Relationship of Metabolic Dysfunction-Associated Steatohepatitis-Related Hepatocellular Carcinoma with Oral and Intestinal Microbiota: A Cross-Sectional Pilot Study.

Background and Objectives: The incidence of metabolic dysfunction-associated steatohepatitis (MASH)-related hepatocellular carcinoma (HCC) is increasing worldwide, alongside the epidemic of obesity and metabolic syndrome. Based on preliminary reports regarding the potential association of HCC and periodontitis, this study aimed to analyze the involvement of periodontal bacteria as well as the oral and intestinal bacterial flora in MASH-related HCC (MASH-HCC). Materials and Methods: Forty-one patients with MASH and nineteen with MASH-HCC participated in the study, completing survey questionnaires, undergoing periodontal examinations, and providing samples of saliva, mouth-rinsed water, feces, and peripheral blood. The oral and fecal microbiome profiles were analyzed by 16S ribosomal RNA sequencing. Bayesian network analysis was used to analyze the causation between various factors, including MASH-HCC, examinations, and bacteria. Results: The genus Fusobacterium had a significantly higher occupancy rate (p = 0.002) in the intestinal microflora of the MASH-HCC group compared to the MASH group. However, Butyricicoccus (p = 0.022) and Roseburia (p < 0.05) had significantly lower occupancy rates. The Bayesian network analysis revealed the absence of periodontal pathogenic bacteria and enteric bacteria affecting HCC. However, HCC directly affected the periodontal bacterial species Porphyromonas gingivalis, Tannerella forsythia, Fusobacterium nucleatum, and Prevotella intermedia in the saliva, as well as the genera Lactobacillus, Roseburia, Fusobacterium, Prevotella, Clostridium, Ruminococcus, Trabulsiella, and SMB53 in the intestine. Furthermore, P. gingivalis in the oral cavity directly affected the genera Lactobacillus and Streptococcus in the intestine. Conclusions: MASH-HCC directly affects periodontal pathogenic and intestinal bacteria, and P. gingivalis may affect the intestinal bacteria associated with gastrointestinal cancer.

Spontaneous differentiation of periodontal ligament stem cells into myofibroblast during ex vivo expansion.

Periodontitis is characterized by the chronic inflammation and destruction of tooth-supporting tissues. Periodontal ligament stem cell (PDLSC) is the mesenchymal stem cell (MSC) population isolated from periodontal ligament, which is the key tissue for regeneration of periodontal tissues. Although transplantation of PDLSCs is proposed as novel regenerative therapy, limited information is available, regarding the characteristic change of PDLSCs during ex vivo expansion. In this study, we encountered morphological change of PDLSCs during standard cell culture and aimed to investigate the change of PDLSCs in stem cell characteristics and to search for the culture condition to maintain stem cell properties. Characteristics of PDLSCs were examined using in vitro osteoblast and adipocyte differentiation. Myofibroblast differentiation was confirmed using immunohistochemistry and collagen gel contraction assay. Replicative senescence was examined by ß-gal staining. PDLSCs changed their morphology from spindle to flat and wide during ex vivo expansion. After the morphological change, PDLSCs showed several features of myofibroblast including extensive stress fiber formation, contraction activity, and myofibroblast marker expression. Upon the morphological change, osteoblastic and adipocyte differentiation capacity were reduced and expression of stem cell-related genes were decreased. ß-Gal staining was not always correlated with the morphological change of PDLSCs. Moreover, exogenous addition of bFGF and PDGF-BB served to maintain spindle shape and osteoblastic differentiation potential of PDLSCs. This study demonstrates that spontaneous differentiation of PDLSCs during ex vivo expansion and may provide the important information of cell culture condition of PDLSCs for clinical use.

Pericytes in the Periodontal Ligament.

