Surface modification of vascular endothelial growth factor-loaded silk fibroin to improve biological performance of ultra-high-molecular-weight polyethylene via promoting angiogenesis

Abstract

Ultra-high-molecular-weight polyethylene (UHMWPE) has been applied in orthopedics, as the materials of joint prosthesis, artificial ligaments, and sutures due to its advantages such as high tensile strength, good wear resistance, and chemical stability. However, postoperative osteolysis induced by UHMWPE wear particles and poor bone-implant healing interface due to scarcity of osseointegration is a significant problem and should be solved imperatively. In order to enhance its affinity to bone tissue, vascular endothelial growth factor (VEGF) was loaded on the surface of materials, the loading was performed by silk fibroin (SF) coating to achieve a controlled-release delivery. Several techniques including field emission scanning electron microscopy (FESEM) and attenuated total reflectance (ATR)-Fourier transform infrared (FTIR) and water contact angle measurement were used to validate the effectiveness of introduction of SF/VEGF. The result of ELISA demonstrated that the release of VEGF was well maintained up to 4 weeks. The modified UHMWPE was evaluated by both in vitro and in vivo experiments. According to the results of FESEM and cell counting kit-8 (CCK-8) assay, bone marrow mesenchymal stem cells cultured on the UHMWPE coated with SF/VEGF and SF exhibited a better proliferation performance than that of the pristine UHMWPE. The model rabbit of anterior cruciate ligament reconstruction was used to observe the graft-bone healing process in vivo. The results of histological evaluation, microcomputed tomography (micro-CT) analysis, and biomechanical tests performed at 6 and 12 weeks after surgery demonstrated that graft-bone healing could be significantly improved due to the effect of VEGF on angiogenesis, which was loaded on the surface by SF coating. This study showed that the method loading VEGF on UHMWPE by SF coating played an effective role on the biological performance of UHMWPE and displayed a great potential application for anterior cruciate ligament reconstruction.

Keywords: UHMWPE; VEGF; graft–bone healing; silk fibroin; surface modification.

Figure 1

Schematic preparation of SF/VEGF coating and animal experiment model. Abbreviations: SF, silk fibroin; UHMWPE, ultra-high-molecular-weight polyethylene; VEGF, vascular endothelial growth factor.

Figure 2

Characterizations of the UHMWPE group, the UHMWPE–SF group, and the UHMWPE–SF/VEGF group. Notes: Scanning electron microscopy observation of UHMWPE (A), UHMWPE–SF (C), and UHMWPE–SF/VEGF (E). Partial magnifications of the black rectangle area are displayed in the right column (B, D, and F). (G) ATR-FTIR spectrum of UHMWPE (a), UHMWPE–SF (b), and UHMWPE–SF/VEGF (c), UHMWPE–SF after immersion for 24 h (d), and UHMWPE–SF/VEGF after immersion for 24 h (e). (H) Water contact angle of three groups. Water contact angle images of UHMWPE (a), UHMWPE–SF (b), and UHMWPE–SF/VEGF (c). ^**P<0.01. (I) Tensile strength of three groups. Abbreviations: ATR-FTIR, attenuated total reflectance Fourier transform infrared; SF, silk fibroin; UHMWPE, ultra-high-molecular-weight polyethylene; VEGF, vascular endothelial growth factor.

Figure 2

Characterizations of the UHMWPE group, the UHMWPE–SF group, and the UHMWPE–SF/VEGF group. Notes: Scanning electron microscopy observation of UHMWPE (A), UHMWPE–SF (C), and UHMWPE–SF/VEGF (E). Partial magnifications of the black rectangle area are displayed in the right column (B, D, and F). (G) ATR-FTIR spectrum of UHMWPE (a), UHMWPE–SF (b), and UHMWPE–SF/VEGF (c), UHMWPE–SF after immersion for 24 h (d), and UHMWPE–SF/VEGF after immersion for 24 h (e). (H) Water contact angle of three groups. Water contact angle images of UHMWPE (a), UHMWPE–SF (b), and UHMWPE–SF/VEGF (c). ^**P<0.01. (I) Tensile strength of three groups. Abbreviations: ATR-FTIR, attenuated total reflectance Fourier transform infrared; SF, silk fibroin; UHMWPE, ultra-high-molecular-weight polyethylene; VEGF, vascular endothelial growth factor.

