The use of growth differentiation factor 5 (GDF-5) in damaged tendons has been shown to improve tendon repair. It has been hypothesized that further improvements may be achieved when GDF-5 is used to promote cell proliferation and induce tenogenic differentiation in human bone marrow-derived mesenchymal stem cells (hMSCs). However, the optimal conditions required to produce these effects on hMSCs have not been demonstrated in previous studies. A study to determine cell proliferation and tenogenic differentiation in hMSCs exposed to different concentrations of GDF-5 (0, 5, 25, 50, 100 and 500 ng/ml) was thus conducted. No significant changes were observed in the cell proliferation rate in hMSCs treated at different concentrations of GDF-5. GDF-5 appeared to induce tenogenic differentiation at 100 ng/ml, as reflected by (1) a significant increase in total collagen expression, similar to that of the primary native human tenocyte culture; (2) a significant upregulation in candidate tenogenic marker gene expression, i.e. scleraxis, tenascin-C and type-I collagen; (3) the ratio of type-I collagen to type-III collagen expression was elevated to levels similar to that of human tenocyte cultures, and (4) a significant downregulation of the non-tenogenic marker genes runt-related transcription factor 2 and sex determining region Y (SRY)-box 9 at day 7 of GDF-5 induction, further excluding hMSC differentiation into other lineages. In conclusion, GDF-5 does not alter the proliferation rates of hMSCs, but, instead, induces an optimal tenogenic differentiation response at 100 ng/ml.
Chondrogenic differentiated mesenchymal stem cells (CMSCs) have been shown to produce superior chondrogenic expression markers in vitro. However, the use of these cells in vivo has not been fully explored. In this study, in vivo assessment of cartilage repair potential between allogenic-derived chondrogenic pre-differentiated mesenchymal stem cells and undifferentiated MSCs (MSCs) were compared. Bilateral full thickness cartilage defects were created on the medial femoral condyles of 12 rabbits (n ¼ 12). Rabbits were divided into two groups. In one group, the defects in the right knees were repaired using alginate encapsulated MSCs while in the second group, CMSCs were used. The animals were sacrificed and the repaired and control knees were assessed at 3 and 6 months after implantation. Quantitative analysis was performed by measuring the Glycosaminoglycans (GAGs)/total protein content. The mean Brittberg score was higher in the transplanted knees as compared to the untreated knee at 6 months (p < 0.05). Quantitative analysis of GAGs was consistent with these results. Histological and immunohistochemical analysis demonstrated hyaline-like cartilage regeneration in the transplanted sites. Significant differences between the histological scores based on O'Driscoll histological grading were observed between contralateral knees at both 3 and 6 months (p < 0.05). No significant differences were observed between the Britberg, O'Driscoll scores, and GAGs/total protein content when comparing defect sites treated with MSC and CMSC (p > 0.05). This study demonstrates that the use of either MSC or CMSC produced superior healing when compared to cartilage defects that were untreated. However, both cells produced comparable treatment outcomes. ß
BackgroundIn many pre-clinical studies of cartilage tissue, it has been generally assumed that the major difference of the tissue between the species is the tissue thickness, which is related to the size of the animal itself. At present, there appear to be lack of studies demonstrating the relationship between chondrocyte densities, protein content, gene expressions and cartilage thickness in the various animal models that are commonly used. The present study was conducted to determine whether or not chondrocyte density, proteoglycan/protein content and selective chondrocyte gene expression are merely related to the cartilage thickness (thus animal size), and not the intrinsic nature of the species being investigated. Mature animals (rabbit, rats and goats) were sacrificed for their hind knee cartilages. Image analyses were performed on five consecutive histological sections, sampled from three pre-defined locations at the lateral and medial femoral condyles. Cartilage thickness, chondrocyte density, Glycosaminoglycan (GAGs)/protein content and gene expression levels for collagen II and SOX-9 were compared across the groups. Correlation analysis was done between cartilage thickness and the other variables.ResultsThe mean cartilage thickness of rats, rabbits and goats were 166.5 ± 10.9, 356.2 ± 25.0 907.5 ± 114.6 μm, respectively. The mean cartilage cell densities were 3.3 ± 0.4×10-3 for rats, 2.6 ± 0.3×10-3 for rabbits and 1.3 ± 0.2×10-3 cells/μm2 for goats. The mean μg GAG/mg protein content were 23.8 ± 8.6 in rats, 20.5 ± 5.3 in rabbits and 328.7 ± 64.5 in goats; collagen II gene expressions were increased by 0.5 ± 0.1 folds in rats; 0.6 ± 0.1 folds in rabbits, and 0.1 ± 0.1 folds in goats, whilst the fold increase of SOX-9 gene expression was 0.5 ± 0.1 in rats, 0.7 ± 0.1 in rabbits and 0.1 ± 0.0 in goats. Cartilage thickness correlated positively with animals’ weight (R2 =0.9856, p = 0.001) and GAG/protein content (R2 =0.6163, p = <0.001). Whereas, it correlates negatively with cell density (R2 = 0.7981, p < 0.001) and cartilage gene expression levels (R2 = 0.6395, p < 0.001).ConclusionThere are differences in the composition of the articular cartilage in diverse species, which are not directly dependent on the cartilage thickness of these animals but rather the unique characteristics of that species. Therefore, the species-specific nature of the cartilage tissue should be considered during any data interpretation.
