Enhancing bone regeneration through 3D printed biphasic calcium phosphate scaffolds featuring graded pore sizes

Abstract

Human long bones exhibit pore size gradients with small pores in the exterior cortical bone and large pores in the interior cancellous bone. However, most current bone tissue engineering (BTE) scaffolds only have homogeneous porous structures that do not resemble the graded architectures of natural bones. Pore-size graded (PSG) scaffolds are attractive for BTE since they can provide biomimicking porous structures that may lead to enhanced bone tissue regeneration. In this study, uniform pore size scaffolds and PSG scaffolds were designed using the gyroid unit of triply periodic minimal surface (TPMS), with small pores (400 μm) in the periphery and large pores (400, 600, 800 or 1000 μm) in the center of BTE scaffolds (designated as 400-400, 400-600, 400-800, and 400-1000 scaffold, respectively). All scaffolds maintained the same porosity of 70 vol%. BTE scaffolds were subsequently fabricated through digital light processing (DLP) 3D printing with the use of biphasic calcium phosphate (BCP). The results showed that DLP 3D printing could produce PSG BCP scaffolds with high fidelity. The PSG BCP scaffolds possessed improved biocompatibility and mass transport properties as compared to uniform pore size BCP scaffolds. In particular, the 400-800 PSG scaffolds promoted osteogenesis in vitro and enhanced new bone formation and vascularization in vivo while they displayed favorable compressive properties and permeability. This study has revealed the importance of structural design and optimization of BTE scaffolds for achieving balanced mechanical, mass transport and biological performance for bone regeneration.

Keywords: 3D printing; Bone tissue engineering; Mass transport property; Mechanical property; Osteogenesis; Pore size graded scaffold; Vascularization.

Graphical abstract

Fig. 1

Design, manufacture and application of PSG BCP scaffolds: (A) Geometrical features and definitions of pore size and thickness of the gyroid structure. Scaffold models with two layers of graded pore distributions are shown for (B, D) in vitro biological evaluations and for (C, E) compressive properties, mass transport characteristics, and in vivo biological evaluations. (F) Schematic diagram illustrating the fabrication process of BCP scaffolds via digital light processing (DLP) 3D printing. (G) Schematic diagram illustrating bone regeneration by using DLP-formed PSG BCP scaffolds.

Fig. 2

Morphological characteristics of uniform pore size and PSG BCP scaffolds. Scaffolds for in vitro biological evaluations: (A) Top view. (B) 3D view. (C) SEM images of central pores. Scaffolds for compressive properties, mass transport characteristics and in vivo biological evaluations: (D) Top view. (E) 3D view. (F) SEM images of central pores.

Fig. 3

Compression behavior and properties of uniform pore size and PSG BCP scaffolds analyzed through compression tests and finite element analysis (FEA): (A) Compressive stress-strain curves. (B) Compressive strength and modulus (NS: no significant difference. ∗∗∗p < 0.001). (C) Weibull plot of the compressive strength. Stress distribution from (D) front view and (E) cross-sectional view.

Fig. 4

Computational fluid dynamics (CFD) analysis of uniform pore size and PSG BCP scaffolds: (A) Boundary conditions and (B) schematic representation of models. (C) Pressure contours on scaffold surfaces and middle cross-sections. (D) Velocity distributions in middle cross-sections.

Fig. 5

Mass transport properties of uniform pore size and PSG BCP scaffolds: (A) Custom-made set up for permeability tests. (B) Pressure drop and computational permeability based on CFD analysis. (C) Experimental results of fluid flow versus pressure drop. (D) Experimental permeability versus fluid heights. (E) Correlation factors at different fluid heights and their fitting curves.

Fig. 6

In vitro biological assessment of uniform pore size and PSG BCP scaffolds: (A) DAPI & Phalloidin stained MC3T3-E1 cells after being cultured for 1, 3 and 5 days. (B) Morphology of MC3T3-E1 cells on BCP scaffolds after being cultured for 5 days (C) Proliferation of MC3T3-E1 cells on BCP scaffolds after incubation of 1, 3 and 5 days on BCP scaffolds (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, N=3).

Fig. 7

In vitro osteogenesis of uniform pore size and PSG BCP scaffolds. ALP activities of (A) staining images and (B) quantitative analyses of MC3T3-E1 cells on four types of scaffolds after being cultured for 7 and 14 days. (B) Alizarin red activity of (C) staining images and (D) quantitative analyses of MC3T3-E1 cells on four types of scaffolds after being cultured for 14 and 21 days. Expression of osteogenic genes of (E) ALP and (F) COL 1 of MC3T3-E1 cells at day 14 of culture. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Fig. 8

Histological analysis of uniform pore size 400-400 scaffolds and graded 400–600, 400–800 and 400–1000 scaffolds after 2-week and 4-week subcutaneous implantation in mice (high magnification): (A) Illustrations for ROIs. (B) Samples after H&E staining (black star: blood vessels). (C) Samples after Goldner's Trichrome staining (black arrow: osteoid).

Fig. 9

Immunohistochemical analysis of uniform pore size and PSG BCP scaffolds after 2-week and 4-week subcutaneous implantation in mice: (A) Illustrations for ROIs. Results for VEGF: (B) Staining results with VEGF. (C) Number of VEGF + vessels. (D) VEGF positive area percentage. Results for CD 31: (E) Staining results with CD 31. (F) Number of CD 31 + vessels. (G) CD 31 positive area percentage (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).