Collagen Membranes Adsorb the Transforming Growth Factor‐β Receptor I Kinase‐Dependent Activity of Enamel Matrix Derivative

Background: Enamel matrix derivative (EMD) and collagen membranes (CMs) are simultaneously applied in regenerative periodontal surgery. The aim of this study is to evaluate the ability of two CMs and a collagen matrix to adsorb the activity intrinsic to EMD that provokes transforming growth factor (TGF)‐β signaling in oral fibroblasts. Methods: Three commercially available collagen products were exposed to EMD or recombinant TGF‐β1, followed by vigorous washing. Oral fibroblasts were either seeded directly onto collagen products or were incubated with the respective supernatant. Expression of TGF‐β target genes interleukin (IL)‐11 and proteoglycan 4 (PRG4) was evaluated by real time polymerase chain reaction. Proteomic analysis was used to study the fraction of EMD proteins binding to collagen. Results: EMD or TGF‐β1 provoked a significant increase of IL‐11 and PRG4 expression of oral fibroblasts when seeded onto collagen products and when incubated with the respective supernatant. Gene expression was blocked by the TGF‐β receptor I kinase inhibitor SB431542. Amelogenin bound most abundantly to gelatin‐coated culture dishes. However, incubation of palatal fibroblasts with recombinant amelogenin did not alter expression of IL‐11 and PRG4. Conclusion: These in vitro findings suggest that collagen products adsorb a TGF‐β receptor I kinase‐dependent activity of EMD and make it available for potential target cells.     

Enamel Matrix Derivative,Alone or Associated With a Synthetic Bone Substitute,in the Treatment of 1‐ to 2‐Wall Periodontal Defects

Background: In this study, we compare the effects of enamel matrix derivative (EMD) associated with a hydroxyapatite and β‐tricalcium phosphate (HA/β‐TCP) implant to EMD alone and to open‐flap debridement (OFD) when surgically treating 1‐ to 2‐wall intrabony defects. Methods: Thirty‐four patients, exhibiting ≥3 intraosseous defects in different quadrants, were each treated by OFD, EMD, or EMD + HA/β‐TCP in each defect. At baseline and 12 and 24 months, a complete clinical and radiographic examination was done. Pre‐therapy and post‐therapy clinical (probing depth [PD], clinical attachment level [CAL], and gingival recession [GR]) and radiographic (defect bone level [DBL] and radiographic bone gain [RBG]) parameters for the different treatments were compared. Results: After 12 and 24 months, almost all the clinical and radiographic parameters showed significant changes from baseline within each group (P <0.001). Differences in PD, CAL, and DBL scores were also seen among the three groups at the 12‐ and 24‐month visits (P <0.001). At 12 and 24 months after treatment, the EMD + HA/β‐TCP group showed significantly greater PD reduction (4.00 ± 0.42 mm; 4.25 ± 0.63 mm), CAL gain (3.47 ± 0.65 mm; 3.63 ± 0.91 mm), and RBG (3.17 ± 0.69 mm; 3.35 ± 0.80 mm) and less GR increase (0.56 ± 0.37 mm; 0.63 ± 0.42 mm) compared with the OFD and EMD groups (P <0.05). Conclusion: Our data support the hypothesis that the adjunct of an HA/β‐TCP composite implant with EMD may improve the clinical and radiographic outcomes of the surgical treatment of unfavorable intrabony defects.     

The Effect of α‐Tocopherol and Selenium on Human Gingival Fibroblasts and Periodontal Ligament Fibroblasts In Vitro

Background: The aim of the present study is to evaluate the effect of α‐tocopherol and selenium on gingival fibroblasts (GFs) and periodontal ligament fibroblasts (PDLFs) in terms of proliferation, basic fibroblast growth factor (bFGF) release, collagen type I synthesis, and wound healing. Methods: Primary cultures of human GFs and PDLFs were isolated. Four test groups and a control group free of medication was formed. In group E, 60 μM α‐tocopherol was used, and in groups ES1, ES2, and ES3, the combination of 60 μM α‐tocopherol with 5 × 10^?9 M, 10 × 10^?9 M, and 50 × 10^?9 M selenium was used, respectively. Viability, proliferation, bFGF, and collagen type I synthesis from both cell types were evaluated at 24, 48, and 72 hours, and healing was compared on a new wound‐healing model at 12, 24, 36, 48, and 72 hours. Results: α‐Tocopherol alone significantly increased the healing rate of PDLFs at 12 hours and increased bFGF and collagen type I release from GFs and PDLFs at 24, 48, and 72 hours. The α‐tocopherol/selenium combination significantly enhanced the proliferation rate of both cells at 48 hours, decreased the proliferation of PDLFs at 72 hours, and increased the healing rate of GFs at 12 hours and PDLFs at 12 and 48 hours. bFGF and collagen type I synthesis was also increased in both cell types at 24, 48, and 72 hours by α‐tocopherol/selenium combination. Conclusion: α‐Tocopherol and α‐tocopherol/selenium combination is able to accelerate the proliferation rate and wound‐healing process and increase the synthesis of bFGF and collagen type I from both GFs and PDLFs.     

