PCL/β-TCP Composite Scaffolds Exhibit Positive Osteogenic Differentiation with Mechanical Stimulation

Abstract

We investigated the use of Polycaprolactone (PCL)/ β-tricalcium phosphate (β-TCP) composites with applied mechanical stimulation as scaffold for bone tissue engineering. PCL-based three-dimensional (3D) structures were fabricated in a solvent-free process using a 3D-printing technique. The mass fraction of β-TCP was varied in the range 0-30%, and the structure and compressive modulus of the specimens was characterized. The shape and interconnectivity of the pores was found to be satisfactory, and the compressive modulus of the specimens was comparable with that of human trabecular bone. Human mesenchymal stem cells were seeded on the composites, and various biological evaluations were performed over 9 days. With a mass fraction of β-TCP of 30%, differentiation began earlier; however, the cell proliferation rate was lower. Through the use of mechanical stimulation, however, the proliferation rate recovered, and was comparable with that of the other groups. This stimulation effect was also observed in ECM generation and other biological assays. With mechanical stimulation, expression of osteogenic markers was lower on samples with a β-TCP content of 10 wt% than without β-TCP; however, with mechanical stimulation, the sample with a β-TCP content of 30 wt% exhibited significantly greater expression of those markers than the other samples. We found that mechanical stimulation and the addition of β-TCP interacted closely, and that a mass fraction of β-TCP of 30% was particularly useful as a bone tissue scaffold when accompanied by mechanical stimulation.

Keywords: 3D-printing system; Human mesenchymal stem cell; Polycaprolactone (PCL); Scaffold; β-tricalcium phosphate (β-TCP).

Fig. 1

The macroscopic and microscopic structure of A PCL, B PCL/β-TCP(10) and C PCL/β-TCP(30) scaffolds (Bar = 400 μm)

Fig. 2

A FE-SEM images of scaffolds, EDS analyses: B SEM-EDS mappings (P, phosphorous; O, oxygen; C, carbone; Ca, calcium) and typical spectrum of C PCL/β-TCP(10) scaffold and D PCL/β-TCP(30) scaffold

Fig. 3

FTIR spectra of the specimens: control_PCL, PCL scaffold, PCL/β-TCP(10), and PCL/β-TCP(30)

Fig. 4

A The stress-strain curves and B compressive modulus (n = 4)

Fig. 5

The measured cell proliferation based on DNA contents (n = 5). Here SX refers to without IHP and SO refers to with IHP

Fig. 6

A Fluorescence images showing the cell viability. Live cells are shown in green and dead cells in red. The scale bar is 100 μm. B SEM images showing the ECM that was generated in the different samples

Fig. 7

Immunofluorescence images showing osteopontin (OPN) at A day 1, 4 and B day 9. The scale bar is 100 μm

Fig. 8

The results of the RT-PCR analysis (n = 3). A Core binding factor-α1 (CBFA-1), B osteonectin (ONN), and C osteocalcin (OCN)