Building Osteogenic Microenvironments with a Double-Network Composite Hydrogel for Bone Repair

Abstract

The critical factor determining the in vivo effect of bone repair materials is the microenvironment, which greatly depends on their abilities to promote vascularization and bone formation. However, implant materials are far from ideal candidates for guiding bone regeneration due to their deficient angiogenic and osteogenic microenvironments. Herein, a double-network composite hydrogel combining vascular endothelial growth factor (VEGF)-mimetic peptide with hydroxyapatite (HA) precursor was developed to build an osteogenic microenvironment for bone repair. The hydrogel was prepared by mixing acrylated β-cyclodextrins and octacalcium phosphate (OCP), an HA precursor, with gelatin solution, followed by ultraviolet photo-crosslinking. To improve the angiogenic potential of the hydrogel, QK, a VEGF-mimicking peptide, was loaded in acrylated β-cyclodextrins. The QK-loaded hydrogel promoted tube formation of human umbilical vein endothelial cells and upregulated the expression of angiogenesis-related genes, such as Flt1, Kdr, and VEGF, in bone marrow mesenchymal stem cells. Moreover, QK could recruit bone marrow mesenchymal stem cells. Furthermore, OCP in the composite hydrogel could be transformed into HA and release calcium ions facilitating bone regeneration. The double-network composite hydrogel integrated QK and OCP showed obvious osteoinductive activity. The results of animal experiments showed that the composite hydrogel enhanced bone regeneration in skull defects of rats, due to perfect synergistic effects of QK and OCP on vascularized bone regeneration. In summary, improving the angiogenic and osteogenic microenvironments by our double-network composite hydrogel shows promising prospects for bone repair.

Fig. 1.

Schematic illustration of double-network composite hydrogels that build an angiogenic and osteogenic microenvironment for bone repair. The QK released from the composite hydrogel recruits BMSCs to the sites of bone injury and directly promotes angiogenesis, then Ca²⁺ and HA induce osteogenic differentiation of BMSCs and promote bone formation.

Fig. 2.

Characteristics of hydrogels. (A) SEM images of the cross section of hydrogels. (B) Compressive modulus of hydrogels. (C) QK release curves of hydrogels. (D) XRD measurement of the GP composite hydrogel. (E) Ca²⁺ release curves of hydrogels. *P < 0.05.

Fig. 3.

Growth of BMSCs and HUVECs on the surface of hydrogels. (A) Dil-labeled BMSCs cultured on the hydrogel surface after 3 days. (B) Morphology of HUVECs on the hydrogel surface after 2 days.

Fig. 4.

Characterizations of cell migration. (A) Crystal violet staining of BMSCs that migrated to the lower chamber of the transwell plate after 24-h culture. (B) Manual random counting in 5 random fields under 100× magnification. (C) Wound-healing assay of a monolayer of BMSCs after 0, 6, and 24 h. Wound boundary was marked with a yellow dashed line. (D) Calculation of wound closure area after 6 and 24 h. *P < 0.05. ns, no significant difference.

Fig. 5.

Characterizations of vascularization. (A) Tube formation assay of HUVECs in different groups. (B) Quantification of average parameters of tube formation. (C) H&E staining of hydrogels implanted into subcutaneous tissue of rat after 5 and 10 days. Blood vessels were marked with yellow arrowheads. (D) Quantification of the area of blood vessel formation. (E) Fluorescein isothiocyanate labeling of blood vessels formation in hydrogels that were implanted into subcutaneous tissue of rat after 10 days. *P < 0.05. ns, no significant difference.

Fig. 6.

Expression of angiogenesis-related genes of BMSCs cultured with hydrogels after 7 and 14 days. *P < 0.05. ns, no significant difference.

Fig. 7.

Characterizations of in vitro osteogenic differentiation of BMSCs. (A) ALP staining of BMSCs in different groups after 7 days. (B) Alizarin red staining of BMSCs in different groups after 21 days. (C) Expression of osteogenesis-related genes of BMSCs cultured with hydrogels: Spp1, Runx2, and Alpl after 7 days; Col1a1 and Bglap after 21 days. *P < 0.05. ns, no significant difference.

Fig. 8.

Characterizations of in vivo bone repair by double-network composite hydrogels. (A) Micro-computed tomography images at 8 and 16 weeks. (B) Bone volume fraction. (C) H&E staining of calvarial critical-sized defects repaired by hydrogels. S, scaffold; NB, new bone. *P < 0.05. ns, no significant difference.