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. 2022 Feb 28:10:831288.
doi: 10.3389/fbioe.2022.831288. eCollection 2022.

Fabrication of In Situ Grown Hydroxyapatite Nanoparticles Modified Porous Polyetheretherketone Matrix Composites to Promote Osteointegration and Enhance Bone Repair

Ningning Wang  1 , Desheng Qi  2 , Lu Liu  3 , Yanlin Zhu  4 , Hong Liu  5 , Song Zhu  1
Affiliations

Fabrication of In Situ Grown Hydroxyapatite Nanoparticles Modified Porous Polyetheretherketone Matrix Composites to Promote Osteointegration and Enhance Bone Repair

Ningning Wang et al. Front Bioeng Biotechnol. .

Abstract

The repairment of critical-sized bone defects is a serious problem that stimulates the development of new biomaterials. In this study, nanohydroxyapatite (nHA)-doped porous polyetheretherketone (pPEEK) were successfully fabricated by the thermally induced phase separation method and hydrothermal treatment. Structural analysis was performed by X-ray diffraction. The water contact angles and scanning electron microscopy were measured to assess physical properties of surfaces. The mechanical strength of the composites is also determined. Microcomputed tomography is used to characterize the nHA content of the composites. The in vitro bioactivity of the composites with or without nHA was investigated by using murine pre-osteoblasts MC3T3-E1, and the results of cytotoxicity and cell proliferation assays revealed that the cytocompatibility of all specimens was good. Adherence assays were employed to examine the adhesion and morphology of cells on different materials. However, nHA-doped composites induced cell attachment and cell spreading more significantly. Osteogenic differentiation was investigated using alkaline phosphatase activity and alizarin red staining, and these in vitro results demonstrated that composites containing nHA particles enhanced osteoblast differentiation. Its effectiveness for promoting osteogenesis was also confirmed in an in vivo animal experiment using a tibial defective rat model. After 8 weeks of implantation, compared to the pure PEEK and pPEEK without nHA groups, the nHA-pPEEK group showed better osteogenic activity. The results indicate that the nHA-pPEEK composites are possibly a well-designed bone substitute for critical-sized bone defects by promoting bone regeneration and osteointegration successfully.

Keywords: animal model; differentiation; nanohydroxyapatite; osteointegration; polyetheretherketone.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

SCHEME 1
SCHEME 1
Design of the experiment. (A) Schematic diagram of synthesis process, including the Po-PEEK with thermally induced phase separation and nHA with hydrothermal methods. (B) Implantation procedure of rat tibial defect model and cellular responses around the host bones after the operation.
FIGURE 1
FIGURE 1
Characterization. (A) 3D schematic of the implant material (B,C) SEM images of porous materials with different magnification. (D) Density and porosity of all groups with different solid content and compressive degrees. (E) Mechanical strength of all groups with different solid content and compressive degrees. (F) Micro-CT image of the material in the PK50-20% group. (G) SEM picture of synthetic nHA. (H) XRD diffractogram for samples of synthetic nHA. (I) The water contact angle analysis of the different samples.
FIGURE 2
FIGURE 2
Biocompatibility of prepared materials. (A) Live/dead cell staining for evaluation of different extracts from all groups. (B) Cell cytotoxicity quantitative measurement by the MTT assay. (C) Analysis of cell proliferation on different surfaces of all groups. (D) Seeding efficiency for MC3T3-E1 cells on the different materials. (E) Cell morphology in SEM of MC3T3-E1 osteoblast cells after co-culture with different samples. (F) Fluorescent staining of cells with FITC-phalloidin (actin cytoskeleton, green) and DAPI (nucleus, blue).
FIGURE 3
FIGURE 3
Osteogenic differentiation of MC3T3-E1 cells. (A) ALP staining assays of cells on the different materials after 7 and 14 days co-culture time. (B) ALP activity in cell lysates collected from cells co-cultured with different materials. (C) ARS staining of cells on the different materials. (D) Quantification of ARS staining in (C).
FIGURE 4
FIGURE 4
In vivo analysis on osseointegration. (A) Three-dimensional images obtained from μCT reconstruction. Overall pattern of tibia (upper); new bone in the defect part (lower). (B) BV/TV, and (C) Tb. N of the defective aera calculated using accessory software. (D) Pull-out strength of the implanted materials in all groups, having different effects on osseointegration. (E) The maximum values of pull-out load taken for quantitative analysis. (F) Quantitative analyses of phosphorus and calcium. (G) The surface composition of each sample analysis by SEM-EDX system, including SEM images and distribution maps of the elements of carbon, oxygen, phosphorus, and calcium.
FIGURE 5
FIGURE 5
Histological analysis. (A) Representative displays of hematoxylin–eosin (HE) staining of tibia tissue sections, revealing that nHA-pPEEK composites markedly promoted new bone formation (black arrows). (B) Results of MTC staining for the collagenous fiber distinguished as blue (black arrows), showing that the experimental group had a large number of blue collagen fibrils and fibrous tissue. (C) Results of BMP-2 protein immunolocalization via immunohistochemistry staining to make a successful differential diagnosis (black arrows). (D) OCN immunolocalization via immunohistochemistry staining to make a successful differential diagnosis (black arrows).
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