Evaluation of zinc-doped mesoporous hydroxyapatite microspheres for the construction of a novel biomimetic scaffold optimized for bone augmentation

Abstract

Biomaterials with high osteogenic activity are desirable for sufficient healing of bone defects resulting from trauma, tumor, infection, and congenital abnormalities. Synthetic materials mimicking the structure and composition of human trabecular bone are of considerable potential in bone augmentation. In the present study, a zinc (Zn)-doped mesoporous hydroxyapatite microspheres (Zn-MHMs)/collagen scaffold (Zn-MHMs/Coll) was developed through a lyophilization fabrication process and designed to mimic the trabecular bone. The Zn-MHMs were synthesized through a microwave-hydrothermal method by using creatine phosphate as an organic phosphorus source. Zn-MHMs that consist of hydroxyapatite nanosheets showed relatively uniform spherical morphology, mesoporous hollow structure, high specific surface area, and homogeneous Zn distribution. They were additionally investigated as a drug nanocarrier, which was efficient in drug delivery and presented a pH-responsive drug release behavior. Furthermore, they were incorporated into the collagen matrix to construct a biomimetic scaffold optimized for bone tissue regeneration. The Zn-MHMs/Coll scaffolds showed an interconnected pore structure in the range of 100-300 μm and a sustained release of Zn ions. More importantly, the Zn-MHMs/Coll scaffolds could enhance the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells. Finally, the bone defect repair results of critical-sized femoral condyle defect rat model demonstrated that the Zn-MHMs/Coll scaffolds could enhance bone regeneration compared with the Coll or MHMs/Coll scaffolds. The results suggest that the biomimetic Zn-MHMs/Coll scaffolds may be of enormous potential in bone repair and regeneration.

Keywords: biomimicry; bone regeneration; drug delivery; mesoporous hydroxyapatite microspheres; scaffold; zinc.

Figure 1

SEM (top and middle rows) and TEM (bottom row) images: (A–C) MHMs, (D–F) Zn2-MHMs, (G–I) Zn5-MHMs. Inset in (H) shows the EDS element mapping for the distribution of Zn, Ca, P, and O in Zn5-MHMs. Abbreviations: SEM, scanning electron microscopy; TEM, transmission electron microscopy; MHM, mesoporous hydroxyapatite microsphere; EDS, energy dispersive spectroscopy.

Figure 2

Physical characteristics of the MHMs, Zn2-MHMs, and Zn5-MHMs. (A) XRD patterns. (B) Nitrogen adsorption-desorption isotherms. (C) BJH pore size distributions. Abbreviations: MHM, mesoporous hydroxyapatite microsphere; XRD, X-ray diffraction; BJH, Barrett-Joyner-Halenda; dV/dD, d(volume adsorbed)/d(diameter).

Figure 3

Drug release curves of (A) DOX-loaded MHMs and (B) DOX-loaded Zn5-MHMs in PBS solutions at different pH values. Cumulative release of DOX from (C) MHMs and (D) Zn5-MHMs as a function of the square root of the release time. Abbreviations: DOX, doxorubicin hydrochloride; MHM, mesoporous hydroxyapatite microsphere; PBS, phosphate-buffered solution.

Figure 4

Physical characteristics of the Coll, MHMs/Coll, Zn2-MHMs/Coll, and Zn5-MHMs/Coll scaffolds. (A) SEM micrographs of the Coll (a–c), MHMs/Coll (d–f), Zn2-MHMs/Coll (g–i), and Zn5-MHMs/Coll scaffolds (j–l). (B) Release of Zn ions from the MHMs/Coll, Zn2-MHMs/Coll, and Zn5-MHMs/Coll scaffolds. Abbreviations: MHMs/Coll, mesoporous hydroxyapatite microspheres/collagen scaffold; SEM, scanning electron microscopy.

Figure 5

Cell morphology observation and viability assay on the Coll, MHMs/Coll, Zn2-MHMs/Coll, and Zn5-MHMs/Coll scaffolds. (A and B) CLSM and SEM images of rBMSCs on each type of scaffold after 7 days of culture. (C) Quantitative evaluation of the viability of rBMSCs on the four types of scaffolds at days 1, 3, and 7. *P<0.05 compared to Coll scaffolds. Abbreviations: MHMs/Coll, mesoporous hydroxyapatite microspheres/collagen scaffold; CLSM, confocal laser scanning microscope; SEM, scanning electron microscopy; rBMSC, rat bone marrow–derived mesenchymal stem cell.

Figure 6

RT-qPCR analysis of the expression of osteogenesis-related genes (Runx2, Alp, and Ocn) in rBMSCs cultured on the Coll, MHMs/Coll, Zn2-MHMs/Coll, and Zn5-MHMs/Coll scaffolds for 7 and 14 days. *P<0.05 compared to Coll scaffolds; ^#P<0.05 compared to MHMs/Coll scaffolds; ^%P<0.05 compared to Zn2-MHMs/Coll scaffolds. Abbreviations: RT-qPCR, real-time quantitative polymerase chain reaction; MHMs/Coll, mesoporous hydroxyapatite microspheres/collagen scaffold; rBMSC, rat bone marrow–derived mesenchymal stem cell.

Figure 7

Micro-CT analysis of bone regeneration for the Coll, MHMs/Coll, and Zn5-MHMs/Coll groups at 8 weeks postimplantation. (A) 3D reconstructed superficial and interior images of femoral condyle defects implanted with different scaffolds. Morphometric analysis of the BV/TV (B), Tb.N (C), Tb.Sp (D), and Tb.Th (E) for each group. *P<0.05 compared to Coll group; ^#P<0.05 compared to MHMs/Coll group. Abbreviations: micro-CT, micro-computed tomography; MHMs/Coll, mesoporous hydroxyapatite microspheres/collagen scaffold; 3D, three dimensional; BV/TV, bone volume to total volume ratio; Tb.N, trabecular number; Tb.Sp, trabecular spacing; Tb.Th, trabecular thickness.

Figure 8

Histological evaluation of bone regeneration in each group. (A) HE staining for newly formed bone (black arrowhead), connective tissue (blue arrowhead), and scaffold remnants (green arrowhead). (B) The percentage of the new bone area assessed by histomorphometric analysis. *P<0.05 compared to Coll group; ^#P<0.05 compared to MHMs/Coll group. Abbreviations: MHMs/Coll, mesoporous hydroxyapatite microspheres/collagen scaffold; HE, hematoxylin and eosin.