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Abstract
Introduction Tendons connect muscles to bone and transmit the force generated during muscle contraction to the skeleton, which are highly prone to injury. Surgical repair is common but natural healing is extremely slow and inefficient. Human-induced pluripotent stem cells (hiPSCs) are highly promising cell source for realising personalised treatments in regenerative medicine [Lian, Q.2010]. Nevertheless, the utility of these cells for tendon tissue engineering has yet not been adequately explored. This study developed a stepwise strategy to induce hiPSCs differentiation into tenocytes and assessed the efficacy of this tissue-engineered construct in promoting tendon regeneration
Methods Clonogenicity and multi-differentiation potential were revealed by colony-forming unit (CFU) and different mesodermal lineages differentiation assay respectively. Surface markers were detected by flow cytometry. Well-aligned chitosan-based ultrafine fibers were fabricated with stable jet electrospinning (SJES) technique. Gene expressions were analysed by Q-PCR. A rat Achilles tendon defect model was created and implanted with AC (i.e., aligned fibre scaffold with hiPSC-MSCs) or RC (i.e., random fibre scaffold with hiPSC-MSCs)in vivo. The morphology of repaired tissues were analysed by histological examination and transmission electron microscope. The amount of deposited collagen was quantified using a collagen quantitative assay kit. Mechanical testing was performed for mechanical properties
Results and discussion hiPSCs were first induce to mesenchymal stem cells (hiPSC-MSCs) as confirmed by differentiation into three mesenchymal lineages. Flow cytometry and CFU assay showed the expression of characteristic MSC surface markers and clonogenicity. Subsequently, hiPSC-MSCs were differentiated into tenocytes by cultivation on the chitosan-based well-aligned ultrafine fiber scaffold. SEM micrographs and immunofluorescence assays showed that hiPSC-MSCs exhibited tenocyte-like morphology and significantly high expression of tendon-specific genes in the hiPSC-MSCs on well-aligned fibre scaffold. ALP and alizarin red staining showed that the random fibre scaffold induced osteogenesis, while the aligned fibre scaffold hindered the process. In addition, aligned cells expressed significantly higher levels of integrin a1, a2, a5 and b1 subunits, myosin IIB, TGFb3 and SDF-1. In rat Achilles tendon repair model, AC-treated tendon had superior structural and mechanical properties than RC-treated tendon. Cell labelling and extracellular matrix expression assays demonstrated that the transplanted hiPSC-MSCs contributed directly to tendon regeneration. Moreover, no teratoma was found in any samples. These findings present a strategy combining well-aligned fibre scaffold with iPSC-MSCs for tendon regeneration and may assist in clinical regenerative medicine to treat tendon diseases.
(a) Confocal micrograph of F-actin exhibiting elongated hiPSC-MSCs on the aligned scaffold and morphological change of hiPSC-MSCs on the randomly-oriented scaffold. (b) Histology morphology of the repair tendon after 4 weeks post-surgery. (c) Histology score of repaired tendon (d) Quantitative analyses of the collagen content in the repaired tendon.
Reference Lian Q. et al. Circulation. 2010;121:1113–1123