We further evaluated the efficacy of Ci-I gelatin hydrogels encapsulated with MSCs and icaritin to boost bone regeneration in a SAON model. Figure 4 a showed that the injection of Ci-I gelatin hydrogels helped to prevent a decrease in BMD around the tunnel compared to the nontreated control group (no hydrogel implantation, “Blank”). The “icaritin+MSC+Gel” group showed significantly enhanced new bone formation in the tunnel ( Figure 4 a). Quantitative measurement of the micro-CT images showed that the “icaritin+MSC+Gel” group exhibited significant elevation in BV/TV, trabecular number, while trabecular thickness and a reduction in trabecular separation, as compared to the other groups, in both the peri-tunnel native bone ( Figure 4 b) and neobone tissue inside the tunnel ( Figure 4 c). Using Goldener’s Trichrome staining, we also further confirmed that the bone tunnel was filled with neobone in the “icaritin+MSC+Gel” group ( Figure 4 d). In contrast, only fibrous tissue was formed in the other groups without supplementation of icaritin ( Figure 4 d).

We further examined the harvested samples to assess the bone regeneration within the drilled tunnel. Immunofluorescent staining against Ki67, a marker widely used to identify proliferating cells, showed significantly more Ki67 positive cells in the tunnel region injected with the Ci-I gelatin hydrogels, which were loaded with both MSCs and icaritin (“icaritin+MSC+Gel”), as compared to that of either hydrogel alone (“Gel”) or hydrogel plus icaritin (“icaritin+Gel”) treatment groups ( Figure 5 a). Moreover, the “icaritin+MSC+Gel” group also exhibited the most intense staining against key osteogenic markers, Runx2 and Osterix ( Figure 5 b,c). At week 3 after the hydrogel implantation, the expressions of active β-catenin and OCN were upregulated in the “icaritin+MSC+Gel” group ( Figure 5 d and Figure S6a ) compared to those of the other groups, and this may have contributed to the enhanced bone formation. In contrast, PPARγ expression was dramatically decreased ( Figure 5 e and Figure S6b ), indicating suppressed adipogenesis, and this is consistent with our previousfindings ( Figure 3 d). The icaritin-laden hydrogels (“icaritin+MSC+Gel” and “icaritin+Gel”) also exhibited significantly elevated expression of another proliferation marker, proliferating cell nuclear antigen (PCNA), in addition to Ki67 at week 6 ( Figure 5 a and Figure S4 ), regardless of MSC supplementation. In addition, the “icaritin+MSC+Gel” group showed slightly reduced expression of c-Src, a detrimental factor that has been identified as a therapeutic target for osteonecrosis. (55) CSF-1R, a tyrosine-kinase transmembrane receptor, (76) was greatly reduced in the “icaritin+MSC+Gel” and “icaritin+Gel” groups, indicating the potential immunoregulative effect of icaritin, as reported by a previous study. (77) More importantly, this finding suggests that icaritin and MSCs delivered by Ci-I hydrogels can potentially mitigate the foreign body response to biomaterial implantations, thereby facilitating further translational applications. (76) By using calcein green (CG) and xylenol orange (XO) to dynamically assess the bone formation rate, at week 3 and 6 postsurgery we found that treatment with hydrogel alone (“Gel”) did not affect bone formation within the bone tunnel ( Figure 5 f–i). Incorporation of icaritin in the injected hydrogels (“icaritin+Gel”) significantly increased the bone formation rate compared to either blank repair or the “Gel” group ( Figure 5 f–i). The encapsulation of both exogenous MSCs and icaritin in the hydrogels (“icaritin+MSC+Gel”) further enhanced bone formation in the defect site ( Figure 5 f–i). These data demonstrate that the Ci-I gelatin hydrogel is an excellent carrier of cells and hydrophobic drugs for promotingbone regeneration and treating SAON.