Biomimetic Ti-6Al-4V alloy/gelatin methacrylate hybrid scaffold with enhanced osteogenic and angiogenic capabilities for large bone defect restoration

Abstract

Titanium-based scaffolds are widely used implant materials for bone defect treatment. However, the unmatched biomechanics and poor bioactivities of conventional titanium-based implants usually lead to insufficient bone integration. To tackle these challenges, it is critical to develop novel titanium-based scaffolds that meet the bioadaptive requirements for load-bearing critical bone defects. Herein, inspired by the microstructure and mechanical properties of natural bone tissue, we developed a Ti-6Al-4V alloy (TC4)/gelatin methacrylate (GelMA) hybrid scaffold with dual bionic features (GMPT) for bone defect repair. GMPT is composed of a hard 3D-printed porous TC4 metal scaffold (PT) backbone, which mimics the microstructure and mechanical properties of natural cancellous bone, and a soft GelMA hydrogel matrix infiltrated into the pores of PT that mimics the microenvironment of the extracellular matrix. Ascribed to the unique dual bionic design, the resultant GMPT demonstrates better osteogenic and angiogenic capabilities than PT, as confirmed by the in vitro and rabbit radius bone defect experimental results. Moreover, controlling the concentration of GelMA (10%) in GMPT can further improve the osteogenesis and angiogenesis of GMPT. The fundamental mechanisms were revealed by RNA-Seq analysis, which showed that the concentration of GelMA significantly influenced the expression of osteogenesis- and angiogenesis-related genes via the Pi3K/Akt/mTOR pathway. The results of this work indicate that our dual bionic implant design represents a promising strategy for the restoration of large bone defects.

Keywords: 3D printing porous titanium alloys; Angiogenesis; Gelatin methacrylate; Osteogenesis.

Graphical abstract

Fig. 1

Schematic illustrations of the biomimetic GMPT with dual-bionic features. The GMPT is composed of a “hard” 3D-printed porous TC4 metal scaffold (PT) backbone which mimics the microstructure and mechanical property of natural cancellous bone, and a “soft” GelMA hydrogel matrix infiltrated in the pores of the PT that mimics the microenvironment of ECM.

Fig. 2

The fabrication process and characterization of GMPT. (a) Schematic illustration of the fabrication of GMPT. TMSPMA was used as a linker to immobilize GelMA hydrogels onto the surface of 3D-printed PT, thus generating hard-soft hybrid 3D scaffolds. (b, c) The presence of Ti, Al, V, C, O, and N elements in the EDS indicated that GelMA was successfully immobilized on the scaffolds. (d) Schematic illustration of the binding force experiment. (e) The binding force of physically attached GMPT (GM-PT) and chemically anchored GMPT (GM-TMSPMA-PT) (n = 3). (f) SEM images of GelMA and PT scaffolds presented in PT, GM-PT, GM-TMSPMA-PT. Scale bar, 500 μm.

Fig. 3

In vitro viability, proliferation and attachment of PT&GMPT hybrid scaffolds (BMSCs as a cell model). (a) Live/dead assay of BSMCs cultured on PT, 5% GMPT, 10% GMPT and 15% GMPT for 1 day. Scale bar, 200 μm. (b) Phalloidin-DAPI staining of HUVECs attached to PT, 5% GMPT, 10% GMPT and 15% GMPT on day 1. Scale bar, 50 μm. (c) SEM of BMSCs attached to PT, 5% GMPT, 10% GMPT and 15% GMPT for 1 day. Scale bar, 20 μm. (d) CCK-8 assay of BMSCs on PT, 5% GMPT, 10% GMPT and 15% GMPT for 1, 3, 5 and 7 days. All scaffolds exhibited excellent cytocompatibility and provided a desirable environment for cell attachment and ingrowth. Asterisks indicate significant differences, * represents P＜0.05, ** represents P＜0.01.

