Comparative study of porous hydroxyapatite/chitosan and whitlockite/chitosan scaffolds for bone regeneration in calvarial defects

Abstract

Hydroxyapatite (HAP; Ca₁₀(PO₄)₆(OH)₂) and whitlockite (WH; Ca₁₈Mg₂(HPO₄)₂(PO₄)₁₂) are widely utilized in bone repair because they are the main components of hard tissues such as bones and teeth. In this paper, we synthesized HAP and WH hollow microspheres by using creatine phosphate disodium salt as an organic phosphorus source in aqueous solution through microwave-assisted hydrothermal method. Then, we prepared HAP/chitosan and WH/chitosan composite membranes to evaluate their biocompatibility in vitro and prepared porous HAP/chitosan and WH/chitosan scaffolds by freeze drying to compare their effects on bone regeneration in calvarial defects in a rat model. The experimental results indicated that the WH/chitosan composite membrane had a better biocompatibility, enhancing proliferation and osteogenic differentiation ability of human mesenchymal stem cells than HAP/chitosan. Moreover, the porous WH/chitosan scaffold can significantly promote bone regeneration in calvarial defects, and thus it is more promising for applications in tissue engineering such as calvarial repair compared to porous HAP/chitosan scaffold.

Keywords: chitosan; hydroxyapatite; osteogenic differentiation; rat critical calvarial defect; tissue engineering; whitlockite.

Figure 1

XRD patterns (A and E), SEM micrographs (B and F), TEM micrographs (C and G) and DLS size distributions (D and H) of HAP (A–D) and WH (E–H) porous hollow microspheres that were prepared by using creatine phosphate disodium salt as an organic phosphorus source through the microwave-assisted hydrothermal method at 120°C for 10 min. Insets of (C) and (G) are selected-area electron diffraction patterns. Abbreviations: XRD, X-ray diffraction; SEM, scanning electron microscope; TEM, transmission electron microscopy; DLS, dynamic light scattering; WH, whitlockite; HAP, hydroxyapatite.

Figure 2

Optical photographs (first column) and SEM micrographs (middle and last columns) of the HAP/chitosan (top row) and WH/chitosan (bottom row) composite membranes prepared by drying at 37°C for 24 h. Abbreviations: SEM, scanning electron microscope; WH, whitlockite; HAP, hydroxyapatite.

Figure 3

Optical photographs (A and E), SEM micrographs (B and F), EDS element mapping (C and G), and pore size statistical analysis (D and H) of the porous HAP/chitosan (A–D), and WH/chitosan (E–H) scaffolds prepared by freeze drying. Abbreviations: SEM, scanning electron microscope; WH, whitlockite; HAP, hydroxyapatite; EDS, energy dispersive spectrometer.

Figure 4

The amounts of HAP and WH dissolved (A) and the cumulative release amount of Mg (B) from porous HAP/chitosan and WH/chitosan scaffolds after being immersed in physiological saline at different times. The amounts of HAP and WH were calculated according to the Ca²⁺ ions released into the physiological saline and measured by ICP analysis. No Mg release could be detected from HAP/chitosan. Abbreviations: WH, whitlockite; HAP, hydroxyapatite; ICP, inductively coupled plasma.

Figure 5

SEM images of hBMSCs on the surface of HAP/chitosan (A) and WH/chitosan (B) scaffolds after culturing for 3 days. Abbreviations: WH, whitlockite; HAP, hydroxyapatite; SEM, scanning electron microscope; hBMSCs, human mesenchymal stem cells.

Figure 6

Cell proliferation of hBMSCs cultured on HAP/chitosan and WH/chitosan membranes and in control membrane at different times. Data are expressed as mean ± SD. *Significant difference between HAP/chitosan and control (P<0.05); **significant difference between WH/chitosan and HAP/chitosan or the control (P<0.05). Abbreviations: WH, whitlockite; HAP, hydroxyapatite; SD, standard deviation; hBMSCs, human mesenchymal stem cells.

Figure 7

Quantitative ALP activity of hBMSCs cultured on WH/chitosan and HAP/chitosan membranes for 7 and 14 days. Data are expressed as mean ± SD. *Significant difference between HAP/chitosan and control (P<0.05); **significant difference between WH/chitosan and HAP/chitosan or the control (P<0.05). Abbreviations: ALP, alkaline phosphatase; WH, whitlockite; HAP, hydroxyapatite; SD, standard deviation; hBMSCs, human mesenchymal stem cells.

Figure 8

Osteogenic gene expression of collagen 1, ALP, Runx2, and OCN after hBMSCs were cultured on WH/chitosan and HAP/chitosan membranes for 7 days. Data are expressed as mean ± SD. *Significant difference between HAP/chitosan and control (P<0.05); **significant difference between WH/chitosan and HAP/chitosan or the control (P<0.05). Abbreviations: ALP, alkaline phosphatase; WH, whitlockite; HAP, hydroxyapatite; SD, standard deviation; hBMSCs, human mesenchymal stem cells; OCN, osteocalcin.

Figure 9

Western blot analysis of protein expression of Runx2 in hBMSCs cultured on the control, WH/chitosan, and HAP/chitosan membranes for 7 days. Abbreviations: WH, whitlockite; HAP, hydroxyapatite; hBMSCs, human mesen-chymal stem cells.

Figure 10

Micro-CT measurement of bone formation in the rat calvarial defects implanted with HAP/chitosan and WH/chitosan scaffold at 8 weeks after implantation. (A) Top and cross-sectional views of reconstructed images; red arrows point to the HAP/chitosan and WH/chitosan scaffold. (B, C) BMD and BV/TV in the defects implanted with the scaffolds. Mean ± SD; n=3. *Significant difference between groups (P<0.05). Abbreviations: CT, computed tomography; WH, whitlockite; HAP, hydroxyapatite; hBMSCs, human mesenchymal stem cells; BMD, bone mineral density; BV/TV, bone volume to total bone volume.

Figure 11

New bone formation and mineralization measured histomorphometrically by fluorochrome-labeling analysis and Van Gieson’s picrofuchsin in rat calvarial defects implanted with HAP/chitosan and WH/chitosan. Tetracycline (yellow), alizarin red (red), and calcein (green) were intraperitoneally injected at week 2, week 4, and week 6, respectively. Column 4 represents merged images of the three fluorochromes of the same sample and column 5 represents Van Gieson’s picrofuchsin as a comparison. Abbreviations: WH, whitlockite; HAP, hydroxyapatite.

Figure 12

H&E staining and immunohistochemical staining of OCN in the defects implanted with HAP/chitosan and WH/chitosan scaffolds at 8 weeks post-implantation. A higher number of new bone were found in WH/chitosan. The OCN (stained brown) secreted from the new bone was also greater in WH/chitosan. Abbreviations: WH, whitlockite; HAP, hydroxyapatite; H&E, hematoxylin and eosin; OCN, osteocalcin.