Teeth are exposed to hundreds of oral bacteria and also challenged by the mastication forces; because teeth are situated in oral cavity, the entrance of the digestive tract, and penetrates through the oral epithelium. The periodontal ligament is a noncalcified tissue that possesses abundant blood vessels, which exist between tooth root and alveolar bone. The ligament is thought to play an important role in absorbing the impact of mastication, in the maintenance of periodontal homeostasis, and in periodontal wound healing. We succeeded in isolating mesenchymal stem cells (MSCs), so-called periodontal stem cells (PDLSCs), with self-renewability and multipotency from the periodontal ligament. We also demonstrated that PDLSCs share some cell surface markers with pericytes and that PDLSCs distribute themselves to stay with the endothelial cell networks and that PDLSCs maintain the endothelial cell networks when added to endothelial cell network formation systems. Pericytes are located in the proximity of microvascular endothelial cells and thought to stabilize and supply nutrients to blood vessels. Recently, it was also reported that pericytes possess multipotency and can be the source of tissue stem cells and/or progenitor cells. This review explores the distinctive features of the periodontal ligament tissue and PDLSCs as well as the puzzling similarities between PDLSCs and pericytes.

The Fate of Transplanted Periodontal Ligament Stem Cells in Surgically Created Periodontal Defects in Rats.

Periodontal disease is chronic inflammation that leads to the destruction of tooth-supporting periodontal tissues. We devised a novel method ("cell transfer technology") to transfer cells onto a scaffold surface and reported the potential of the technique for regenerative medicine. The aim of this study is to examine the efficacy of this technique in periodontal regeneration and the fate of transplanted cells. Human periodontal ligament stem cells (PDLSCs) were transferred to decellularized amniotic membrane and transplanted into periodontal defects in rats. Regeneration of tissues was examined by microcomputed tomography and histological observation. The fate of transplanted PDLSCs was traced using PKH26 and human Alu sequence detection by PCR. Imaging showed more bone in PDLSC-transplanted defects than those in control (amnion only). Histological examination confirmed the enhanced periodontal tissue formation in PDLSC defects. New formation of cementum, periodontal ligament, and bone were prominently observed in PDLSC defects. PKH26-labeled PDLSCs were found at limited areas in regenerated periodontal tissues. Human Alu sequence detection revealed that the level of Alu sequence was not increased, but rather decreased. This study describes a novel stem cell transplantation strategy for periodontal disease using the cell transfer technology and offers new insight for cell-based periodontal regeneration.

High-frequency pulsed low-level diode laser therapy accelerates wound healing of tooth extraction socket: An in vivo study.

BACKGROUND AND OBJECTIVE: This study aimed to evaluate the effects of high-frequency pulsed (HiFP) low-level laser therapy (LLLT) on early wound healing of tooth extraction sockets in rats. STUDY DESIGN/MATERIALS AND METHODS: Bilateral maxillary first molars were extracted from 6-week-old Sprague-Dawley rats. Sockets on the right were treated by HiFP low-level diode laser irradiation (904-910 nm); the left sides served as unirradiated controls. LLLT (0.28 W, 30 kHz, 200-ns pulse, 0.6% duty cycle, 61.2 J/cm2 total power density) was employed immediately after extraction and every 24 hours thereafter. The maxillae including the sockets were resected 3 or 7 days after extraction. Soft-tissue healing was evaluated on days 0, 3, and 7. The bone mineral content (BMC), bone volume (BV), and bone mineral density (BMD) of the extraction sockets were evaluated by microcomputed tomography, and histomorphometric analysis was carried out on day 7. Real-time PCR analysis of osteogenic marker expression and immunohistochemical detection of proliferating cell nuclear antigen (PCNA)-positive cells were performed on day 3. RESULTS: Compared with control sites, the un-epithelialized areas of the extracted sites were significantly reduced by irradiation (P = 0.04), and the BMC, BV, and BMD of laser-treated sites were significantly increased (P = 0.004, 0.006, and 0.009, respectively). On day 7, the mean height of newly formed immature woven bone was higher in laser-treated sites (P = 0.24). On day 3, laser-treated sites showed significantly higher osteocalcin mRNA expression (P = 0.04) and PCNA-positive cell numbers (P = 0.01). CONCLUSION: HiFP low-level diode laser irradiation enhanced soft- and hard-tissue healing of tooth extraction sockets. Lasers Surg. Med. 48:955-964, 2016. © 2016 Wiley Periodicals, Inc.

Gingival tissue healing following Er:YAG laser ablation compared to electrosurgery in rats.

