Abstract
Purpose
The biomechanical role of the meniscus in the knee joint is a function of its extracellular matrix which consists of type I collagen throughout, type II collagen in the inner meniscus region and glycosaminoglynated (GAG) proteins of which aggrecan is the most prevaleet. Meniscus reparative capacity is limited, particularly when a defect is located in the inner avascular portion, and menisectomy predisposes the joint to osteoarthritis. Using meniscus cells in tissue engineering strategies has been advocated to generate functional meniscus substitutes. However, meniscus cells, like chondrocytes of cartilage, lose their matrix-forming phenotype during culture expansion. Co-culture of chondrocytes with stem cells has been shown to result in enhanced matrix formation. We hypothesized that meniscus cells in co-culture with stem cells will result in increased matrix formation.
Method
Tissue specimens were obtained after approval of the local ethical committee and informed consent. Menisci were obtained from 3 patients undergoing total knee arthroplasty; (53–84; mean age 66.6). Meniscus cells were isolated after digestion of menisci with collagenase II. Isolated meniscus cells were plated for 24–48 hr before use. Bone marrow aspirates were obtained from the iliac crest of 3 donors: 1 female (46) and 2 males (15 and 21) undergoing routine orthopaedic procedures. Plastic adherent bone marrow stromal cell populations were isolated and expanded under normal oxygen tension of 21%O2 in a-MEM growth media plus FGF-2 until passage 2. Cells were mixed at a variety of meniscus cells (Men): BMSC ratio including 5/95, 10/90 and 25/75, respectively. Mixed cells were centrifuged to form spherical pellets followed by culture in a defined serum free chondrogenic differentiation medium. Control groups were pure Men and pure BMSCs. Total cell number per pellet was 25×104. Pellets were cultured for 3 weeks under normal oxygen tension. Thereafter, pellets were processed: biochemically for GAG and DNA content, and histologically for Safranin-O staining of sulphated GAG and immunohistochemical analyses for collagen types I and II. Analysis was performed on a minimum of 2 independent pellets.
Results
Relative to pure cell control pellets, co-cultured cell pellets of expanded human BMSCs and meniscus cells had more GAG matrix per DNA content. The amplitude of GAG enhancement in all co-cultures varied with donor and with the Men:BMSC ratio. However, the mean GAG enhancement was 1.8–6 fold. The GAG contents of pellets correlated with Safranin-O staining. Positive staining for collagens types I and II was increased in co-cultured cell pellets.
Conclusion
Co-seeding of meniscus cells and stem cells on a suitable scaffold may aid the generation of functional grafts with improved biomechanical properties relative to those generated via expanded meniscus cells alone or stem cells alone.
