After demineralization the rabbit distal femoral osteochondral tissues were decellularized, separately with SDS and Triton X-100 for 24, 48 and 72 hours, at concentrations of 2%, 1% and 0.5%, respectively. The greatest DNA removal was achieved with Triton X-100 solutions. Cytotoxicity tests with CSM and chondrocytes have shown good and very good results, but a gradual decrease in cell viability related to the duration of treatment with surfactants compared to the control was registered. The same trend was observed in the cells population test after 7 days, while there was no difference at the 14th day. It was also determined that samples decellularized with SDS have a higher resistance to enzymatic degradation than the control and the decellularized tissue with Triton X-100. The swelling test and elasticity modulus measurements did not show values dependent of the surfactant nature.
Background: The existing surgical techniques used to regenerate articular cartilage fail. Utilisation of hierarchical, biphasic structures obtained from osteochondral tissue, through demineralisation, decellularization, longitudinal perforation and combination with chondroprogenitor cells, presents a high potential in cartilage defects regeneration. Material and methods: The research was performed on 36 rabbits, separated equally in two experimental and one control group. In the experimental groups, the experimental osteochondral defects of 4-4.5 mm in depth, were performed with a 3.7 drill bit at the level of weight bearing surface of the medial femoral condyle. In the 1st group the defects were treated with grafts combined with autologous chondrocytes, and in the 2nd group with grafts combined with autologous mesenchymal stem cells. In the control group, cartilaginous defects were treated by transferring the osteochondral plugs taken from the trochlear groove. The rabbits were removed from the experiment at 6 and 12 weeks. The results were evaluated by Unified Histological Score of Regenerated Cartilage (UHSRC). Results: At 6 weeks, according to UHSRC, the 1st group had 28.33±1.53 points, the 2nd group –27.67±2.08 points and the control group –26.33±1.53 points (p˃0.1; p˃0.2). At 12 weeks the 1st group had 18.68±5 points, the 2nd group –14.89±3.76 points and the control group –17.22 ±4.84 points (p˃0.5; p˃0.2). Conclusions: According to UHSRC, the experimental groups don’t show a significant difference compared to the control group at 6 and 12 weeks, also the quality of regenerated cartilage is poor.
Background: Articular cartilage has poor regenerative capacities. Numerous cartilage repair techniques are known, including implantation of autologous chondrocytes. Material and methods: From 18 rabbits pieces of cartilage were harvested from femoral condyle. Minced cartilage was treated with 0.25% trypsin-EDTA. In the 1st group (n=9) the cartilage was digested with 0.6% collagenase in 15 ml tubes by shaking in incubator at 37°C, 5%CO2 . In the 2nd group (n=9) digestion was performed in 25cm2 cell culture flasks placed on the lateral side, monitoring the process under a microscope after 120 minutes. The isolated cells were cultured to a 80-90% confluence. The chondrocytes were identified using histochemical staining after culturing for 16 days in overconfluence. Results: Chondrocytes isolation in the 1st group lasted a fixed 360 minutes, in the 2nd group – 140±10 minutes. In the 1stgroup were isolated 9.2x104 ±3.1x104 chondrocytes with a viability of 85.36±16.41%, but in the 2nd group – 1.6x105 ±3.4x104 chondrocytes with a viability of 98.09±3.85%. The mean period of cell culture in the 1st group was 15±2 days, in the 2nd group – 11±3 days. In first passage of the 1st group were obtained – 1.2x106 ±4.3x105 chondrocytes and in the 2nd group – 2.92x106 ±3.6x105 chondrocytes. The secreted extracellular matrix by chondrocytes was stained specifically for cartilaginous tissue. Conclusions: The method used for chondrocytes isolation has a direct impact on the number of isolated cells, their viability, but also upon the culture period and the number of cells obtained during the first passage.
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