Effect of growth and differentiation factor 6 on the tenogenic differentiation of bone marrow-derived mesenchymal stem cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Recent studies showed that bone marrow-derived mesenchymal stem cells (BMSCs) had risk of ectopic bone formation. In this study, we aimed to investigate the effect of growth and differentiation factor 6 (GDF-6) on the tenogenic differentiation of BMSCs in vitro, and then combined with small intestine submucous (SIS) to promote tendon regeneration in vivo.</p><p><b>METHODS</b>The BMSCs were isolated from the green fluorescent protein (GFP) rats, and were characterized by multi-differentiation assays following our previous study protocol. BMSCs cultured with different concentrations of GDF-6, without growth factors served as control. After 2 weeks, mRNA expression and protein expression of tendon specific markers were examined by qRT-PCR and Western blotting to define an optimal concentration of GDF-6. Mann-Whitney U-test was used to compare the difference in relative mRNA expression among all groups; P ≤ 0.05 was regarded as statistically significant. The GDF-6 treated BMSCs combined with SIS were implanted in nude mice and SD rat acute patellar tendon injury model, the BMSCs combined with SIS served as control. After 12 and 4 weeks in nude mice and tendon injury model, the samples were collected for histology.</p><p><b>RESULTS</b>After the BMSCs were treated with different concentration of GDF-6 for 2 weeks, the fold changes of the specific markers (Tenomodulin and Scleraxis) mRNA expression were significantly higher in GDF-6 (20 ng/ml) group (P ≤ 0.05), which was also confirmed by Western blotting result. The BMSCs became parallel in orientation after GDF-6 (20 ng/ml) treatment, but the BMSCs in control group were randomly oriented. The GDF-6 (20 ng/ml) treated BMSCs were combined with SIS, and were implanted in nude mice for 12 weeks, the histology showed neo-tendon formation. In the SD rat patellar tendon window injury model, the histology also indicated the GDF-6 (20 ng/ml) treated BMSCs combined with SIS could promote tendon regeneration.</p><p><b>CONCLUSIONS</b>GDF-6 has tenogenic effect on the tenogenic differentiation of BMSCs, and GDF-6 (20 ng/ml) has better tenogenic effect compared to other concentrations. The GDF-6 (20 ng/ml) treated BMSCs combined with SIS can form neo-tendons and promote tendon regeneration.</p>

Does erroneous differentiation of tendon-derived stem cells contribute to the pathogenesis of calcifying tendinopathy? / 中华医学杂志(英文版)

Calcifying tendinopathy is a tendon disorder with calcium deposits in the mid-substance presented with chronic activity-related pain, tenderness, local edema and various degrees of incapacitation. Most of current treatments are neither effective nor evidence-based because its underlying pathogenesis is poorly understood and treatment is usually symptomatic. Understanding the pathogenesis of calcifying tendinopathy is essential for its effective evidence-based management. One of the key histopathological features of calcifying tendinopathy is the presence of chondrocyte phenotype which surrounds the calcific deposits, suggesting that the formation of calcific deposits was cell-mediated. Although the origin of cells participating in the formation of chondrocyte phenotype and ossification is still unknown, many evidences have suggested that erroneous tendon cell differentiation is involved in the process. Recent studies have shown the presence of stem cells with self-renewal and multi-differentiation potential in human, horse, mouse and rat tendon tissues. We hypothesized that the erroneous differentiation of tendon-derived stem cells (TDSCs) to chondrocytes or osteoblasts leads to chondrometaplasia and ossification and hence weaker tendon, failed healing and pain, in calcifying tendinopathy. We present a hypothetical model on the pathogenesis and evidences to support this hypothesis. Understanding the key role of TDSCs in the pathogenesis of calcifying tendinopathy and the mechanisms contributing to their erroneous differentiation would provide new opportunities for the management of calcifying tendinopathy. The re-direction of the differentiation of resident TDSCs to tenogenic or supplementation of MSCs programmed for tenogenic differentiation may be enticing targets for the management of calcifying tendinopathy in the future.

Bone morphogenetic protein 2 promotes transforming growth factor β3-induced chondrogenesis of human osteoarthritic synovium-derived stem cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Synovium-derived stem cells (SDSCs) with higher chondrogenic potential are attracting considerable attention as a cell source for cartilage regeneration. We investigated the effect of bone morphogenetic protein 2 (BMP-2) on transforming growth factor beta3 (TGF-β3)-induced chondrogenesis of SDSCs isolated from human osteoarthritic synovium in a pellet culture system.</p><p><b>METHODS</b>The clonogenicity, stem cell marker expression and multi-differentiation potential of isolated SDSCs were determined by colony forming unit assay, flow cytometry and specific staining including alizarin red S, Oil red O and alcian blue staining, respectively. SDSCs pellet was cultured in chondrogenic medium with or without TGF-β3 or/and BMP-2. At day 21, the diameter and the weight of the pellets were measured. Chondrogenic differentiation of SDSCs was evaluated by Safranin O staining, immunohistochemical staining of collagen type II, sulfated glycosaminoglycan (sGAG) synthesis and mRNA expression of collagen type II, aggrecan, SOX9, link-protein, collagen type X and BMP receptor II.</p><p><b>RESULTS</b>Cells isolated under the optimized culturing density (10(4)/60 cm(2)) showed clonogenicity and multi-differentiation potential. These cells were positive (> 99%) for CD44, CD90, CD105 and negative (< 10%) for CD34 and CD71. SDSCs differentiated to a chondrocytic phenotype in chondrogenic medium containing TGF-β3 with or without BMP-2. Safranin O staining of the extracellular matrix was positive and the expression of collagen type II was detected. Cell pellets treated with TGF-β3 and BMP-2 were larger in diameter and weight, produced more sGAGs, and expressed higher levels of collagen type II and other chondrogenic markers, except COL10A1, than medium with TGF-β3 alone.</p><p><b>CONCLUSIONS</b>SDSCs could be isolated from human osteoarthritic synovium. Supplementation with BMP-2 significantly promoted the in vitro TGF-β3-induced chondrogenic differentiation of SDSCs.</p>