The erbium-doped yttrium aluminum garnet (Er:YAG) laser is currently used for periodontal soft tissue management with favorable outcomes. However, the process of wound healing after Er:YAG laser (ErL) treatment has not been fully elucidated yet. The aim of this study was to investigate the gingival tissue healing after ErL ablation in comparison with that after electrosurgery (ElS). Gingival defects were created in 28 rats by ablation with ErL irradiation or ElS. The chronological changes in wound healing were evaluated using histological, histometrical, and immunohistochemical analyses. The ErL-ablated gingival tissue revealed much less thermal damage, compared to the ElS. In the ElS sites, the postoperative tissue destruction continued due to thermal damage, while in the ErL sites, tissue degradation was limited and the defects were re-epithelialized early. Heat shock protein (Hsp) 72/73 expression was detected abundantly remote from the wound in the ElS, whereas it was slightly observed in close proximity to the wound in the ErL sites. Hsp47 expression was observed in the entire connective tissue early in the wound healing and was found limited in the wound area later. This phenomenon proceeded faster in the ErL sites than in the ElS sites. Expression of proliferating cell nuclear antigen (PCNA) persisted in the epithelial tissue for a longer period in the ElS than that in the ErL. The ErL results in faster and more favorable gingival wound healing compared to the ElS, suggesting that the ErL is a safe and suitable tool for periodontal soft tissue management.

Relationship between aging and periodontal disease severity in gauge-raised cynomolgus monkeys (Macaca fascicularis).

The study aimed to evaluate the periodontal disease status in different age groups and clarify the relationship between aging and the severity of periodontal disease. The test animals were cynomolgus monkeys that were born and raised at the Tsukuba Primate Research Center of the National Institutes of Biomedical Innovation, Health, and Nutrition. The participants were divided into three groups: young (5-10 years old), middle (10-19 years old), and old (≥20 years old). The plaque Index (PLI), Gingival Index (GI), probing pocket depth (PPD), and Bleeding on probing (BOP) were used for the periodontal examination. Representative teeth were also examined. Polymerase chain reaction (PCR) was used to identify Porphyromonas macacae in dental plaque. Multiple comparisons and regression analyses were used to analyze the relationship between each age group and each oral examination index. Statistically significant differences were found between the age groups and periodontal examination index. Multiple regression analysis revealed that age was strongly correlated with each oral examination index. Based on these results, oral examinations of cynomolgus monkeys kept in the same environment confirmed an association between aging and periodontal disease severity. Monkeys at this facility are expected to serve as new experimental models for elucidating the mechanisms underlying the progression of age-related periodontal disease.

Oral and Intestinal Bacterial Flora in Patients with Increased Periodontal Inflamed Surface Area: A Cross-Sectional Study.

Background/Objectives: Periodontitis is caused by bacterial plaque. The oral microflora may interact with the intestinal microflora and play a role in the development of periodontitis. The periodontal inflamed surface area (PISA) has been shown to be a useful indicator of periodontal disease related to systemic diseases; however, few studies have shown an association between PISA and the bacterial flora. This study aimed to determine the association between PISA and oral and intestinal bacteria. Methods: Participants were recruited between 2018 and 2021 at the Medical and Dental Collaboration Center of Kanagawa Dental University Hospital. A periodontal clinical examination was performed, and the PISA was calculated. Salivary tests were conducted, and leukocyte scores in the saliva were calculated. Moreover, 16S rRNA amplicon sequencing was performed using saliva and stool samples to analyze oral and intestinal bacteria, respectively. Results: Higher PISA levels resulted in an increased presence of Bacteroides and a decreased presence of Proteobacteria and Actinobacteria in the saliva. An increase in Bacteroides was detected in the saliva of patients with high leukocyte scores. No correlation was observed between PISA and intestinal bacteria. Conclusions: Bacteroides was highly abundant in the saliva of patients with worsened periodontal conditions, as indicated by PISA. No association was found between PISA and intestinal bacteria.

Decreased Tongue-Lip Motor Function in Japanese Population with Low Taste Sensitivity: A Cross-Sectional Study.