Effect of Enamel Matrix Derivative on Periodontal Wound Healing and Regeneration in an Osteoporotic Model

Background : Despite the worldwide increased prevalence of osteoporosis, no data are available evaluating the effect of an enamel matrix derivative (EMD) on the healing of periodontal defects in patients with osteoporosis. This study aims to evaluate whether the regenerative potential of EMD may be suitable for osteoporosis‐related periodontal defects. Methods: Forty female Wistar rats (mean body weight: 200 g) were used for this study. An osteoporosis animal model was carried out by bilateral ovariectomy (OVX) in 20 animals. Ten weeks after OVX, bilateral fenestration defects were created at the buccal aspect of the first mandibular molar. Animals were randomly assigned to four groups of 10 animals per group: 1) control animals with unfilled periodontal defects; 2) control animals with EMD‐treated defects; 3) OVX animals with unfilled defects; and 4) OVX animals with EMD‐treated defects. The animals were euthanized 28 days later, and the percentage of defect fill and thickness of newly formed bone and cementum were assessed by histomorphometry and microcomputed tomography (micro‐CT) analysis. The number of osteoclasts was determined by tartrate‐resistant acid phosphatase (TRAP), and angiogenesis was assessed by analyzing formation of blood vessels. Results: OVX animals demonstrated significantly reduced bone volume in unfilled defects compared with control defects (18.9% for OVX animals versus 27.2% for control animals) as assessed by micro‐CT. The addition of EMD in both OVX and control animals resulted in significantly higher bone density (52.4% and 69.2%, respectively) and bone width (134 versus 165μm) compared with untreated defects; however, the healing in OVX animals treated with EMD was significantly lower than that in control animals treated with EMD. Animals treated with EMD also demonstrated significantly higher cementum formation in both control and OVX animals. The number of TRAP‐positive osteoclasts did not vary between untreated and EMD‐treated animals; however, a significant increase was observed in all OVX animals. The number of blood vessels and percentage of new vessel formation was significantly higher in EMD‐treated samples. Conclusions: The results from the present study suggest that: 1) an osteoporotic phenotype may decrease periodontal regeneration; and 2) EMD may support greater periodontal regeneration in patients suffering from the disease. Additional clinical studies are necessary to fully elucidate the possible beneficial effect of EMD for periodontal regeneration in patients suffering from osteoporosis.     

Tumor Necrosis Factor‐α and Porphyromonas gingivalis Lipopolysaccharides Decrease Periostin in Human Periodontal Ligament Fibroblasts

Background: Periostin is a matricellular protein essential for tissue integrity and maturation and is believed to have a key function as a modulator of periodontal ligament (PDL) homeostasis. The aim of this study is to evaluate whether periodontal disease‐associated pathogen‐related virulence factors (endotoxins/lipopolysaccharides [LPS]) and proinflammatory cytokines alter the expression of periostin in PDL cells. Methods: Human PDL cultures were exposed to inflammatory mediators (tumor necrosis factor‐α [TNF‐α]), bacterial virulence factors (Porphyromonas gingivalis LPS) or a combination in a biomechanically challenged environment. Culture conditions were applied for 24 hours, 4 days, and 7 days. Periostin and TGF‐β inducible gene clone H3 (βIGH3) mRNA expression from cell lysates were analyzed. Periostin and βIGH3 proteins were also detected and semiquantified in both cell lysates and cell culture supernatants by Western blot. In addition, periostin localization by immunofluorescence was performed. Analysis of variance and Fisher tests were used to define the statistical differences among groups (P <0.05). Results: In a mechanically challenged environment, periostin protein was more efficiently incorporated into the matrix compared to the non‐loaded controls (higher levels of periostin in the supernatant in the non‐loaded group). Interestingly, chronic exposure to proinflammatory cytokines and/or microbial virulence factors significantly decreased periostin protein levels in the loaded cultures. There was greater variability on βIGH3 levels, and no particular pattern was clearly evident. Conclusions: Inflammatory mediators (TNF‐α) and bacterial virulence factors (P. gingivalis LPS) decrease periostin expression in human PDL fibroblasts. These results support a potential mechanism by which periostin alterations could act as a contributing factor during periodontal disease progression.     