The results have an implication of supporting the potential use of MSCs for cartilage repair after sports injuries or diseases, in view of similar efficacy but less patient morbidity and potential cost savings as compared with conventional autoC therapy.
Monosodium -iodoacetate (MIA)-induced animal model of osteoarthritis (OA) is under-utilised despite having many inherent advantages. At present, there is lack of studies that directly compare the degenerative changes induced by MIA with the surgical osteoarthritis induction method and human osteoarthritis, which would further verify a greater use of this model. Therefore, we compared the histological, biochemical and biomechanical characteristics in rat model using MIA against the anterior cruciate ligament transection (ACLT) and human cartilage with clinically established osteoarthritis. The right knees of Sprague-Dawley rats were subjected to either MIA or ACLT (n=18 in each group). Six rats were used as controls. Human cartilage samples were collected and compared from patients clinically diagnosed with (n=7) and without osteoarthritis (n=3). Histological, biochemical (Glycosaminoglycans/total protein) and biomechanical (cartilage stiffness) evaluations were performed at the end of the 1st and 2nd week after OA induction. For human samples, evaluations were performed at the time of sampling. Histopathological changes in the MIA group were comparable to that observed in the ACLT group and human OA. The Mankin scores of the 3 groups were comparable (MIA: 11.5±1.0; ACLT: 10.1±1.1; human OA: 13.2±0.8). Comparable reduction in Glycosaminoglycan/total protein content in the intervention groups were observed (MIA: 7±0.6; ACLT: 6.6±0.5; human OA: 3.1±0.7). Cartilage stiffness score were 24.2±15.3 Mpa for MIA, 25.3±4.8 for ACLT and 0.5±0.0 Mpa for human OA. The MIA model produces comparable degenerative changes to ACLT and human OA with the advantage of being rapid, minimally invasive and reproducible. Therefore, wider utilisation of MIA as animal translational OA model should perhaps be advocated.
Human amniotic membrane (HAM) is an established biomaterial used in many clinical applications. However, its use for tissue engineering purposes has not been fully realized. A study was therefore conducted to evaluate the feasibility of using HAM as a chondrocyte substrate/carrier. HAMs were obtained from fresh human placenta and were process to produced air dried HAM (AdHAM) and freeze dried HAM (FdHAM). Rabbit chondrocytes were isolated and expanded in vitro and seeded onto these preparations. Cell proliferation, GAG expression and GAG/cell expression were measured at days 3, 6, 9, 12, 15, 21, and 28. These were compared to chondrocytes seeded onto plastic surfaces. Histological analysis and scanning electron microscopy was performed to observe cell attachment. There was significantly higher cell proliferation rates observed between AdHAM (13-51%, P=0.001) or FdHAM (18-48%, p = 0.001) to chondrocytes in monolayer. Similarly, GAG and GAG/cell expressed in AdHAM (33-82%, p = 0.001; 22-60%, p = 0.001) or FdHAM (41-81%, p = 0.001: 28-60%, p = 0.001) were significantly higher than monolayer cultures. However, no significant differences were observed in the proliferation rates (p = 0.576), GAG expression (p = 0.476) and GAG/cell expression (p = 0.135) between AdHAM and FdHAM. The histology and scanning electron microscopy assessments demonstrates good chondrocyte attachments on both HAMs. In conclusion, both AdHAM and FdHAM provide superior chondrocyte proliferation, GAG expression, and attachment than monolayer cultures making it a potential substrate/carrier for cell based cartilage therapy and transplantation.
