The repair of articular cartilage defects is a difficult clinical treatment. During repair, it is difficult for the newborn cartilage to form a whole with the surrounding of the defect cartilage and the subchondral bone, which often leads to poor repair effect [23]. In recent years, the repair of cartilage defects with artificial cartilage scaffolds has received much attention in the field of orthopaedics. The three elements of scaffold materials, seed cells and bioactive substances work together to better promote the repair of cartilage defects [24, 25]. In this study, we constructed a novel scaffold of icariin/porous magnesium alloy to repair cartilage defects in the non-weight-bearing area of the intercondylar fossa of the knee joint in rats. The effect and possible mechanism of the novel scaffold in repairing knee articular cartilage defect in rats were preliminarily discussed.
The metal material we selected - porous magnesium alloy, acts as a scaffold for the repair of cartilage defects, providing support for the attachment of newly generated cartilage tissue, and the existence of pores is also conducive to the growth of BMSCs, chondrocytes, and new blood vessels. As the porous magnesium scaffold degrades in vivo, the release of large amounts of magnesium ions can promote the differentiation of BMSCs towards chondrogenesis. Since icariin not only promotes chondrocyte proliferation and inhibits the degradation of extracellular matrix, but also promotes the differentiation of BMSCs towards chondrogenesis, which is an effective promoter of cartilage formation [26]. It has been shown that 1 µmol/L icariin has the most significant effect on promoting chondrocyte proliferation, and can significantly enhance the expression of Aggrecan, Col2α1 and Sox9, which leads to promote the cartilage tissue differentiation [27, 28]. Wang et al. found that the magnesium ions generated by the degradation of magnesium-based materials in vivo can be routinely metabolized by the circulatory system, which is often inconsistent with the optimum magnesium ion concentrations derived from in vitro studies of material extracts. Therefore, they conclude that magnesium-based extracts should be diluted 6–10 times in concentration [29]. Based on the above reasons, we prepared a novel icariin/porous magnesium alloy scaffold used in this study. In this scaffold, the concentration of icariin − 10 µmol/L, which is 10 times higher than the optimum concentration, is used, which is loaded on the porous magnesium alloy by physical adsorption. The scaffolds degrade in vivo while releasing magnesium ions and icariin, and in this process, the concentrations of magnesium ions and icariin are diluted.
Through the macroscopic observation and micro-CT scanning analysis of the cartilage tissue, we found that the novel icariin/porous magnesium alloy scaffold has a strong ability to repair the cartilage defect of the rat knee joint. The cartilage defect in the femoral intercondylar fossa of the posterior rat knee joint was filled with cartilage-like tissue, and the overall integrity from the cartilage to the subchondral bone was good; And the effect of pure porous magnesium alloy scaffolds was the second. The cartilage defect was not completely repaired, but the defect was reduced compared with the non-scaffold group, and the surrounding of the defect was filled with cartilage-like tissue, and had the integrity of subchondral bone. The cartilage defect in the no scaffold group was not completely repaired, and the new tissue was mostly similar to fibrous tissue, and an obvious repair outline could be seen. Safranin O staining can specifically bind to proteoglycan in the cartilage matrix, so that the morphology and content of articular cartilage can be observed microscopically. Toluidine blue staining can also specifically bind to hyaline cartilage, thereby reflecting the content of hyaline cartilage in tissue samples. And the depth of staining color is proportional to the content of hyaline cartilage. In the results of staining, we found that the novel scaffold of icariin/porous magnesium alloy can repair articular cartilage well, with dark staining of safranin O and toluidine blue, and there were more cartilage tissues; Although the cartilage defect was not completely repaired in the pure porous magnesium alloy scaffold group, the staining of safranin O and toluidine blue around the defect was deep, and the cartilage defect was significantly smaller than that in the no scaffold group; However, in the no scaffold group, the cartilage defect was larger, and there was basically no safranine O staining in the defect, and the toluidine blue staining in the defect was too light. Previous studies were similar to the results of the present experiment, where magnesium hydroxide loaded on PLGA promoted the repair of cartilage defective parts in rats [30]. Combining the macroscopic and microscopic observations, we found that the novel scaffold of icariin/porous magnesium alloy repaired cartilage defects with better regenerated cartilage tissue structure, more satisfactory repair results, and better integration of cartilage and osteochondral interfaces than the other two groups.
