miRNA-Based Early Healing Mechanism of Extraction Sockets: miR-190a-5p, a Potential Enhancer of Bone Healing.

Objective: Tooth extraction causes a wound with hard and soft tissue defects in the alveolar ridge. Few studies have reported the function of microRNAs (miRNAs) in the healing of extraction sockets. This study used bioinformatics analysis to reveal the possible relevance and role of miRNAs during the early stages following tooth extraction. Materials and Methods: Socket tissues from beagle dogs (Canis familiaris; two males and two females) were collected 1 and 12 hours after extraction of premolars on both sides of the mandible. miRNA expression was profiled through miRNA sequencing, and hub miRNAs showing characteristic expression patterns were selected and subjected to target enrichment analysis. Alkaline phosphatase (ALP) activity analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were performed to verify the effect of hub miRNA on osteoblast differentiation and bone regeneration in vivo. Results: Five miRNAs were identified to have consistently high expression levels, with cfa-miR-451 showing the highest expression. Additionally, 20 hub miRNAs were selected as candidates expected to play an important role in the healing process. Pathways, such as the MAPK, axon guidance, TGF-ß, and Wnt signaling, were significantly enriched. Among hub miRNAs, miR-190a-5p increased ALP activity and mRNA expression of osteogenic markers and increased new bone formation in vivo. Conclusions: Our findings suggest that miRNAs may be involved in the earliest stages of socket healing after tooth extraction and can play an important role in moderating the entire socket healing mechanism in the extraction socket.

A Study on the Correlation between C-Reactive Protein Concentration and Teeth with a ≥5 mm Periodontal Pocket in Chronic Periodontitis Patients.

OBJECTIVE: To evaluate the relationship between CRP levels and teeth with ≥5 mm PD in chronic periodontitis patients. MATERIALS AND METHODS: We evaluated 49 patients with chronic periodontitis who visited the Department of Periodontology at Wonkwang University Dental Hospital. At the first visit, high-sensitive CRP testing of venous blood samples was performed, and correlations were statistically evaluated. RESULTS: The mean hs-CRP level of patients diagnosed with severe periodontitis was 2.0 mg/L (0.13-13.95 mg/L). Statistically, patients with a high rate of teeth diagnosed with severe periodontitis are more likely to have higher hs-CRP level. CONCLUSION: Within the limits of this study, the number and proportion of teeth showing ≥5 mm PD was positively correlated with CRP concentration.

Alveolar Crestal Approach for Maxillary Sinus Membrane Elevation with <4 mm of Residual Bone Height: A Case Report.

INTRODUCTION: For maxillary sinus membrane elevation (MSME), the lateral window approach and crestal approach are available, and high success rates have been achieved with low residual bone height as a development of technology. OBJECTIVE: To evaluate MSME using the crestal approach with a rotary-grind bur (RGB (including reamer or sinus bur)) in patients with residual bone height of <4 mm. MATERIALS AND METHODS: Ten implants were placed in 10 patients with residual bone height of <4 mm, by sinus elevation using an RGB. The implant stability quotient (ISQ) was measured immediately after implant placement (ISQ 1) and before taking impression for the final prosthesis (ISQ 2). The extent of marginal bone loss was measured on periapical radiographs. RESULTS: The mean residual bone height before implant placement was 3.41 ± 0.53 mm; no complications, including membrane perforation, severe postoperative pain, or discomfort, occurred either during or after surgery. The mean ISQ 1 was 63.4 ± 12.1, whereas the mean ISQ 2 was 77.6 ± 5.8. The mean marginal bone resorption was 0.23 ± 0.18 mm on periapical radiographs. CONCLUSIONS: MSME using the crestal approach with an RGB is a reliable technique for implant placement in sites where available bone is insufficient.

BST2 Mediates Osteoblast Differentiation via the BMP2 Signaling Pathway in Human Alveolar-Derived Bone Marrow Stromal Cells.

