Enhancement of osseointegration of polyethylene terephthalate artificial ligament by coating of silk fibroin and depositing of hydroxyapatite

Abstract

Background: Application of artificial ligament in anterior cruciate ligament reconstruction is one of the research focuses of sports medicine but the biological tendon-bone healing still remains a problem. The preliminary study of hydroxyapatite (HAP) coating on the polyethylene terephthalate (PET) surface could effectively induce the osteoblast differentiation, but the tendon-bone healing was still not stable. As a green synthesis process, the biomimetic mineralization can simulate the natural bone growth in vitro and in vivo.

Methods: HAP crystals were grown under the guide of silk fibroin (SF) PET surface by biomimetic route. Several techniques including scanning electron microscopy, attenuated total reflectance Fourier transform infrared spectroscopy, X-ray diffraction, and energy-dispersive X-ray spectroscopy were utilized for proving the introduction of both SF and HAP. The viability and osseointegration of bone marrow stromal cells on the surface of three kinds of ligament, including PET group (non-coating group), PET+SF group (SF-coating group), and PET+SF+HAP group (combined HAP- and SF-coating group), were analyzed by CCK-8 assays and alkaline phosphatase (ALP) detection. Seventy-two mature male New Zealand rabbits were randomly divided into three groups. Among them, 36 rabbits were sacrificed for mechanical testing, and histological examination for the others.

Results: The SF and SF+HAP were successfully coated on the surface of PET fiber. The CCK-8 assay showed that the cell proliferation on PET+SF+HAP group was better than the other two groups from 24 to 120 hours. After 14 days of culture, the cells in the PET+SF+HAP group delivered higher levels of ALP than the other two groups. After 3 days of culture, the expression level of integrin β1 in the PET+SF+HAP group and PET+SF group were higher than in the PET group. The mean load to failure and the stiffness value of the PET+SF+HAP group were both higher than the other two groups. Hematoxylin and eosin staining showed that new bone tissue formation was only found in the PET+SF+HAP group 8 weeks postoperatively. Masson staining showed that in the PET+SF+HAP group 8 weeks postoperatively, the PET fibers were almost completely encircled by collagen. Histomorphometric analysis showed that the width of the graft-bone interface in the PET+SF+HAP group was narrower than that in the other two groups 4 and 8 weeks postoperatively. The mRNA level of BMP-7 in the PET+SF+HAP groups was significantly higher than those in the other two groups 4 and 8 weeks postoperatively.

Conclusion: The study showed that the combined SF and HAP coating by biomimetic route on the surface of PET artificial ligament could induce graft osseointegration in the bone tunnel, providing theoretical and experimental foundation for manufacturing novel artificial ligaments meeting the clinical needs.

Keywords: biomineralization; ligament reconstruction; tendon–bone healing.

Figure 1

Scanning electron microscopy observation of PET (A), PET+SF (B), and PET+SF+HAP (C); inset pictures are amplified. Three-dimensional topography of PET (D), PET+SF (E), and PET+SF+HAP (F), acquired by atomic force microscopy in tapping mode. Abbreviations: HAP, hydroxyapatite; PET, polyethylene terephthalate; SF, silk fibroin.

Figure 2

ATR-FTIR spectrum of three kinds of ligaments sample. Abbreviations: ATR-FTIR, attenuated total reflectance Fourier transform infrared; HAP, hydroxyapatite; PET, polyethylene terephthalate; SF, silk fibroin.

Figure 3

Energy-dispersive X-ray spectrum (A) and X-ray diffraction pattern (B) of PET+SF+HAP. Abbreviations: HAP, hydroxyapatite; PET, polyethylene terephthalate; SF, silk fibroin.

Figure 4

Measurement of BMSC proliferation, differentiation, and adhesion among the PET group, PET+SF group, and PET+SF+HAP group. Notes: (A) BMSC proliferation was measured by CCK-8 assays. (B) Cell differentiation was monitored by measuring the ALP activity. (C) Cell adhesion was assessed by measuring the expression level of integrin β1. *P<0.05; **P<0.01. Abbreviations: ALP, alkaline phosphatase; BMSC, bone marrow stromal cell; CCK, cell counting kit; HAP, hydroxyapatite; PET, polyethylene terephthalate; SF, silk fibroin.

Figure 5

Mechanical examinations for graft–bone healing in a rabbit model at each time point after surgery. Notes: (A) Comparison of maximal failure load among the PET group, PET+SF group, and SF+HAP group. (B) Comparison of stiffness at failure among the PET group, PET+SF group, and PET+SF+HAP group. **P<0.01. Abbreviations: HAP, hydroxyapatite; PET, polyethylene terephthalate; SF, silk fibroin.

Figure 6

Histological characterization of the PET group (A and D), PET+SF group (B and E), and PET+SF+HAP group (C and F) at 4 weeks (A, B, and C) and 8 weeks (D, E, and F) after surgery. Notes: The figure shows the interface between the host bone and the graft (H&E staining, ×200 magnification). In the PET+SF+HAP group 8 weeks after surgery, new bone growth into the interface was observed (as indicated by black arrows). Bar =200 μm. Abbreviations: G, artificial ligament graft; H&E, hematoxylin and eosin; HAP, hydroxyapatite; HB, host bone; IF, interface; PET, polyethylene terephthalate; SF, silk fibroin.

Figure 7

Collagen fiber status of the PET group (A and D), PET+SF group (B and E), and PET+SF+HAP group (C and F) at 4 weeks (A, B, and C) and 8 weeks (D, E, and F) after surgery. Notes: The figure shows the formation and infiltration of the collagen fibers and bone formation at the interface of the graft and the host bone (Masson staining, ×40 magnification). In the PET+SF+HAP group 8 weeks after surgery, plenty of collagen fibers and new bone (as indicated by black arrows) was found. Bar =500 μm. Abbreviations: G, artificial ligament graft; HAP, hydroxyapatite; HB, host bone; PET, polyethylene terephthalate; SF, silk fibroin.

Figure 8

Interface width between the host bone and the graft of the PET group, PET+SF group, and PET+SF+HAP group 4 and 8 weeks after surgery. Notes: *P<0.05; **P<0.01. Abbreviations: HAP, hydroxyapatite; PET, polyethylene terephthalate; SF, silk fibroin.

Figure 9

RT-PCR analysis shows mRNA expression levels of BMP-7 (A) and VEGF (B) 4 and 8 weeks after surgery. Notes: *P<0.05; **P<0.01. Abbreviations: mRNA, messenger RNA; RT-PCR, real-time polymerase chain reaction; VEGF, vascular endothelial growth factor.