Fig 1.
Simplified diagram of the overall research design.
Fig 2.
Changes in GAG contents and cell viability after chondroitinase treatment.
A Biochemical analysis of each group of OCA GAG content and B normalized percentage (n = 10). C Live-dead Confocal microscope images of different groups of OCAs, where dead cells are stained in red and live cells are stained in green. D Quantitative analysis of cell viability and e) viable cell density of each group by Image J software (n = 4). Statistical analysis was conducted using One-way ANOVA test (*p < 0.05, **p < 0.01, ***p < 0.001). Magnification x50.
Fig 3.
Changes in GAG content analysis according to μCT analysis in OCA grafts after chondroitinase treatment.
A Representative μCT images and safranin-o-stained images of control and degraded OCA (2h, 4h, and 8h exposure to chondroitinase), the trends of the two analyzed images are inversely proportional to each other (n = 4). Scale bar = 500 μm. B, C Comparison of μCT value and GAG content of each group of OCAs (including in vitro and preliminary in vivo experimental OCAs; 64 in total). With increasing chondroitinase treatment time, the CT value of OCA increased whereas the GAG content decreased. D Linear regression plots: μCT value vs GAG content measured by biochemical assay (n = 64). The R2 in the linear regression graph is 0.8856. E Biomechanical analysis of each group after chondroitinase treatment (n = 4) and F histological score of OCA in each group (n = 4). Statistical analysis was conducted using One-way ANOVA test (*p < 0.05, **p < 0.01, ***p < 0.001).
Table 1.
Quality assessment of OCA before animal experiment.
Fig 4.
In vivo experimental analysis of OCA transplant sites.
A Representative gross morphological image, representative Safranin-O staining (n = 5), and μCT analysis images of the OCA transplantation site at 4 and 12 weeks postoperatively. Scale bar = 1 mm. B The scores of the International Cartilage Research Society (ICRS) macroscopic evaluation of each transplantation site. Statistical analysis was determined by Kruskal-Wallis test with Dunn’s tests. C Intergroup comparison of compressive modulus was used to evaluate the biomechanical properties (n = 5). D Histological scores for the OCA transplantation site at 4 and 12 weeks after surgery (n = 5). Data represent the means ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 between control group and remaining group. #P < 0.05, ## P < 0.01, ### P < 0.001 between 2h group and remaining group. + P < 0.05, +++ P < 0.001 between 4h group and 8h group. Data were analyzed by two-way ANOVA with Tukey’s test if not described.
Table 2.
Quality assessment of OCA after animal experiment.
Fig 5.
Changes in OCA effect after 4 and 12 weeks of transplantation.
A Quantification of GAG content at the graft site by μCT analysis, B histological score and C compressive modulus were compared at 4 and 12 weeks postoperatively(*p<0.05 **p < 0.01, ***p < 0.001).
Fig 6.
Cell viability at the OCA transplantation site at 12 weeks postoperative (n = 5).
A Representative live/dead stained images of the OCA transplantation sites at 12 weeks after surgery. Dead cells are marked in red whereas live cells are marked in green. B Percentage of cell viability in transplantation site. C Quantitative analysis of the available cell density in the transplantation site (*p < 0.05, **p < 0.01, ***p < 0.001). Magnification x20.