Figure 3

VEGF release profile of the UHMWPE–SF/VEGF group and cell proliferation and morphology of the UHMWPE group, the UHMWPE–SF group, and the UHMWPE–SF/VEGF group. Notes: (A) Cumulative release of VEGF from SF coating displaying a sustained release of VEGF during 28 days. (B) BMSC viability in the scaffolds was detected with CCK-8. ^**P<0.01 compared to the UHMWPE group. ^#P<0.05 compared to the UHMWPE–SF group. (C) Scanning electron microscopic images of cell morphology in three groups at 3, 7, and 14 days. Abbreviations: CCK-8, cell counting kit-8; SF, silk fibroin; UHMWPE, ultra-high-molecular-weight polyethylene; VEGF, vascular endothelial growth factor.

Figure 4

Histological characterization of the UHMWPE group, the UHMWPE–SF group, and the UHMWPE–SF/VEGF group at the time of 6 and 12 weeks after surgery. Notes: (A, E, I) HE staining and (B, F, J) Masson staining evaluation of the graft–bone interface at 6 weeks after surgery. (C, G, K) HE staining and (D, H, L) Masson staining evaluation of the graft–bone interface at 12 weeks after surgery. Yellow imaginary line shows the border of graft and host bone. Newly formed blood vessels at the interface were indicated by white arrowheads. Bar =50 μm. Abbreviations: G, graft; HB, host bone; IF, interface; SF, silk fibroin; UHMWPE, ultra-high-molecular-weight polyethylene; VEGF, vascular endothelial growth factor; HE, hematoxylin and eosin.

Figure 5

PicroSirius red staining evaluation results of the UHMWPE group, the UHMWPE–SF group, and the UHMWPE–SF/VEGF group at the time of 12 weeks after surgery. Notes: Images observed under normal condition are shown on the first row. Corresponding sections observed under polarizing condition are shown on the second row. Partial magnifications of the red rectangle area are displayed on the third row. Dense and parallel collagen bundles with red, orange, and yellow birefringent fibers suggest the presence of type I collagen (marked by *symbols). Loose collagen bundles with green birefringent fibers suggest the presence of type III collagen (marked by ^#symbols). Bar =50 μm. Abbreviations: G, graft; HB, host bone; IF, interface; SF, silk fibroin; UHMWPE, ultra-high-molecular-weight polyethylene; VEGF, vascular endothelial growth factor.

Figure 6

Micro-CT analysis of at the UHMWPE group, the UHMWPE–SF group, and the UHMWPE–SF/VEGF group at the time of 12 weeks after surgery. Notes: (A) Micro-CT scans of specimens from three groups at the time of 12 weeks after surgery. The cross-sectional areas of the bone tunnels at the depth of 5 mm from the femur joint surface and tibial joint surface were measured. Representative axial micro-CT images: the upper and lower panels show images obtained from femur and tibia, respectively. Areas within the red dotted circles represent the cross-sectional areas that were measured for each bone tunnel. (B) Quantification of cross-sectional areas of the bone tunnels. (C) The trabecular bone volume fraction of the total tissue volume of interest (BV/TV) value. ^*P<0.05; ^**P<0.01. Abbreviations: micro-CT, microcomputed tomography; SF, silk fibroin; UHMWPE, ultra-high-molecular-weight polyethylene; VEGF, vascular endothelial growth factor.

Figure 7

Mechanical examinations for graft–bone healing in a rabbit model at 12 weeks after surgery. Notes: (A) Digital camera image of biomechanical test experiment of implanted graft. Red arrow points to the graft. (B) Comparison of maximal failure load among three groups. (C) Comparison of stiffness among three groups. ^*P<0.05; ^**P<0.01. Abbreviations: SF, silk fibroin; UHMWPE, ultra-high-molecular-weight polyethylene; VEGF, vascular endothelial growth factor.