Anti-hypertrophic effect of synovium-derived stromal cells on costal chondrocytes promotes cartilage repairs

Abstract

Background: Costal chondrocytes (CCs), as a promising donor cell source for cell-based therapy for cartilage repair, have strong tendency of hypertrophy and calcification, which limited CCs from further application in cartilage regenerative medicine. Synovium-derived stromal cells (SDSCs), have shown their beneficial effect for chondrocytes to maintain phenotype. This study aims to investigate whether SDSCs could help CCs to maintain chondrogenic phenotype and suppress hypertrophic differentiation in cartilage repairs.

Methods: CCs were directly cocultured with SDSCs in pellet or indirectly cocultured using a conditioned medium in vitro for 3 weeks. Cartilage matrix formation and hypertrophic differentiation of CCs were analyzed by RT-PCR, biochemical assays, and histological staining. Cocultured pellets were implanted into the osteochondral defects made on the femoral groove of the rats. Then, macroscopic and histological evaluations were performed.

Results: Pellets formed by CCs alone and CCs cocultured with SDSCs reveal equal cartilage matrix deposition. However, the gene expression of type X collagen was significantly downregulated in cocultured pellets. Immunohistochemistry analysis revealed suppressed expression of type X collagen in cocultured pellets, indicating SDSCs may suppress hypertrophic differentiation of chondrocytes. Further in indirect coculture experiment, SDSCs suppressed type X collagen expression as well and promoted the proliferation of CCs, indicating SDSCs may influence CCs by paracrine mechanism. The pellets implanted in the osteochondral defects showed good restoration effects, whereas the grafts constructed with CCs and SDSCs showed lower type X expression levels.

Conclusion: These results suggest that SDSCs may maintain the phenotype of CCs and prevent the hypertrophic differentiation of CCs in cartilage repair.The Translational Potential of this Article: CCs is a promising donor cell source for cell-based therapy for cartilage repair. Based on our study, cocultured with SDSCs weakened the tendency of hypertrophy and calcification of CCs, which provide a potential usage of SDSCs in CCs-based cartilage repair therapy to suppress newly formed cartilage calcification and improve clinical outcomes.

Keywords: ACI, Autologous chondrocyte implantation; CCs, costal chondrocytes; CM, conditioned medium; Costal chondrocyte; GAG, glycosaminoglycan; Hypertrophic differentiation; RT-PCR, reverse transcription-polymerase chain reaction; SDSCs, synovium-derived mesenchymal stromal cells; Synovium-derived mesenchymal stromal cell.

Figure 1

Effect of SDSCs on CCs in direct coculture. A) Schematic of mixed pellet coculture experimental outline. In light blue: CCs; In yellow: SDSCs; In dark blue: CCs pellet; In green: CCs+SDSCs pellet B) Macroscopic appearance of pellets. C) RT-PCR analysis for chondrogenesis and hypertrophic-related gene expression after 3 weeks of culture. D) H&E staining of pellets. Black arrows: lacuna-like structures. E) Alcian blue staining. F, G) Safranin-O staining. H) Biochemical evaluation of GAG content and GAG/DNA ratio of pellets. I, J) Immunohistochemistry analysis of type II collagen. K, L) Immunohistochemistry analysis of type X collagen. (For interpretation of the references to color/colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 2

Paracrine effect of SDSCs toward CCs in indirect coculture. A) Schematic of conditioned medium coculture experimental outline. In light blue: CCs; In yellow: SDSCs; In dark blue: CCs pellet. B) Macroscopic appearance of pellets. C) RT-PCR analysis for chondrogenesis and hypertrophic-related gene expression after 3 weeks of culture. D) H&E staining. E) Alcian blue staining. F, G) Safranin-O staining. H) Biochemical evaluation of GAG content and GAG/DNA ratio of pellets. I, J) Immunohistochemistry analysis of type II collagen. K, L) Immunohistochemistry analysis of type X collagen. M) Western blot analysis of type X collagen. Crtl: Control group. CM: SDSCs-conditioned medium group. (For interpretation of the references to color/colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 3

Chondrogenic induction of SDSCs and its promotion of CCs proliferation. A) Schematic of chondrogenic induction of SDSCs experimental outline. In light blue: CCs; In yellow: SDSCs; In dark blue: CCs pellet; In orange: SDSCs pellet. B) Macroscopic appearance of pellets. C) Quantitative analysis of diameters of pellets constructed by CCs and SDSCs. D) H&E staining. E) Alcian blue staining. F, G) Safranin-O staining. H) Biochemical evaluation of GAG content and GAG/DNA ratio of pellets. I, J) Immunohistochemistry analysis of type II collagen. K, L) Immunohistochemistry analysis of type X collagen. M) Schematic of cell proliferation experimental outline. N) Cell quantities of CCs transwell cocultured with SDSCs. (For interpretation of the references to color/colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 4

In vivo cartilage repair of SDSC cocultured pellets. A) Macroscopic appearance of operated knees at 4 weeks. B) ICRS macroscopic scores for the defect-only and pellet-implanted groups. C) Histological and immunohistochemical findings. a-f are Safranin-O staining image; g-i are immunohistochemistry analysis of type II collagen; j-o are immunohistochemistry analysis of type X collagen; D) ICRS histological scores for the defect-only and pellet-implanted groups.