Sustained Release of VEGF to Promote Angiogenesis and Osteointegration of Three-Dimensional Printed Biomimetic Titanium Alloy Implants

Abstract

Tumor resection and treatment of trauma-related regional large bone defects have major challenges in the field of orthopedics. Scaffolds that treat bone defects are the focus of bone tissue engineering. 3D printing porous titanium alloy scaffolds, prepared via electron beam melting technology, possess customized structure and strength. The addition of a growth factor coating to the scaffold introduces a specific form of biological activation. Vascular endothelial growth factor (VEGF) is key to angiogenesis and osteogenesis in vivo. We designed a porous titanium alloy scaffold/thermosensitive collagen hydrogel system, equipped with VEGF, to promote local osseointegration and angiogenesis. We also verified the VEGF release via thermosensitive collagen and proliferation and induction of the human umbilical vein endothelial cells (HUVECs) via the composite system in vitro. In vivo, using microscopic computed tomography (Micro-CT), histology, and immunohistochemistry analysis, we confirmed that the composite scaffold aids in angiogenesis-mediated bone regeneration, and promotes significantly more bone integration. We also discovered that the composite scaffold has excellent biocompatibility, provides bioactive VEGF for angiogenesis and osteointegration, and provides an important theoretical basis for the restoration of local blood supply and strengthening of bone integration.

Keywords: 3D-printed porous titanium alloy scaffold; VEGF; angiogenesis; bioactive interface; osseointegration.

FIGURE 1

Microscopic characterization of eTi (A) and cTi (B), using SEM scanning images (C) VEGF release curve in vitro within 15 days (D) Analysis of the HUVECs proliferation in each group, using the eTi of the first day as a reference (n = 3, *indicates significant differences between groups, *p < 0.05; **p < 0.01; ***p < 0.001).

FIGURE 2

(A) Live/dead HUVECs staining was done in a co-culture of HUVECs with eTi, cTi, and cTi/VEGF for 1 and 4 days, respectively (n = 3) (B) Total cellular count was evaluated via gross observation, under an inverted microscope (C) Tube formation of HUVECs cultured with Extracts of eTi, cTi, or cTi/VEGF for 6 h (n = 3) (D) The total tubular lengths of HUVECs in different scaffolds.

FIGURE 3

(A) Effects of eTi, cTi, and cTi/VEGF on HUVECs invasion (B) HUVECs stained with crystal violet were quantitatively analyzed (n = 3) (C) The effects of eTi, cTi, and cTi/VEGF on wound healing, as observed under a phase contrast microscope (D) Assessment of wound healing rates (n = 3).

FIGURE 4

RT-qPCR analysis of (A) MMP-2 (B) Bcl-2, and (C) Bax gene expression in HUVECs cultured with eTi, cTi, and cTi/VEGF for 1 and 4 days, respectively (n = 3).

FIGURE 5

Representative 3D reconstruction images of scaffold implantation in each group at 6 (A) and 12 (B) weeks after operation (yellow: bone tissue; white: scaffolds). Micro-CT image analysis of BV/TV (C), Tb.Th (D), Tb. Sp (E), and Tb.N (F) in each group (n = 3).

FIGURE 6

(A) The eTi, cTi, and cTi/VEGF scaffolds were separately implanted into rabbit femoral epicondyles and allowed to remain for 6 and 12 weeks. Subsequently, hard sections were prepared and stained with VG (black: Ti6Al4V scaffold; red: new bone) (B) the proportion of new bone area to scaffolds pores (C) maximum load in the detaching test of eTi, cTi, and cTi/VEGF after implantation into rabbit femoral epicondyles for 6 and 12 weeks (n = 3).

FIGURE 7

Immunofluorescence staining of COL I (A) and CD 31 (B) expression in bone surrounding scaffolds of eTi, cTi, and cTi/VEGF after implantation for 6 and 12 weeks (n = 3).