Dual functional hydrogel of osteoclastic-inhibition and osteogenic-stimulation for osteoporotic bone defect regeneration

Abstract

Osteoporotic bone regeneration poses significant challenges due to the complexity of the condition. Osteoporosis, a degenerative disorder, results from an imbalance in bone homeostasis driven by dysregulation of osteoblast and osteoclast activity. This complicates the treatment of osteoporosis and its related bone injuries in clinical practice. Despite the development of various polymer scaffolds for bone defect repair, achieving effective regeneration in osteoporotic bones-especially when combined with osteoporosis medications-remains difficult. In this study, we designed a drug delivery system composed of mesoporous bioactive glass (MBG) and photo-crosslinked hyaluronic acid methacrylate (HAMA). This system, loaded with the osteogenesis-promoting peptide DWIVA (D5) and the osteoclastogenesis-inhibiting drug alendronate (ALN), is gelled using a light initiator and 405 nm wavelength light. The MBG@D5-Gel complex enables the controlled spatiotemporal release of these agents, markedly enhancing bone regeneration in osteoporotic conditions within ovariectomized rats by inhibiting osteoclastogenesis and bone resorption while promoting osteogenic differentiation and mineralization. This dual-action system synergistically regulates osteoblast and osteoclast activity, optimizing the pathological microenvironment of osteoporosis and facilitating the repair of osteoporotic bone defects. MBG@D5-Gel holds great potential as an effective organic-inorganic hybrid biomimetic implant material for the treatment of osteoporotic bone defects.

Keywords: Alendronate; Bone regeneration; Hydrogel; Osteoclastic differentiation; Osteogenic differentiation; Osteoporosis.

Graphical abstract

Scheme 1

The synthesis and preparation of the composite hydrogel enables the repair and regeneration of osteoporotic bone defects by promoting osteogenic differentiation and inhibiting osteoclastic differentiation.

Fig. 1

Fabrication and characterization of HAMA-ALN and MBG-Gel. (A) Schematic diagram of MBG synthesis. (B) TEM image of MBG. (C) Mapping analysis. (D) Schematic diagram of HAMA-ALN synthesis. (E) ¹H NMR spectra (600 MHz) of HA, HAMA and HAMA-ALN in D₂O. (F) Schematic diagram of MBG@D5-Gel. (G) Solution containing HAMA-ALN and MBG@D5 underwent gelation at LAP and 405 nm wavelength illumination. (H) SEM images of MBG-Gel. (I) Spectrum of elastic (G′) and viscous (G″) modulus of MBG-Gel with varying strain, frequency, and time applied. (J) Drug release of ALN and D5 peptide.

Fig. 2

In vitro biocompatibility evaluations of HAMA, HAMA-ALN, MBG-Gel and MBG@D5-Gel. (A) Live (green) and dead (red) cells of BMSCs after cultured with the extract. (B) Cell viability of BMSCs after cultured with the extract, (n = 5). (C) Live (green) and dead (red) cells of BMMs after cultured with the extract. (D) Cell viability of BMMs after cultured with the extract, (n = 5). (E) Hemolysis Rate of red blood cells in different treated with HAMA, HAMA-ALN, MBG-Gel and MBG@D5-Gel. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

In vitro osteogenic properties of the dual functional hydrogel. (A) ALP staining. (B) Quantitative evaluation of ALP activity. (C) Alizarin red S staining. (D) Semiquantitative evaluation of ECM mineralization. (E) Gene levels of ALP, Runx2, SP7, OCN, and OPN. (F) Immunofluorescence staining of Runx2 (green). (G) Western blotting of COL1, Runx2, ALP and β-actin in BMSCs after different treatments. (∗∗∗ denotes p < 0.001 compared to the control group). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

In vitro osteoclastic properties of the dual functional hydrogel. (A) TRAP staining. (B) Bone resorption assays. (The red dashed square encloses bone resorption area) (C) Quantification of area of TRAP + multinuclear cells (D) Quantification of the area of resorption pits per bone slice. (E) Immunofluorescence staining using phalloidin (green) for F-Actin ring formation assays. (F) Gene levels of TRAcP, MMP9, and Nfatc1. (G) Western blotting of CTSK, MMP-9, and NFATc1 in BMMs after different treatments. (H) Quantitative analysis of Western blot results. (∗∗∗ denotes p < 0.001 compared to the RANKL group). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

In vivo evaluation of bone regeneration effect of dual functional hydrogel implanted in osteoporotic rat model with bone defect. (A) Schematic illustration showing the design of animal experiments. (B) Representative 3D reconstructed images of the OVX rat distal femurs and quantitative parameters of trabecular bone, BMD, bone mineral density; BV/TV, bone volume/tissue volume ratio; Tb. Sp, trabecular separation; Tb. N, trabecular number. (C) Representative 3D reconstructed images of bone defect of the 4 and 12 weeks. (Red arrow refers to the area of defect) (D) Quantitative parameters of trabecular bone in the defect area. (∗∗∗ denotes p < 0.001 compared to the OVX group). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Histological analysis in the bone defect area. (A) H&E staining and Masson staining of bone defect area of 4 weeks. (B) H&E staining and Masson staining of bone defect area of 12 weeks. (C) TRAP staining. (D) Quantitative analysis of TRAP positive cell number. (∗∗∗ denotes p < 0.001 compared to the OVX group).

Fig. 7

Representative H&E stained images of kidneys, hearts, spleens, lungs, and livers in rats from each group.