Local bone metabolism balance regulation via double-adhesive hydrogel for fixing orthopedic implants

Abstract

The effective osteointegration of orthopedic implants is a key factor for the success of orthopedic surgery. However, local metabolic imbalance around implants under osteoporosis condition could jeopardize the fixation effect. Inspired by the bone structure and the composition around implants under osteoporosis condition, alendronate (A) was grafted onto methacryloyl hyaluronic acid (H) by activating the carboxyl group of methacryloyl hyaluronic acid to be bonded to inorganic calcium phosphate on trabecular bone, which is then integrated with aminated bioactive glass (AB) modified by oxidized dextran (O) for further adhesion to organic collagen on the trabecular bone. The hybrid hydrogel could be solidified on cancellous bone in situ under UV irradiation and exhibits dual adhesion to organic collagen and inorganic apatite, promoting osteointegration of orthopedic implants, resulting in firm stabilization of the implants in cancellous bone areas. In vitro, the hydrogel was evidenced to promote osteogenic differentiation of embryonic mouse osteoblast precursor cells (MC3T3-E1) as well as inhibit the receptor activator of nuclear factor-κ B ligand (RANKL)-induced osteoclast differentiation of macrophages, leading to the upregulation of osteogenic-related gene and protein expression. In a rat osteoporosis model, the bone-implant contact (BIC) of the hybrid hydrogel group increased by 2.77, which is directly linked to improved mechanical stability of the orthopedic implants. Overall, this organic-inorganic, dual-adhesive hydrogel could be a promising candidate for enhancing the stability of orthopedic implants under osteoporotic conditions.

Keywords: Dual-functional; Hydrogels; Osseointegration; Osteoporosis; Peri-implant.

Graphical abstract

Scheme 1

Schematic illustration of double-adhesive hydrogel for Fixing Orthopedic Implants. A) Synthesis of oxidized dextran; B) Synthesis of AB; C) Synthesis of A-H; D) Fixing orthopedic implants and promoting cancellous bone reconstruction via regulating local bone metabolism in animal experiment.

Fig. 1

Physical characterization of the double adhesive hydrogel. A-B) FTIR spectra of DEX, ODEX, BG and AB; C) SEM images of AB; D) Particle size analysis of AB; E) FTIR spectra of H and A-H; F) 1H NMR of A, H, and A-H; G-I) Swelling test, mechanical test, and weight loss analysis of A-H, A-H + AB, and A-H + AB-O (n = 3); J) ions release behavior of the A-H + AB-O hydrogels (n = 3); K) interior SEM images of A-H,A-H + AB,A-H + AB-O; L) digital images of adhesion between the hydrogel and rat's femur (Plastic ring was 1.01 g, rat's femur was 0.83 g).

Fig. 2

Evaluation of the biocompatibility of the organic-inorganic, double-bonded hydrogels in vitro. A-B) Live/dead staining of BMMs and MC3T3-E1 cells on different hydrogels after 3 days' co-culture. C-D) CCK-8 assay of BMMs and MC3T3-E1 cells. (n = 3, all values were mean ± std. dev., NS, not significant, *p < 0.05, **p < 0.01 when comparing A-H + AB-O and other groups via two-way ANOVA analysis followed by Tukey's multiple comparison test by GraphPad Prism 7.0 Software (USA)).

Fig. 3

Osteogenesis evaluation. A-B) ALP and alizarin red staining of MC3T3-E1 cells cultured in osteogenic medium with different hydrogel groups. C-D) immunofluorescence staining of RUNX2 on day 7 and OCN on day 14, with the nuclei stained blue (DAPI), the proteins stained green and the cytoskeletal structure stained red. E-F) quantitative analysis of ALP activity and alizarin red staining results (n = 3, all values were mean ± std. dev., NS, not significant, **p < 0.01, ***p < 0.001 when comparing Control and other groups via one-way ANOVA analysis followed by Tukey's multiple comparison test by GraphPad Prism 7.0 Software (USA)). G-H) semiquantitative analysis of the fluorescence intensity of RUNX2 and OCN (n = 3, all values were mean ± std. dev., NS, not significant, *p < 0.05, **p < 0.01, ***p < 0.001 when comparing A-H and other groups; ^#p < 0.05 when comparing A-H + AB and A-H + AB-O via one-way ANOVA analysis followed by Tukey's multiple comparison test by GraphPad Prism 7.0 Software (USA)). I-J) ELISA results of the concentration of collagen I and OPN in the cell supernatant (n = 3, all values were mean ± std. dev., NS, not significant, *p < 0.05, **p < 0.01 when comparing Control and other groups via one-way ANOVA analysis followed by Tukey's multiple comparison test by GraphPad Prism 7.0 Software (USA)). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Osteoclast evaluation. A) TRAP staining of BMMs cells cultured in RANKL-containing medium with different hydrogel groups. B) Immunofluorescence assays for MMP-9 on day 5, with the nuclei stained blue (DAPI), the protein stained green and the cytoskeletal structure stained red. C-D) quantitative analysis of TRAP positive cell number; Quantitative analysis of TRAP positive staining area. E) semiquantitative analysis of the fluorescence intensity in MMP-9. F-G) ELISA analysis of the concentration of MMP-9 and TRAP concentrations on day 5 in the cell supernatant. (n = 3, all values were mean ± std. dev., NS, not significant, *p < 0.05, **p < 0.01, ***p < 0.001 when comparing A-H + AB-O and other groups via one-way ANOVA analysis followed by Tukey's multiple comparison test by GraphPad Prism 7.0 Software (USA)). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