A Synthetic Oligopeptide Derived From Enamel Matrix Derivative Promotes the Differentiation of Human Periodontal Ligament Stem Cells Into Osteoblast‐Like Cells With Increased Mineralization

Background: In a previous study, the authors obtained a synthetic peptide (SP) for useful periodontal tissue regeneration. Periodontal ligament stem cells (PDLSCs) have multiple potentiality to contribute to tissue regeneration. The aim of this experiment is to investigate the effect of SP on human PDLSCs. Methods: Periodontal ligament cells were obtained from healthy adult human third molars and used to isolate single PDLSC‐derived colonies. The mesenchymal stem cell nature of the PDLSCs was confirmed by immunohistochemical evaluation of STRO‐1 expression. Proliferation and osteoblastic differentiation were investigated by culturing PDLSCs in normal or osteogenic medium with and without SP (100 ng/mL). Osteoblast differentiation was assessed by measuring alkaline phosphatase (ALP) activity, osteocalcin production, mRNA expression of osteonectin, mineralization, and calcium deposition. Results: Isolated PDLSCs were immunohistochemically positive for vimentin and STRO‐1 and negative for cytokeratin. A greater number of calcified nodules were observed in osteogenic medium culture with SP than without. In the early and later stages of PDLSC culture with SP, osteonectin production and osteocalcin production were increased. SP in culture with osteogenic medium significantly enhanced proliferation of PDLSCs, as well as ALP activity, expression of osteonectin, osteocalcin production, formation of calcified nodules, and mineralization. Conclusions: SP enhances the formation of calcified nodules and osteocalcin production in the culture of PDLSCs into osteoblast‐like cells and is a useful material for periodontal tissue regeneration.     

Tumor Necrosis Factor‐α Induces Matrix Metalloproteinases‐3, ‐10, and ‐13 in Human Periodontal Ligament Cells

Background: Various biologic mediators, including matrix metalloproteinases (MMPs), that are implicated in periodontal tissue breakdown can be induced by cytokines. MMPs are known to degrade periodontal ligament attachment, and bone matrix proteins and tissue inhibitors of metalloproteinase (TIMPs) inhibit the activity of MMPs. The aim of this study is to investigate the effect of tumor necrosis factor (TNF)‐α on the expression of MMPs in human periodontal ligament (PDL) cells in vitro and establish which MMPs are expressed specifically in response to that stimulus. Methods: Cultured PDL cells were stimulated with TNF‐α and analyzed with an MMP antibody array. Real‐time polymerase chain reaction (PCR), enzyme‐linked immunosorbent assay (ELISA), and western blot with cell lysate and zymography were used to measure messenger RNA (mRNA) and protein levels of MMP‐3, ‐10, and ‐13. To examine TNF receptor (TNFR) expression, PDL cells were examined by flow cytometry, and expression of MMP‐3, ‐10, and ‐13 was observed after blocking the TNFR with an antagonist. Results from real‐time PCR, ELISA, and western blot were analyzed by paired t test. Results: The antibody array showed that the protein most strongly upregulated by TNF‐α stimulation was MMP‐3, followed by MMP‐13 and MMP‐10. The TNF‐α receptor blocker specifically inhibited expression of MMP‐3 and ‐13. In addition, TNF‐α increased levels of MMP mRNAs in MMP‐3, ‐13, and ‐10 (in decreasing order). However, ELISAs showed that MMP‐13 was the most upregulated protein, followed by MMP‐10 and MMP‐3. Western blotting indicated that TNF‐α increased MMP‐3 and ‐13 levels but had no significant effect on the level of MMP‐10, and zymography showed that TNF‐α increased the activities of all forms of MMP‐3 and ‐13, but MMP‐10 was not detected. Flow cytometry demonstrated that the majority of PDL cells expressed TNFR1. Conclusions: TNF‐α (10 ng/mL) upregulates levels of MMP‐3, ‐10, and ‐13 in human PDL cells. These results suggest that these proteins play an important role in the inflammation of PDLs.     