The molecular mechanisms controlling the differentiation of bone marrow stromal stem cells into osteoblasts remain largely unknown. In this study, we investigated whether bone marrow stromal antigen 2 (BST2) influences differentiation toward the osteoblasts lineage. BST2 mRNA expression in human alveolar-derived bone marrow stromal cells (hAD-BMSCs) increased during differentiation into osteoblasts. hAD-BMSCs differentiation into osteoblasts and the mRNA expression of the bone-specific markers alkaline phosphatase, collagen type α 1, bone sialoprotein, osteocalcin, and osterix were reduced by BST2 knockdown using siRNA. Furthermore, BST2 knockdown in hAD-BMSCs resulted in decreased RUNX2 mRNA and protein expression. We hypothesized that BST2 is involved in differentiation of into osteoblasts via the BMP2 signaling pathway. Accordingly, we evaluated the mRNA expression levels of BMP2, BMP receptors (BMPR1 and 2), and the downstream signaling molecules SMAD1, SMAD4, and p-SMAD1/5/8 in BST2 knockdown cells. BMP2 expression following the induction of differentiation was significantly lower in BST2 knockdown cells than in cells treated with a non-targeting control siRNA. Similar results were found for the knockdown of the BMP2 receptor- BMPR1A. We also identified significantly lower expression of SMAD1, SMAD4, and p-SMAD1/5/8 in the BST2 knockdown cells than control cells. Our data provide the first evidence that BST2 is involved in the osteogenic differentiation of bone marrow stromal cells via the regulation of the BMP2 signaling pathway.

Functionalization of scaffolds with chimeric anti-BMP-2 monoclonal antibodies for osseous regeneration.

Recent studies have demonstrated the ability of murine anti-BMP-2 monoclonal antibodies (mAb) immobilized on an absorbable collagen sponge (ACS) to mediate de novo bone formation, a process termed antibody-mediated osseous regeneration (AMOR). The objectives of this study were to assess the efficacy of a newly generated chimeric anti-BMP-2 mAb in mediating AMOR, as well as to evaluate the suitability of different biomaterials as scaffolds to participate in AMOR. Chimeric anti-BMP-2 mAb was immobilized on 4 biomaterials, namely, titanium microbeads (Ti), alginate hydrogel, macroporous biphasic calcium phosphate (MBCP) and ACS, followed by surgical implantation into rat critical-size calvarial defects. Animals were sacrificed after 8 weeks and the degree of bone fill was assessed using micro-CT and histomorphometry. Results demonstrated local persistence of chimeric anti-BMP-2 mAb up to 8 weeks, as well as significant de novo bone regeneration in sites implanted with chimeric anti-BMP-2 antibody immobilized on each of the 4 scaffolds. Ti and MBCP showed the highest volume of bone regeneration, presumably due to their resistance to compression. Alginate and ACS also mediated de novo bone formation, though significant volumetric shrinkage was noted. In vitro assays demonstrated cross-reactivity of chimeric anti-BMP-2 mAb with BMP-4 and BMP-7. Immune complex of anti-BMP-2 mAb with BMP-2 induced osteogenic differentiation of C2C12 cells in vitro, involving expression of RUNX2 and phosphorylation of Smad1. The present data demonstrated the ability of chimeric anti-BMP-2 mAb to functionalize different biomaterial with varying characteristics to mediate osteogenesis.

Association of gingival biotype with the results of scaling and root planing.

