HYPOXIC CULTURE OF BONE MARROW AND ADIPOSE-DERIVED MESENCHYMAL STROMAL CELLS FOR LIGAMENT TISSUE ENGINEERING

Anterior cruciate ligament (ACL) injuries are among the most common knee injuries. A damaged ACL is unable to properly heal and regenerate after injury due to an absence of blood supply and relative hypoxia in the native environment. The current gold standard for ACL repair is a reconstruction using tendon auto/allografts. The graft failure rate is high, thus creating a need for alternative tissue reconstruction strategies. The application of human bone marrow and adipose-derived mesenchymal stromal cells (hBM and hAD-MSCs) for bioengineering ACL tissue is an emerging field, however, bioengineered ACL grafts fail at the site of implantation due to the mismatch in their culture conditions and the native tissue environment, which is relatively hypoxic. hBM-MSCs and hAD-MSCS grown and primed under hypoxic conditions are potentially the optimal cell sources for cell-seeded bioengineered ligament implants to reduce graft rejections. This would enhance surgical outcomes and the quality of life of individuals and populations at risk of ACL reinjuries, such as athletes. In this study, we cultured bone marrow and adipose-derived mesenchymal stromal cells (hBM-MSCs and hAD-MSCs) at 3 different oxygen tensions (2%, 5%, and 20%) using a modular incubator hypoxia chamber to mimic the physiological environment. Cell culture media containing TGF-b and bFGF was used to help the cells differentiate into tenocytes/ligamentocytes. We measured cell viability using a trypan blue exclusion assay, metabolic activity using Alamar Blue reagent, and gene expression using qPCR.  Our data shows lower cell viability and growth rate under 2% oxygen tension, however, no difference in cell viability and growth rate under 5% and 20% oxygen tension for hBM-MSCs. No differences were observed for hAD-MSCs. Higher metabolic activity was observed under 2% and 5% oxygen tensions for both cell types. A higher expression of ligament/tendon markers, collagen type I and III, scleraxis, and tenascin-c, was observed when the cells were cultured in hypoxia for both cell types. Our study investigated the differentiation of bone marrow and adipose-derived mesenchymal stromal cells (MSCs) into tenocytes/ligamentocytes under varying oxygen tensions. By optimizing the conditions for MSC differentiation, bioengineered ligament implants hold the potential to improve surgical outcomes and enhance the quality of life for individuals at risk of ACL rupture. Further research in this field may pave the way for innovative approaches to tackle graft failure and ultimately improve the management of ACL injuries.