Enhanced osseointegration of three-dimensional supramolecular bioactive interface through osteoporotic microenvironment regulation

Abstract

Purpose: Osteoporosis is more likely to cause serious complications after joint replacement, mainly due to physiological defects of endogenous osteogenic cells and the pathological osteoclast activity. It is a feasible solution to design a prosthetic surface interface that specifically addresses this troublesome situation. Methods: A novel "three-dimensional (3D) inorganic-organic supramolecular bioactive interface" was constructed consisting of stiff 3D printing porous metal scaffold and soft multifunctional, self-healable, injectable, and biodegradable supramolecular polysaccharide hydrogel. Apart from mimicking the bone extracellular matrix, the bioactive interface could also encapsulate bioactive substances, namely bone marrow mesenchymal stem cells (BMSCs) and bone morphogenetic protein-2 (BMP-2). A series of in vitro characterizations, such as topography and mechanical characterization, in vitro release of BMP-2, biocompatibility analysis, and osteogenic induction of BMSCs were carried out. After that, the in vivo osseointegration effect of the bioactive interface was investigated in detail using an osteoporotic model. Results: The administration of injectable supramolecular hydrogel into the inner pores of 3D printing porous metal scaffold could obviously change the morphology of BMSCs and facilitate its cell proliferation. Meanwhile, BMP-2 was capable of being sustained released from supramolecular hydrogel, and subsequently induced osteogenic differentiation of BMSCs and promoted the integration of the metal microspores-bone interface in vitro and in vivo. Moreover, the osteoporosis condition of bone around the bioactive interface was significantly ameliorated. Conclusion: This study demonstrates that the 3D inorganic-organic supramolecular bioactive interface can serve as a novel artificial prosthesis interface for various osteogenesis-deficient patients, such as osteoporosis and rheumatoid arthritis.

Keywords: bioactive interface; bone morphogenetic protein 2; osseointegration; osteoporotic microenvironment; supramolecular hydrogel.

Scheme 1

pTi filled with BMSCs and BMP-2 dual-loaded supramolecular hydrogels as bioactive composite scaffolds for enhancing osteoporotic bone defect osseointegration. BMP-2 can promote osteogenic differentiation of exogenous BMSCs and endogenous BMSCs. With the degradation of hydrogel, bone tissue grows into the pores of the pTi scaffold, thus achieving good osseointegration.

Figure 1

(A) Bioactive interface constructed by BMSCs and BMP-2 loaded hydrogel filled the pTi in a cell culture plate; (B) Representative optical image and 3D micro-CT image of pTi; (C) Mixed hydrogel and gelation; SEM images of (D) pTi, (E) supramolecular hydrogel, and (F) pTi filled with hydrogel.

Figure 2

(A) Calcein AM/PI staining of live cells (green) and dead cells (red), and fluorescent imaging with rhodamine-DAPI staining of Con, S, SG, and SGB groups; (B) Quantitative analysis of cell survival rate by Calcein AM/PI staining; (C) Cell proliferation within the different scaffolds at 1, 7, and 14 d (*p < 0.05, **p < 0.01).

Figure 3

Evaluation of the osteogenic differentiation of BMSCs seeding in the Con, S, SG, and SGB groups. (A) Images of different samples after staining with alizarin red S; (B) Statistical analysis of semi-quantitative analysis of alizarin red staining; (C-F) The expressions of osteogenic-related genes, such as ALP, RUNX-2, OCN, and COL-1 (*p < 0.05, **p < 0.01).

Figure 4

Immunohistochemical staining and quantitative analysis of osteogenic related proteins in BMSCs cultured in different groups for 14 d, namely (A, B) ALP, (C, D) Runx-2, (E, F) OCN, and (G, H) COL-1 (*p < 0.05, **p < 0.01).

Figure 5

(A) Visual appearance and (B) radiographic images of the distal femurs; (C) 3D reconstruction images of ROI (bone was yellow and scaffolds were white); Quantitative analysis of (D) BV/TV, (E) Tb.Th, (F) Tb.Sp, and (G) Tb.N of the S, SG, SGC, SGB, and SGCB groups derived from micro-CT (*p < 0.05, **p < 0.01).

Figure 6

(A) Representative histological photos in the region of bone defect by Van-Gieson staining (The black areas stand for titanium alloys and the red areas stand for bones); (B) The ratio of regenerated bone area to total defect area (BA/TA) was analyzed using Image Pro Plus 6.0; (C) Evaluation of osseointegration through pull-out biomechanical testing after implantation for 3 months (*p < 0.05, **p < 0.01).