Ectopic study of tissue-engineered bone complex with enamel matrix proteins, bone marrow stromal cells in porous calcium phosphate cement scaffolds, in nude mice

Abstract

Objective: This study aimed to investigate the potential of enamel matrix proteins (EMPs) on promoting osteogenic differentiation of porcine bone marrow stromal cells (pBMSCs), as well as new bone formation capabilities, in a tissue-engineered bone complex scaffold of EMPs, pBMSCs and porous calcium phosphate cement (CPC).

Materials and methods: Effects of EMPs on pBMSCs in vitro was first determined by alkaline phosphatase (ALP) activity, von Kossa staining assay and mRNA expression of ALP, bone sialoprotein (BSP) and osteocalcin (OCN) genes. Next, an ectopic new bone formation test was performed in a nude mouse model with four groups: CPC scaffold alone; CPC scaffold + EMPs; CPC scaffold + pBMSCs; and CPC scaffold + EMPs + pBMSCs, for 2 or 4 weeks.

Results: ALP activity, von Kossa assay and mRNA expressions of ALP, BSP and OCN genes were all significantly higher with 150 μg/ml EMP treatment in vitro. In nude mice, new bone formation was detected only in the CPC scaffold + EMPs + pBMSCs group at 2 weeks. At 4 weeks, in the tissue-engineered construct there was significantly higher bone formation ability than other groups.

Conclusions: EMPs promoted osteogenic differentiation of pBMSCs, and the tissue-engineered complex of EMPs, pBMSCs and CPC scaffold may be a valuable alternative to be used in periodontal bone tissue engineering and regeneration.

Figure 1

SEM observation of microstructure of the three‐dimensional porous structure of CPC scaffold (×50).

Figure 2

Cell culture and EMP treatment. Cell morphology of pBMSCs treated with 0, 50 and 150 μg/ml EMPs at 3 days (a, b, c) and 6 days (d, e, f) respectively. pBMSCs without being treated with EMPs always maintained short fibroblasts‐like morphology, while pBMSCs treated with EMPs displayed spindle and polygonal morphology at day 3, and showed a morphology of polygonal or squarish by day 6 (reverse phase contrast microscope, ×100).

Figure 3

In vitro analysis of osteogenic differentiation of pBMSCs with or without EMP treatment. (a, b, c) Alkaline phosphatase expression of pBMSCs treated with 0, 50 and 150 μg/ml EMPs for 14 days (reverse phase contrast microscope, ×100). (d, e, f) von Kossa staining for mineralized nodules of pBMSCs treated with 0, 50 and 150 μg/ml EMPs for 21 days (reverse phase contrast microscope, ×16).

Figure 4

Real‐time PCR analysis of osteogenic differentiation gene expression in pBMSCs treated with 0, 50 and 150 μg/ml EMPs at 3 and 6 days. (a) alkaline phosphatase (ALP), (b) bone sialoprotein (BSP), (c) osteocalcin (OCN). All values normalized to GAPDH (*P < 0.05).

Figure 5

Representative histological images of implants at 2 weeks post‐surgery. (a) and (e) CPC alone group, (b) and (f) CPC + 150 μg/ml EMPs group, (c) and (g) CPC + pBMSCs group, (d) and (h) CPC + 150 μg/ml EMPs + pBMSCs group (a, b, c, d ×100; e, f, g, h ×200). A slight amount of new bone formation was found in the CPC + 150 μg/ml EMPs + pBMSCs group, whereas no new bone was found in other groups.

Figure 6

Representative histological image and new bone area assessment of implants at 4 weeks post‐surgery. (a) and (e) CPC alone group, (b) and (f) CPC + 150 μg/ml EMPs group, (c) and (g) CPC + pBMSCs group, (d) and (h) CPC + 150 μg/ml EMPs + pBMSCs group (a, b, c, d ×100, e, f, g, h ×200). Apparent new bone formation was observed in CPC + pBMSCs group and CPC + 150 μg/ml EMPs + pBMSCs group, but still no new bone was found in CPC alone and CPC + 150 μg/ml EMPs groups. (i) Percentage of new bone area in CPC + 150 μg/ml EMPs + pBMSCs group was significantly higher than that in CPC + pBMSCs group by histomorphometric analysis (n = 4, *P < 0.05).