3D Collagen Nanofibrous Scaffold's Pore Size Affected HDPCs’ Biological Behavior

Objectives: This study was to fabricate 3D collagen nanofibrous scaffolds with different pore size and evaluate its effect on cell proliferation and odontoblastic differentiation of human dental pulp cells (hDPCs).
Methods: The collagen nanofibrous scaffolds with different pore size were prepared by electrospinning combined with freeze drying technique. The surface morphology and pore sizes of the scaffolds were analyzed by scanning electron microscopy (SEM). HDPCs were seeded in the scaffolds with different pore size, the cell proliferation ability was detected by CCK-8 assay. Besides the odontoblasts differentiation related gene (DSPP, DMP-1, ALP, OCN) expression of the hDPCs was analyzed with Real-Time RT-PCR at 1, 2, 4 weeks, the expression of the odontoblasts differentiation related proteins at 4 weeks was further validated using immunofluorescence staining. Furthermore , Von-Kossa staining, SEM and Energy Dispersive X-ray (EDX) were used to analyze the formation of mineralized nodules.
Results: Three types of scaffolds were prepared, whose pore size is 146.93±24.53μm (Large), 76.68±22.71μm (Medium) and 19.95±4.43μm (Small), respectively. Cell proliferation rate was similar among these scaffolds, while hDPCs in Large group showed higher proliferation rate after 7 days (p<0.05). After cultured with odontoblastic medium for 4 weeks, hDPCs in all groups could differentiated into odontoblasts, while Medium group presented the highest odontogenic gene and protein expressions. In addition, larger mineral nodules were observed on Medium and Large group, and calcium content on the surface of scaffolds in Medium group was the highest.
Conclusions: Our study indicated that 3D collagen nanofibrous scaffolds with different pore size provide a suitable environment for cell proliferation and odontogenic differentiation of hDPCs, and the medium pore size scaffolds could further stimulate the odontogenic differentiation.