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2008 Annual Meeting Abstracts


PHOTO-CROSSLINKING COLLAGEN GEL FOR TISSUE ENGINEERED CARTILAGE
Xing Zhao, M.D.1, David A. Bichara, M.D.1, Shinichi Ibusuki, M.D., Ph.D.2, Mark A. Randolph, M.A.S.1, Robert W. Redmond, Ph.D.3, Irene E. Kochevar, Ph.D.3, Thomas J. Gill, M.D.2.
1Plastic Surgery Research Lab, Massachusetts General Hospital, Boston, MA, USA, 2Laboratory for Musculoskeletal Tissue Engineering, Massachusetts General Hospital, Boston, MA, USA, 3Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA, USA.

INTRODUCTION: Numerous hydrogels have been developed as three-dimensional scaffolds for chondrogenesis. Solubilized collagen is an ideal natural material that can be used as a scaffold because of its biocompatibility and biodegradability. Although spontaneously forming collagen gels can conform to a cartilage defect, they are soft and unstable. Kochevar et al. reported that type I collagen can be crosslinked using photoreactive dyes, such as riboflavin (vitamin B2), and exposure to visible light. In situ gel crosslinking could induce molecular interactions with the native cartilage surrounding the lesions to stabilize the gel during cartilage formation. The governing hypotheses of this work are: 1) photochemical crosslinking can be used to generate stable collagen hydrogels; 2) Chondrocytes encapsulated in the hydrogels form neocartilage; 3) the neocartilage will integrate with existing cartilage.
METHODS: Chondrocytes were suspended in 4 test concentrations of riboflavin solution (0.1-1 mM). The cell suspension was mixed with an equal volume of 0.5% type I collagen solution. The suspension with a final cell concentration of 40×106 cells/ml was poured onto 6 well culture plates and photocrosslinked using 4 irradiation test doses of visible light. Control samples were not subjected to irradiation. Construct digestion using 0.01% collagenase type II was performed to assay the stability and mechanical property of photochemically cross-linked gels.
Implantation of photocrosslinked constructs (n=8) was performed to determine whether this novel method would allow the construct to make hyaline cartilage in the in vivo environment. Specimens were evaluated histologically (Safranin-O and immunohisto-chemically for COL I and COL II) and biochemically (collagen and GAG content). To evaluate the ability of the gel to permit cartilage formation and integration with the surrounding native cartilage, photocrosslinked gels with cells were placed between discs of knee cartilage and implanted in mice.
RESULTS: Cell viability remained high with short irradiation times at all concentrations of riboflavin. Specimens placed in mice showed neocartilage formation as evidenced on specimens stained with Toluidine blue (Fig 1A) and Safranin-O and produced GAG (Fig 1B) and type II collagen (Fig 1C). Neocartilage between cartilage discs formed tight bonds with existing cartilage (Fig 1D).
DISCUSSION & CONCLUSIONS: Crosslinking collagen into hydrogels can be achieved using benign light sensitive photreactive dyes like riboflavin and visible light. Photchemically crosslinking the collagen solutions containing chondrocytes permits cell survival and neocartilage formation. As such, collagen containing chondrocytes could be injected into a defect site and polymerized in situ. The crosslinking process could stabilize the hydrogel in the defect and permit new cartilage formation to restore the joint surface. The results from this study encourages further study in large animal joint models.
ACKNOWLEDGEMENTS: These studies were supported in part by the AO foundation and PSEF.