Periodontal ligament stem cells (PDLSCs) are known to play a pivotal role in regenerating the periodontium. Amelogenin, which belongs to a family of extracellular matrix (ECM) proteins, is a potential bioactive molecule for periodontal regenerative therapy. However, its downstream target molecules and/or signaling patterns are still unknown. Our recent proteomic study identified glucose‐regulated protein 78 (Grp78) as a new amelogenin‐binding protein. In this study, we demonstrate, for the first time, the cellular responses induced by the biological interaction between amelogenin and Grp78 in the human undifferentiated PDL cell line 1‐17, which possesses the most typical characteristics of PDLSCs. Confocal co‐localization experiments revealed the internalization of recombinant amelogenin (rM180) via binding to cell surface Grp78, and the endocytosis was inhibited by the silencing of Grp78 in 1‐17 cells. Microarray analysis indicated that rM180 and Grp78 regulate the expression profiles of cell migration‐associated genes in 1‐17 cells. Moreover, Grp78 overexpression enhanced rM180‐induced cell migration and adhesion without affecting cell proliferation, while silencing of Grp78 diminished these activities. Finally, binding of rM180 to Grp78 promoted the formation of lamellipodia, and the simultaneous activation of Rac1 was also demonstrated by NSC23766, a widely accepted Rac1 inhibitor. These results suggest that Grp78 is essential for enhancing amelogenin‐induced migration in 1‐17 cells. The biological interaction of amelogenin with Grp78 offers significant therapeutic potential for understanding the biological components and specific functions involved in the signal transduction of amelogenin‐induced periodontal tissue regeneration. J. Cell. Physiol. 231: 414–427, 2016. © 2015 Wiley Periodicals, Inc.

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Sprouty was identified as an inhibitor of the fibroblast growth factor (FGF) receptor, and Sprouty2 (Spry2) functions as a negative regulator of receptor tyrosine kinase signaling. In this study, we investigated how inhibition of Spry2 affects osteoblasts and gingival epithelial cells in periodontal tissue regeneration in vitro. Transduction of a dominant-negative mutant of Spry2 (Y55A-Spry2) enhanced basic fibroblast growth factor (bFGF)- and epidermal growth factor (EGF)-induced ERK activation in MC3T3-E1 osteoblastic cells. In contrast, it decreased their activation in GE1 cells. Consistent with these observations, Y55A-Spry2 increased osteoblast proliferation with bFGF and EGF stimulation, whereas the proliferation of Y55A-Spry2-introduced GE1 cells was decreased via the ubiquitination and degradation of EGF receptors (EGFRs). In addition, Y55A-Spry2 caused upregulation of Runx2 expression and downregulation of Twist, a negative regulator of Runx2, with treatment of bFGF and EGF, resulting in enhanced osteoblastogenesis accompanied by alkaline phosphatase activation and osteocalcin expression in MC3T3-E1 cells. These data suggest that suppression of Spry2 expression induces proliferation and differentiation of osteoblastic cells after the addition of a bFGF and EGF cocktail but inhibits proliferation in gingival epithelial cells. These in vitro experiments may provide a molecular basis for novel therapeutic approaches in periodontal tissue regeneration. Taken together, our study proposes that combined application of an inhibitor for tyrosine 55 of Spry2, bFGF, and EGF may effectively allow alveolar bone growth and block the ingrowth of gingival epithelial cells toward bony defects, biologically mimicking a barrier effect in guided tissue regeneration, with in vivo investigation in the future.

To examine the involvement of transforming growth factor-β₁ (TGF-β₁) in intestinal anastomotic repair, we administered a TGF-β₁-neutralizing antibody to rats after operation, and then examined its influence on the healing process and interaction with other peptide growth factors. Thirty male Sprague-Dawley rats were subjected to primary anastomosis after transection of the small intestine (n = 30) and treated by intraperitoneal administration of IgG (n = 15) or the TGF-β₁ neutralizing antibody (n = 15). Treatment with the antibody against TGF-β₁ resulted in more definite mucosal growth and increased vascularity on day 5 after surgery. Augmented mRNA expression of epidermal growth factor and vascular endothelial growth factor, and an increased number of cells that expressed these peptides in granulation tissue were demonstrated by RT-PCR and immunohistochemical staining. Taken together it was indicated that TGF-β₁ has negative effects on regeneration of the bowel wall mucosa and angiogenesis in the course of intestinal anastomotic wound healing.

