Injectable and Assembled Calcium Sulfate/Magnesium Silicate 3D Scaffold Promotes Bone Repair by In Situ Osteoinduction

Abstract

(1) Background: Osteonecrosis of the femoral head (ONFH), caused by insufficient blood supply, leads to bone tissue death. Current treatments lack effective bone regeneration materials to reverse disease progression. This study introduces an injectable and self-setting 3D porous bioceramic scaffold (Mg@Ca), combining MgO + SiO₂ mixtures with α-hemihydrate calcium sulfate, designed to promote bone repair through in situ pore formation and osteoinduction. (2) Methods: In vitro experiments evaluated human bone marrow mesenchymal stem cell (h-BMSC) proliferation, differentiation, and osteogenic marker expression in Mg@Ca medium. Transcriptome sequencing identified bone development-related pathways. In vivo efficacy was assessed in a rabbit model of ONFH to evaluate bone repair. (3) Results: The Mg@Ca scaffold demonstrated excellent biocompatibility and supported h-BMSC proliferation and differentiation, with significant up-regulation of COL1A1 and BGLAP. Transcriptome analysis revealed activation of the PI3K-Akt signaling pathway, critical for osteogenesis. In vivo results confirmed enhanced trabecular density and bone volume compared to controls, indicating effective bone repair and regeneration. (4) Conclusions: The Mg@Ca scaffold offers a promising therapeutic approach for ONFH, providing a minimally invasive solution for bone defect repair while stimulating natural bone regeneration. Its injectable and self-setting properties ensure precise filling of bone defects, making it suitable for clinical applications.

Keywords: bioceramics; bone regeneration; injectable; osteonecrosis of the femoral head; scaffold.

Figure 1

SEM images of Mg@Ca at (a) 69×, (b) 243×, (c) 1000×, (d) 5000×, and (e) 14,290×. (f) XRD of Mg@Ca. All peaks match to CaSO₄(H₂O)_0.5 and α-CaSO₄.

Figure 2

The osteogenic properties of bioceramic scaffolds. (a) EdU staining for evaluation of the influences of three groups on the proliferation of h-BMSC. The new generation cells were detected via EdU (green). DAPI stained nuclei in blue. Merged view of EdU (green) and DAPI (blue) showing the overlap. (b) Quantitative analysis results of EdU staining using GraphPad Prism 9.0.0 software (La Jolla, CA, USA). (c) Quantitative analysis results of CCK-8 staining. (d) BGLAP and (e) COL1A1 expression of h-BMSCs cultured with blank medium, β-TCP medium, and Mg@Ca medium evaluated by qPCR. Data are presented as mean ± s.d. (n = 5). * p < 0.05, ** p < 0.01 (one-way ANOVA).

Figure 3

(a,b) The heatmap and volcano plot of the differentially expressed genes identified by transcriptome sequencing. The up−regulated genes are marked in red, and the down−regulated genes are marked in green. Cutoff: p−value < 0.05 and |log2 FC| > 1. (c) Top 20 signal pathways enriched by differentially expressed genes using KEGG analysis. The red boxes highlight the PI3K-Akt signaling pathway and ECM-receptor interaction pathway due to their critical roles in osteogenesis. (d) GSEA analysis from DisGeNET also indicated enrichment of DEGs in pathways related to BONE CELL DEVELOPMENT. (e) DEGs specifically expressed in the Mg@Ca group. Red typically indicates higher gene expression levels, while blue indicates lower gene expression levels across different samples. The identified differentially expressed genes enriched in (f) PI3K−Akt signaling pathway on the transcriptome sequencing and IPA 24.0.2 software analysis (created with BioRender.com).

Figure 4

Mg@Ca efficacy in the treatment of rabbit ONFH model: (a1–a6) Micro-CT scans of the osteonecrosis of the femoral head in the blank group at (a1,a2) 4 weeks, (a3,a4) 8 weeks, and (a5,a6) 12 weeks. The tunnels created by Kirschner (shown by the red arrows) wire remained continuously in the subcapital region. (b1–b6) Micro-CT scans of the osteonecrosis of the femoral head in the Mg@Ca group at (b1,b2) 4 weeks, (b3,b4) 8 weeks, and (b5,b6) 12 weeks. Mg@Ca within the tunnels was gradually absorbed and replaced by newly formed trabecular bone (shown by the red arrows). (c) Quantitative analysis results of bone volume using GraphPad Prism 9.0.0 software. (d) Quantitative analysis results of percent bone volume. Data are presented as mean ± s.d. (n = 3). * p < 0.05, ** p < 0.01 (one-way ANOVA).

Figure 5

Bone repair promotion by Mg@Ca in rabbit ONFH model: (a1–a3) HE staining of the osteonecrosis of the femoral head in the blank group at (a1) 4 weeks, (a2) 8 weeks, and (a3) 12 weeks. (b1–b3) HE staining of the osteonecrosis of the femoral head in the Mg@Ca group at (b1) 4 weeks, (b2) 8 weeks, and (b3) 12 weeks. (c1–c3) safranin O-fast green staining of the osteonecrosis of the femoral head in the blank group at (c1) 4 weeks, (c2) 8 weeks, and (c3) 12 weeks. (d1–d3) HE staining of the osteonecrosis of the femoral head in the Mg@Ca group at (d1) 4 weeks, (d2) 8 weeks, and (d3) 12 weeks. The arrows indicated the trabecular bone repair around the tunnel.