Collagenous matrix supported by a 3D-printed scaffold for osteogenic differentiation of dental pulp cells

Abstract

Objective: A systematic characterization of hybrid scaffolds, fabricated based on combinatorial additive manufacturing technique and freeze-drying method, is presented as a new platform for osteoblastic differentiation of dental pulp cells (DPCs).

Methods: The scaffolds were consisted of a collagenous matrix embedded in a 3D-printed beta-tricalcium phosphate (β-TCP) as the mineral phase. The developed construct design was intended to achieve mechanical robustness owing to 3D-printed β-TCP scaffold, and biologically active 3D cell culture matrix pertaining to the Collagen extracellular matrix. The β-TCP precursor formulations were investigated for their flow-ability at various temperatures, which optimized for fabrication of 3D printed scaffolds with interconnected porosity. The hybrid constructs were characterized by 3D laser scanning microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and compressive strength testing.

Results: The in vitro characterization of scaffolds revealed that the hybrid β-TCP/Collagen constructs offer superior DPCs proliferation and alkaline phosphatase (ALP) activity compared to the 3D-printed β-TCP scaffold over three weeks. Moreover, it was found that the incorporation of TCP into the Collagen matrix improves the ALP activity.

Significance: The presented results converge to suggest the developed 3D-printed β-TCP/Collagen hybrid constructs as a new platform for osteoblastic differentiation of DPCs for craniomaxillofacial bone regeneration.

Keywords: 3D-printing; Collagen; Dental pulp cells; Hybrid scaffolds; Osteogenic differentiation; β-TCP.

Figure 1

(A–C) Shear stress as a function of shear rate at various temeartures for β-TCP-based paste formulations described in Table 1 (A: formulation 1, B: formulation 2, and C: formulation 3). (D–F) Dependance of shear viscosity on shear rate at different temeartures for the β-TCP-based paste formulations (D: formulation 1, E: formulation 2, and F: formulation 3).

Figure 2

(A) The X-ray diffraction patterns of TCP samples, as received β-TCP powder in comparison with the ground sintered 3D-printed TCP scaffolds (The inset shows a schematic representation of 3D-printed β-TCP-based scaffolds). (B) Differential scanning calorimetry (DSC) thermogram of fully dehydrated freeze-dried Collagen matrix (The inset displays the corresponding DSC thermogram for the freeze-dried Collagen matrix). (C) Fourier transform infrared spectra of β-TCP, Collagen, and Collagen-TCP composite samples.

Figure 3

(A) Schematic representation of the combinatorial approach developed to fabricate hybrid constructs consisted of freeze-dried collagen matrix supported by 3D-printed β-TCP scaffold. (B–D) Macro- to microscale demonstration of structural features of 3D-printed β-TCP scaffold. (E–G) Macro- to microscale demonstration of structural features of 3D-printed β-TCP/Col hybrid constructs.

Figure 4

(A) 3D laser scanning micrographs of the 3D-printed β-TCP scaffold (The scale bar is 500 μm). Porosity of Collagen and Collagen-TCP matrices compared with β-TCP/Col and β-TCP/Col-TCP hybrid constructs. (C) Compressive modulus of 3D-printed β-TCP scaffolds in dry state and after 1, 2, and 4 weeks immersion in phosphate-buffered saline. (D) Compressive modulus of Collagen and Collagen-β. TCP matrices compared with β-TCP/Col and β-TCP/Col-TCP hybrid constructs (E,F) optical image of 2D cultured DPCs. (G) Alizarin Red S staining of 2D cultured DPCs after three weeks osteogenic differentiation (scale bar is 400 μm). (H) Immunofluorescence images of 2D cultured DPCs using surface marker of CD90 and DAPI (scale bar is 400 μm).

Figure 5

(A) Hematoxylin and eosin (H&E) staining of DPCs seeded onto 3D-printed β-TCP/Col construct after three weeks. Scanning electron micrographs of (B–C) 3D-printed β-TCP scaffold, and (D–E) 3D-printed β-TCP/Col-TCP construct seeded with DPCs after three weeks (B, C: scale bar is 500 μm; D, E: scale bar is 200 μm). The immunofluorescent images of encapsulated DPCs into (F) β-TCP/Col, and (G) β-TCP/Col-TCP constructs after 3 weeks. (H) DPCs viability cultured onto 3D-printed β-TCP scaffold in comparison with β-TCP/Col and β-TCP/Col-TCP constructs after over three weeks. (I) Alkaline phosphatase (ALP) activity of DPCs cultured onto 3D-printed β-TCP scaffold in comparison with β-TCP/Col and β-TCP/Col-TCP constructs after 1, 2, and 3 weeks.