ECM-mimicking composite hydrogel for accelerated vascularized bone regeneration

Abstract

Bioactive hydrogel materials have great potential for applications in bone tissue engineering. However, fabrication of functional hydrogels that mimic the natural bone extracellular matrix (ECM) remains a challenge, because they need to provide mechanical support and embody physiological cues for angiogenesis and osteogenesis. Inspired by the features of ECM, we constructed a dual-component composite hydrogel comprising interpenetrating polymer networks of gelatin methacryloyl (GelMA) and deoxyribonucleic acid (DNA). Within the composite hydrogel, the GelMA network serves as the backbone for mechanical and biological stability, whereas the DNA network realizes dynamic capabilities (e.g., stress relaxation), thereby promoting cell proliferation and osteogenic differentiation. Furthermore, functional aptamers (Apt19S and AptV) are readily attached to the DNA network to recruit bone marrow mesenchymal stem cells (BMSCs) and achieve sustained release of loaded vascular endothelial growth factor towards angiogenesis. Our results showed that the composite hydrogel could facilitate the adhesion of BMSCs, promote osteogenic differentiation by activating focal adhesion kinase (FAK)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/β-Catenin signaling pathway, and eventually enhance vascularized bone regeneration. This study shows that the multifunctional composite hydrogel of GelMA and DNA can successfully simulate the biological functions of natural bone ECM and has great potential for repairing bone defects.

Keywords: Composite hydrogel; DNA hydrogel; Osteogenesis; Stress relaxation; Vascularization.

Graphical abstract

Scheme 1

Schematic of the design of the GDSV-VEGF hydrogel with ECM characteristics to promote vascularized bone regeneration.

Fig. 1

Synthesis and characterization of GDSV hydrogel. A) Schematic of the structure of GDSV hydrogel. B) Sequence information of DNA components (“handles” sequences are labelled as red letters). C) Native PAGE of DNA components. D) Macroscopic appearance and SEM images of the DSV, GelMA, GDSV, GelMA-VEGF, and GDSV-VEGF hydrogels, scale bar = 100 μm. E) FTIR spectra of the DSV, GelMA, GDSV, GelMA-VEGF, and GDSV-VEGF hydrogels. F) Storage modulus (G′) and loss modulus (G″) of hydrogels (n = 4). G) Normalized stress relaxation of the DSV, GelMA, GDSV, GelMA-VEGF, and GDSV-VEGF hydrogels (n = 4). H) Elastic modulus of hydrogels (n = 4, **p < 0.01). I) Swelling profile of the DSV, GelMA, GDSV, GelMA-VEGF, and GDSV-VEGF hydrogels (n = 4). J) Fluorescence images showing FAM labelled DSV and GDSV hydrogels via observation of remaining fluorescent area in serum-supplemented medium, scale bar = 500 μm.

Fig. 2

GDSV hydrogel biocompatibility and promotion of cell proliferation. A) Live/dead staining of BMSCs on GelMA, GelMA-VEGF, GDSV, and GDSV-VEGF hydrogels after 3 days of culture. Viable cells are green and dead cells are red, control group: BMSCs seeded in plate well directly, scale bar = 200 μm. B) CCK-8 assay of BMSCs proliferation and viability on different hydrogels for 1, 4 and 7 days (n = 6, *p < 0.05, **p < 0.01).

Fig. 3

GDSV hydrogel for promoting the homing of BMSCs. A) Schematic of the DNA aptamer (Apt19S), which performs the function of BMSCs recruitment. B) Illustration of the transwell migration test. C) BMSCs that traversed the membranes were stained. Images of cells towards GelMA, GelMA-VEGF, GDSV, GDSV-VEGF, GDV and GDV-VEGF hydrogels were captured, control group: no hydrogel was placed in the lower chamber, scale bar = 100 μm. D) Quantitative analysis of migrated BMSCs (n = 6, **p < 0.01).