Fig. 4

In vitro analysis of osteogenesis on PT and GMPT scaffolds. (a) Schematic of the experiment. Cells were collected from each group after 7 days and 14 days of incubation. HUVECs were sorted into different groups and used in subsequent experiments. (b) Alizarin red staining images. (c) Quantification of Alizarin red staining through the absorbance value of osteogenesis of PT, 5% GMPT, 10% GMPT and 15% GMPT for 14 days. (d) ALP activity of PT, 5% GMPT, 10% GMPT and 15% GMPT on days 7 and 14. (e–h) The RT-PCR results of osteogenesis-associated gene expression of PT, 5% GMPT, 10% GMPT and 15% GMPT for 7 days and 14 days. The results demonstrated that the softer stiffness of GelMA provided a desirable surrounding environment for osteogenesis, among which 10% GMPT induced a higher level of osteogenic gene expression. Asterisks indicate significant differences, * represents P＜0.05, ** represents P＜0.01, *** represents P＜0.001 and **** represents P＜0.0001.

Fig. 5

In vitro angiogenesis of PT and GMPT scaffolds. (a) Schematic of the tubule formation of HUVECs cultured on PT, 5% GMPT, 10% GMPT and 15% GMPT for 24 h. (b) Tubular length of different groups. (c) Immunofluorescence of tube formation. Scale bar, 200 μm. (d–g) The RT-PCR results of angiogenesis-associated gene expression of PT, 5% GMPT, 10% GMPT and 15% GMPT for 3 days and 10 days. The in vitro results proved that 10% GMPT provided a better environment for angiogenesis. Asterisks indicate significant differences, *represents P＜0.05, ** represents P＜0.01, *** represents P＜0.001, **represents P＜0.0001.

Fig. 6

Gene expression and bioinformatic analysis across the 10% GMPT scaffold. (a) Volcano plot of the differentially expressed genes between groups. (≥2-fold difference; red: upregulated genes; blue: downregulated genes). (b) Heat map of angiogenesis-related gene expression. (c) Heat map of osteogenesis-related gene expression. (red: high expression; blue: low expression). (d) The gene enrichment KEGG pathway analysis. (e–g) Intracellular flow cytometry (FCM) for the phosphorylation of Pi3k (e), Akt (f) and mTOR (g) in the PT, 5% GMPT, 10% GMPT and 15% GMPT groups, where (i) are FCM figures and (ii) are their quantitation results. Asterisks indicate significant differences, *represents P＜0.05.

Fig. 7

In situ implantation of PT and GMPT scaffolds, micro-CT 3D reconstruction and biomechanical test of PT and GMPT in critical radius defects of rabbits. (a) Schematic illustration of GMPT in vivo implantation. (b) The surgical process of GMPT implantation in rabbits with critical radius defects. (c) Micro-CT reconstruction of bone regeneration with the scaffolds at 4 and 12 weeks postsurgery. The new bones were brown color. (d) Bone volume/total volume (BV/TV) analysis of the bone defect 4 and 12 weeks after surgery. (e) Bone mineral density (BMD) analysis of regenerated bone 4 and 12 weeks after surgery. (f) Schematic illustration of the direct mechanical testing for in vivo samples. (g) The maximum load of direct mechanical testing for implanted scaffolds. These results demonstrated that the prognosis of in situ implantation of GMPT scaffolds in critical radius defects was preferable to that of PT scaffolds. Overall, 10% GMPT showed the best results, in accordance with the in vitro test. *represents P＜0.05, ** represents P＜0.01, *** represents P＜0.001.

Fig. 8

Histological analysis of implant samples after 4 and 12 weeks in rabbit radius defect sites. Van Gieson's staining of the PT and GMPT scaffolds at 4 and 12 weeks after the operation was carried out to evaluate osteogenesis and angiogenesis. The GMPT group showed thicker and more trabeculae than the PT group at both weeks 4 and 12 (yellow arrows indicate the PT scaffold, white arrows indicate new bone, green arrows reveal new vessels). The 10% GMPT group showed the best osteogenesis and angiogenesis ability.

Fig. 9

CD31 immunofluorescence staining images of the vessel around scaffolds. Yellow arrows indicate the formed blood vessels. The staining area was distributed sporadically in the PT group, while in the GMPT groups, highly concentrated staining was observed. The 10% GMPT group had more mature vessels formed than the other GMPT groups.