[Osteogenic stimulation of human dental pulp stem cells with gelatin-hydroxyapatite-tricalcium phosphate scaffold]

To investigate the osteogenic stimulation effect of gelatin-hydroxyapatite-tricalcium phosphate (gelatin-HA-TCP) scaffold on human dental pulp stem cells(hDPSCs).

Gelatin scaffold and gelatin-HA-TCP scaffold were developed via solution casting/particle washing method, and the physical and mechanical properties of the scaffolds were examined using scanning electron microscopy and a universal testing machine, respectively. The stress intensity(SS) and elastic modulus(E) of the scaffold were measured by vertical electronic universal testing machine. The osteogenic medium was divided into control group (osteogenic medium), experimental group 1 (gelatin scaffold extract + osteogenic medium) and experimental group 2(gelatin-HA-TCP scaffold + osteogenic medium) according to different mediums. The effects of scaffolds on proliferation and alkaline phosphatase(ALP) activity of hDPSCs were evaluated by CCK8 and ALP staining. The expression of osteogenic factors in hDPSCs was detected by alizarin red S staining and reverse transcription-polymerase chain reaction(RT-PCR). Data were processed by SPSS 20.0 software package.

Compared with gelatin scaffold, gelatin-HA-TCP scaffold had higher SS and lower E(P<0.05); CD146 and CD105 were positively expressed in hDPSCs, and CD34 and CD45 were negatively expressed in hDPSCs. The number of cells on the 3rd, 5th and 7th day of experimental group 1 was significantly higher than that of the control group (P<0.05); The number of cells on the 1st, 3rd, 5th and 7th day of experimental group 2 was significantly higher than that of the control group (P<0.05); ALP activities on the 4th, 7th and 12th day of experimental group 2 were significantly higher than those of the control group and experimental group 1(P<0.05). Compared with the control group, visual fields were red in both experimental groups, and the unevenly distributed calcified precipitation was also found in both experimental groups. The mRNA expression level of Runt-related transcription factor 2(Runx2) on the 4th day of experimental group 1 and experimental group 2 was significantly higher than that of the control group(P<0.05). The mRNA expression level of osterix (OSX) on 7th day of experimental group 2 was significantly higher than that of the control group(P<0.05). The mRNA expression level of bone sialoprotein (BSP) on the 4th day of experimental group 2 was significantly higher than that of the control group and experimental group 1 (P<0.05).

The microscopic characterization of gelatin-HA-TCP scaffold is similar to that of natural bone structure with significant osteoinductive effect on the proliferation and differentiation of hDPSCs, and may be a promising biomaterial for bone tissue engineering.