Combination of chondrocytes with chondrons improves Proteoglycan and Collagen-2 production to promote the repairs of defective knee cartilage in rabbits

Background: Chondrons are composed of chondrocytes and the surrounding pericellular matrix (PCM) and function to enhance chondrocyte-mediated cartilage tissue engineering. This study aimed at investigating the potential effect of combined chondrocytes with chondrons on the production of proteoglycan and collagen-II (Col-2) and the repair of defective knee cartilage in rabbits. Methods: Chondrocytes and chondrons were isolated from the knee cartilage of rabbits, and cultured alone or co-cultured for varying periods in vitro. Their morphology was characterized by histology and immunohistochemistry. The levels of aggrecan (AGG), Col-2 and glycosaminoglycan (GAG) expression were quantified by qRT-PCR, Alcian Blue-based precipitation and ELISA. The effect of combined chondrocytes with chondrons in alginate spheres on the repair of defective knee cartilage was examined in rabbits. Results: The isolated chondrocytes and chondrons displayed unique morphology and began to proliferate on day 3 and 6 post culture, respectively, accompanied by completely degenerated PCM on day 6 post culture. Evidently, chondrocytes had stronger proliferation capacity than chondrons. Longitudinal analyses indicated that culture of chondrons, but not chondrocytes, increased AGG mRNA transcripts and GAG levels with time and Col-2 mRNA transcripts only on day 3 post culture. Compared with chondrocytes or chondrons alone, co-culture of chondrocytes and chondrons significantly up-regulated AGG and Col-2 expression and GAG production, particularly at a ratio of 1:1. Implantation with chondrocytes and chondrons at 1:1 significantly promoted the repair of defective knee cartilage in rabbits, accompanied by reduced the Wakiteni scores with time. Conclusion: Combined chondrons with chondrocytes promoted the production of proteoglycan and Col-2 and the repair of defective knee cartilage in rabbits.

trochlear grooves were dissected for subsequent experiments.

Histologic evaluation
The femoral trochlear groove tissues were routinely fixed and decalcified for 4 weeks, followed by paraffin-embedded. The sagittal sections (5 μm) were routinely stained with haematoxylin and eosin or Safranin O. In addition, the sections were subjected to immunohistochemistry using anti-Col2 (1:200). The tissue repairs in the defect areas of each rabbit were evaluated for cell morphology, matrix staining, surface regularity, cartilage thickness, and the donor integration in the recipients using the modified Wakitani grading system [31].

Statistical analysis
Data are present as mean ± standard deviation (SD). The difference between the groups was analyzed by analysis of variance (ANOVA) using SPSS 13.0 software (SPSS, USA). Statistical significance level (α) was set at 0.05.

Characterization of rabbit chondrocytes and chondrons
To understand the role of chondrons in the function of chondrocytes, rabbit chondrocytes and chondrons were isolated and cultured for varying periods. Following culture for one day, both chondrocytes and chondrons were small and rounded ( Figure 1A). The chondrocytes and chondrons displayed like fibroblasts with sharp spindles on day 3 and 6 post culture, respectively. Immunofluorescent staining revealed that the Col-6 was expressed in the cytoplasm of both types of cells and the expression levels increased with culture time.
MTT assays indicated that the chondrocytes began their proliferation on day 3 post culture and gradually increased with the prolonged culture time periods while the chondrons appeared to increase their OD values on day 6 post culture, which were further enlarged at a later time ( Figure 1B). As a result, the proliferation of chondrocytes was significantly stronger than that of chondrons on day 3, 6 and 9 post culture. Furthermore, qRT-PCT revealed that while there was a similar level of AGG mRNA transcripts in the cultured chondrocytes significantly higher levels of AGG mRNA transcripts were detected in the cultured chondrons on day 6 and 9 post culture ( Figure 2A). In contrast, we detected very low levels of Col-2 mRNA transcript in the different groups of cells, except for a dramatically higher level of Col-2 mRNA transcripts in the chondrons on day 3 post culture ( Figure 2B). Moreover, Alcian Blue-based precipitation detected gradually increased levels of GAG in the supernatants of cultured chondrocytes and chondrons beginning on day 3 post culture ( Figure 2C). ELISA detected similarly low levels of Col-2 in the supernatants of cultured chondrocytes and chondrons ( Figure 2D).
Together, such data indicated both rabbit chondrocytes and chondrons exhibited their biological characteristics ex vivo.

Co-culture of chondrocytes and chondrons enhances the production of AGG, Col-2 and GAG in vitro.
It is well known that chondrons can support the function of chondrocytes [33]. To determine the role of rabbit chondrons, we co-cultured chondrocytes with chondrons for 6 days. We found that while culture of chondrocytes or chondrons alone only promoted low levels of AGG mRNA transcription coculture of chondrocytes with chondrons at a ratio of 2:1 or 1:1 significantly increased the relative levels of AGG mRNA transcripts ( Figure 3A). The highest levels of AGG mRNA transcripts were detected in the co-cultured cells at 1:1. A similar pattern of Col-2 mRNA transcripts was observed among these groups of cells ( Figure 3B). In comparison with that in the chrondrocytes or chondrons alone, Alcian Blue-based precipitation detected significantly higher levels of GAG in the supernatants of co-cultured chondrocytes and chondrons, particularly for those with a ratio of 1:1 ( Figure 3C).
However, ELISA revealed that there was no significant difference in the levels of Col-2 in the supernatants of cultured cells, regardless of their culture alone or co-culture ( Figure 3D). Thus, coculture of both chondrocytes with chondrons enhanced the production of AGG, GAG and Col-2 in vitro.

Implantation of both chondrocytes and chondrons significantly accelerates the repairs of cartilage defects in rabbits.
To explore the role of chondrons and chondrocytes in the repairs of cartilage defects, we induced knee cartilage defects in rabbits and implanted with chrondrocytes/alginate, chondrons/alginate or chondrocytes/chondrons/alginate spheres, respectively ( Figure 4A). Six weeks later, we examined the repairs of defective knee cartilage in individual rabbits by histology and immunohistochemistry. As shown in Figure 4B The PCM is primarily composited of collagen VI surrounding chondrocytes and crucial for cartilage tissue engineering [39,40]. We found that the isolated chondrons displayed a round shape and positively staining with anti-Col-6 up to 3 days post culture, suggesting that the PCM surrounded the chondrocytes. In contrast, the isolated chondrocytes were present as fibroblast-like morphology on day 3 post culture with obvious proliferation. In addition, we did not detect anti-Col-6 staining surrounding the chondrocytes in the cultured chondrons, indicating that the PCM had been degraded on day 6 post culture, in which, the enclosed chondrocytes proliferated. Such data suggest that the chondron may be sued as seeding cells, together with chondrocytes for cartilage engineering.
The PCM in the chondrons provides a microenvironment for gene expression and metabolism in chondrocytes [41][42][43]. Actually, Vonk et al [44] found that chondrons expressed higher levels of Col-2, but lower Col-1 than chondrocytes, which may stem from the inhibition of the PCM on lipid peroxidation on the cell membrane surface to reduce active oxidation, leading to increased Col-2 and Col-6 expression and metabolism in chondrocytes and attenuating their hypertrophy and dedifferentiation [45,46]. Although the numbers of chondrons were less than that of chondrocytes, we observed that the relative levels of AGG expression in the cultured chondrons were significantly higher than that in the chondrocytes, but similar levels of GAG and Col-2 were detected in the