Cryopreservation Effect on Proliferative and Chondrogenic Potential of Human Chondrocytes Isolated from Superficial and Deep Cartilage

[Abstract] Objectives: To compare the proliferative and chondrogenic potential of fresh and frozen chondrocytes isolated from superficial and deep articular cartilage biopsies. Materials and Methodology: The study included 12 samples of fresh and frozen healthy human knee articular cartilage. Cell proliferation was tested at 3, 6 and 9 days. Studies of mRNA quantification, protein expression and immunofluorescence for proliferation and chondrogenic markers were performed. Results: Stimulation of fresh and frozen chondrocytes from both superficial and deep cartilage with fetal bovine serum produced an increase in the proliferative capacity compared to the non-stimulated control group. In the stimulated fresh cells group, the proliferative capacity of cells from the deep biopsy was greater than that from cells from the superficial biopsy (0.046 vs 0.028, respectively, p<0.05). There was also a significant difference between the proliferative capacity of superficial zone fresh (0.028) and frozen (0.051) chondrocytes (p<0.05). CCND1 mRNA and protein expression levels, and immunopositivity for Ki67 revealed a higher proliferative capacity for fresh articular chondrocytes from deep cartilage. Regarding the chondrogenic potential, stimulated fresh cells showed higher SOX9 and Col II expression in chondrocytes from deep than from superficial zone (p<0.05, T student test). Conclusions: The highest rate of cell proliferation and chondrogenic potential of fresh chondrocytes was found in cells obtained from deep cartilage biopsies, whereas there were no statistically significant differences in proliferative and chondrogenic capacity between biopsy origins with frozen chondrocytes. These results indicate that both origin and cryopreservation affect the proliferative and chondrogenic potential of chondrocytes.Servizo Galego de Saúde; PS07/84Instituto de Salud Carlos III; CIBER BBN CB06-01-0040Ministerio Ciencia e Innovacion; PLE2009-0144Ministerio Ciencia e Innovación; PI 08/202

Increased expression of nerve growth factor (NGF) and high affinity ngf receptor (p140 TrkA) in human osteoarthritic chondrocytes

Objective. We aimed to investigate the expression of nerve growth factor (NGF) and high affinity NGF receptor (p140 TrkA) on chondrocytes from human healthy and osteoarthritic cartilage. Methods. We recruited 12 patients with osteoarthritis (OA) undergoing surgical knee replacement. Articular cartilage was split into two zones showing macroscopically and histologically the lowest (MIN) and highest (MAX) degree of osteoarthritic damage. Additional specimens of cartilage were obtained from three healthy donors. Chondrocytes were isolated by enzymatic digestion and freshly processed for NGF protein, Trk A detection and mRNA extraction. NGF-b mRNA was determined by a reverse transcriptase–polymerase chain reaction (RT–PCR). NGF-b and TrkA expression was evaluated by immunofluorescence and flow cytometry analysis. Results. NGF-b-specific mRNA was detected in normal and osteoarthritic chondrocytes. NGF-b protein levels were low in normal chondrocytes, increased in MIN osteoarthritic cartilage and further enhanced in MAX osteoarthritic cartilage. Likewise, TrkA was scarcely expressed on normal chondrocytes and progressively increased on osteoarthritic chondrocytes based on the extent of anatomic damage. Conclusions. This is the first study showing that human chondrocytes synthesize NFG-b and express on their surface the high affinity NGFR (p140 TrkA). Of note, NGF-b and TrkA were upregulated in osteoarthritic chondrocytes suggesting a role of NGF in the pathophysiology of OA. We can speculate that NGF, like other growth factors, stimulates chondrocyte metabolism in the osteoarthritic proces