Evaluation of the Viability And Chondrogenic Capability of Cadaveric Chondrocytes For Clinical Application In Cartilage Repair. ANELL OLIVOS-MEZA, MD, CARMINA ORTEGA, Biol, VALENTIN MARTINEZ, Biol, ENRIQUE ALVAREZ-LARA, Tech, CRISTINA VELASQUILLO, PhD, JULIO GRANADOS, PhD, ALDO IZAGUIRRE, PhD, CLEMENTE IBARRA, MD. Instituto Nacional de Rehabilitacion, Mexico, Mexico. Disclosures: A. Olivos-meza: None. C. Ortega: None. V. Martinez: None. E. Alvarez-lara: None. C. Velasquillo: None. J. Granados: None. A. Izaguirre: None. C. Ibarra: None. Introduction: The viability of cryopreserved cartilage has been reported between 20-30% cell survival. Different viability results have been reported between animal and human articular cartilage when stored at 4 oc in different medial for different time periods. Curran and Gibson reported that human hyaline cartilage survived as long as 40 days; Hagerty et al. evaluated rib cartilage founding cartilage survival no longer than 50 days stored in plasma. Donor-site morbidity, limited numbers of cells, loss of phenotype during ex vivo expansion, and age-related decline in chondrogenic activity present critical obstacles to the use of autologous chondrocyte implantation for cartilage repair. Chondrocytes from juvenile cadaveric donors may represent an alternative to autologous cells. Methods: Sixteen osteochondral biopsies harvested from the femoral condyles of 4 cadaveric donors were processed. Articular cartilage biopsies were harvested under sterile conditions from the knee. Samples were transported in DMEM-F12 antibioticantimicotic medium at 4oC. Chondrocyte isolation was realized with 0.1% collagenasa A. Viability and cell number was analyzed with Tripan-Blue stain. Freshly isolated chondrocytes were either cryopreserved in liquid nitrogen or seeded in monolayer culture at low density (250,000 chondrocytes in a T-25 culture box). Cultures were incubated at 37oC and 95% relative humidity supplemented with DMEM-F12-antibiotic-antimicotic 1%-human serum 10%. At a 90% confluency, cells were harvested by 0.5% Trypsin-EDTA and re-seeded in monolayer culture at the same initial density. At the end of every Passage type I Collagen, type II collagen, aggrecan, and Sox-9 evaluation was analyzed by immunofluorescence and end-point-pcr. At the end of Passage-2, three groups of cells were seeded onto a type III collagen membrane (Biogide ). Passage 2 chondrocytes, coculture of criopreserved primary chondrocytes and Passage 2 chondrocytes, or coculture of newly isolated chondrocytes and Passage 2 chondrocytes. These constructs were implanted subcutaneously in nude mice for 12 weeks. After harvest, specimens were fixed in buffered formalin, and paraffin sections were stained with H-E and safranin-o for evaluation of new tissue formation. Results: Mean time between donor s death to Procurement and harvesting was 9 hours. Isolation of the chondrocytes was performed 24 hours after donor s death. Donor s mean age was 26 (±14.7) years. Chondrocyte isolation number per milligram of cartilage was calculated in every sample with a mean of 9,391 (±6,870) viable cells. Expansion rate of the initial cell culture until passage 2 was 12.4 (±2.54) fold (Tab.1). Chondrocyte morphology changed from round shape in primary chondrocytes to fibroblastic phenotype at the end of the passage 2. End-point-PCR showed type I collagen expression in passage 2. Aggrecan, Sox-9 and collagen II evaluation by immunofluorescence although in less amount remain present in monolayer cultures at passage 2, while collagen I content increases at the same passage (Fig.1). Samples from the younger cadaveric donors showed increased expression of cartilage markers and multiplied faster than adult cells. Histology of the tissue formed by the co-cultured passage cells showed a more chondrocytic-like morphology than cells within the tissue formed by passage-2 cells not in coculture. Co-cultured cells also showed more intense safranin-o staining than passage 2- cells alone (Fig 2). Discussion: Cadaveric donor cartilage provides constant amount of viable chondrocytes. Expression of constitutive chondrocyte markers is retained, but we do not know how much, and if those expression levels can be reversed with the addition of appropriate stimulus. Chondrocytes from younger donors would show stronger and more stable chondrogenic activity than adult ones. Cadaveric chondrocytes either cryopreserved or newly isolated have the capacity to form repair tissue. More specific analysis is necessary to identify the quantitative patterns of those cartilage markers. Significance: Chondrocytes from juvenile cadaveric donors may represent an alternative to autologous cells for cell-based cartilage repair. Decreasing the donor site morbidity, and probably obtaining a larger number of primary chondrocytes, and decreasing if not completely avoiding the dedifferentiation process, during cell expansion. Acknowledgments: Procurement Department, Musculoeskeletal Tissue Bank, Biograft Mexico. Enrique Obermeier, Alvarado Paulo, Joel Matus Jimenez, José Raúl Flores Flete. References: Amiel et al. 1989; Bringhton et al. 1979: Curran and Gibson 1956; Hagerty et al. 1960, 1967; Rodrigo et al. 1980; Wayne ey al. 1990.
ORS 2014 Annual Meeting Poster No: 0369