Sonic hedgehog improves redifferentiation of dedifferentiated chondrocytes for articular cartilage repair

Abstract

Sonic hedgehog (Shh) is involved in the induction of early cartilaginous differentiation of mesenchymal cells in the limb. We investigated whether Shh could promote redifferentiation of dedifferentiated chondrocytes and have a favorable effect on the regeneration of cartilage. Articular chondrocytes of rats were separated and cultured. The redifferentiation of dedifferentiated chondrocytes transfected with Shh was evaluated using monolayer and pellet culture system. The signaling molecules (Ptc 1, Gli 1 and Sox9) of the hedgehog pathway were investigated. A rat model of articular cartilage defect was used to evaluate cartilage repair after transplantation with dedifferentiated chondrocytes. After Shh gene transfer, the hedgehog pathway was upregulated in dedifferentiated chondrocytes. Real time-PCR and western blot analysis verified the stronger expression of Ptc1, Gli1 and Sox9 in Shh transfected cells. Shh upregulates the Shh signaling pathway and multiple cytokines (bone morphogenetic protein 2 and insulin-like growth factor 1) in dedifferentiated chondrocytes. After transplantation in the joint, histologic analysis of the regenerative tissues revealed that significantly better cartilage repair in rats transplanted with Shh transfected cells. These data suggest that Shh could induce redifferentiation of dedifferentiated chondrocytes through up-regulating Shh signaling pathway, and have considerable therapeutic potential in cartilage repair.

Figure 1. Redifferentiation of dedifferentiated chondrocytes stimulated by Shh in monolayer and pellet culture.

RT-PCR revealed that the expression of type II collagen and aggrecan in Shh transfected cells after 4 days in monolayer culture in differentiation medium (A). The matrix of Shh transfected chondrocytes was toluidine blue positive after 14 days in the pellet cultured in differentiation medium, and chondrocytes-like cells were found in the pellet. Type II collagen expression was proved by immunological staining (B). Original magnification 10×. *p<0.05

Figure 2. Shh upregulates the Shh signaling pathway and multiple cytokines in Shh transfected chondrocytes.

To explore potential mechanisms responsible for the redifferentiation effect of Shh, we evaluated the Shh signaling pathway. Real time RT-PCR and western blot analysis verified the stronger expression of Ptc1, Gli1 and Sox9 in Shh transfected cells after 48 hours (A, B). The mRNA expression of BMP2 and IGF1 were upregulated by Shh after 48 hours (C). *p<0.05.

Figure 3. Shh expression in vivo. In non-transfected group, Shh expression was not detected (A).

In the defects in Shh group, the repair tissues contained Shh protein proved by immunohistochemical staining 2 weeks after transplantation (B). In the negative control without primary antibody, Shh expression was not detected (data not shown). Original magnification 20×.

Figure 4. Histologic evaluation of the regeneration of cartilage 4 weeks and 8 weeks after transplantation.

Representative results of histologic evaluation of the regenerative tissues stained with toluidine blue: control group (A, B), non-transfected group (E, F) and Shh group (I, J); hematoxylin-eosin: control group (C, D), non-transfected group (G, H) and Shh group (K, L). Original magnification 20×. 4w: 4weeks, 8w: 8weeks.

Figure 5. Representative results of immunohistological staining for type II collagen of repaired tissues after transplantations in treatment group.

Immunohistological staining of regenerative tissues 4 weeks after transplantation of non-transfected chontrocytes (A), and Shh transduced chondrocytes (C). Regenerative tissues 8 weeks after transplantation with immunohistological staining: non-transfected chondrocytes (B), and Shh transfected chondrocytes (D). Original magnification 20×. 4w: 4weeks, 8w: 8weeks.