Enamel Matrix Derivative in Propylene Glycol Alginate for Treatment of Infrabony Defects With or Without Systemic Doxycycline: 12‐ and 24‐Month Results

Background: This aim of this study is to compare regenerative therapy of infrabony defects with and without administration of post‐surgical systemic doxycycline (DOXY) 12 and 24 months after therapy. Methods: In each of 57 patients, one infrabony defect (depth ≥4 mm) was treated regeneratively using enamel matrix derivative at two centers (Frankfurt am Main and Heidelberg). By random assignment, patients received either 200 mg DOXY per day or placebo (PLAC) for 7 days after surgery. Twelve and 24 months after surgery, clinical parameters (probing depths [PDs] and vertical clinical attachment level [CAL‐V]) and standardized radiographs were obtained. Missing data were managed according to the last observation carried forward. Results: Data of 57 patients (DOXY: 28; PLAC: 29) were analyzed (26 males and 31 females; mean age: 52 ± 10.2 years; 13 smokers). In both groups, significant (P <0.01) PD reduction (DOXY: 3.7 ± 2.2 mm; PLAC: 3.4 ± 1.7 mm), CAL‐V gain (DOXY: 2.7 ± 1.9 mm; PLAC: 3.0 ± 1.9 mm), and bone fill (DOXY: 1.6 ± 2.7 mm; PLAC: 1.8 ± 3.0 mm) were observed 24 months after surgery. However, the differences between both groups failed to be statistically significant (PD: P = 0.574; CAL‐V: P = 0.696; bone fill: P = 0.318). Conclusions: Systemic DOXY, 200 mg/day for 7 days, after regenerative therapy of infrabony defects did not result in better PD reduction, CAL‐V gain, or radiographic bone fill compared with PLAC 12 and 24 months after surgery, which may be attributable to low power and, thus, random chance.     

Aspirin Enhances Osteogenic Potential of Periodontal Ligament Stem Cells (PDLSCs) and Modulates the Expression Profile of Growth Factor–Associated Genes in PDLSCs

Background: This study investigates the effects of aspirin (ASA) on the proliferative capacity, osteogenic potential, and expression of growth factor–associated genes in periodontal ligament stem cells (PDLSCs). Methods: Mesenchymal stem cells (MSCs) from PDL tissue were isolated from human premolars (n = 3). The MSCs’ identity was confirmed by immunophenotyping and trilineage differentiation assays. Cell proliferation activity was assessed through 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. Polymerase chain reaction array was used to profile the expression of 84 growth factor–associated genes. Pathway analysis was used to identify the biologic functions and canonic pathways activated by ASA treatment. The osteogenic potential was evaluated through mineralization assay. Results: ASA at 1,000 μM enhances osteogenic potential of PDLSCs. Using a fold change (FC) of 2.0 as a threshold value, the gene expression analyses indicated that 19 genes were differentially expressed, which includes 12 upregulated and seven downregulated genes. Fibroblast growth factor 9 (FGF9), vascular endothelial growth factor A (VEGFA), interleukin‐2, bone morphogenetic protein‐10, VEGFC, and 2 (FGF2) were markedly upregulated (FC range, 6 to 15), whereas pleotropin, FGF5, brain‐derived neurotrophic factor, and Dickkopf WNT signaling pathway inhibitor 1 were markedly downregulated (FC 32). Of the 84 growth factor–associated genes screened, 35 showed high cycle threshold values (≥35). Conclusions: ASA modulates the expression of growth factor–associated genes and enhances osteogenic potential in PDLSCs. ASA upregulated the expression of genes that could activate biologic functions and canonic pathways related to cell proliferation, human embryonic stem cell pluripotency, tissue regeneration, and differentiation. These findings suggest that ASA enhances PDLSC function and may be useful in regenerative dentistry applications, particularly in the areas of periodontal health and regeneration.     