Background/Objectives: Taste disorders have a negative impact on meal enjoyment, which is essential for maintaining adequate nutrition and quality of life. Japan is a rapidly aging society with an increasing number of individuals with taste disorders. However, despite the increasing prevalence of taste disorders, the correlation between oral frailty and taste sensitivity remains largely unknown. The objective of this study was to assess the relationship between oral health status and taste sensitivity among the Japanese population. Methods: Participants were recruited from Kanagawa Dental University Hospital Medical-Dental Collaboration Center between 2018 and 2021. The exclusion criteria were severe systemic infections, pregnancy, or lactation. Clinical examinations, oral function assessments, and taste tests were conducted using tap water and 1% sweet, 0.3% salty, 0.03% umami, and 0.1% umami tastants. The relationships between oral function, systemic indicators, and taste sensitivity were statistically evaluated. Results: Of the 169 participants included in this cross-sectional study, 39.6% were male and 60.4% were female (median age, 68 years). Participants with low taste sensitivity showed a decline in tongue-lip motor function, independent of age, sex, or smoking status. A multiple logistic regression analysis conducted using two age categories-younger than 65 years and older than 65 years-revealed an association between tongue-lip motor function and taste sensitivity among participants younger than 65 years. Conclusions: Decreased taste sensitivity is associated with tongue-lip motor function. Therefore, the early maintenance of oral function and taste sensitivity may be beneficial for optimal tongue-lip motor function.

Carbamazepine-Induced Systemic Lupus Erythematosus in a Patient With Idiopathic Trigeminal Neuralgia: A Case Report.

A 50-year-old woman presented with a mandibular second molar and facial pain and was diagnosed with idiopathic trigeminal neuralgia. Carbamazepine (CBZ) was initiated at 300 mg/day, successfully relieving the pain. However, on the 8th day of CBZ treatment, the patient developed symptoms resembling those of systemic lupus erythematosus with malaise, nausea, and facial erythema. CBZ was immediately discontinued. Subsequently, she experienced numbness in both lower limbs and mild fever, which resolved within a few days. Laboratory tests revealed leukopenia (2.8 × 103/µL), elevated C-reactive protein levels (0.46 mg/dL), and the presence of antinuclear antibodies (ANA) and anti-Sjögren's syndrome-related antigen A antibodies. The clinical course suggested CBZ-induced drug-induced lupus erythematosus (DILE). This case highlights the possibility of DILE onset even after short-term CBZ treatment, the importance of prompt discontinuation of the causative drug in patients suspected of DILE, and the conduct of ANA testing in diagnosing DILE.

Associations between Periodontal Status and Liver Function in the Japanese Population: A Cross-Sectional Study.

A relationship between periodontitis and liver function has been suggested. Indeed, patients with severe periodontal disease have been found to be more prone to liver dysfunction. The periodontal inflammatory surface area (PISA) has been shown to be a useful indicator of periodontal and systemic diseases. However, little information is available regarding whether the PISA is associated with liver function markers, such as gamma-glutamyltransferase (GGT), aspartate aminotransferase (AST), and alanine aminotransferase (ALT). This study aimed to clarify relationship between liver function markers, AST, ALT, and GGT, and PISA level in a cross-sectional study. The subjects were recruited between 2018 and 2021 at the Medical and Dental Collaboration Center of Kanagawa Dental College Hospital. A periodontal clinical examination was performed, and the PISA was calculated. Peripheral blood samples were collected, and serum levels of liver function markers were measured. The levels of liver function markers were examined in different values of PISA. Participants with high PISA scores were more likely to have increased GGT levels while AST and ALT were not changed with PISA. Increased GGT was found in 10.8% and 29.4% (p = 0.0056), increased AST in 48.2% and 52.9% (p = 0.62), and increased ALT in 35.2% and 47.0% (p = 0.20) among <300 mm2 and ≧300 mm2 PISA groups, respectively. It was found that males with a PISA of 300 mm2 or higher had an elevated level of serum GGT. In conclusion, elevated GGT was found in the high PISA group, particularly in males, while AST and ALT did not differ by PISA.

Influence of Meal Sequence and Number of Teeth Present on Nutrient Intake Status: A Cross-Sectional Study.

