Design and 3D Printing of Personalized Hybrid and Gradient Structures for Critical Size Bone Defects

Abstract

Treating critical-size bone defects with autografts, allografts, or standardized implants is challenging since the healing of the defect area necessitates patient-specific grafts with mechanically and physiologically relevant structures. Three-dimensional (3D) printing using computer-aided design (CAD) is a promising approach for bone tissue engineering applications by producing constructs with customized designs and biomechanical compositions. In this study, we propose 3D printing of personalized and implantable hybrid active scaffolds with a unique architecture and biomaterial composition for critical-size bone defects. The proposed 3D hybrid construct was designed to have a gradient cell-laden poly(ethylene glycol) (PEG) hydrogel, which was surrounded by a porous polycaprolactone (PCL) cage structure to recapitulate the anatomical structure of the defective area. The optimized PCL cage design not only provides improved mechanical properties but also allows the diffusion of nutrients and medium through the scaffold. Three different designs including zigzag, zigzag/spiral, and zigzag/spiral with shifting the zigzag layers were evaluated to find an optimal architecture from a mechanical point of view and permeability that can provide the necessary mechanical strength and oxygen/nutrient diffusion, respectively. Mechanical properties were investigated experimentally and analytically using finite element analysis (FEA), and computational fluid dynamics (CFD) simulation was used to determine the permeability of the structures. A hybrid scaffold was fabricated via 3D printing of the PCL cage structure and a PEG-based bioink comprising a varying number of human bone marrow mesenchymal stem cells (hBMSCs). The gradient bioink was deposited inside the PCL cage through a microcapillary extrusion to generate a mineralized gradient structure. The zigzag/spiral design for the PCL cage was found to be mechanically strong with sufficient and optimum nutrient/gas axial and radial diffusion while the PEG-based hydrogel provided a biocompatible environment for hBMSC viability, differentiation, and mineralization. This study promises the production of personalized constructs for critical-size bone defects by printing different biomaterials and gradient cells with a hybrid design depending on the need for a donor site for implantation.

Keywords: 3D printing; gradient structures; hybrid printing; large bone defects; personalized scaffolds.

Figure 1

Schematic representation of CAD modeling and calculation of the 3D hybrid printing path using different PCL cage geometries. (i) Extracting a CAD model of the defect area, (ii) determining the inner and outer contours of the PCL cage, (iii) designing different models for the hybrid structure, (iv) sequential printing PCL and cell-laden hydrogel structures in the hybrid design, and (v) showing the first two layers of the PCL cage.

Figure 2

Fluid domain for the CFD simulation and boundary conditions for the (A) axial flow and (B) radial flow.

Figure 3

Digital images of the 3D printed PCL cages of (A) zigzag, (B) zigzag/spiral, and (C) zigzag/spiral-shifted patterns at (i) top view, (ii) perspective angle, and (iii) side view. Scale bars are 3 mm.

Figure 4

Characterization of the mechanical properties and porosity of the alternative PCL cage structures. (A) Compressive stress–strain curves for the 3D printed structures of zigzag, zigzag/spiral, and zigzag/spiral-shifted patterns. Micro-CT images of (B) zigzag, (C) zigzag/spiral, and (D) zigzag/spiral-shifted structures at a (i) perspective angle, (ii) cross-sectional perspective angle, (iii) top view, and (iv) cross-sectional side view.

Figure 5

Mechanical strength analysis for the PCL scaffolds. (A) Comparison of the FE analysis and experimental compressive stress values of the 3D printed PCL scaffolds. von Mises stress distribution of the scaffolds with (B) zigzag, (C) zigzag/spiral, and (D) zigzag/spiral-shifted patterns.

Figure 6

CFD simulation for the mass transport at (A) axial and (B) radial pressure distribution contours of the 3D printed scaffolds of (i) zigzag, (ii) zigzag/spiral, and (iii) zigzag/spiral-shifted patterns.

Figure 7

WSS distribution in (A) axial and (B) radial contours of the 3D printed scaffolds of (i) zigzag, (ii) zigzag/spiral, and (iii) zigzag/spiral-shifted patterns.

Figure 8

Demonstration of the 3D printed hybrid structures. (A) PCL cage structure, (B) PEG-based bioink bioprinted inside the PCL cage, and (C) multilayer bioprinting of the PEG-based bioink. Four-layers were printed by extrusion of fibers from the microcapillaries. The bioink was separately aspirated in each layer. The side view projections of the multilayered structure are given in (Ci) and (Cii). Scale bars: 2 mm.

Figure 9

Biocompatibility evaluation of the PEG-based bioink for hBMSCs. (A) Calcein AM (green) and PI (red) staining were performed on (i) day 1, (ii) day 3, and (ii) day 7 after bioprinting to show live and dead cells inside the PEG-based hydrogel. (B) Osteogenic differentiation of hBMSCs in the PEG-based bioink. ALP activity assay was performed to show osteogenic differentiation depending on the varying concentrations of hBMSCs in the bioprinted constructs. Monolayers of PEG-based hydrogel constructs with 1 × 10⁶, 2 × 10⁶, and 5 × 10⁶ hBMSCs/mL were treated with MM and OM for 21 days. The constructs treated with OM were evaluated for osteogenic differentiation while MM-treated cells were used as the control. Student’s t-test was used to show the significant levels between the groups (*p < 0.05). The analysis was performed for at least three different samples in a group. (C) Alizarin red staining was performed to show mineralization in monolayers of the PEG-based hydrogel containing 1 × 10⁶, 2 × 10⁶, and 5 × 10⁶ cells (Ci, Cii, Ciii), separately and in a three-layered construct (Civ). Scale bars for (A) and (Civ): 500 μm and (Ci, Cii, Ciii): 200 μm.