Periodontal tissue engineering and regeneration: Consensus Report of the Sixth European Workshop on Periodontology

Aim: To review the scientific preclinical background and clinical studies of current methods of periodontal regeneration in the treatment of infrabony defects and soft tissue deficiencies
Method: Five commissioned review papers including two systematic reviews were scrutinized by a group of experts in order to derive consensus conclusions, clinical relevance/implications and to propose future research requirements.
Results: The following five papers were assessed:
  1. Biological mediators and periodontal regeneration: a review of enamel matrix proteins at the cellular and molecular levels.
  2. Regeneration of periodontal tissues: combination of barrier membranes and grafting materials – Biological foundation and preclinical evidence.
  3. Clinical outcomes with bioactive agents alone or in combination with grafting or GTR
  4. Treatment of gingival recession with coronally advanced flap procedures. A systematic review.
  5. Soft tissue management at implant sites     

Tumor Necrosis Factor‐α Inhibits Transforming Growth Factor‐β–Stimulated Myofibroblastic Differentiation and Extracellular Matrix Production in Human Gingival Fibroblasts

Background: Fibroblasts play a critical role during wound healing and chronic inflammation through the synthesis and assembly of extracellular matrix (ECM) molecules. These responses may be modulated by soluble cytokines and growth factors present in tissues. In the present study, we evaluate whether transforming growth factor‐β1 (TGF‐β1) and tumor necrosis factor‐α (TNF‐α) modulate myofibroblastic differentiation and the production of ECM components. Methods: Primary cultures of human gingival fibroblasts (HGFs) were stimulated with recombinant TGF‐β1 and TNF‐α. Protein levels of α‐smooth muscle actin (α‐SMA), type I collagen, heat shock protein‐47 (HSP‐47), fibronectin (FN), ED‐A‐FN, and periostin and activation of the Smad pathway were evaluated through Western blot analysis. α‐SMA and actin fibers were identified by immunofluorescence. TGF‐β1, TNF‐α, and α‐SMA were identified by immunohistochemistry in biopsies of inflamed human gingival tissues. TGF‐β1 activity was evaluated using a plasminogen activator inhibitor‐1 (PAI‐1) reporter transfected in HGFs. Results: TGF‐β1 stimulated the differentiation of myofibroblasts as evidenced by strong expression of α‐SMA and ED‐A‐FN. Moreover, TGF‐β1 induced the production of type I collagen, HSP‐47, FN, and periostin. Costimulation with TNF‐α and TGF‐β1 significantly reduced the expression of all the above‐mentioned proteins. TNF‐α also inhibited the activation of the Smad2/3 pathway and the activity of the PAI‐1 reporter. Conclusions: TNF‐α inhibits several cell responses induced by TGF‐β1, including the differentiation of myofibroblasts, the activation of the Smad signaling pathway, and the production of key molecules involved in tissue repair, such as type I collagen, FN, and periostin. The interaction between cytokines may explain the delayed tissue repair observed in chronic inflammation of gingival tissues.     

Proline‐Rich Peptide Mimics Effects of Enamel Matrix Derivative on Rat Oral Mucosa Incisional Wound Healing

Background: Proline‐rich peptides have been shown to promote periodontal regeneration. However, their effect on soft tissue wound healing has not yet been investigated. The aim of this study is to evaluate the effect of enamel matrix derivative (EMD), tyrosine‐rich amelogenin peptide (TRAP), and a synthetic proline‐rich peptide (P2) on acute wound healing after a full‐thickness flap procedure in an incisional rat model. Methods: This experimental study has a split‐mouth, randomized, placebo‐controlled design. Test and control wounds were created on the palatal mucosa of 54 Sprague‐Dawley rats. Wounds were histologically processed, and reepithelialization, leukocyte infiltration, and angiogenesis were assessed at days 1, 3, and 7 post‐surgery. Results: EMD and P2 significantly promoted early wound closure at day 1 (P <0.001 and P = 0.004, respectively). EMD maintained a significant acceleration of reepithelialization at day 3 (P = 0.004). Wounds treated by EMD and P2 showed increased angiogenesis during the first 3 days of healing (P = 0.03 and 0.001, respectively). Leukocyte infiltration was decreased in EMD‐treated wounds at day 1 (P = 0.03), and P2 and TRAP induced a similar effect at days 3 (P = 0.002 and P <0.0001, respectively) and 7 (P = 0.005 and P <0.001). Conclusion: EMD and P2 promoted reepithelialization and neovascularization in full‐thickness surgical wounds on rat oral mucosa.     

