*2.4. The IA Administration of 7 rh Decreases the Expression of Hypertrophic Markers (Col X, MMP13, and IHH) after OA Induction*

IHC staining was performed to investigate whether 7 rh alleviated OA progression by reducing the chondrocyte hypertrophic differentiation. The representative images of IHC staining of chondrocyte hypertrophic markers, including Col X, IHH, and MMP13 at 5 weeks, are shown in Figure 4A–C and the results of the quantitative analysis are shown in Figure 4D–F. Col X, IHH, and MMP13 were well-established markers for detecting the chondrocyte during the hypertrophic differentiation. The ensity of immunolocalized Col X was significantly increased in the ACLT group, and the treatment of 7 rh (13.8 nM) in ACLT mice at 5 weeks significantly inhibited the expression of Col X (Figure 4A,D). As shown in Figure 4B,E, the levels of IHH expression also increased after OA induction and reduced significantly in the 7 rh-treated mice (13.8 nM group) compared to ACLT-OA mice at 5 weeks. As shown in Figure 4C,F, the immunolocalized MMP13 protein was predominantly found in the articular chondrocytes in the ACLT-OA mice and the expressions were decreased significantly after 7 rh treatment (13.8 nM group). These results revealed that chondrocytes undergo terminal differentiation after OA induction, but 7 rh (13.8 nM) can prevent chondrocytes from terminal differentiation.

**Figure 4.** Intra-articular injections of 7 rh reduced the chondrocyte hypertrophic differentiation in anterior cruciate ligament transection (ACLT)-induced osteoarthritis (OA) cartilage. (**A**) The representative micrographs of immunolocalized type X collagen (Col X) in articular cartilage of the sham control groups (sham+ saline and sham+ 7 rh 13.8 nM), ACLT, and two 7 rh treating groups. (**B**) The representative micrographs of immunolocalized Indian hedgehog (IHH). (**C**) The representative micrographs of immunolocalized matrix metallopeptidase 13 (MMP13). (**D**) Quantitative analysis of the immunohistochemically (IHC) staining of Col X. (**E**) Quantitative analysis of the IHC staining of IHH. (**F**) Quantitative analysis of the immunohistochemical IHC of MMP13. In quantitative analysis, each bar represents the mean±SEM of 12 samples in each group. (\* *p* < 0.05 versus the sham control groups; # *p* < 0.05 versus the ACLT group).