PURPOSE: The concept of gingival biotype has been used as a predictor of periodontal therapy outcomes since the 1980s. In the present study, prospective and controlled experiments were performed to compare periodontal pocket depth (PPD) reduction and gingival shrinkage (GSH) after scaling and root planing (SRP) according to gingival biotype. METHODS: Twenty-five patients diagnosed with chronic periodontitis participated in the present study. The PPD and GSH of the labial side of the maxillary anterior teeth (from the right canine to the left canine) were evaluated at baseline and 3 months after SRP. Changes in the PPD following SRP were classified into 4 groups according to the gingival thickness and initial PPD. Two more groups representing normal gingival crevices were added in evaluation of the GSH. The results were statistically analyzed using the independent t-test. RESULTS: In the end, 16 patients participated in the present study. With regard to PPD reduction, there were no significant differences according to gingival biotype (P>0.05). Likewise, sites with a PPD of over 3 mm failed to show any significant differences in the GSH (P>0.05). However, among the sites with a PPD of under 3 mm, those with the thin gingival biotype showed more GSH (P<0.05). CONCLUSIONS: PPD changes after SRP were not affected by gingival biotype with either shallow or deep periodontal pockets. GSH also showed equal outcomes in all the groups without normal gingival crevices. The results of SRP seem not to differ according to gingival biotype.

IFITM1 increases osteogenesis through Runx2 in human alveolar-derived bone marrow stromal cells.

The exact molecular mechanisms governing the differentiation of bone marrow stromal stem/progenitor cells (BMSCs) into osteoblasts remain largely unknown. In this study, a highly expressed protein that had a high degree of homology with interferon-induced transmembrane protein 1 (IFITM1) was identified using differentially expressed gene (DEG) screening. We sought to determine whether IFITM1 influenced osteoblast differentiation. During differentiation, IFITM1 expression gradually increased from 5 to 10days and subsequently decreased at 15 days in culture. Analysis of IFITM1 protein expression in several cell lines as well as in situ studies on human tissues revealed its selective expression in bone cells and human bone. Proliferation of human alveolar-derived bone marrow stromal cells (hAD-BMSCs) was significantly inhibited by IFITM1 knockdown by using short hairpin RNA, as were bone specific markers such as alkaline phosphatase, collagen type I α 1, bone sialoprotein, osteocalcin, and osterix were decreased. Calcium accumulation also decreased following IFITM1 knockdown. Moreover, IFITM1 knockdown in hAD-BMSCs was associated with inhibition of Runx2 mRNA and protein expression. Collectively, the present data provide evidence for the role of IFITM1 in osteoblast differentiation. The exact mechanisms of IFITM1's involvement in osteoblast differentiation are still under investigation.

In Vitro and In Vivo Osteogenic Activity of Largazole.

Due to their capability of modifying chromatin structure and thereby regulating gene transcription, histone deacetylases (HDACs) have been reported to play important roles in osteogenesis and considered a promising potential therapeutic target for bone diseases, including osteoporosis. We showed that the novel marine-derived HDAC inhibitor largazole exhibits in vitro and in vivo osteogenic activity. Largazole significantly induced the expression of ALP and OPN. The osteogenic activity of largazole was mediated through the increased expression of Runx2 and BMPs. Importantly, largazole showed in vivo bone-forming efficacy in the mouse calvarial bone formation assay and the rabbit calvarial bone fracture healing model. The dual action of largazole to stimulate bone formation and inhibit bone resorption would be a useful feature in drug development for bone-related disorders.

Healing pattern of the mucous membrane after tooth extraction in the maxillary sinus.

PURPOSE: To investigate the healing pattern of the mucous membrane after tooth extraction necessitated by periodontal disease in the maxillary sinus. METHODS: One hundred and three patients with 119 maxillary sinuses were investigated. Before implant placement, cone-beam computed tomography (CT) scanning was performed. The causes of extraction, the time elapsed since extraction, smoking, periodontal disease in adjacent teeth, and gender were recorded. In addition, the thickness of the mucous membrane of the maxillary sinus and the height of residual alveolar bone at the extracted area were calculated from CT images. RESULTS: The thickness of the mucous membrane in the periodontal disease group (3.05±2.71 mm) was greater than that of the pulp disease group (1.92±1.78 mm) and the tooth fracture group (1.35±0.55 mm; P<0.05). The causes of extraction, the time elapsed since extraction, and gender had relationships with a thickening of the mucous membrane of the maxillary sinus (P<0.05). In contrast, the height of the residual alveolar bone at the extracted area, periodontal disease in adjacent teeth, and smoking did not show any relation to the thickening of the mucous membrane of the maxillary sinus. CONCLUSIONS: The present study revealed distinct differences in healing patterns according to the causes of extraction in the maxillary sinus, especially periodontal disease, which resulted in more severe thickening of the mucous membrane.

