To investigate whether Pinch proteins have a role in adult cartilage, we generated the Aggrecan^CreERT2; Pinch1^flox/flox; Pinch2^−/− mice by crossing the Aggrecan^CreERT2 mice with floxed Pinch1 (Pinch1^flox/flox) mice and Pinch2-null (Pinch2^−/−) mice, as indicated in Figure 1A. At the age of 2 months, the male Aggrecan^CreERT2; Pinch1^flox/flox; Pinch2^−/− mice were administrated with 5 daily intraperitoneal injections of tamoxifen (100 mg/kg body weight, dissolved in corn oil) to induce Pinch1 deletion in aggrecan-expressing chondrocytes. Notably, age-matched male Aggrecan^CreERT2; Pinch1^flox/flox; Pinch2^−/− mice were treated with corn oil and served as a control group. Immunofluorescent staining analyses showed that, at 1 week after tamoxifen treatment, the expression of Pinch1 was significantly decreased in articular chondrocytes of tamoxifen-treated mice, as compared with those in corn-oil-treated controls (Figure 1B, C). Since the Pinch2 gene is globally inactivated in Aggrecan^CreERT2; Pinch1^flox/flox; Pinch2^−/− mice; as expected, the expression of Pinch2 was barely detectable in articular chondrocytes in both groups (Figure 1B, D).

To determine whether Pinch1/2 deficiency could induce structural abnormalities in adult articular cartilage, six 2-month-old male Aggrecan^CreERT2; Pinch1^flox/flox; Pinch2^−/− mice were treated with tamoxifen. Another six age-matched Aggrecan^CreERT2; Pinch1^flox/flox; Pinch2^−/− mice were treated with corn oil as controls. Eight months later, all mice were sacrificed, and the knee joint samples were collected (Figure 2A). Immunofluorescent staining analyses confirmed that the protein expression of Pinch1 was markedly decreased in the articular cartilage of tamoxifen-treated mice, as compared with control mice (Figure 2B). We performed safranin O and fast green staining and quantitative histological analyses to assess the joint structure and OA-related parameters, including the OARSI (osteoarthritis research society international) score, cartilage area, synovitis score, and osteophyte score. The results showed no marked difference in articular cartilage and other joint tissues, including growth plate cartilage, synovium, and meniscus (Figure 2C). Moreover, the OA-related parameters were comparable between tamoxifen- and corn oil-treated Aggrecan^CreERT2; Pinch1^flox/flox; Pinch2^−/− mice (Figures 2D–G).

Next, we tested if Pinch1/2 deficiency could promote spontaneous OA defects in aged mice. Briefly, six 2-month-old male Aggrecan^CreERT2; Pinch1^flox/flox; Pinch2^−/− mice were treated with tamoxifen, and another six age-matched Aggrecan^CreERT2; Pinch1^flox/flox; Pinch2^−/− mice were treated with corn oil. At the age of 17 months, all mice were sacrificed, and the knee joint samples were collected (Figure 3A). The Pinch1 expression was markedly decreased in the articular cartilage of tamoxifen-treated mice, as compared with control mice (Figure 2B). Histological analyses demonstrated no significant alterations in knee joints and OA-related parameters between tamoxifen- and corn oil-treated groups (Figures 3C–G).

Next, we investigated whether Pinch1/2 deficiency could exacerbate the OA lesions in a destabilization of the medial meniscus (DMM) mouse model of OA. Six 2-month-old male Aggrecan^CreERT2; Pinch1^flox/flox; Pinch2^−/− mice were treated with tamoxifen, and another six age-matched Aggrecan^CreERT2; Pinch1^flox/flox; Pinch2^−/− mice were treated with corn oil. One week later, all mice were subjected to DMM surgery. Ten weeks after DMM surgery, all mice were sacrificed, and the knee joint samples were collected (Figure 4A). The Pinch1 expression was markedly decreased in the articular cartilage of tamoxifen-treated mice, as compared with control mice (Figure 2B). Histological analyses showed that DMM surgery successfully induced multiple OA lesions, including cartilage degradation and osteophyte formation (Figure 4C); nonetheless, the severity of OA lesions was comparable between tamoxifen- and corn oil-treated groups (Figures 4D–G).

The generation of Pinch1^flox/flox and Pinch2^−/− mice was previously described (Lei et al., 2020). To obtain the Aggrecan^CreERT2; Pinch1^flox/flox; Pinch2^−/− mice, Pinch1^flox/flox mice were first crossed with Pinch2^−/− mice to generate Pinch1^flox/+; Pinch2^+/− mice. The Pinch1^flox/+; Pinch2^+/− mice were then bred with each other to generate Pinch1^flox/flox; Pinch2^−/− mice. Meanwhile, the Pinch1^flox/flox mice were crossed with the Aggrecan^CreERT2 mice to generate Aggrecan^CreERT2; Pinch1^flox/+ mice. The Aggrecan^CreERT2; Pinch1^flox/+ mice were crossed with each other to obtain Aggrecan^CreERT2; Pinch1^flox/flox mice. Finally, the Aggrecan^CreERT2; Pinch1^flox/flox mice were bred with the Pinch1^flox/flox; Pinch2^−/− mice to generate the Aggrecan^CreERT2; Pinch1^flox/flox; Pinch2^+/− mice. Then the Aggrecan^CreERT2; Pinch1^flox/flox; Pinch2^+/− mice were bred with each other to generate Aggrecan^CreERT2; Pinch1^flox/flox; Pinch2^−/− mice. It should be noted that the Pinch2 gene is globally inactivated in the Aggrecan^CreERT2; Pinch1^flox/flox; Pinch2^−/− mice. For inducible deletion of the Pinch1 gene in aggrecan-expressing chondrocytes, 2-month-old male Aggrecan^CreERT2; Pinch1^flox/flox; Pinch2^−/− mice were administrated with tamoxifen (Sigma T5648, 100 mg/kg per body weight/day) via intraperitoneal injections as previously described (Wu et al., 2022a; Wu et al., 2022b; Chen et al., 2022). Age-matched male Aggrecan^CreERT2; Pinch1^flox/flox; Pinch2^−/− mice were administrated with corn oil and used as the control group. All research protocols in this study were approved by the Institutional Animal Care and Use Committees (IACUC) of the Southern University of Science and Technology.

The histological analyses of mouse knee joints were performed according to our previously established protocols (Liu et al., 2021; Wu et al., 2022a; Wu et al., 2022b; Lai et al., 2022). The 5-μm thick knee joint sections were stained with safranin O and fast green using a commercial kit (Solarbio, Cat#G1371). The OA-related parameters, including the OARSI score, synovitis score, and osteophyte score were quantified using the histological scoring systems as previously described (Wu et al., 2022a). The areas of safranin O-stained articular cartilage were measured by ImageJ (version 1.53k) as previously described (Wu et al., 2022a). Representative images of safranin O and fast green staining were selected based on the mean values of histological scores. Immunofluorescent (IF) staining was performed using antibodies against Pinch1 (Abcam, ab108609) and Pinch2 (Abcam, ab173008) according to our previously established protocol (Wang et al., 2019; Gao et al., 2021; Qu et al., 2022).

All mice used in this study were randomly assigned to each group. Statistical analyses were completed using the Prism GraphPad. Results were expressed as mean ± standard deviation (s.d.). Two-tailed unpaired Student’s t-test was employed to compare the differences between the two groups. Differences with p < 0.05 were considered statistically significant.