gms | German Medical Science

Objectives: Cell-based tendon engineering is an attractive alternative therapeutic approach to established treatments of tendon injuries. Numerous cell types are promising sources 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 (Chu et al., 2020). Furthermore, pilot clinical studies suggested a promising therapeutic potential of autologous DFs for discorded tendons (Connell et al., 2009&2011), but the underlying repair mechanisms remain unclarified. To investigate tenogenic differentiation process in great detail, we have previously established a three-dimensional (3D) sell-assembling organoid model, comprising of three consecutive steps leading to the formation of 3D tendon-like rod organoids (Hsieh et al., 2018; Yan et al., 2020). Hence, the aim of this study was to carry out a pilot examination of the tenogenic potential of human DFs (hDFs) by implementing the 3D organoid model.

Methods: hDFs and hBMSCs (human bone marrow mesenchymal stem cells) and hTSPCs (human tendon stem/progenitor cells, n=1) were used and subjected to the 3D model. In 2D culture, semi-qPCR was used 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, respectively. After harvesting the 3D cell sheets, wet weigh measurements, H&E and collagen I stainings, and semi-qPCR of Scleraxis and tenomodulin were performed.

Results and Conclusion: Semi-qPCR of DF markers validated the dermal origin of both donor-derived hDFs; however, the data suggested that donor 1 was a mixed cell pool of papillary and reticular DFs, whilst donor 2 consisted of reticular dermis cells. In FACS analysis, the expression levels of CD73 and CD90 were comparable among all cell types. For CD105, ca. 20% of the cells were negative in both hDF and hTSPC cultures, but only 2% in hBMSCs. As expected, all three cell types were vimentin-positive. 3D cell sheet formation was successful for all cell types. Interestingly, the hDF organoids were thicker and ca. 2-fold heavier than that of hBMSCs and hTSPCs. Next, H&E and collagen I analyses revealed higher cellularity as well as higher collagen I deposition in the hDF organoids compared to the other two cell types. Last, semi-qPCR for Scleraxis and tenomodulin revealed upregulation of both genes in hDF and hTSPC sheets versus 2D culture.

Our pilot data suggests that hDFs perform well and even exceed hBMSCs and hTSPCs in the 3D model in terms of size, cellularity and collagen I expression. However, increasing the cohort size and further detailed molecular and histomorphometric analyses are necessary to conclude on the promising tenogenic potential of hDFs.