Effects of the dichloromethane fraction of Dipsaci Radix on the osteoblastic differentiation of human alveolar bone marrow-derived mesenchymal stem cells.

Dipsaci Radix is the dried root of Dipsacus asper Wall. It has been used in Korean herbal medicine to treat bone fractures. In this study, we examined the effect of the dichloromethane fraction of Dipsaci Radix (DR(DM)) on the osteoblastic differentiation of human alveolar bone marrow-derived MSCs (ABM-MSCs). The ABM-MSCs were isolated from healthy subjects and cultured in vitro, followed by phenotypic characterization. They showed a fibroblast-like morphology and expressed CD29, CD44, CD73, and CD105, but not CD34. Calcified nodules were generated in response to both dexamethasone (DEX) and DR(DM). There was a significant increase in the alkaline phosphatase (ALP) activity and protein expression of bone sialoprotein (BSP) and osteocalcin (OC) in response to DEX and DR(DM) as compared to control. These results provide evidence for the osteogenic potential of cultured ABM-MSCs in response to DR(DM). Also, an active single compound was additionally isolated from DR(DM). The single compound (hederagenin 3-O-(2-O-acetyl)-α-L-arabinopyranoside) also significantly increased ALP activity and the level of protein expression of BSP and OC. These results highlight the possible clinical applications of DR(DM) and hederagenin 3-O-(2-O-acetyl)-α-L-arabinopyranoside in bone regeneration.

The anti-inflammatory role of heme oxygenase-1 in lipopolysaccharide and cytokine-stimulated inducible nitric oxide synthase and nitric oxide production in human periodontal ligament cells.

BACKGROUND: Although heme oxygenase-1 (HO-1) is involved in anti-inflammation, the mechanisms of its activity in regulating periodontal inflammation are largely unclear. Therefore, the aim of this study is to investigate the anti-inflammatory properties of HO-1 in lipopolysaccharide (LPS)- and proinflammatory cytokine-stimulated inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in human periodontal ligament (PDL) cells. METHODS: PDL cells were treated with LPS plus a combination of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta in serum-free media for 1 day. The production of NO was evaluated using a Griess reagent kit. The expression of iNOS and HO-1 proteins and mRNAs was evaluated using Western blotting and reverse transcriptase-polymerase chain reaction, respectively. RESULTS: Proinflammatory cytokines and LPS triggered iNOS and HO-1 expression and NO production in PDL cells. HO-1 inhibitor and HO-1 small interfering RNA (siRNA) attenuated the LPS- and cytokine-stimulated NO release and iNOS and HO-1 expression. Specific inhibitors of p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases phosphatidylinositol 3-kinase (PI3K), nuclear factor-kappa B (NF-kappaB), and protein kinase C delta (PKC-delta) greatly reduced the levels of iNOS and HO-1 expression induced by LPS plus cytokines. CONCLUSIONS: Collectively, these data suggested that HO-1 inhibition blocked LPS- and proinflammatory cytokine-stimulated iNOS expression and NO production in PDL cells via a mechanism that involves p38, ERK, PI3K, NF-kappaB, and PKC-delta. Thus, the regulation of HO-1 activity may be a therapeutic strategy for periodontal disease.

Comparative histomorphometric analysis of extraction sockets healing implanted with bovine xenografts, irradiated cancellous allografts, and solvent-dehydrated allografts in humans.

