Preparation, characterization, and in vitro osteoblast functions of a nano-hydroxyapatite/polyetheretherketone biocomposite as orthopedic implant material

Abstract

A bioactive composite was prepared by incorporating 40 wt% nano-hydroxyapatite (nHA) into polyetheretherketone (PEEK) through a process of compounding, injection, and molding. The mechanical and surface properties of the nHA/PEEK composite were characterized, and the in vitro osteoblast functions in the composite were investigated. The mechanical properties (elastic modulus and compressive strength) of the nHA/PEEK composite increased significantly, while the tensile strength decreased slightly as compared with PEEK. Further, the addition of nHA into PEEK increased the surface roughness and hydrophilicity of the nHA/PEEK composite. In cell tests, compared with PEEK and ultra-high-molecular-weight polyethylene, it was found that the nHA/PEEK composite could promote the functions of MC3T3-E1 cells, including cell attachment, spreading, proliferation, alkaline phosphatase activity, calcium nodule formation, and expression of osteogenic differentiation-related genes. Incorporation of nHA into PEEK greatly improved the bioperformance of PEEK. The nHA/PEEK composite might be a promising orthopedic implant material.

Keywords: biocomposite; nano-hydroxyapatite; orthopedic implant material; osteoblast functions; polyetheretherketone.

Figure 1

Transmission electron microscope image of nano-hydroxyapatite.

Figure 2

Scanning electron microscope image (A) and X-ray diffraction pattern (B) of the nano-hydroxyapatite (nHA)/polyetheretherketone (PEEK) composite.

Figure 3

Water contact angles of the nano-hydroxyapatite/polyetheretherketone (nHA/PEEK) composite and polyetheretherketone (PEEK). Note: **Significant difference compared with PEEK (P<0.01). Abbreviations: PEEK, polyetheretherketone; nHA/PEEK, nano-hydroxyapatite/polyetheretherketone.

Figure 4

Cell attachment on the samples assessed by cell counting kit-8 assay. Notes: *Significant difference compared with PEEK (P<0.05); ^#significant difference compared with UHMWPE (P<0.05). Abbreviations: nHA/PEEK, nano-hydroxyapatite/polyetheretherketone; OD, optical density; PEEK, polyetheretherketone; UHMWPE, ultra-high-molecular-weight polyethylene.

Figure 5

Cell spreading on the samples at 24 hours, as observed by (A) confocal laser scanning microscope and (B) scanning electron microscope. Note: Arrows indicate cells. Abbreviations: nHA/PEEK, nano-hydroxyapatite/polyetheretherketone; PEEK, polyetheretherketone; UHMWPE, ultra-high-molecular-weight polyethylene.

Figure 6

Cell proliferation on the samples assessed by cell counting kit-8 assay. Notes: (A) proliferative tendency from 1 to 7 days; (B) relative proliferation rate. The modified optical densities at 3 and 7 days were normalized to the values at 1 day. *Significant difference compared with PEEK (P<0.05); ^#significant difference compared with UHMWPE (P<0.05). Abbreviations: nHA/PEEK, nano-hydroxyapatite/polyetheretherketone; OD, optical density; PEEK, polyetheretherketone; UHMWPE, ultra-high-molecular-weight polyethylene.

Figure 7

Cell proliferation in the samples assessed by 4,6-diamidino-2-phenylindole (DAPI) staining. Notes: (A) MC3T3-E1 cells stained with DAPI after 1, 3, and 7 days; (B) quantitative analysis of DAPI staining. **Significant difference compared with PEEK (P<0.01); ^##significant difference compared with UHMWPE (P<0.01); scale bar =50 μm. Abbreviations: nHA/PEEK, nano-hydroxyapatite/polyetheretherketone; PEEK, polyetheretherketone; UHMWPE, ultra-high-molecular-weight polyethylene.

Figure 8

Scanning electron microscope images of MC3T3-E1 cells on the samples at 3 and 7 days. Abbreviations: nHA/PEEK, nano-hydroxyapatite/polyetheretherketone; PEEK, polyetheretherketone; UHMWPE, ultra-high-molecular-weight polyethylene.

Figure 9

Alkaline phosphatase (ALP) staining and quantification of ALP activity on the samples after osteogenic induction for 7, 10, and 14 days. Notes: (A) ALP staining on the samples; (B) quantitative analysis of ALP activity. ALP activity was normalized to the corresponding total protein content. *Significant difference compared with PEEK (P<0.05); **significant difference compared with PEEK (P<0.01); ^##significant difference compared with UHMWPE (P<0.01). Abbreviations: nHA/PEEK, nano-hydroxyapatite/polyetheretherketone; PEEK, polyetheretherketone; UHMWPE, ultra-high-molecular-weight polyethylene.

Figure 10

Alizarin-red staining and quantitative analysis of mineralization on the samples. Notes: (A) samples, stained samples without cells, and stained samples with cells at 21 and 28 days; (B) colorimetric quantification of the extracellular matrix mineralization at 21 and 28 days. **Significant difference compared with PEEK (P<0.01); ^##significant difference compared with UHMWPE (P<0.01). Abbreviations: OD, optical density; nHA/PEEK, nano-hydroxyapatite/polyetheretherketone; PEEK, polyetheretherketone; UHMWPE, ultra-high-molecular-weight polyethylene.

Figure 11

Expression of osteogenic differentiation-related genes of MC3T3-E1 cells on the samples assessed by real-time polymerase chain reaction. Notes: (A) ALP, (B) COL1, (C) OPN, and (D) OC. The data shown are the mean expression relative to β-actin expression and are normalized with respect to the expression on PEEK at 7 days. *Significant difference compared with PEEK (P<0.05); **significant difference compared with PEEK (P<0.01); ^#significant difference compared with UHMWPE (P<0.05); ^##significant difference compared with UHMWPE (P<0.01). Abbreviations: ALP, alkaline phosphatase; COL1, type I collagen; mRNA, messenger ribonucleic acid; nHA/PEEK, nano-hydroxyapatite/polyetheretherketone; OPN, osteoprotein; OC, osteocalcin; PEEK, polyetheretherketone; UHMWPE, ultra-high-molecular-weight polyethylene.