Background In sinus augmentation, when remaining bone height is ≤5 mm, a lateral window approach is often the preferred choice; nonetheless, patients prefer to have a less invasive approach such as crestal sinus augmentation (CSA). Prior case reports have described the use of various staged approaches of a CSA technique in cases of limited bone height. The aim of this report was to describe the results of a case series in which a two‐stage CSA technique was used in patients with 4 to 6 mm of bone height. Methods Nineteen subjects with 28 sinuses of initial vertical bone height of 4 to 6 mm were included in which a two‐stage CSA technique was used in place of a lateral window approach. In the first surgery, 0.3 mL graft material was inserted into all sites. In the second surgery, 13 sites were filled with 0.2 mL graft material and remaining 15 sites were filled with 0.4 mL. Results No damage was observed in the maxillary sinus floor membrane after first 0.2 mL filling; however, one case had Schneiderian membrane perforation after filling 0.4 mL. The average elevation height (EH) after first surgery was 5.81 ± 0.7 mm, 5.15 ± 0.91 mm before second surgery, 6.69 ± 0.89 mm with 0.2 mL filling (total 0.5 mL) and 8.11 ± 1.24 mm with 0.4 mL filling (total 0.7 mL). The thickness of maxillary sinus membrane before first surgery was 2.6 ± 2.59 mm; however, it has become 0.97 ± 1.59 mm before second surgery, with a decrease of 1.6 mm estimate. Conclusion This case series that assessed outcomes of staged crestal maxillary sinus augmentation was an effective approach to elevating 6 or 8 mm alveolar bone height without causing major membrane perforation. However, the two‐stage approach was used in the limited residual bone height (4 to 6 mm) and required two separate surgical procedures.

Periodontitis is a chronic inflammatory disorder caused by specific bacteria residing in the biofilm, particularly Porphyromonas gingivalis (Pg). Sprouty2 (Spry2) functions as a negative regulator of the fibroblast growth factor (FGF) signaling pathway. We previously demonstrated that sequestration of Spry2 induced proliferation and osteogenesis in osteoblastic cells by basic FGF (bFGF) and epidermal growth factor (EGF) stimulation in vitro, but diminished cell proliferation in gingival epithelial cells. In addition, Spry2 knockdown in combination with bFGF and EGF stimulation increases periodontal ligament cell proliferation and migration accompanied by prevention of osteoblastic differentiation. In this study, we investigated the mechanisms through which Spry2 depletion by interferon (IFN) γ and Pg lipopolysaccharide (LPS) stimulation affected the physiology of macrophages in vitro. Transfection of macrophages with Spry2 small‐interfering RNA (siRNA) promoted the expression of genes characteristic of M2 alternative activated macrophages, induced interleukin (IL)‐10 expression, and enhanced arginase activity, even in cells stimulated with IFNγ and Pg LPS. In addition, we found that phosphoinositide 3‐kinase (PI3K) and AKT activation by Spry2 downregulation enhanced efferocytosis of apoptotic cells by increasing Rac1 activation and decreasing nuclear factor kappa B (NFκB) p65 phosphorylation but not signal transducer and activator of transcription 1 (STAT1) phosphorylation. Collectively, our results suggested that topical administration of Spry2 inhibitors may efficiently resolve inflammation in periodontal disease as macrophage‐based anti‐inflammatory immunotherapy and may create a suitable environment for periodontal wound healing. These in vitro findings provide a molecular basis for new therapeutic approaches in periodontal tissue regeneration.

Spry2 combined with bFGF and EGF stimulation reduced PDL cell migration and proliferation with inducing osteoblastic differentiation. These in vitro findings may provide a molecular basis for novel therapeutic approaches for establishing periodontal tissue regeneration.