Cell-based tendon engineering is an attractive alternative therapeutic approach to established treatments of tendon injuries. Numerous cell types are promising source of tendon engineering; however, there are certain disadvantages for each cell type. Interestingly, dermal fibroblasts (DFs) are able to transdifferentiate into other cell types, they are widely distributed in dermis and easy to harvest and isolate. Furthermore, pilot clinical studies suggested a promising therapeutic potential of autologous DFs for discorded tendons (Connell et al., 2009&2011), but the underlining repair mechanisms remain unclarified. To investigate tenogenic differentiation process in great detail, we have previously established a three-dimensional (3D) cell sheet model, comprising of three consecutive step (expansion, stimulation and maturation) leading to the formation of 3D tendon-like tube (Hsieh et al., 2018; Yan et al., 2020). Hence, the aim of this study was to carry out pilot examination of the tenogenic potential of human DFs (hDFs) by implementing the 3D cell sheet model. hDFs (company purchased, n=2), hBMSCs (human bone marrow mesenchymal stem cells, n=1) and hTSPCs (human tendon stem/progenitor cells, n=1) were used and subjected to the 3D model. In 2D culture, semi-qPCR was performed to validate the expression of DF markers in hDFs, namely NTN1, PDPN and CD26 for papillary dermis layer, and PPARG, ACTA2 and CD36 for reticular dermis layer). FACS analysis and immunofluorescence were employed to validate expression of CD73, CD90, CD105 and vimentin (mesenchyme marker), respectively. After harvesting the 3D cell sheets, wet weigh measurements, H&E and collagen type I stainings, and semi-qPCR for Scleraxis and tenomodulin were executed.Introduction
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Tendons are dense connective tissues and critical components of the musculoskeletal system with known long repair process. Tissue engineering is a promising approach for achieving complete recovery of ruptured tendons. The most studies have focused on the combination of cells with various carriers; however, frequent times the biomaterials do not match the tissue organization and strength. For this reason, we first reviewed the literature for an alternative scaffold-free strategy for tendon engineering and second, we compared the cell sheet formation of two different cell types: bone marrow-derived mesenchymal stem cells (BM-MSCs) and tendon stem/progenitor cells (TSPCs). Methods: Literature search was performed in Pubmed using “tendon tissue engineering” and “scaffold-free” keywords and was limited to the last ten years. By using a 3-step protocol, BM-MSCs and TSPCs were induced to form cell sheets in 5 weeks. The sheets were compared by analysis for weight, diameter, cell density, tissue morphology (H&E and scoring) and cartilaginous matrix (DMMB and S.O. staining). Results: Scaffold-free models (cell sheets and pellet cultures) are available; however, further optimization is needed. Comparison between the two cell types clearly demonstrated that TSPCs form more mature cell sheet, while BMSCs form larger but less organized and differentiated sheet as judged by higher cell density and lower scoring outcome. Future efforts will focus on identifying mechanisms to speed BM-MSC sheet formation and maturation, which can in turn lead to development of new methodology for scaffold-free tendon tissue engineering.
Human Mesenchymal stem cells (hMSCs) are a promising source for articular cartilage repair. Unfortunately, under Pellets of passage 2 hMSCs were formed in V-bottom well plates by centrifugation and pre-differentiated in a chemically defined medium containing 10ng/mL TGFß (CM+) for 14 days. Thereafter, pellets were cultured for an additional 14 days under 6 conditions: CM+, CM- (w/out TGFß), and hypertrophic medium (CM- with 25 ng/ml BMP 4, w/out dexamethasone). Each of these first three conditions was additionally supplemented with the RA receptor (RAR) inverse agonist BMS493 (BMS) at 2μM after 14 days of chondrogenic pre-differentiation. One additional BMP4 group was supplemented with BMS from the beginning of chondrogenic differentiation until day 14. The pellets were assessed for gene expression (Col 2, Col 10, Col 1 and MMP13) and histologically using dimethyl methylene blue (DMMB), alkaline phosphatase staining (ALP) and collagen II and X immunohistochemistry.Introduction
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