Evaluation of Recession Defects Treated With Coronally Advanced Flaps and Either Recombinant Human Platelet‐Derived Growth Factor‐BB Plus β‐Tricalcium Phosphate or Connective Tissue: Comparison of Clinical Parameters at 5 Years

Background: In a previously reported split‐mouth, randomized controlled trial, Miller Class II gingival recession defects were treated with either a connective tissue graft (CTG) (control) or recombinant human platelet‐derived growth factor‐BB + β‐tricalcium phosphate (test), both in combination with a coronally advanced flap (CAF). At 6 months, multiple outcome measures were examined. The purpose of the current study is to examine the major efficacy parameters at 5 years. Methods: Twenty of the original 30 patients were available for follow‐up 5 years after the original surgery. Outcomes examined were recession depth, probing depth, clinical attachment level (CAL), height of keratinized tissue (wKT), and percentage of root coverage. Within‐ and across‐treatment group results at 6 months and 5 years were compared with original baseline values. Results: At 5 years, all quantitative parameters for both treatment protocols showed statistically significant improvements over baseline. The primary outcome parameter, change in recession depth at 5 years, demonstrated statistically significant improvements in recession over baseline, although intergroup comparisons favored the control group at both 6 months and 5 years. At 5 years, intergroup comparisons also favored the test group for percentage root coverage and change in wKT, whereas no statistically significant intergroup differences were seen for 100% root coverage and changes to CAL. Conclusions: In the present 5‐year investigation, treatment with either test or control treatments for Miller Class II recession defects appear to lead to stable, clinically effective results, although CTG + CAF resulted in greater reductions in recession, greater percentage of root coverage, and increased wKT.     

Stimulation of Periodontal Ligament Stem Cells by Dentin Matrix Protein 1 Activates Mitogen‐Activated Protein Kinase and Osteoblast Differentiation

Background: Periodontitis can ultimately result in tooth loss. Many natural and synthetic materials have been tried to achieve periodontal regeneration, but the results remain variable and unpredictable. We hypothesized that exogenous treatment with dentin matrix protein 1 (DMP1) activates specific genes and results in phenotypic and functional changes in human periodontal ligament stem cells (hPDLSCs). Methods: hPDLSCs were isolated from extracted teeth and cultured in the presence or absence of DMP1. Quantitative polymerase chain reactions were performed to analyze the expression of several genes involved in periodontal regeneration. hPDLSCs were also processed for immunocytochemical and Western blot analysis using phosphorylated extracellular signal‐regulated kinase (pERK) and ERK antibodies. Alkaline phosphatase and von Kossa staining were performed to characterize the differentiation of hPDLSCs into osteoblasts. Field emission scanning electron microscopic analysis of the treated and control cell cultures were also performed. Results: Treatment with DMP1 resulted in the upregulation of genes, such as matrix metalloproteinase‐2, alkaline phosphatase, and transforming growth factor β1. Activation of ERK mitogen‐activated protein kinase signaling pathway and translocation of pERK from the cytoplasm to the nucleus was observed. Overall, DMP1‐treated cells showed increased expression of alkaline phosphatase, increased matrix, and mineralized nodule formation when compared with untreated controls. Conclusion: DMP1 can orchestrate a coordinated expression of genes and phenotypic changes in hPDLSCs by activation of the ERK signaling pathway, which may provide a valuable strategy for tissue engineering approaches in periodontal regeneration.     

Activation of Receptor Activator of Nuclear Factor‐κB Ligand and Matrix Metalloproteinase Production in Periodontal Fibroblasts by Endothelin Signaling

