Abstract
The aim of the present study was to investigate the effect of Sonic hedgehog (Shh) on human dental pulp cells (hDPCs) and the potential of complexes with Shh gene modified hDPCs and porous calcium phosphate cement (CPC) for mineralized tissue formation. hDPCs were cultured and transfected with adenoviral mediated human Shh gene (AdShh). Overexpression of Shh and cell proliferation was tested by real-time PCR analysis, western blotting analysis, and MTT analysis, respectively. The odontoblastic differentiation was assessed by alkaline phosphatase (ALP) activity and real-time PCR analysis on markers of Patched-1 (Ptc-1), Smoothened (Smo), Gli 1, Gli 2, Gli 3, osteocalcin (OCN), dentin matrix protein-1 (DMP-1), and dentin sialophosphoprotein (DSPP). Finally, AdShh-transfected hDPCs were combined with porous CPC and placed subcutaneously in nude mice for 8 and 12 weeks, while AdEGFP-transfected and untransfected hDPCs were treated as control groups. Results indicated that Shh could promote proliferation and odontoblastic differentiation of hDPCs, while Shh/Gli 1 signaling pathway played a key role in this process. Importantly, more mineralized tissue formation was observed in combination with AdShh transfected hDPCs and porous CPC, moreover, the mineralized tissue exhibited dentin-like features such as structures similar to dentin-pulp complex and the positive staining for DSPP protein similar to the tooth tissue. These results suggested that the constructs with AdShh-transfected hDPCs and porous CPC might be a better alternative for dental tissue regeneration.
Highlights
Tissue engineering technology provides an approach to achieve dentin regeneration to potentially replace or repair the impaired dental tissues caused by caries, trauma, or clinical errors [1,2]
As shown in fig. 1E, Sonic hedgehog (Shh) mRNA in adenoviral mediated human Shh gene (AdShh)-transfected human Dental pulp cells (DPCs) (hDPCs) was upregulated to a peak at day 3, it gradually decreased with significant difference until 15 days after gene transduction (p,0.05)
Western blotting analysis demonstrated that significant increase of Shh protein expression in AdShh-transfected hDPCs was detected from 3 days to 18 days, as compared with control group (p,0.05)
Summary
Tissue engineering technology provides an approach to achieve dentin regeneration to potentially replace or repair the impaired dental tissues caused by caries, trauma, or clinical errors [1,2] This procedure can be defined as the new dental structures including dentin, root structures, and the dentin-pulp complex, are constructed in vitro or in vivo by using scaffolds, cells, and growth factors, alone or in combination, based on the basic principles of biology and engineering [3,4]. To enhance the efficacy of DPCs application for dental tissue regeneration, it is important to replicate the permissive signals that induct terminal events in odontoblastic differentiation during tooth development [6] Among these signals, Sonic hedgehog (Shh) was required at multiple stages of tooth development, such as early tooth initiation and later morphogenesis. These studies indicated that Shh had multiple functions in tooth development; whether Shh could enhance proliferation and odontoblastic differentiation of human DPCs (hDPCs), and promote mineralized tissue formation was largely unknown
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