Bone defects resulting from trauma, tumor resection, or infection pose significant challenges in orthopedic surgery. While autologous and allogeneic bone grafts are used clinically, limitations exist including donor site morbidity, limited bone availability, and immune rejection. Bone tissue engineering offers a promising alternative by using scaffolds to guide bone regeneration. Bioactive ceramics, particularly mesoporous bioactive glass (MBG) scaffolds, are attractive due to their biocompatibility and osteoconductivity. However, in osteoporosis, MBG alone may not provide sufficient bone induction. The current research aimed to improve bone regeneration in osteoporosis by incorporating strontium (Sr) into a modified MBG scaffold. Strontium ranelate, a drug containing strontium, is used to treat osteoporosis due to strontium's ability to inhibit osteoclast activation and promote osteoblast differentiation. This study hypothesizes that incorporating Sr into an amino-functionalized MBG scaffold (Sr-N-MBG) will enhance bone regeneration in an osteoporosis model. Furthermore, the study aimed to investigate the underlying molecular mechanisms using bioinformatics analysis to understand the interaction between the biomaterial and osteoporotic bone marrow mesenchymal stem cells (BMSCs). This approach aimed to provide new insights into designing biomaterials that improve osteogenesis and address the challenges of bone regeneration in osteoporosis.

The existing literature highlights the challenges in treating bone defects caused by osteoporosis. Autologous and allogeneic bone grafts are limited by donor site morbidity, availability of bone tissue, and potential immune rejection. Bone tissue engineering, using bioactive ceramic scaffolds such as MBG, has emerged as a potential solution. While MBG shows good biocompatibility and osteoconductivity, its bone induction capability is insufficient in osteoporotic conditions. Various strategies have been explored to enhance the performance of these scaffolds, including surface modification and chemical component regulation. However, these methods face challenges in terms of efficacy, complexity, cost, and potential side effects. Strontium ranelate has shown promise in osteoporosis treatment by releasing Sr ions which prevent osteoclast activation and stimulate osteoblast differentiation. However, systemic administration of strontium ranelate can lead to unwanted side effects. Incorporating strontium directly into scaffolds offers a potential solution for localized delivery of Sr, avoiding systemic side effects, while promoting bone regeneration.

The study involved fabricating strontium-incorporated amino-functional mesoporous bioactive glass (Sr-N-MBG) scaffolds. The scaffolds were characterized using SEM to analyze their surface morphology and inductively coupled plasma-mass spectrometry (ICP-MS) to determine the released Sr ion concentration over time. An ovariectomized (OVX) rat model was used to induce osteoporosis. Bone marrow mesenchymal stem cells (OVX BMSCs) were isolated from the osteoporotic rats. In vitro biocompatibility assays, including live/dead staining, SEM for cell morphology, and CCK-8 assay for cell proliferation, were conducted. Osteogenic potential was evaluated via ALP activity assays, real-time PCR for osteogenic markers (Runx2, OCN), and Alizarin red S staining for calcium deposition. Angiogenic capacity was assessed using a tube formation assay with human umbilical vein endothelial cells (HUVECs) and Western blotting for VEGF expression. In vivo studies were performed using ectopic osteogenesis in nude mice and a critical-sized calvarial defect model in osteoporotic rats. Bone regeneration was assessed using micro-CT to quantify bone volume, bone mineral density, trabecular thickness and number, and histology (HE staining) to visualize bone formation. Immunohistochemistry (IHC) was used to assess angiogenesis markers (CD31 and VEGF). RNA sequencing was performed to identify differentially expressed genes and investigate underlying molecular mechanisms involved in the enhanced bone regeneration. Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were used for functional annotation. Intracellular and mitochondrial ROS levels were measured using fluorescence probes (DCFH-DA and MitoSOX Red), and Western blotting was used to confirm cAMP/PKA signaling pathway activation. Statistical analysis was performed using ANOVA and t-tests.

The study found that Sr-N-MBG scaffolds exhibited excellent biocompatibility, supporting cell proliferation and attachment without cytotoxicity. In vitro, Sr incorporation significantly enhanced osteogenic differentiation of OVX BMSCs, as demonstrated by increased ALP activity and upregulation of osteogenic markers (Runx2, OCN). The scaffolds also promoted angiogenesis, indicated by increased VEGF expression and enhanced tube formation by HUVECs. In vivo, Sr-N-MBG scaffolds significantly improved bone regeneration in both ectopic osteogenesis and critical-sized calvarial defect models. Micro-CT analysis showed a dose-dependent increase in bone volume, bone mineral density (BMD), trabecular thickness, and trabecular number in the Sr-N-MBG groups compared to the control N-MBG group. Histological analysis confirmed enhanced bone formation and angiogenesis in the Sr-N-MBG groups. RNA sequencing revealed significant differences in gene expression between the N-MBG and 2Sr-N-MBG groups. GO analysis suggested changes in cellular components related to extracellular matrix and cell-cell junctions. Analysis showed a reduction in oxidative stress and activation of the cAMP/PKA signaling pathway by Sr-N-MBG scaffolds, leading to decreased intracellular and mitochondrial ROS levels. Western blotting confirmed increased cAMP and p-PKA expression in OVX BMSCs cultured with Sr-N-MBG extracts.

The findings of this study demonstrate that incorporating Sr into amino-functionalized MBG scaffolds significantly enhances bone regeneration in an osteoporotic model. The improved osteogenic and angiogenic effects are likely attributed to the synergistic effects of the scaffold's structure and the local release of Sr ions. The reduced ROS levels and activation of the cAMP/PKA signaling pathway further support the anti-osteoporotic and bone regenerative effects of Sr. These results align with previous studies showing that strontium promotes osteoblast proliferation and inhibits osteoclast activity. The local delivery of Sr through the scaffold avoids the systemic side effects associated with strontium ranelate. This study provides a mechanistic understanding of how Sr-incorporated scaffolds enhance bone regeneration by reducing oxidative stress and activating the cAMP/PKA pathway. The findings are highly significant for the development of new biomaterials for treating bone defects in osteoporotic patients.

This study successfully demonstrated the efficacy of Sr-incorporated amino-functional MBG scaffolds in enhancing bone regeneration in an osteoporotic model. The improved biocompatibility, osteogenesis, and angiogenesis, combined with the mechanistic insights into ROS reduction and cAMP/PKA pathway activation, highlight the potential of this biomaterial for treating osteoporosis-related bone defects. Future research could focus on optimizing the Sr concentration and scaffold design for further improved bone regeneration and exploring the application of this biomaterial in other bone defects. Investigating the long-term effects and clinical translation of this biomaterial would also be valuable.

The study primarily used a rat model, which may not fully represent the complexity of human bone regeneration. The sample size for the in vivo studies could be considered relatively small. The long-term effects of the Sr-N-MBG scaffolds on bone regeneration are yet to be determined. Further studies are needed to assess the potential for different types of bone defects and the long-term biocompatibility and efficacy.