Background: Periodontitis is a group of inflammatory diseases affecting the tissues supporting the teeth that will progressively cause the loss of alveolar bone and periodontal ligaments and eventually the dentition. Activation of osteoclast activity by receptor activator of nuclear factor‐κB ligand (RANKL) and released enzymes such as matrix metalloproteinases (MMPs) are among the factors involved in the breakdown of the periodontium. However, the mechanisms regulating their production in periodontitis are poorly understood. Endothelin signaling via the activation of the endothelin‐A receptor (EDNRA) by endothelin‐1 may play a role in the disease because the expression of the receptor and ligand is elevated in the periodontal tissues of patients with periodontitis. Methods: Cultured primary human periodontal fibroblasts were treated with 20 and 100 nM endothelin‐1 for 6 and 24 hours and then collected to assess MMP and RANKL production by immunoblotting. Inhibitors were used to identify the molecular pathways activated by EDNRA in these cells. Results: Endothelin‐1 stimulated the production of MMP1, MMP8, and RANKL in a dose‐ and time‐dependent manner; blocking EDNRA function with the antagonist TBC3214 inhibited the response, although EDNRA activation had no effects on osteoprotegerin production. These mechanistic studies indicate that EDNRA activates phospholipase C, which then 1) increases the MMP1 protein levels through activation of the extracellular signal‐regulated kinase mitogen‐activated protein kinase‐dependent pathway and 2) upregulates RANKL by a different pathway. Conclusion: These results suggest that EDNRA may function in the breakdown of the periodontal tissues associated with periodontitis by promoting the protein expression of MMPs and RANKL via the phospholipase C pathway.     

Clinical Outcomes After Treatment of Non‐Contained Intrabony Defects With Enamel Matrix Derivative or Guided Tissue Regeneration: A 12‐Month Randomized Controlled Clinical Trial

Background: The purpose of this study is to compare the healing of deep, non‐contained intrabony defects (i.e., with a ≥80% 1‐wall component and a residual 2‐ to 3‐wall component in the most apical part) treated with either an enamel matrix derivative (EMD) or guided tissue regeneration (GTR) after 12 months. Methods: In this randomized, controlled clinical trial, 40 subjects with 40 defects affecting single‐rooted teeth were treated. The defects were treated with EMD alone or with a non‐resorbable titanium‐reinforced membrane. No grafting materials were used. At baseline and after 12 months, clinical parameters including probing depths (PDs) and clinical attachment levels (CAL) were recorded. The difference in CAL gain was the primary outcome. Results: At baseline, the intrabony component of the defects amounted to 8.5 ± 2.2 mm at EMD‐treated sites and 8.6 ± 1.7 mm at GTR‐treated sites (P = 0.47). The mean CAL gain at sites treated with GTR was significantly greater (P <0.001) than that at sites treated with EMD (4.1 ± 1.4 mm versus 2.4 ± 2.2 mm, respectively). GTR therapy, compared to EMD application alone, significantly (P = 0.01) increased the probability of CAL gain ≥4 mm (79.2% versus 11.3%, respectively) and significantly (P = 0.01) decreased the probability of residual PDs ≥6 mm (3% versus 79.3%, respectively). Conclusion: Although the outcomes of open‐flap debridement alone were not investigated, the application of EMD alone appeared to yield less PD reduction and CAL gain compared to GTR therapy in the treatment of deep, non‐contained intrabony defects.     

Quantitative Analysis of Interdental Gingiva in Patients With Chronic Periodontitis and Transforming Growth Factor‐β1 29C/T Gene Polymorphisms

Background: The association of transforming growth factor (TGF)‐β1 29C/T gene polymorphisms with level of tissue breakdown and periodontal disease progression is not clear. In this study, quantitative parameters of interdental papilla are investigated in patients with chronic periodontitis (CP) and TGF‐β1 29C/T gene polymorphisms. Methods: Sixty gingiva samples were included. After determination of TGF‐β1 29C/T gene polymorphisms using tetra‐primer amplification refractory mutation system/polymerase chain reaction (T‐ARMS‐PCR), 15 gingival tissue samples from patients with CP in each genotype (TT, TC, and CC) were considered as case groups. Fifteen control samples were also collected from healthy individuals. After tissue processing, interdental gingiva tissues were exhaustively sectioned into 4‐μm‐thick sections. Ten to 13 sections were sampled by systematic uniform random sampling and stained with Masson trichrome, and the volume density (V_v) of the gingival components was estimated using Cavalieri estimation. Results: Statistically significant differences were found in V_v of epithelium, connective tissue, collagenous and non‐collagenous matrix, and blood vessels between control and CP groups (P <0.0001). There was a corresponding decrease in the collagenous matrix V_v in patients with the TT genotype compared with those with CT and CC genotypes. Collagenous matrix and blood vessel V_v values were statistically correlated with the number of T alleles (r = ?0.74, r² = 54.8%, P = 0.0001 and r = 0.84, r² = 70.6%, P = 0.0001, respectively). Conclusion: This study shows that there is a strong association between TGF‐β1 29C/T gene polymorphisms and quantitative parameters of interdental papilla in patients with CP.