Strontium-incorporated bioceramic scaffolds for enhanced osteoporosis bone regeneration

Bone defects from trauma, tumor resection, and infection are challenging to treat. Traditional autografts and allografts face limitations including limited donor tissue, complications, and immune rejection. Bone tissue engineering using bioactive ceramic scaffolds, particularly mesoporous bioactive glass (MBG), offers promise due to biocompatibility and osteoconductivity, but MBG shows insufficient osteoinduction in osteoporotic conditions. Strategies to enhance scaffold performance include surface morphology modification and chemical component regulation, but each has drawbacks related to efficacy, complexity, cost, and side effects. Prior work transformed MBG into amino-functionalized MBG (N-MBG), which showed some osteogenic capability when loaded with anti-osteoporosis drugs; however, robust regeneration in osteoporotic bone remains difficult. Strontium ranelate, which releases Sr ions that inhibit osteoclasts and promote osteoblasts, suggests that Sr incorporation into N-MBG (Sr-N-MBG) could enhance regeneration in osteoporosis. The study aims to fabricate Sr-incorporated N-MBG scaffolds and evaluate their bioactivity in an osteoporotic model, including investigating gene expression and signaling pathways to better understand biomaterial–cell interactions.

The paper reviews bioactive ceramics and MBG scaffolds as leading candidates for bone defect repair due to their absorbability and interfacial reactivity. While surface morphology alterations (micro/nanostructures, functional coatings) and chemical regulation (growth factors, polymers) have been used to enhance performance, these approaches often show limited in vivo efficacy or face practical barriers. Strontium ranelate is an anti-osteoporosis agent with dual actions: promoting osteoblast differentiation and inhibiting osteoclast activation. Previous studies mostly provided phenomenological evidence for Sr-incorporated scaffolds improving osteogenesis, with limited exploration of gene expression and signaling pathways; bioinformatics analyses have been lacking. This study addresses these gaps by combining Sr incorporation with amino-functional MBG and conducting transcriptomic and pathway analyses.

- Scaffold fabrication: A one-step synthesis using P123 in HNO3/water, with SrCl2·6H2O added at 0 g, 0.67 g, or 1.34 g to generate three mixtures; tetraethoxysilane, Ca(NO3)2·4H2O, and triethyl phosphate were added with stirring. Mixtures were transferred to an autoclave and heated at 700 °C for 5 h. Scaffolds were formed by powder pressing using polyvinylpyrrolidone and polyethylene glycol as binders/pore formers. Amino groups were grafted using 3-aminopropyltrimethoxysilane. Groups: N-MBG (no Sr), 1Sr-N-MBG, 2Sr-N-MBG. - Characterization: SEM assessed surface morphology; ICP-MS quantified Sr ion release from extracts collected on Days 1, 3, 5, 7, and 14. - Osteoporotic model and OVX BMSC culture: Female Sprague–Dawley rats underwent bilateral ovariectomy. Three months later, bone marrow was harvested from femora to isolate OVX BMSCs (passages 2–4) for experiments. - In vitro biocompatibility and proliferation: OVX BMSCs (2.0×10^4 cells/sample) were seeded on scaffolds. Live/dead staining (Calcein AM/PI) after 3 days; SEM observed morphology. Proliferation measured by CCK-8 on Days 3 and 7 (absorbance at 490 nm). - In vitro osteogenesis: ALP activity quantified after 7 days using PNPP (OD 405 nm), normalized by protein (Bradford). Gene expression (Runx2, OCN, VEGF) assessed by RT–PCR (β-actin control) after culture (methods note 14 days). Mineralization evaluated by ARS staining after culture in scaffold extracts for 14 days. - In vitro angiogenesis: HUVEC tube formation on Matrigel after 4 h in scaffold extracts; quantification of mesh size and isolation branch length. Western blot for VEGF expression in HUVECs after 7 days in extracts. - Ectopic osteogenesis: OVX BMSC–seeded scaffolds implanted subcutaneously into nude mice for 4 weeks; HE staining assessed ectopic bone formation. - Critical-sized calvarial defect repair: 5-mm calvarial defects in osteoporotic rats implanted with 5-mm scaffolds. After 8 weeks, micro-CT quantified BMD, BV/TV, Tb.Th, Tb.N; HE histology and IHC for CD31 and VEGF evaluated bone formation and neovascularization. - Mechanism studies: RNA-Seq on OVX BMSCs cultured on N-MBG vs 2Sr-N-MBG; DEGs defined by fold change >2 or <0.5, P<0.05; GO and KEGG enrichment analyses performed. Intracellular ROS measured by DCFH-DA; mitochondrial ROS by MitoSOX Red with MitoTracker Green. Western blot for cAMP and p-PKA in OVX BMSCs cultured in material extracts for 7 days. - Statistics: SPSS; ANOVA and t-tests; P<0.05 considered significant; annotations: * vs N-MBG, # vs 1Sr-N-MBG.