PURPOSE: Bovine-derived bone xenograft and mineralized cancellous bone allograft have been successfully used as bone substitutes in dental surgery, but few clinical studies in humans have been reported. The objective of this study was to compare the osteoconductive effects of deproteinized bovine bone mineral (DBBM), irradiated cancellous allograft (ICA), and solvent-dehydrated allograft (SDA) when used to preserve extraction sockets. MATERIALS AND METHODS: Twenty patients received bone grafting in extraction sockets with DBBM (n = 7), ICA (n = 8), or SDA (n = 5). Core biopsies were taken from each graft site 4 to 6 months after grafting and were evaluated histomorphometrically. One-way analysis of variance was used to compare each variable. P values less than .05 were considered significant. RESULTS: DBBM induced more new bone deposition in the periphery of the native bone particles than ICA or SDA, whereas ICA and SDA were more frequently surrounded by fibrous tissue than DBBM. In addition, DBBM retained more residual graft bony particles than ICA or SDA. CONCLUSIONS: Based on these findings, the DBBM showed more of an osteoconductive effect than ICA or SDA, producing a more rigid bony structure. It is therefore suggested that DBBM may be more favorable for the preservation of extraction sockets than allogeneic graft materials.

Effects of nicotine on antioxidant defense enzymes and RANKL expression in human periodontal ligament cells.

BACKGROUND: This study examined the effects of nicotine on osteoblastic differentiation and the osteoclastogenesis regulatory molecules receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG). In addition, we investigated the mechanism by which nicotine induced antioxidant defense enzyme expression as a protective response. METHODS: The expression of osteoblast markers, RANKL, OPG, and antioxidant defense enzymes were examined in nicotine-treated human periodontal ligament (PDL) cells by reverse transcription-polymerase chain reaction and Western blotting. RESULTS: Nicotine treatment concomitantly downregulated the expression of OPG and osteoblastic differentiation markers, such as alkaline phosphatase, osteocalcin, and osteopontin, and upregulated the expression of RANKL. Nicotine induced the synthesis of the transcription factor NF-E2-related factor-2 (Nrf2) as well as a number of cellular antioxidants and phase II enzymes, such as heme oxygenase-1. Pretreatment with antioxidants inhibited the upregulation of RANKL, the downregulation of OPG expression, and cytotoxicity by nicotine in PDL cells. CONCLUSIONS: Nicotine upregulated RANKL and antioxidant defense enzymes. These data suggest that Nrf2-mediated induction of cellular antioxidants and phase II enzymes could contribute to the cellular defense against nicotine-induced cytotoxicity and osteoclastic differentiation in PDL cells.

Characterization of the surface immobilized synthetic heparin binding domain derived from human fibroblast growth factor-2 and its effect on osteoblast differentiation.

Fibroblast growth factor (FGF)-2 regulates a variety of cellular functions, such as proliferation and differentiation, by binding to cell surface FGF receptors (FGFRs) in the presence of heparin proteoglycans. FGF-2 is known as a heparin-binding growth factor, but the localization of the heparin binding site has not been fully investigated until now. We used two potential heparin binding domains of FGF-2, the residues 105-111 (F105, YKRSRYT) and 119-135 (F119, KRTGQYKLGSKTGPGQK). Peptides could be stably immobilized onto the surface of tissue culture plates. Using solid phase binding assays, we demonstrated that both peptides had higher binding affinity toward heparin compared with nonbinding control sequence. The biological significance of these sites was tested by cell attachment and osteoblast differentiation studies. Cell attachment to the peptides F105 and F119 increased in a dose-dependent manner. Heparin and heparinase treatments decreased cell adhesion to both F105 and F119. This demonstrates that both F105 and F119 interact with cell-surface heparan sulfate proteoglycans, suggesting that FGF-2 has two heparin binding sites. In addition, osteoblast differentiation, confirmed by ALPase activity and mineralization, was increased by surface immobilized peptide F105 and F119. Taken together, these heparin binding peptides could be applied as biological agents enhancing osteoblast differentiation as well as surface modification tools in the tissue regeneration area, especially for bone regeneration.

Human pulp cells response to Portland cement in vitro.

