RESUMO
PURPOSE: The entry of bacteria or harmful substances through the epithelial seal of human gingival keratinocytes (HGKs) in the junctional epithelium (JE) is blocked by specialized intercellular junctions such as E-cadherin junctions (ECJs). However, the influence of roughened substrates, which may occur due to apical migration of the JE, root planing, or peri-implantitis, on the development of the ECJs of HGKs remains largely unknown. METHODS: HGKs were cultured on substrates with varying levels of roughness, which were prepared by rubbing hydrophobic polystyrene dishes with silicon carbide papers. The activity of c-Jun N-terminal kinase (JNK) was inhibited with SP600125 or by transfection with JNK short hairpin RNA. The development of intercellular junctions was analyzed using scanning electron microscopy or confocal laser scanning microscopy after immunohistochemical staining of the cells for E-cadherin. The expression level of phospho-JNK was assessed by immunoblotting. RESULTS: HGKs developed tight intercellular junctions devoid of wide intercellular gaps on smooth substrates and on rough substrates with low-nanometer dimensions (average roughness [Ra]=121.3±13.4 nm), although the ECJs of HGKs on rough substrates with low-nanometer dimensions developed later than those of HGKs on smooth substrates. In contrast, HGKs developed short intercellular junctions with wide intercellular gaps on rough substrates with mid- or high-nanometer dimensions (Ra=505.3±115.3 nm, 867.0±168.6 nm). Notably, the stability of the ECJs was low on the rough substrates, as demonstrated by the rapid destruction of the cell junction following calcium depletion. Inhibition of JNK activity promoted ECJ development in HGKs. JNK was closely associated with cortical actin in the regulation of ECJs in HGKs. CONCLUSIONS: These results indicate that on rough substrates with nanometer dimensions, the ECJs of HGKs develop slowly or defectively, and that this effect can be reversed by inhibiting JNK.
RESUMO
The periodontium, consisting of gingiva, periodontal ligament (PDL), cementum, and alveolar bone, is necessary for the maintenance of tooth function. Specifically, the regenerative abilities of cementum with inserted PDL are important for the prevention of tooth loss. Periodontal ligament stem cells (PDLSCs), which are located in the connective tissue PDL between the cementum and alveolar bone, are an attractive candidate for hard tissue formation. We investigated the effects of recombinant human plasminogen activator inhibitor-1 (rhPAI-1) on cementogenic differentiation of human PDLSCs (hPDLSCs) in vitro and in vivo. Untreated and rhPAI-1-treated hPDLSCs mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) and dentin matrix were transplanted subcutaneously into the dorsal surface of immunocompromised mice to assess their capacity for hard tissue formation at 8 and 10 weeks posttransplantation. rhPAI-1 accelerated mineral nodule formation and increased the mRNA expression of cementoblast-associated markers in hPDLSCs. We also observed that rhPAI-1 upregulated the levels of osterix (OSX) and cementum protein 1 (CEMP1) through Smad2/3 and p38 pathways, whereas specific inhibitors of Smad3 and p38 inhibited the enhancement of mineralization of hPDLSCs by rhPAI-1. Furthermore, transplantation of hPDLSCs with rhPAI-1 showed a great ability to promote cementogenic differentiation. Notably, rhPAI-1 induced hPDLSCs to regenerate cementum-like tissue with PDL fibers inserted into newly formed cementum-like tissue. These results suggest that rhPAI-1 may play a key role in cementogenic differentiation of hPDLSCs. rhPAI-1 with hPDLSCs may be a good candidate for future clinical applications in periodontal tissue regeneration and possibly in tooth root bioengineering.
