483 A NOVEL CELLULAR IMPLANTATION SYSTEM USING AN INJECTABLE ULTRA-PURIFIED ALGINATE GEL FOR ARTHROSCOPIC REPAIR OF OSTEOCHONDRAL DEFECTS

Abstract

Purpose: To determine the superiority of chondrogenic potential from BMSCs using an ultra-purified alginate gel over a commercial grade one and to assess the reparative tissues treated with BMSCs and a purified alginate in rabbit cartilage defect model. Methods: Ultra-purified alginate (Sea Matrix, Mochida Pharma., Japan, endotoxin level of 5.76 EU/g) and commercial grade one (Sodium Alginate 500, Wako, Japan, endotoxin level of 75,950 EU/g) were prepared. Cytotoxicity of CaCl2 : BMSCs in alginate were exposed to 50, 100, 200, and 400mM CaCl2. The living cells were counted with Cell Counting Kit-8 (CCK-8). Proliferation assay : BMSCs encapsulated in purified and commercial grade alginate beads were cultured in DMEM with 10% FBS. At 0, 1, 2, 3, and 7 days, the viable cells were counted with CCK-8. Alginate beads culture: Alginate beads containing BMSCs were cultured in chondrogenic medium containing TGF-b3. At 7 and 14 days, relative gene expressions were analyzed by real-time RT-PCR. At 14 and 21 days, each alginate bead was histologically evaluated. Rabbit model: The defects (O5mm) were created in the patella groove and divided into five groups: defect (no treatment); CAlg, commercial grade alginate alone; CAlgC, commercial grade alginate with BMSCs; PAlg, purified alginate alone; PAlgC, purified alginate with BMSCs. To avoid excessive exposure to a cytotoxic CaCl2, CaCl2 was injected to the surface of alginate. It needed no any fixation. In-vivo evaluation: At 4 and 12 weeks, the macroscopic and histological findings were scored with a grading scale, and the mechanical properties were measured by indentation test. Cadaver model: The stained alginate gel was injected into the defect with our novel technique. After the operation, manual mobilization was intermittently performed. Statistical Analysis: Comparison was assessed by an ANOVA followed by multiple-comparison post hoc tests. P< 0.05 was statistically considered significant. Results: Cytotoxicity of CaCl2 : The cell number in the 200 and 400mM groups significantly decreased, compared to the other groups (p< 0.05). Proliferation assay : From 1 to 7 days, the number of living cells in the purified alginate gel was significantly higher (p< 0.05). Alginate beads culture: The levels of aggrecan and sox 9 genes in the purified alginate at 14 days were significantly higher (p< 0.05). The proliferated BMSCs and a newly synthesized matrix were observed within pore spaces in the both alginate. Rabbit model: The surface of the defect group was rough and depressed. In the PAlg and PAlgC groups, the defects were substituted with firm and white cartilage-like tissue. The macroscopic score at 12 weeks in the PAlgC group was significantly superior to the CAlg and CAlgC groups (p< 0.05). Histological findings: In the CAlg and CAlgC groups, residual alginate gels were found, and inflammatory reactions were found in subchondral bone lesion. The PAlgC group exhibited the normal cartilaginous structures. The histological scores at 12 weeks were significantly higher in the PAlg and PAlgC groups than in the defect and CAlg groups (p< 0.05). Mechanical properties: From 4 to 12 weeks, the modulus of the CAlgC and PAlgC groups significantly improved (p< 0.05) and approximately reached to 80% of normal. Cadaver model: After mobilization, the alginate gel was not detached and maintained the initial shape and hardness. Conclusions: We confirm that the purified alginate gel has a great promise as a cell-carrier material for a clinical immnoisolated implantation of cells into cartilage defects. This study showed a clinical possibility of arthroscopic implantation using the current injectable system to the articular cartilaginous lesions.