In Vitro and In Vivo Evaluation of 3D Printed Poly(Ethylene Glycol) Dimethacrylate-Based Photocurable Hydrogel Platform for Bone Tissue Engineering

Abstract

This study focuses to develop a unique hybrid hydrogel bioink formulation that incorporates poly(ethylene glycol) dimethacrylate (PEGDMA), gelatin (Gel), and methylcellulose (MC). This formulation achieves the necessary viscosity for extrusion-based three-dimensional (3D) printing of scaffolds intended for bone regeneration. After thorough optimization of the hybrid bioink system with Gel, three distinct scaffold groups are investigated in vitro: 0%, 3%, and 6% (w/v) Gel. These scaffold groups are examined for their morphology, mechanical strength, biodegradation, in vitro cell proliferation and differentiation, and in vivo bone formation using a rat cranial defect model. Among these scaffold compositions, the 3% Gel scaffold exhibits the most favorable characteristics, prompting further evaluation as a rat mesenchymal stem cell (rMSC) carrier in a critical-size cranial defect within a Lewis rat model. The compressive strength of all three scaffold groups range between 1 and 2 MPa. Notably, the inclusion of Gel in the scaffolds leads to enhanced bioactivity and cell adhesion. The Gel-containing scaffolds notably amplify osteogenic differentiation, as evidenced by alkaline phosphatase (ALP) and Western blot analyses. The in vivo results, as depicted by microcomputed tomography, showcase augmented osteogenesis within cell-seeded scaffolds, thus validating this innovative PEGDMA-based scaffold system as a promising candidate for cranial bone defect healing.

Keywords: 3D printing; gelatin; in vivo; methylcellulose; photocross‐linking; poly(ethylene glycol) dimethacrylate; scaffold.

Figure 1.

Scaffold design and preliminary studies of hydrogel; (a)(i) 3D model of 500μm pore scaffold with 4 layers; (ii) G-code file containing the printing path of the scaffold; (iii) 3D printer; (b) (i) Printability of the hydrogel with increasing Gel %; (ii) Continuous extrusion of the bioink-good; (iii) Discontinuous extrusion of bioink-bad; (iv) PEGDMA only gel in the 10 ml syringe with the 27 gauge nozzle; (v) PEGDMA + 3% Gel; (vi) Printed multilayer scaffolds – side view- (A) 4 layers (1.2 mm), (B) 8 layers, (C) 16 layers, (D) 32 layers (c) Appearance of the 3D printed scaffolds according to the Gel % and the live cell assay images of scaffolds after 7 days of culture using murine preosteoblasts. Scale bar = 1 mm.

Figure 2.

(a) Swelling study of the scaffolds for 7 day period, n=3; (b) pH variation of the scaffold in 2 ml of PBS solution compared to the control (PBS only), n=3.

Figure 3.

(a) SEM micrographs of the scaffolds; (b) Compressive modulus study (i) testing apparatus with 10 lbf load cell (ii) Dry scaffolds (iii) Wet scaffolds from the left PEGDMA only, PEGDMA + 3% Gel, and PEGDMA + 6% Gel (iv) Compressive modulus of dry scaffolds (v) Compressive modulus of wet scaffolds; * indicates the significance of p<0.05; n=7

Figure 4.

Fluorescence images of the live-dead cell assay at days 4, 7, and 14, indicating live cells in green-calcein, and red cells in red-ethidium homodimer-1

Figure 5.

(a) WST-1 assay optical density values at 440 nm at day 4, day 7, and day 14; (b) Amount of DNA obtained from attached and the proliferated cells on the scaffolds at days 7, and 14; * indicates the significance of p<0.05; n=3

Figure 6.

(a) ALP activity of the scaffolds at days 7,14, and 21; * indicates the significance of p < 0.05; n=3; (b) Qualitative western blot analysis of rMSCs following the induction of osteogenic differentiation at day 10, BMP-2 and osteopontin intensities are relative to the β-actin.

Figure 7.

In vivo study and microCT analysis (a) 4.5 mm diameter circular scaffold G-code file; (b) Printed scaffold; (c) Live cell assay image after day 3; (d) Two cranial defects on rat skull including the scaffolds; (e) Appearance of the scaffold in the harvested bone sample after 12 weeks; (f) MicroCT images of scaffolds at 6 weeks and 12 weeks; (g) Quantitative analysis of new bone volume at 6 weeks; (h) Quantitative analysis of new bone at 12 weeks; indicated the significance of p < 0.05 with Control and scaffolds + rMSCS groups; n=10