3D printing of dual-cell delivery titanium alloy scaffolds for improving osseointegration through enhancing angiogenesis and osteogenesis

Abstract

Background: The repair of large bone defects is a great challenge for orthopedics. Although the development of three-dimensional (3D) printed titanium alloy (Ti6Al4V) implants with optimized the pore structure have effectively promoted the osseointegration. However, due to the biological inertia of Ti6Al4Vsurface and the neglect of angiogenesis, some patients still suffer from postoperative complications such as dislocation or loosening of the prosthesis.

Methods: The purpose of this study was to construct 3D printed porous Ti6Al4V scaffolds filled with bone marrow mesenchymal stem cells (BMSC) and endothelial progenitor cells (EPC) loaded hydrogel and evaluate the efficacy of this composite implants on osteogenesis and angiogenesis, thus promoting osseointegration.

Results: The porosity and pore size of prepared 3D printed porous Ti6Al4V scaffolds were 69.2 ± 0.9 % and 593.4 ± 16.9 μm, respectively, which parameters were beneficial to bone ingrowth and blood vessel formation. The BMSC and EPC filled into the pores of the scaffolds after being encapsulated by hydrogels can maintain high viability. As a cell containing composite implant, BMSC and EPC loaded hydrogel incorporated into 3D printed porous Ti6Al4V scaffolds enhancing osteogenesis and angiogenesis to repair bone defects efficiently. At the transcriptional level, the composite implant up-regulated the expression levels of the osteogenesis-related genes alkaline phosphatase (ALP) and osteocalcin (OCN), and angiogenesis-related genes hypoxia-inducible factor 1 alpha (HIF-1α), and vascular endothelial growth factor (VEGF).

Conclusions: Overall, the strategy of loading porous Ti6Al4V scaffolds to incorporate cells is a promising treatment for improving osseointegration.

Keywords: 3D printed; Angiogenesis; Osseointegration; Osteogenesis; Titanium alloy implant.

Fig. 1

A Gross view and SEM image of empty porous scaffold. B Schematic diagram of poloxamer 407 hydrogel filled scaffolds. C Gross view and SEM image of hydrogel incorporated scaffold. After hydrogel incorporation, the scaffolds (blue arrow) were completely filled with interconnected hydrogel networks (red arrow)

Fig. 2

A BMSC proliferation and B EPC proliferation cultured with Con, eTi, and hTi scaffolds at 1, 3, and 5 days. C The schematic diagram of the cell-loaded hydrogel incorporated scaffolds. D BMSC and/or EPC proliferation cultured within hTi scaffolds at 1, 3, and 5 days. E Calcein AM/PI staining of live cells (green) and dead cells (red) of BMSC/hTi, EPC/hTi, and Dual/hTi groups. F Quantitative analysis of cell survival rate (**p < 0.01, compared with the Day 1; ^#p < 0.05 and ^##p < 0.01 compared with the Day 3)

Fig. 3

A 3D reconstruction images of porous scaffolds after 12 weeks of implantation. Newly formed bone is indicated in yellow, and the scaffolds are seen in white. B Bone volume/total volume (BV/TV, %). C Trabecular thickness (Tb.Th, mm). D Trabecular number (Tb.N, 1/mm). E Trabecular separation (Tb.Sp, mm) parameters analysis according to Micro-CT after 12 weeks of implantation. F Representative Masson staining images of new bone in and around the porous scaffolds (*p < 0.05, **p < 0.01 compared with the eTi Group; ^#p < 0.05, ^##p < 0.01 compared with the hTi Group; ^†p < 0.05, ^††p < 0.01 compared with the EPC/hTi Group; ^‡p < 0.05 compared with the BMSC/hTi Group)

Fig. 4

A Microangiography images of newly formed blood vessel around the scaffolds. B Percentages of BVV/TV in these scaffolds was calculated (*p < 0.05, **p < 0.01 compared with the eTi Group; ^#p < 0.05, ^##p < 0.01 compared with the hTi Group; ^†p < 0.05, with the EPC/hTi Group; ^‡p < 0.05, ^‡‡p < 0.05 compared with the BMSC/hTi Group)

Fig. 5

Relative mRNA expression of osteogenesis-related genes (A) ALP and (B) OCN. Relative mRNA expression of angiogenesis-related genes (C) HIF-1α, and (D) VEGF in vivo at 12 weeks after implantation (*p < 0.05, **p < 0.01 compared with the eTi Group; ^#p < 0.05, ^##p < 0.01 compared with the hTi Group; ^†p < 0.05, ^†p < 0.01 with the EPC/hTi Group; ^‡p < 0.05, ^‡‡p < 0.05 compared with the BMSC/hTi Group)