0.0001; Figure 1a(A)). The presence of BMP-2 led to an initial significant increase of metabolic cell activity in both biomaterials at day 1 (HA vs. HA + BMP-2 and HA vs. HA + BMP-2 + IL-10 day 1: P < 0.0001; CS vs. CS + BMP-2 and CS vs. CS + BMP-2 + IL-10 day 1: P < 0.0001; Figure 1a(A)). Cell quantification of live and dead cells revealed a high proportion of viable hCh in HA and CS during 4 weeks of in vitro cultivation (HA day 1: 93% ± 1.76%; CS day 1: 84.33% ± 8.48%; Figure 1a(B)). At day 28, significantly more viable hCh were detected in HA (P = 0.0006), but still more than 74% ± 8.85% hCh were viable in CS. Supplementing BMP-2 and IL-10 had no significant influence on cell viability (data not shown). Morphologically, hCh embedded in hydrogel showed a spherical phenotype in contrast to a fibroblastic-like cell phenotype in the 3D collagen matrix (Fig. 1b(A1 and C1)). The ramified cell phenotype was paralleled by greater F-actin accumulation in cells embedded in CS (Fig. 1c(A and B)). Chondrocytes in Hydrogel Show Superior Chondrogenic Re-Differentiation Cells cultivated in CPM showed a time-dependent increase of chondrogenic marker expression in both biomaterials (Fig. 2(A-F)), except COL2A1 in untreated HA. Cell redifferentiation was strongly enhanced by BMP-2 treatment, while supplementation of IL-10 was less effective. Overall, a trend for marked chondrogenic marker expression was observed in the hydrogel groups as demonstrated by shift from COL1A1 to COL2A1 (Fig. 2(F)). In detail, BMP-2 supplementation significantly increased relative gene expression of COL2A1 in HA by 3.4 (P = 0.0039; Fig. 2(A)). After 28 days, significantly increased COL2A1 expression was detected in those experimental groups that had received the addition of BMP-2 and the co-treatment of IL-10 and BMP-2 (HA day 28 vs. HA + BMP-2 day 28: P < 0.0001; HA day 28 vs. HA + BMP-2 + IL-10 day 28: P < 0.0001). For CS groups, a comparable COL2A1 induction was observed but the expression levels remained significantly lower compared with the hydrogel groups (HA + BMP-2 day 28 vs. CS + BMP-2 day 28: P < 0.0001). Transcription levels of ACAN showed a comparable pattern (Fig. 2(B)). In HA groups, relative mRNA expression was significantly amplified under BMP-2 treatment (HA day 28 vs. HA + BMP-2 day 28: P = 0.0002; HA day 28 vs. HA + BMP-2 + IL-10 day 28: P < 0.0001) and was also significantly higher than of CS groups after 28 days (HA + BMP-2 day 28 vs. CS + BMP-2 day 28: P = 0.0004). For SOX9, mRNA expression tended to be higher in HA groups without significant effects of IL-10 and BMP-2 (Fig. 2(C)). Furthermore, there was a trend for lower mRNA 224 CARtIlAgE 14(2) Figure 1. (a) Metabolic cell activity and viable quantification: (A) Celltiter-blue assay of hChs embedded in Ha and CS after 1 and 28 days with and without supplementation of il-10 (100 pg/ml) and BMP-2 (250 ng/ml). (B) Quantification of viable cells (%) after 1 and 28 days. Cells were cultured in chondropermissive medium without il-10 and BMP-2 supplementation. Ha = hyaluronic acid–based hydrogel; CS = collagen scaffold; il-10 = interleukin-10; BMP-2 = bone morphogenetic protein–2; aNOVa = analysis of variance; SD = standard deviation. asterisks indicate significant differences with *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way aNOVa. Data are presented as mean + SD (n = 3). (b) Chondrocyte viability and morphology in Ha and CS: (A-D) representative 3-dimensional reconstructions of cell-laden Ha and CS after l/D staining at day 1 and day 28. Cells were cultured in chondropermissive medium without il-10 and BMP-2 supplementation. an increase in cell density indicates proliferation in both biomaterials. (A1-D1) representative 2-dimensional sections. Chondrocytes in Ha appear spheric while cells in CS displayed a ramified phenotype. (c) Cytoskeletal changes: expression of actin stress-fibers. Phalloidin/DaPi staining of human articular chondrocytes embedded in CS (A) and Ha (B) after 7 days of culture in chondropermissive medium (b and c). Ha = hyaluronic acid–based hydrogel; CS = collagen scaffold; il-10 = interleukin-10; BMP-2 = bone morphogenetic protein–2; aNOVa = analysis of variance; DaPi = 4',6-diamidino-2-phenylindole. Bar 25 µm Weitkamp et al. 225 expression of the cell de-differentiation marker COL1A1 in untreated HA compared with CS groups (Fig. 2(E)). Treatment with BMP-2 led to positive effects by significantly lowering COL1A1 expression levels in HA compared with CS groups after 28 days of culture (HA + BMP-2 day 28 vs. CS + BMP-2 day 28: P = 0.003). The cell hypertrophy marker COL10A1 was similar in HA similar to monolayer chondrocytes but reduced in CS groups (Fig. 2(D)). Figure 2. Chondrogenic potential of human articular chondrocytes embedded in Ha and CS. transcription levels of messenger ribonucleic acid (mrNa) of chondrogenic markers ((A) COl1a1, (B) aCaN, (C) SOX9) and markers of de-differentiation ((D) COl1a1, (E) COl10a1) were measured after 1 and 28 days incubation with il-10 (100 pg/ml), BMP-2 (250 ng/ml), and cotreatment. COl2a1/COl1a1 ratio is displayed in (F). gene expression levels were normalized to that of gaPDH reference gene and then normalized to day 1 monolayer chondrocytes, which had an expression level = 1. Ha = hyaluronic acid–based hydrogel; CS = collagen scaffold; COl10a1 = type 10 collagen; aCaN = human aggrecan; SOX9 = transcription factor SOX-9; il-10 = interleukin-10; BMP-2 = bone morphogenetic protein–2; gaPDH = glyceraldehyde-3-phosphate dehydrogenase; aNOVa = analysis of variance; SD = standard deviation. asterisks indicate significant differences with *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way aNOVa. Data are presented as mean + SD (n = 3). 226 CARtIlAgE 14(2) Figure 3. (a) glycosaminoglycan content and release in cell-laden Ha and CS normalized to DNa content (A-C). Cell-laden biomaterials after 28 days of culture with and without il-10 (100 pg/ml), BMP-2 (250 ng/ml), and co-treatment, respectively. (A) total sgag content and cumulative gag release were analyzed by DMMB assay and normalized to corresponding sample’s DNa content (gag/DNa ratio). (B) total DNa content and (C) total sgag content in Ha and CS groups. asterisks indicate significant differences with *P < 0.05, **P < 0.01, one-way aNOVa. Data are presented as mean + SD (n = 3). (b) glycosaminoglycan biosynthesis in cellularized Ha (A-D) and CS (E-H). representative images of tB staining after 28 days of in vitro culture in chondropermissive medium with and without il-10 and BMP-2 supplementation (enhanced metachromasia due to acidophilic sgags). Ha = hyaluronic acid–based hydrogel; CS = collagen scaffold; il-10 = interleukin-10; BMP-2 = bone morphogenetic protein–2; sgag = sulphated glycosaminoglycans; DNa = deoxyribonucleic acid; DMMB = dimethylmethylene blue; tB = toluidine blue. Bar 200 μm (A1-D1, E1-H1: 100 μm). Weitkamp et al. 227 Figure 4. (a) type 2 collagen biosynthesis in cellularized Ha (A-D) and CS (E-H). representative images of type 2 collagen immunohistochemistry after 28 days of culture in chondropermissive medium with and without il-10 and BMP-2 supplementation (brownish staining indicating type 2 collagen). (b) type i collagen biosynthesis in cellularized Ha (A-D) and CS (E-H). representative images of type i collagen immunohistochemistry after 28 days of in vitro culture in chondropermissive medium with and without il-10 and BMP-2 supplementation (brownish staining indicating type 1 collagen) (a and b). Ha = hyaluronic acid–based hydrogel; CS = collagen scaffold; Col1 = type 1 collagen; il-10 = interleukin-10; BMP-2 = bone morphogenetic protein–2; Col1 = type 2 collagen. Bar 200 μm (A1-D1, E1-H1: 100 μm). 228 CARtIlAgE 14(2) In summary, hCh encapsulated in the HA showed favorable chondrogenic phenotype alterations, which was paralleled by chondrocyte morphology as previously observed. Deposition of Cartilage-Specific ECM Is Enhanced in CSs Quantification of sGAG in digested scaffolds normalized to DNA content revealed higher sGAG synthesis in HA in all treatment groups without reaching statistical significance (Fig. 3a(A)). DNA content in HA samples was generally lower in all groups (HA + BMP-2 vs. CS + BMP-2: P = 0.0232; Figure 3a(B)). There was a trend for higher sGAG content in IL-10 and BMP-2-treated samples with pronounced increase in biosynthesis in the co-treatment group (Fig. 3a(C)). sGAG loss in the medium was less pronounced in CS groups, which was paralleled by higher amount of retained sGAG in CS groups, observed in histology studies (Fig. 3b(A-H)). Toluidine blue staining of HA and CS showed more enhanced metachromasia within the CS grafts with BMP-2 treatment and co-treatment (Fig. 3b(G and H). No distinct effects of IL-10 were detected. A similar staining Figure 5. live/dead images of cell invasion assay. (A-C) representative tile scans after l/D staining of bovine cartilage rings with agarose (negative control), Ha and CS embedded after 28 days in vitro culture in chondropermissive medium. (A1-C1) Higher magnifications of the biomaterial/cartilage interface. (C2) Morphology of bovine chondrocytes after invasion into CS. (D and D1) Cellular invasion into CS from human osteoarthritic articular cartilage. Bar (A-D) 1 mm, (C2) 100 µm. Ha = hyaluronic acid–based hydrogel; CS = collagen scaffold; Oa cartilage = osteoarthritic cartilage.