A bioceramic scaffold composed of strontium-doped three-dimensional hydroxyapatite whiskers for enhanced bone regeneration in osteoporotic defects

Abstract

Reconstruction of osteoporotic bone defects is a clinical problem that continues to inspire the design of new materials. Methods: In this work, bioceramics composed of strontium (Sr)-doped hydroxyapatite (HA) whiskers or pure HA whiskers were successfully fabricated by hydrothermal treatment and respectively named SrWCP and WCP. Both bioceramics had similar three-dimensional (3D) porous structures and mechanical strengths, but the SrWCP bioceramic was capable of releasing Sr under physiological conditions. In an osteoporotic rat metaphyseal femoral bone defect model, both bioceramic scaffolds were implanted, and another group that received WCP plus strontium ranelate drug administration (Sr-Ran+WCP) was studied for comparison. Results: At week 1 post-implantation, osteogenesis coupled blood vessels were found to be more common in the SrWCP and Sr-Ran+WCP groups, with substantial vascular-like structures. After 12 weeks of implantation, comparable to the Sr-Ran+WCP group, the SrWCP group showed induction of more new bone formation within the defect as well as at the implant-bone gap region than that of the WCP group. Both the SrWCP and Sr-Ran+WCP groups yielded a beneficial effect on the surrounding trabecular bone microstructure to resist osteoporosis-induced progressive bone loss. While an abnormally high blood Sr ion concentration was found in the Sr-Ran+WCP group, SrWCP showed little adverse effect. Conclusion: Our results collectively suggest that the SrWCP bioceramic can be a safe bone substitute for the treatment of osteoporotic bone defects, as it promotes local bone regeneration and implant osseointegration to a level that strontium ranelate can achieve.

Keywords: bone regeneration; calcium phosphate bioceramics; osteoporosis; strontium; whiskerization..

Figure 1

Synthesis and physiochemical properties of the WCP and SrWCP bioceramics. (A) The schematic diagram of the fabrication process for the WCP and SrWCP bioceramics. (B) Scanning electron images of the WCP and SrWCP bioceramics. (C) The porosity, mean pore diameter, specific surface area and maximum load of the WCP and SrWCP bioceramics.

Figure 2

Phase composition analysis of the WCP and SrWCP bioceramics. (A) EDS mapping for the major elements of the SrWCP bioceramics. (B) XRD spectra of the WCP and SrWCP bioceramics. Standard spectra of hydroxyapatite was provided below. (C) Comparison XPS spectra (including characteristic peaks of Ca2p, P2s, O1s and Sr3p) of the WCP and SrWCP bioceramics.

Figure 3

In vitro degradation rates of the WCP and SrWCP bioceramics and the viability of cells cultured on the WCP and SrWCP bioceramics. (A) Time-dependent change of Ca, P and Sr ionic concentrations released from the WCP and SrWCP bioceramics after immersion into in Tris-HCl buffer solution (pH = 7.4) and citric acid buffer solution (pH = 3.0): *p < 0.05 vs the WCP group; **p < 0.01 vs the WCP group. (B) FDA/PI staining of MSCs growth on the different bioceramics at days 1, 3 and 7.

Figure 4

In vivo assessment of the WCP and SrWCP bioceramics. (A) The timeline of the study to evaluate the WCP and SrWCP bioceramics with Sprague-Dawley rats. (B) Time-dependent changes of the body weights, serum ion concentrations (strontium, calcium) and serum levels of the bone formation (PINP, osteocalcin) and resorption (CTX-I) biomarkers (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 5

Micro-CT rendered images and data of the bone formation. (A) Reconstructed micro-CT images of the coronal sections from the metaphyseal femur at weeks 1, 8 and 12. The materials (white) were embedded in newly formed bone (grey) after implantation. Right corner of each sample: 3D reconstructed images of the newly formed bone inside the gap (100 μm) between the implants and host bone (scale bar = 2 mm). (B) Quantitative analysis of micro-CT data: the new bone formation (nBV, mm³) within the drilled hole (nTV, mm³) and the volume of circular new bone formation (cBV, mm³) within the circular gap (cTV, mm³). The original volume of each material (MV) was measured before implantation and the volume of the remaining material (RMV) within the bone defect at each time point can be calculated. Thus, the degraded material (DV) is equal to MV minus RMV. The bone ingrowth rate (nBV/nTV), bone-implant osseointegration rate (cBV/cTV) and bone substitution rate (nBV/DV) were then obtained, respectively (*p < 0. 05, **p < 0.01).

Figure 6

Evaluation for the effect of the implantation on the trabecular bone adjacent to the implants. (A) The reconstruction of the transverse sections from the femur samples and the trabecular bone of a cubic volume of interest (VOI) adjacent to the defect (scale bar = 2 mm). (B) Trabecular microarchitecture parameters of VOI, including bone mineral density (BMD, mg HA/ccm), bone volume fraction (BV/TV, %), specific bone surface (BS/BV, mm^-1), trabecular number (Tb.N, mm^-1), trabecular thickness (Tb.Th, mm) and trabecular separation (Tb.Sp, mm) (*p < 0.05, ** p < 0.01).

Figure 7

Evaluation of the in vivo angiogenesis, osteogenesis and osteoclastogenesis after implantation for 1 week. (A) H&E histological images of the bioceramics with the infiltrated tissue: the first row: general view of the section; the second row: magnification of the center area of the bioceramics from each group. (B) Immunofluorescence staining: Green fluorescence indicates active CD31; red fluorescence indicates active Emcn, TRAP and OCN respectively; blue fluorescence indicates PI bound to the nuclei of cells. Quantitative analyses of the mean fluorescence intensity values of CD31, Emcn and TRAP were provided below (*p < 0.05 vs the WCP group; **p < 0.01 vs the WCP group).

Figure 8

Histological staining for the bone regeneration and elemental analysis at the bone-bioceramics interface. (A) Hematoxylin and eosin (H&E) staining of the different groups at week 8 and 12. The magnified images showed osteocyte residence and new bone formation on the wall of the pores. (B) EDS mapping for the major elements at the tissue-implants interface (yellow box in the upper row) and composition determination of the new bone adjacent to the implants (*p < 0.05, **p < 0.01).

Figure 9

Nanoindentation tests on bone and material: representative loading-unloading curve, average elastic modulus of the newly formed bone (E_b), the hardness of the newly formed bone (H_b), the average elastic modulus of the undegraded material (E_m) and the hardness of the undegraded material (H_m). Optical images of the indentation residues of each group were obtained (*p < 0.05, **p < 0.01). Optical images of the indentation residues of each group were shown below.