- Biocompatibility: Live/dead assays showed no cytotoxicity across groups; OVX BMSCs adhered and spread well on all scaffolds. Proliferation increased on Sr-N-MBG scaffolds, with 2Sr-N-MBG showing the greatest increase by Day 7 (P<0.05 vs N-MBG; 2Sr > 1Sr). - Osteogenesis in vitro: Sr-N-MBG significantly increased ALP activity at Day 7 (P<0.05). Runx2 and OCN expression were significantly upregulated on 2Sr-N-MBG (P<0.05); 1Sr-N-MBG showed a trend but not significant vs N-MBG. VEGF expression increased in a Sr dose-dependent manner. ARS staining showed higher calcium deposition in cells cultured with Sr-N-MBG extracts. - Angiogenesis in vitro: HUVEC tube formation improved with Sr-N-MBG extracts, with 2Sr-N-MBG producing the largest mesh areas and isolation branch lengths (P<0.05 vs N-MBG; and vs 1Sr for 2Sr). VEGF protein levels were higher with Sr-containing extracts. - Ectopic osteogenesis: HE staining revealed new bone formation in Sr-N-MBG groups with more mature bone as Sr content increased; N-MBG showed fibrous connective tissue predominance. - Calvarial defect repair (8 weeks): Micro-CT showed greater new bone formation with Sr-N-MBG vs N-MBG. Quantitatively, 2Sr-N-MBG had significantly higher BMD, BV/TV, Tb.Th, and Tb.N than N-MBG and 1Sr-N-MBG (P<0.05); 1Sr trended higher than N-MBG without significance. Histology showed more extensive, better-connected new bone and more blood vessels in 2Sr-N-MBG. IHC demonstrated higher CD31 and VEGF in 2Sr-N-MBG (P<0.05 vs N-MBG). - Mechanisms: RNA-Seq identified 127 DEGs between N-MBG and 2Sr-N-MBG (59 up, 68 down). GO enrichment highlighted changes in collagen trimer and collagen-containing extracellular matrix components and processes related to detection of oxidative stress. Sr-N-MBG reduced intracellular and mitochondrial ROS levels (lower DCF and MitoSOX fluorescence). KEGG enrichment implicated cAMP signaling; Western blots showed higher cAMP and PKA phosphorylation in Sr-N-MBG groups, suggesting activation of cAMP/PKA as a mechanism for ROS reduction and enhanced osteogenesis. A Sr-induced nanoneedle surface topography was observed, potentially contributing via mechanotransduction.

The study demonstrates that locally delivering Sr via amino-functional MBG scaffolds addresses the insufficient osteoinductivity of conventional MBG in osteoporotic environments. Sr incorporation improved OVX BMSC proliferation, osteogenic differentiation, and paracrine pro-angiogenic signaling in vitro, and enhanced both bone regeneration and neovascularization in vivo in ectopic and critical-sized defect models. Transcriptomics and functional assays indicate that Sr-N-MBG modulates extracellular matrix-related gene programs and reduces oxidative stress through activation of cAMP/PKA signaling, aligning with the pathophysiology of osteoporosis where elevated ROS impairs osteogenesis. The synergistic effects of Sr ion release and Sr-induced nanoneedle topography likely enhance cytoskeletal activity and mechanotransduction, further promoting osteogenic pathways. These findings support Sr-incorporated, amino-functional MBG as a promising scaffold design for osteoporotic bone repair, offering benefits over systemic Sr therapies by localizing effects and potentially minimizing systemic side effects.

Sr-incorporated amino-functional MBG scaffolds are biocompatible and significantly enhance osteogenesis and angiogenesis in osteoporotic models, improving bone quantity and quality in critical-sized defects. Mechanistically, Sr-N-MBG reduces intracellular and mitochondrial ROS and activates cAMP/PKA signaling, contributing to anti-osteoporotic effects while promoting osteogenesis; scaffold surface nanoneedle topography may further aid mechanotransduction. This work validates incorporating trace elements into biomaterial scaffolds and offers design principles for antioxidative, pro-osteogenic scaffolds for osteoporosis-related bone repair. Future research should optimize Sr dosing and release profiles, delineate long-term safety and efficacy, dissect the interplay between topography and ion release on mechanotransduction pathways, and evaluate performance in large animal models and load-bearing sites.