A) Expression level of osteogenesis-related genes including RUNX2, OCN, OPN (n = 3, all values were mean ± std. dev., NS, not significant, **p < 0.01, ***p < 0.001, ****p < 0.0001 when comparing A-H + AB-O and other groups; ^#p < 0.05, ^##p < 0.01 when comparing Control and A-H via two-way ANOVA analysis followed by Tukey's multiple comparison test by GraphPad Prism 7.0 Software (USA)). B) Expression level of osteoclastogenesis-related genes including MMP-9, c-FOS, NFATc1 on day 5 (n = 3, all values were mean ± std. dev., NS, not significant, **p < 0.01, ***p < 0.001, ****p < 0.0001 when comparing A-H + AB-O and other groups; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 when comparing Control and A-H; ^&p < 0.05, ^&&p < 0.01, ^&&&p < 0.01 when comparing A-H and A-H + AB via one-way ANOVA analysis followed by Tukey's multiple comparison test by GraphPad Prism 7.0 Software (USA)).

Fig. 6

Establishment of the animal model. A) Ovariectomy image of SD rats. B) Micro-CT 3D reconstructed images. C-D) H&E and Masson's trichrome staining. E-J) quantitative analysis of Micro-CT results including BV, BMD, Conn.D, Tb.N, Tb.Th, and Tb.Sp (n = 5, all values were mean ± std. dev., *p < 0.05, **p < 0.01 when comparing OVX and SHAM via student's t-test analysis by GraphPad Prism 7.0 Software (USA)).

Fig. 7

In vivo application of the double-bonded hydrogel. A) The process of screw implantation in SD rats. B) Micro-CT 3D reconstructed images. C-E) Micro-CT analysis of BV/TV, Conn.D and Tb.N (n = 4, all values were mean ± std. dev., NS, not significant, *p < 0.05, **p < 0.01, ***p < 0.001 when comparing A-H + AB-O and other groups via two-way ANOVA analysis followed by Tukey's multiple comparison test by GraphPad Prism 7.0 Software (USA)). F) The results of pull-out testing (n = 3, all values were mean ± std. dev., NS, not significant, *p < 0.05, **p < 0.01, ***p < 0.001 when comparing A-H + AB-O and other groups via two-way ANOVA analysis followed by Tukey's multiple comparison test by GraphPad Prism 7.0 Software (USA)). G) Histomorphometric results of BIC (n = 4, all values were mean ± std. dev., NS, not significant, *p < 0.05, **p < 0.01, ***p < 0.001 when comparing A-H + AB-O and other groups via two-way ANOVA analysis followed by Tukey's multiple comparison test by GraphPad Prism 7.0 Software (USA)). H) Representative samples stained with toluidine.

Fig. 8

H&E and Masson staining. A-B) H&E and Masson staining in each group, 4- and 8- weeks post-surgery. C-D) quantitative results of H&E and Masson staining in the proportion of bone collagen (n = 3, all values were mean ± std. dev., NS, not significant, *p < 0.05, **p < 0.01, ***p < 0.001 when comparing A-H + AB-O and other groups via two-way ANOVA analysis followed by Tukey's multiple comparison test by GraphPad Prism 7.0 Software (USA)).

Fig. 9

Immunohistochemistry. A-D) immunohistochemistry of collagen type I, OPN, TRAP, and CD31. E-H) quantitative results in the proportion of positive areas in collagen I, OPN, TRAP, and CD31 (n = 3, all values were mean ± std. dev., NS, not significant, *p < 0.05, **p < 0.01, ***p < 0.001 when comparing A-H + AB-O and other groups via two-way ANOVA analysis followed by Tukey's multiple comparison test by GraphPad Prism 7.0 Software (USA)).