Mimicking biophysical and biochemical cues of tendon tissue for ASCs

摘要

Mesenchymal stem cells are a candidate cell source for engineered tendon repair including BMSCs, ASCs and other cell types. We hypothesized that elongated cell morphology and uniaxial mechanical strain are the main physiological niche factors to determine tenocyte phenotype. In this study, we employed a parallel microgrooved silicone membrane (10m in groove width, 3m in groove depth) to enforce an elongated cell morphology. Mouse adipose derived stem cells (mASCs) were isolated from vascular stromal fraction followed by cell sorting with CD34 as a marker. Flow cytometry analysis showed that CD34 positive cells were highly overlapped with the cell portion positive for CD29 and Sca-1. Additionally, CD34 positive ASCs revealed much potent multiple differentiation potentials along with greater colony forming efficiency when compared to CD34 negative ASCs. When seeded on microgrooved membrane, ASCs were forced into an elongated morphology with apparent shift towards tenogenic phenotype, as these cells exhibited significantly upregulated gene expression of tenomodulin, scleraxis, collagens Ⅰ, Ⅲ, Ⅺ. By contrast, elongated cells revealed inhibited differentiation towards other lineages as the cells expressed lower levels of SOX9, Aggrecan, collagen Ⅱ, AP2, Adiponectin, C/EBP, PPAR, RNX2, AKP and Osteocalcin when compared to non-elongated cells. Nevertheless, when ASCs derived from scleraxis knock-out mice were seeded on the topographical patterned surface, the tenogenic induction effect was abrogated. Next, we seeded ASCs on a knitted scaffold followed by in vitro mechanical loading to engineer a neo-tendon tissue, which was further implanted in vivo to repair Achilles tendon with mechanical loading. Interestingly, with long term mechanical loading for 45 weeks, ASC seeded scaffold eventually was transformed into a mature tendon tissue with mechanical property and histology similar to those of native tendon. Thus, our hypothesis was approved by this combined in vitro and in vivo study.