ATF2-driven osteogenic activity of enoxaparin sodium-loaded polymethylmethacrylate bone cement in femoral defect regeneration

Abstract

Background: Polymethylmethacrylate (PMMA) bone cement loaded with enoxaparin sodium (PMMA@ES) has been increasingly highlighted to affect the bone repair of bone defects, but the molecular mechanisms remain unclear. We addressed this issue by identifying possible molecular mechanisms of PMMA@ES involved in femoral defect regeneration based on bioinformatics analysis and network pharmacology analysis.

Methods: The upregulated genes affecting the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) were selected through bioinformatics analysis, followed by intersection with the genes of ES-induced differentiation of BMSCs identified by network pharmacology analysis. PMMA@ES was constructed. Rat primary BMSCs were isolated and cultured in vitro in the proliferation medium (PM) and osteogenic medium (OM) to measure alkaline phosphatase (ALP) activity, mineralization of the extracellular matrix, and the expression of RUNX2 and OCN using gain- or loss-of-function experiments. A rat femoral bone defect model was constructed to detect the new bone formation in rats.

Results: ATF2 may be a key gene in differentiating BMSCs into osteoblasts. In vitro cell assays showed that PMMA@ES promoted the osteogenic differentiation of BMSCs by increasing ALP activity, extracellular matrix mineralization, and RUNX2 and OCN expression in PM and OM. In addition, ATF2 activated the transcription of miR-335-5p to target ERK1/2 and downregulate the expression of ERK1/2. PMMA@ES induced femoral defect regeneration and the repair of femoral defects in rats by regulating the ATF2/miR-335-5p/ERK1/2 axis.

Conclusion: The evidence provided by our study highlighted the ATF2-mediated mechanism of PMMA@ES in the facilitation of the osteogenic differentiation of BMSCs and femoral defect regeneration.

Keywords: ATF2; Bone marrow mesenchymal stem cells; Enoxaparin sodium; Femoral defect regeneration; Femoral defect repair; Osteogenic differentiation; Polymethylmethacrylate bone cement; miR-335-5p.

Fig. 1

Identification of key genes involved in the osteogenic differentiation of BMSCs. A Volcano map of differential analysis for microarray data GSE9451. Red dots indicate upregulated genes, green dots indicate downregulated genes and black dots indicate genes without significant difference (BMSCs group, n = 3; BMSCs_osteogenesis group, n = 3). B The heat map of differential analysis for microarray data GSE9451 dataset (BMSCs group, n = 3; BMSCs_osteogenesis group, n = 3). C Venn map of the intersection between CTD database and microarray data GSE9451. D GO enrichment analysis for candidate target genes. Abscissa indicates GeneRatio. E KEGG enrichment analysis for candidate target genes. Abscissa indicates GeneRatio. F Protein interaction among the 31 candidate genes. Nodes represent genes, and edges represent the interactions between genes. G Candidate target genes according to Degree values

Fig. 2

Effects of ATF2 on the osteogenic differentiation of BMSCs. A JASPAR website was used to predict the binding site of the ATF2 transcription factor to miR-335-5p. B ChIP assay was used to detect ATF2 enrichment in the promoter region of miR-335-5p. C RT-qPCR for ATF2 and miR-335-5p expression in BMSCs after 0, 7, and 14 days of osteogenic differentiation. D RT-qPCR for miR-335-5p expression in BMSCs after exogenous interference with ATF2. E Western blot analysis and RT-qPCR were used to detect miR-335-5p and ATF2 expression in BMSCs treated with oe-ATF2 or in combination with miR-335-5p inhibitor. F ALP staining and quantification of BMSCs treated with oe-ATF2 or in combination with miR-335-5p inhibitor in PM and OM. G ARS staining and quantification of BMSCs treated with oe-ATF2 or in combination with miR-335-5p inhibitor in PM and OM.H Western blot analysis and RT-qPCR were used to detect RUNX2 expression in BMSCs treated with oe-ATF2 or combined with miR-335-5p inhibitor in PM and OM. I Western blot analysis, and RT-qPCR were used to detect OCN expression in BMSCs treated with oe-ATF2 or in combination with miR-335-5p inhibitor in PM and OM. J Venn diagram of the miRDB, TargetScan, and DIANA TOOLS databases results. K RT-qPCR for CALU and ERK1/2 expression in BMSCs after 0, 7, and 14 days of osteogenic differentiation. L TargetScan database was used to predict the target binding sites of miR-335-5p to miR-335-5p to ERK1/2. M Dual-luciferase reporter assay was used to verify the binding of miR-335-5p to ERK1/2. N Western blot analysis, and RT-qPCR were used to detect ERK1/2 expression in BMSCs transfected with miR-335-5p mimic. O Western blot analysis and RT-qPCR were used to detect ATF2, miR-335-5p, and ERK1/2 expression in BMSCs treated with oe-ATF2 alone or combined with miR-335-5p inhibitor. *p < 0.05. Cell experiments were repeated three times

Fig. 3

Effects of ATF2 on the osteogenic differentiation of BMSCs via regulation of ERK1/2. BMSCs were treated with oe-ATF2 or in combination with oe-ERK1/2. A Western blot analysis and RT-qPCR were used to detect miR-335-5p, ERK1/2, and ATF2 expression in BMSCs. B ALP staining for BMSCs in PM and OM. C ARS staining for BMSCs in PM and OM. D Western blot analysis and RT-qPCR were used to detect RUNX2 expression in BMSCs in PM and OM. E Western blot analysis and RT-qPCR were used to detect OCN expression in BMSCs in PM and OM. *p < 0.05. Cell experiments were repeated three times

Fig. 4

Effects of PMMA@ES on the osteogenic differentiation of BMSCs. A Western blot analysis for ATF2 protein levels in BMSCs treated with PMMA, ES, or PMMA@ES. B ALP staining and quantification of BMSCs treated with PMMA, ES, or PMMA@ES in PM and OM. C ARS staining and quantification of BMSCs treated with PMMA, ES, or PMMA@ES in PM and OM. D Western blot analysis and RT-qPCR were used to detect RUNX2 expression in BMSCs treated with PMMA, ES, or PMMA@ES in PM and OM. E Western blot analysis and RT-qPCR were used to detect OCN expression in BMSCs treated with PMMA, ES, or PMMA@ES in PM and OM. *p < 0.05. Cell experiments were repeated three times

Fig. 5

Effects of PMMA@ES on femoral defect regeneration in rats via the ATF2/miR-335-5p/ERK1/2 axis. Rats with femoral defects were treated with PMMA, ES, or PMMA@ES (n = 8). A micro-CT scanning quantitative results of the site of the femoral defect in rats. B Immunohistochemical staining for positive expression of RUNX2, OCN, ATF2, and ERK1/2 at the new bone formation site of rats with femoral defects. Dark-brown granules indicating positive staining are marked by black arrows. *p < 0.05

Fig. 6

Schematic representation of the molecular mechanism of PMMA@ES in femoral defects. PMMA@ES-induced osteogenic differentiation of BMSCs involved in femoral defect regeneration by elevating ATF2 expression and activating the miR-335-5p/ERK1/2 axis