The aim of this study was to investigate the cellular effects of Portland cement on cultured human pulp cells. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, no cytotoxicity was observed in the Portland cement group in comparison with the negative control group, whereas the glass ionomer cement, intermediate restorative material, and Dycal groups showed a survival rate of less than 40% at 12 hours. Scanning electron microscopy revealed that human pulp cells attached to the Portland cement were flat and had numerous cytoplasmic extensions. In the groups in which other materials were used, a few rounded cells were observed on the material but no living cells were observed. The expression of both osteonectin and dentin sialophosphoprotein mRNAs was induced in the Portland cement-treated group. These results suggest that Portland cement is biocompatible, allows the expression of mineralization-related genes on cultured human pulp cells, and has the potential to be used as a proper pulp-capping material.

Heme oxygenase-1 mediates cytoprotection against nitric oxide-induced cytotoxicity via the cGMP pathway in human pulp cells.

OBJECTIVE: This study examined the effects of exogenous nitric oxide (NO) on human pulp cells and the involvement of cyclic 3',5'-monophosphate (cGMP) in pulpal protection induced by heme oxygenase-1 (HO-1) against NO-induced cytotoxicity. STUDY DESIGN: This study investigated cytotoxicity and HO-1 induction in pulp cells induced by the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP), by using Western blotting and a cell viability assay. It also investigated whether HO-1 contributes to the cytoprotective effect against the cytotoxicity caused by NO and the relationship between HO-1 and cGMP in the signaling pathway. RESULTS: S-nitroso-N-acetyl-D,L-penicillamine decreased cell viability, but increased HO-1 expression in a concentration- and time-dependent manner in human pulp cells. NO-induced cytotoxicity was inhibited in the presence of hemin (inducer of HO-1), whereas it was enhanced in the presence of zinc protoporphyrin IX (ZnPP IX, HO-1 inhibitor); therefore, the NO-induced cytotoxicity was correlated with HO-1 expression. Pretreatment with a membrane-permeable cGMP analog, 8-bromo-cGMP, restored cell death and enhanced the HO-1 protein expression induced by SNAP. By contrast, 1 mM SNAP inhibited guanylate cyclase in pulp cells pretreated with 1H-[1,2,4]oxadiazole[4,3-alpha]quinoxalin-1-one (ODQ), resulting in marked cytotoxicity. CONCLUSION: These findings of a link between HO-1, regulated via the cGMP system and NO-induced cytotoxicity in human pulp cells, suggest a protective role for HO-1 in pulpal inflammation.

Roles of p38 and ERK MAP kinases in IL-8 expression in TNF-alpha- and dexamethasone-stimulated human periodontal ligament cells.

Orthodontic tooth movement is recognized as a pro-inflammatory stressor of human periodontal ligament (hPDL) cells. However, the cell-signaling pathways linking interleukin-8 (IL-8), intercellular adhesion molecule-1 (ICAM-1), pro-inflammatory cytokines, and dexamethasone in hPDL cells have not been well elucidated. In this study, we investigated the role of mitogen-activated protein (MAP) kinases in dexamethasone- and TNF-alpha-induced IL-8 and ICAM-1 expression in hPDL cells. IL-8 production was measured by enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. MAP kinase activation and IkappaB degradation were determined by Western blot analysis, and ICAM-1 expression was determined by RT-PCR and FACS analysis. TNF-alpha increased IL-8 mRNA expression and protein secretion in a dose- and time-dependent manner. Dexamethasone suppressed TNF-alpha-induced IL-8 production in a dose-dependent manner. In addition, dexamethasone inhibited TNF-alpha-induced phosphorylation of p38 MAP kinase and extracellular-regulated kinases (ERKs), IkappaB degradation, and NF-kappaB activation. Selective inhibitors for ERKs and p38 attenuated TNF-alpha-induced IL-8 and ICAM-1 expression in the presence and absence of dexamethasone, indicating that MAP kinases play a role in the response of hDPL cells to TNF-alpha. Furthermore, these results suggest that inflammatory cytokine- and dexamethasone-induced IL-8 and ICAM-1, produced via a MAP kinase pathway, may serve as an important mediator of PDL immunoregulation involved in bone remodeling during orthodontic tooth movement.