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

Meniscus tears in adult patients do not heal spontaneously and represent a risk factor for OA development. PDGF is well known as an enhancer of meniscal cell biosynthetic activity and also has chemotactic activity for mesenchymal cells. PDGF incorporation into scaffolds should be efficient for recruitment of cells to initiate repair in the injured meniscus. We recently developed decellularized meniscus sheet for use in the treatment of meniscus tears. The aim of this study is to examine the potential of PDGF-coated decellularized meniscus scaffold in mediating integrative healing by endogenous cell migration. Fresh bovine meniscus was chemically decellularized. Round sheets were made from the decellularized tissue. Heparin was covalently conjugated with decellularized meniscus scaffold (DMS). PDGF-BB was immobilized by binding to the heparin-conjugated DMS. In vitro, PDGF release kinetics was analyzed by ELISA. DMS was transplanted into the injured meniscus explants and cultured for 2 and 4 weeks. The numbers of migrated cells at the border between DMS and injured explant were counted on DAPI stained sections and PDGFRb expressing cells were counted after immunohistochemical staining. The newly produced ECM and collagen fiber alignment was detected by histology on Safranin-O and picrosirius red stained sections. The explants were also tested for tensile properties. PDGF release kinetics showed sustained slow release in heparin-conjugated DMS, with 11.2% release at day- 16th compared to 26.1% release from the DMS without heparin. Insertion of the PDGF-treated DMS into the meniscus tears in bovine meniscus explants led to the migration of endogenous meniscus cells to the defect zone. The migrated cells expressed PDGFRb and produced new ECM in the defect area. Safranin-O and pircrosirius red staining showed tissue integration between DMS and injured explants. Moreover, the higher concentration of PDGF promoted cell integration into the DMS. Tensile properties of injured explants treated with PDGF coated DMS were significantly higher than in DMS without PDGF. Heparin-conjugated DMS showed strong immobilization of PDGF, which was released slowly. PDGF coated DMS promoted migration of endogenous meniscus cells to the defect area and into the scaffold. New matrix was formed that bridged the space between the native meniscus and the scaffold and this was associated with improved biomechanical properties. The PDGF coated DMS is a novel, feasible and efficient approach for the treatment of meniscus tears