Previous studies have shown that platelet concentrates used in conjunction with appropriate growth media enhance osteogenic differentiation of human mesenchymal stromal cells (hMSCs). However, their potential in inducing osteogenesis of hMSCs when cultured in serum free medium has not been explored. Furthermore, the resulting osteogenic molecular signatures of the hMSCs have not been compared to standard osteogenic medium. We studied the effect of infrequent supplementation (8-day interval) of 15% non-activated platelet-rich concentrate (PRC) in serum free medium on hMSCs proliferation and differentiation throughout a course of 24 days, and compared the effect with those cultured in a standard osteogenic medium (OM). Cell proliferation was analyzed by alamar blue assay. Gene expression of osteogenic markers (Runx2, Collagen1, Alkaline Phosphatase, Bone morphogenetic protein 2, Osteopontin, Osteocalcin, Osteonectin) were analyzed using Q-PCR. Immunocytochemical staining for osteocalcin, osteopontin and transcription factor Runx2 were done at 8, 16 and 24 days. Biochemical assays for the expression of ALP and osteocalcin were also performed at these time-points. Osteogenic differentiation was further confirmed qualitatively by Alizarin Red S staining that was quantified using cetylpyridinium chloride. Results showed that PRC supplemented in serum free medium enhanced hMSC proliferation, which peaked at day 16. The temporal pattern of gene expression of hMSCs under the influence of PRC was comparable to that of the osteogenic media, but at a greater extent at specific time points. Immunocytochemical staining revealed stronger staining for Runx2 in the PRC-treated group compared to OM, while the staining for Osteocalcin and Osteopontin were comparable in both groups. ALP activity and Osteocalcin/DNA level were higher in the PRC group. Cells in the PRC group had similar level of bone mineralization as those cultured in OM, as reflected by the intensity of Alizarin red stain. Collectively, these results demonstrate a great potential of PRC alone in inducing proliferation of hMSCs without any influence from other lineage-specific growth media. PRC alone has similar capacity to enhance hMSC osteogenic differentiation as a standard OM, without changing the temporal profile of the differentiation process. Thus, PRC could be used as a substitute medium to provide sufficient pool of pre-differentiated hMSCs for potential clinical application in bone regeneration.
Platelet-rich concentrate (PRC), used in conjunction with other chondroinductive growth factors, have been shown to induce chondrogenesis of human mesenchymal stromal cells (hMSC) in pellet culture. However, pellet culture systems promote cell hypertrophy and the presence of other chondroinductive growth factors in the culture media used in previous studies obscures accurate determination of the effect of platelet itself in inducing chondrogenic differentiation. Hence, this study aimed to investigate the effect of PRC alone in enhancing the chondrogenic differentiation potential of human mesenchymal stromal cells (hMSC) encapsulated in three-dimensional alginate constructs. Cells encapsulated in alginate were cultured in serum-free medium supplemented with only 15% PRC. Scanning electron microscopy was used to determine the cell morphology. Chondrogenic molecular signature of hMSCs was determined by quantitative real-time PCR and verified at protein levels via immunohistochemistry and enzyme-linked immunosorbent assay. Results showed that the cells cultured in the presence of PRC for 24 days maintained a chondrocytic phenotype and demonstrated minimal upregulation of cartilaginous extracellular matrix (ECM) marker genes (SOX9, TNC, COL2, ACAN, COMP) and reduced expression of chondrocyte hypertrophy genes (Col X, Runx2) compared to the standard chondrogenic medium (p < 0.05). PRC group had correspondingly higher levels of glycosaminoglycan and increased concentration of chondrogenic specific proteins (COL2, ACAN, COMP) in the ECM. In conclusion, PRC alone appears to be very potent in inducing chondrogenic differentiation of hMSCs and offers additional benefit of suppressing chondrocyte hypertrophy, rendering it a promising approach for providing abundant pool of chondrogenic MSCs for application in cartilage tissue engineering.
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