Osteogenic differentiation of human amniotic epithelial cells and its application in alveolar defect restoration

Abstract

The present study investigated the detailed in vitro osteogenic differentiation process and in vivo bone regenerative property of human amniotic epithelial cells (hAECs). The in vitro osteogenic differentiation process of hAECs was evaluated by biochemical staining, real-time polymerase chain reaction, and immunofluorescence. Next, β-tricalcium phosphate (β-TCP) scaffolds alone or loaded with hAECs were implanted into the alveolar defects of rats. Micro-computed tomography evaluation and histologic studies were conducted. Our results validated the in vitro osteogenic capacity of hAECs by upregulation of Runx2, osterix, alkaline phosphatase, collagen I, and osteopontin, with positive biochemical staining for osteoblasts. An epithelial-mesenchymal transformation process might be involved in the osteogenic differentiation of hAECs by increased expression of transforming growth factor-β1. Our data also demonstrated that in vivo implantation of hAECs loaded on β-TCP scaffolds, not only improved bone regeneration by direct participation, but also reduced the early host immune response to the scaffolds. The presented data indicate that hAECs possess proper osteogenic differentiation potential and a modulatory influence on the early tissue remodeling process, making these cells a potential source of progenitor cells for clinical restoration of the alveolar defect.

Keywords: Alveolar defect; Host immune response; Human amniotic epithelial cells; Osteogenesis.

©AlphaMed Press.

Figures

Figure 1.

Characterization of hAECs in vitro. (A, B): hAECs at passages 0 and 1 displayed a cobblestone-like morphology. (C): Some hAECs changed into fibroblast-like cells after 7 days of osteoblastic culture. (D): hAECs showed significant morphological changes and settled on superimposed layers after 21 days of osteoblastic culture. (E): Cell proliferation of hAECs at passage 1 was significantly higher than at passages 0 and 5 from day 4 to day 12; hAECs at passage 5 displayed the lowest proliferation rate from day 6 to day 14 (☆, p < .05). (F): Flow cytometry analysis showed that hAECs expressed CD44, CD90, CD105, and SSEA-4 and did not express CD34, CD45, and HLA-DR. Values represent the percentages of all assessed cells positively stained by the indicated antigens (bottom of each graph). Nonspecific fluorescence was determined as the blank control using isotype-matched monoclonal antibodies (PE blank, FITC blank). (G): Representative images of microscopic and general photographs for ALP and ARS staining in osteogenic and control groups indicated the osteogenic differentiation of hAECs in vitro. Scale bar = 200 μm. Abbreviations: ALP, alkaline phosphatase; ARS, alizarin red S; FITC, fluorescein isothiocyanate; hAEC, human amniotic epithelial cell; OD, optical density; p, passage; PE, phycoerythrin.

Figure 2.

Real-time polymerase chain reaction (PCR) and immunofluorescence study for osteogenic differentiation of hAECs in vitro. (A): Real-time PCR assay of osteoblastic marker genes showed significant upregulation of Runx2, Osx, ALP, Col I, and OPN in hAECs at days 5, 10, and 14 after osteogenic induction compared with those in the control group (☆, p < .05). (B): Representative images of OPN and Col I immunofluorescence staining at 10 days of osteogenic differentiation in osteogenic and control groups under epifluorescence microscope. Image magnification, ×200. Scale bar = 50 μm. Abbreviations: ALP, alkaline phosphatase; Col I, collagen I; CTR, control (defect grafting with β-tricalcium phosphate scaffolds); DAPI, 4′6-diamidino-2-phenylindole; hAECs, human amniotic epithelial cells; OPN, osteopontin; Osx, osterix.

Figure 3.

Real-time polymerase chain reaction (PCR), Western blot, and immunofluorescence study for epithelial-mesenchymal transformation (EMT) process during osteogenic differentiation of hAECs in vitro. (A): Real-time PCR assay of EMT marker genes showed significant upregulation of vimentin, Snai1, TGF-βR1, and TGF-β1 and downregulation of Klf4 and E-cadherin in hAECs at days 5, 10, and 14 after osteogenic induction compared with those in the control group (☆, p < .05). (B): Representative images of E-cadherin and vimentin immunofluorescence staining at 10 days of osteogenic differentiation in osteogenic and control groups under epifluorescence microscope. Image magnification, ×200. Scale bar = 50 μm. (C): Qualitative and semiquantitative Western blot analysis of TGF-β1 expression in hAECs at days 5, 10, and 14 after osteogenic induction compared with those in the control group (☆, p < .05). Abbreviations: CTR, control (defect grafting with β-tricalcium phosphate scaffolds); DAPI, 4′6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hAECs, human amniotic epithelial cells; TGF-β1, transforming growth factor-β1; TGFBR1, transforming growth factor-β receptor type 1.

Figure 4.

In vivo healing process in alveolar defect at 4 and 8 weeks postoperatively. (A): Representative three-dimensional micro-computed tomography (CT) reconstruction images of hAECs+β-TCP scaffold (EXP) and β-TCP scaffold (CTR) at 4 and 8 weeks postoperatively. Scale bar = 1 mm. (B): Micro-CT parameters acquired among β-TCP scaffold in vitro, hAECs+β-TCP scaffold in vivo, and β-TCP scaffold alone in vivo at 4 and 8 weeks postoperatively (☆, p < .05). (C): Hematoxylin and eosin staining of the rat alveolar defect at 4 and 8 weeks postoperatively revealed more active new bone formation in the EXP group than in the CTR group (×50 and ×200 magnification). Alveolar defect treated with hAECs+β-TCP scaffold exhibited a more mature lamellae-bone formation at 8 weeks postoperatively. Scale bar = 200 μm. (D): ANA-positive cells, visible as green fluorescence in the nuclei, were observed within the newly deposited OCN, and OPN-positive bone tissue, visible as red fluorescence, in the EXP group at 4 weeks postoperatively, indicating a mature osteoblastic function of these hAEC-derived cells. Scale bar = 50 μm. (E): Representative images of immunohistochemical staining of sections with anti-VEGF antibody and anti-CD68 antibody in hAECs+β-TCP scaffold (EXP) and β-TCP scaffold (CTR) at 4 and 8 weeks postoperatively. Scale bar = 200 μm. (F): The histomorphometric quantification of the relative new bone area, VEGF-positive area, and CD68-positive area showed that more bone tissue regeneration was observed in the EXP group than in the CTR group at 4 and 8 weeks postoperatively. The positive signal of VEGF and CD68 in the EXP group was much weaker at 4 weeks postoperatively and became more intense at 8 weeks postoperatively compared with the CTR group (☆, p < .05). Abbreviations: ANA, anti-nuclear antibody; BV/TV, bone volume/tissue volume ratio; BMD, bone mineralization density; hAECs, human amniotic epithelial cells; OPN, osteopontin; post op, postoperatively; SMI, structure model index; Tb.Th., trabecular thickness; Tb.N., trabecular number; Tb.Sp., trabecular separation; β-TCP, β-tricalcium phosphate; VEGF, vascular endothelial growth factor.