Capacity of muscle derived stem cells and pericytes to promote tendon graft integration and ligamentization following anterior cruciate ligament reconstruction

Abstract

Purpose: The aim of this study is to examine the capacity of muscle tissue preserved on hamstring tendons forming candy-stripe grafts in order to improve tendon to bone ingrowth and ligamentization. We hypothesized that muscle tissue does possess a stem cell population that could enhance the healing process of the ACL graft when preserved on the tendons. Methods: Human samples from gracilis and semitendinosus muscles were collected during ACL surgery from ten patients and from these tissue samples human muscle-derived stem cells and tendon-derived stem cells were isolated and propagated. Both stem cell populations were in-vitro differentiated into osteogenic lineage. Alkaline phosphatase activity was determined at days zero and 14 of the osteogenic induction and von Kossa staining to assess mineralization of the cultures. Total RNA was collected from osteoblast cultures and real time quantitative PCR was performed. Western-blot for osteocalcin and collagen type I followed protein isolation. Immunofluorescence double labeling of pericytes in muscle and tendon tissue was performed. Results: Mesenchymal stem cells from muscle and tendon tissue were isolated and expanded in cell culture. More time was needed to grow the tendon derived culture compared to muscle derived culture. Muscle derived stem cells exhibited more alkaline phosphatase actvity compared to tendon derived stem cells, whereas tendon derived stem cells formed more mineralized nodules after 14 days of osteoinduction. Muscle derived stem cells exhibited higher expression levels of bone sialoprotein, and tendon derived stem cells showed higher expression of dental-matrix-protein 1 and osteocalcin. Immunofluorescent staining against pericytes indicated that they are more abundant in muscle tissue. Conclusions: These results indicate that muscle tissue is a better source of stem cells than tendon tissue. Achievement of this study is proof that there is vast innate capacity of muscle tissue for enhancement of bone-tendon integration and ligamentization of ACL hamstring grafts and consequently muscle tissue should not be treated as waste after harvesting.