"Introduction: Tendons are dense connective tissues, necessary for the integrity and function of musculoskeletal system. Poor tendon repair remains a serious clinical problem, due to the difficulties in reorganising the complex multiscale hierarchical structure of native tendons. Tendon stem/progenitor cells (TSPCs) were recently identified as tendon-specific adult stem cells that exhibit universal characteristics of stem cells and play a key role in tendon homeostasis, repair or regeneration once tissue is injuried. Moreover, it has been recently showed that TSPCs regulate inflammation during healing of acute tendon injuries. In this study, we used a 3D scaffold to provide an in-vitro model for tenogenesis. This 3D environment was bioengineered with TSPCs obtained from both healthy and tendinophatic human samples to compare their differentiation potential under cyclic strain and a proper tenogenic medium1. Furthermore, a 3D co-culture with human Peripheral Blood Mononuclear Cells (hPBMCs) was set to explore a potential cross-talk activity in both healthy and pathological conditions.
Methodology: Healthy and tendinophatic tendon samples were obtained from patients undergoing surgery after traumas. hTSPCs were extracted in a period of 3 weeks, collected and characterized by flow cytometry2. hTSPCs were then cultured within 3D scaffolds for 21 days, alone or in combination with hPBMCs, under dynamic conditions provided by a custom-made bioreactor3,4. Evaluation of cell density, proliferation, senescence, histomorphometric analysis, as well as quantitative gene expression of different target genes (related to tenogenesis and immunomodulation) were performed.
Results: The results revealed that the 3D matrix exerted a rejuvenating effect on pathological TSPCs, in a comparable manner to healthy TSPCs, supporting simultaneously cell survival and proliferation. Moreover, coupled to a dynamic environment, it promoted hTSPCs differentiation events, with tendon related genes overexpression (SCX-A, DCN, TNC, TNMD). Interestingly, the co-culture with hPBMCs induced an upregulation of target genes, as confirmed by histomorphometric analysis, but in a more significant way for pathological hTSPCs, that also showed a more marked overexpression of anti-inflammatory cytokines (IL-10, TGF-β1), when compared to healthy hTSPCs.
Conclusions: The 3D scaffold plus dynamic culture seemed an extremely favorable biomimetic environment that allowed cells rejuvenation and differentiation. Furthermore, both tenogenic and anti-inflammatory cytokines gene expression of hTSPCs were modulated by the presence of hPBMCs, suggesting a cooperative effect between resident stem cells and hPBMCs during tendon healing events. The study opens perspectives on TSPCs 3D culture (healthy and pathological) into an in vitro model matrix for the understanding of their behavior in impaired extra-cellular matrix secretion. The model also provided the possibility to study the role of PBMCs in tenogenic events.
1. Ciardulli M.C. et al., Int. J. Mol. Sci. 21(16):5905 (2020).
2. Ciardulli M.C. et al., Submitted to Front. Cell Dev. Biol. (Dec 2021).
3. Ciardulli M.C. et al., Pharmaceutics. 13(9):1448 (2021).
4. Ciardulli M.C. et al., Cells. 9(5):1268 (2020).