In immunohistochemical staining for chondrogenic-related factors, we found that the expression of chondrogenic-related proteins Aggrecan, Col2α1 and Sox9 were significantly increased in the icariin/porous magnesium alloy novel scaffold repair compared to the porous magnesium alloy group and the no scaffold group. The expression of chondrogenic-related proteins was significantly upregulated in the group repaired by the porous magnesium scaffold compared with the group without the scaffold, demonstrating that the porous magnesium scaffold could promote the expression of chondrogenic-related proteins around the cartilage defect, so as to enhance the repair of the cartilage defect. Previous studies have demonstrated that magnesium alloys promote the expression of chondrogenic markers (Aggrecan, Col2α1 and Sox9) [31], Liao et al. previously confirmed that JDBM alloys are highly biocompatible with chondrocytes in vitro [32], Zhao found that JDBM scaffolds recruited hBMSCs and promoted their chondrogenic effects [33]. All of these studies are similar to our experimental results, and magnesium alloy scaffolds can promote the expression of cartilage-related proteins in vivo, thereby accelerating the process of cartilage defect repair. When comparing the novel icariin/porous magnesium alloy scaffold with the results of the pure porous magnesium alloy group, we found that icariin further promoted the effect of porous magnesium alloy in repairing cartilage defects in vivo, and the synergistic effect of the two promoted the cartilage repair. We believe that the novel scaffold of icariin/porous magnesium alloy can be used as a material in cartilage defect repair, and with the continuous degradation of magnesium alloy material, icariin is released to promote chondrogenic differentiation of BMSCs in synergy with magnesium ions, which in turn contributes to cartilage and subchondral bone repair.
In this study, we also conducted a preliminary discussion on the mechanism of the neovel icariin/porous magnesium alloy scaffold to repair articular cartilage defects. We used immunohistochemical staining to detect the expression of Wnt/β-catenin signaling pathway related factors β-catenin, Wnt5a, and Wnt1 proteins. We found that the expression of β-catenin, Wnt5a, and Wnt1 proteins were significantly higher in the new articular cartilage tissues after repair with the novel icariin/porous magnesium scaffold, compared to the porous magnesium scaffold, the no scaffold group and shame group. The expression levels of the three proteins in the pure porous magnesium alloy scaffold group were significantly higher than those in the no scaffold group. It shows that the implantation of porous magnesium alloy can activate the Wnt/β-catenin signaling pathway in cartilage defects to a certain extent, and the icariin/porous magnesium alloy scaffold further promotes this activation. In detecting the expression of sFRP1, we found no statistical difference between the groups. Tanthaisong et al. found in vitro studies that the Wnt/β-catenin pathway was activated when Gsk3β inhibitors synergistically stimulated chondrogenic differentiation of human BMSCs with TGF-β3 [34]. Moreover, Wnt5a and Wnt5b promote chondrogenic differentiation of BMSCs, and upregulation of the Wnt5a gene delays chondrocyte hypertrophy and abnormal ossification of the bone [35]. Hung et al. found that magnesium ions induce osteogenesis in BMSCs by activating the Wnt/β-catenin signaling pathway [36], and icariin also activates this pathway as previously described, so the chondrogenic effect of the novel scaffold of icariin/porous magnesium alloy in our results may also be accomplished by the Wnt/β-catenin signaling pathway. These studies are consistent with the results that Wnt/β-catenin pathway-related factors were significantly up-regulated in this study. However, the study found that secreted frizzled-related protein 1 (sFRP1) was not significantly expressed in each group. We analyzed that the reason may be that sFRP1 is an antagonist of the Wnt/β-catenin signaling pathway, which can compete with Wnt ligands related to the signaling pathway, thereby affecting the expression of Wnt/β-catenin pathway. Im G et al. also found that in the process of chondrogenic differentiation of hMSCs, increasing the expression of sFRP1 did not promote the expression of chondrogenic-related factors in MSCs although it inhibited the Wnt/β-catenin pathway, which also demonstrated that the Wnt/β-catenin pathway does not have an inhibitory effect on cartilage defect repair [37]. In our study, we found that while the novel icariin/porous magnesium alloy scaffold could repair cartilage defects well, Wnt/β-catenin signaling pathway-related factors were significantly up-regulated in newborn cartilage, so we believe that the novel scaffold of icariin/porous magnesium alloy promote the repair of articular cartilage defects through activation of the Wnt/β-catenin pathway.