Fig. 4

GDSV-VEGF hydrogel for promoting tube formation via sustained-release of VEGF. A) Schematic of the DNA aptamer (AptV) which performs the function of the sustained-release of VEGF. B) Illustration of extract from medium with hydrogel to detect release of VEGF and tube formation. C) VEGF cumulative release curve of GelMA-VEGF, GDSV-VEGF, and GDS-VEGF hydrogels (n = 4). D) Images of tube formation of HUVECs under extracts from GDSV, GelMA-VEGF, GDSV-VEGF and GDS-VEGF hydrogels at time points of 1, 6, and 10 days, control group: extract from medium without hydrogel, scale bar = 200 μm. E) Quantitative analysis of total lengths, nodes and meshes numbers (n = 4, *p < 0.05, **p < 0.01).

Fig. 5

GDSV hydrogel for enhancing BMSCs osteogenic differentiation in vitro. A) ALP and ARS staining of BMSCs on GelMA, GelMA-VEGF, GDSV and GDSV-VEGF hydrogels, scale bar = 1 mm. B) Semi-quantitative analysis of ALP and ARS activity (n = 6, **p < 0.01). C) Expression of osteogenic-related genes (Runx2, Osterix, Col1, Alp, and Ocn) in BMSCs on different hydrogels (n = 6, **p < 0.01).

Fig. 6

GDSV-VEGF hydrogel for promoting vascularized bone formation in vivo. A) Micro-CT reconstruction of cranium defects implanted with different hydrogels (Groups: control, GelMA, GDSV, GelMA-VEGF, and GDSV-VEGF) 8 weeks after the operation. The new bone formation in the defect regions (indicated by white rectangle or circle) was observed from side view, top view and bottom view, control group: cranium defects without hydrogel, scale bar = 1 mm. B) Quantitative analysis of BV/TV and BMD (n = 6, **p < 0.01). C) Undecalcified sections with VG staining were performed to observe new bone formation in the defect regions (indicated by blue rectangle) as histological evaluation, scale bar = 1 mm. D) Quantitative analysis of new bone area (n = 6, **p < 0.01). E) Sequential fluorescent labeling for dynamic bone mineralization by ARS, (red, 4 weeks) and CA (green, 6 weeks), scale bar = 100 μm. F) Quantitative analysis of ARS and CA labelled area (n = 6, *p < 0.05, **p < 0.01). G) Micro-CT reconstruction of new blood vessels perfused with Microfil in defect areas (indicated by white circle) 8 weeks after the operation, scale bar = 1 mm. H) Quantitative analysis of new blood vessels area (n = 6, **p < 0.01). I) Decalcified sections with immunofluorescent staining of CD31 were performed to observe new vessels formation (CD31: red, cell nuclei: blue), scale bar = 100 μm. J) Quantitative analysis of CD31 labelled area (n = 6, **p < 0.01).

Fig. 7

Mechanism and role of GDSV hydrogel for promoting BMSCs osteogenic differentiation. A) Volcano diagram of DEGs between GelMA and GDSV groups. B) Gene enrichment GO-BP (Biological process) analysis of GDSV group vs. GelMA group. C) Gene enrichment KEGG pathways analysis of GDSV group vs. GelMA group. D) Heat map of DEGs related to cell adhesion and osteogenesis in GDSV group vs. GelMA group, such as Ptk2 encoding FAK, Ctnnb1 encoding β-Catenin (n = 4). E) Immunofluorescent staining of F-actin to observe BMSCs adhesion on different hydrogels (F-actin: green, cell nuclei: blue), scale bar = 100 μm and 20 μm, quantitative analysis of cell spreading area (n = 6, **p < 0.01). F) Western blot analysis of protein expression of FAK, p-FAK, Akt, p-Akt, β-Catenin, Runx2 and GAPDH between GelMA and GDSV groups. G) Quantitative analysis of FAK, Akt, β-Catenin, Runx2 normalized to GAPDH, p-FAK normalized to FAK, and p-Akt normalized to Akt (n = 6, **p < 0.01).