Background
Although there are numerous studies and extensive data on tissue engineering of cartilage using young healthy swine, rabbit, or sheep cells for chondrogenesis, there is a paucity of data on generating engineered cartilage from human chondrocytes. While cells from immature animals possess a high capacity for proliferating and forming new cartilage matrix, it is unknown whether chondrocytes obtained from middle to older aged humans can produce new matrix similarly. We hypothesize that cartilage can be engineered from human chondrocytes with morphological, biochemical and biomechanical properties compared to native cartilage. In this study, we used articular chondrocytes from healthy (non-arthritic) human donors and fibrin gel which was composed of bovine fibrinogen and thrombin to determine whether: 1) human chondrocytes encapsulated in hydrogel could form neocartilage; 2) the neocartilage could integrate with existing human cartilage. Results were compared to neocartilage made from swine chondrocytes as controls.
Methods and Materials
Articular chondrocytes from healthy human donors and 3-month-old swine were isolated and placed in culture for proliferation. The cells were mixed with a bovine fibrinogen saline solution, and then the cell/fibrinogen mixture was polymerized with an equal volume of thrombin resulting in a final cell concentration of 60 x 106cells/ml. Two 6mm diameter discs of native devitalized human articular cartilage were placed on the top and bottom of 100ul cells/fibrin gel mixture, creating a cartilage-cells/hydrogel-cartilage construct (“sandwich” model, n=36) to determine cartilage integration. The same cell concentration was used to create human cells/fibrin gel nodules (n=30) to determine engineered cartilage formation. All constructs were implanted into a subcutaneous pocket on the dorsum of nude mice for 12, 18 and 24 weeks. The specimens were evaluated histologically (H&E, Safranin-O, Toluidine Blue and IHC for Col I and Col II), biochemically (Collagen and GAG).
Results
The nodules placed in mice showed neocartilage formation by the human chondrocytes as evidenced on specimens stained with H&E that was morphologically similar to that observed in the swine chondrocytes specimens. Safranin-O and Toluidine Blue demonstrated intense staining showing the production of proteoglycans in both swine and human specimens. Immunostaining the specimens showed low amounts of type I collagen and intense staining for type II collagen. Neocartilage formed between the cartilage discs and showed tight bonds and integration with existing cartilage.
Conclusion
Whereas previous in vitro and animal studies have demonstrated the feasibility for chondrogenesis utilizing animal cells, the results from this study demonstrate the production of new cartilage matrix by human articular chondrocytes in predictable and reliable manner. Neocartilage formation was comparable to that found using immature animal cells and that there was integration between the engineered cartilage and the native cartilage. This study is a crucial step in translating tissue engineered cartilage into clinical application.