Intake of fiber, as well as protein, and lipid preloading help to control postprandial glycemic elevation in people with type 2 diabetes and in healthy individuals. However, there are few studies on the awareness of meal sequence and nutrient intake status that consider oral conditions. This cross-sectional study aimed to determine the effects of meal sequences on nutrient intake status and whether these relationships were related to the number of teeth present. The subjects were recruited from the Medical and Dental Collaboration Center of Kanagawa Dental University Hospital between 2018 and 2021. Medical and dental examinations were performed, and a questionnaire was used to determine whether the diet consisted of vegetables, meat or fish, and carbohydrates in that order. Nutrient intake status was assessed using the brief-type self-administered diet history questionnaire. Data were collected from 238 participants. The group with awareness of meal sequence ingested increased nutrients such as n-3 fatty acids, total dietary fiber, calcium, and vitamin C. Saturated fatty acid intake increased in those with fewer teeth, while it was not significantly related to meal sequence. In conclusion, our results showed that meal sequence was associated with nutrient intake status. In addition, the intake of saturated fatty acids increased when many teeth were lost, regardless of meal sequence.

Er:YAG laser-assisted comprehensive periodontal pocket therapy for residual periodontal pocket treatment: A randomized controlled clinical trial.

BACKGROUND: This study evaluated the effectiveness of a novel pocket therapy (Er:YAG laser-assisted comprehensive periodontal pocket therapy [Er-LCPT]) for residual pocket treatment, compared with conventional mechanical treatment alone, in a randomized controlled clinical trial. METHODS: Two sites in 18 patients having residual periodontal pockets of ≥5 mm depth, extant following initial active therapy, or during supportive therapy, were randomized into two groups in a split mouth design: the control group received scaling and root planing (SRP) by curette, and the test group received Er-LCPT using curette and laser. With Er-LCPT, after root debridement, inflamed connective tissue on the inner gingival surface and on the bone surface/within extant bone defects was thoroughly debrided. Furthermore, removal of proximate oral epithelium and coagulation of the blood clot in the pocket entrance were performed with laser. Clinical parameters were evaluated, before and after treatment, through 12 months. RESULTS: Both groups showed significant improvements in clinical parameters. With Er-LCPT, pocket debridement was thoroughly and safely performed, without any adverse side effects and complications, and favorable healing was observed in most of the cases. At 12 months, Er-LCPT demonstrated significantly higher probing pocket depth reduction (2.78 mm vs. 1.89 mm on average; p = 0.012, Wilcoxon signed-rank test), clinical attachment gain (1.67 mm vs. 1.06 mm; p = 0.004) as primary outcomes, and reduced BOP value (0.89 vs. 0.56; p = 0.031), compared with SRP alone. CONCLUSION: The results of this study indicate that Er-LCPT is more effective for residual pocket treatment, compared with SRP alone.

Cementum protein 1 (CEMP1) induces a cementoblastic phenotype and reduces osteoblastic differentiation in periodontal ligament cells.

Cementum is a calcified tissue covering the tooth root surface, which functions as rigid tooth-anchoring structure. Periodontal ligament is a unique non-mineralized connective tissue, and is a source of mineralized tissue forming cells such as cementoblasts and osteoblasts. The CEMP1 is a novel cementum component the presence of which appears to be limited to cementoblasts and their progenitors. In order to understand the function of CEMP1, we investigated CEMP1 expression during the differentiation of human periodontal ligament cells. Immunomagnetically enriched alkaline phosphatase (ALP)-positive periodontal ligament cells preferentially expressed CEMP1. CEMP1 expression was reduced when periodontal ligament cells differentiated to osteoblasts in vitro. Over-expression of CEMP1 in periodontal ligament cells enhanced cementoblast differentiation and attenuated periodontal and osteoblastic phenotypes. Our data demonstrate for the first time that the CEMP1 is not only a marker protein for cementoblast-related cells, but it also regulates cementoblast commitment in periodontal ligament cells.

Effect of Locally Delivered Minocycline on the Profile of Subgingival Bacterial Genera in Patients with Periodontitis: A Prospective Pilot Study.

This prospective pilot study aimed to evaluate the effect of minocycline-HCl ointment (MO), locally delivered as an adjunct to scaling and root planing (SRP), on subgingival microflora. A total of 59 periodontitis patients received SRP as an initial periodontal therapy. In the selected periodontal pockets with probing depths (PD) of 6−9 mm, the sites that exhibited a positive reaction following a bacterial test using an immunochromatographic device were subsequently treated with MO (SRP + MO group, n = 25). No additional treatment was performed at sites showing a negative reaction (SRP group, n = 34). In addition to subgingival plaque sampling, measurement of clinical parameters including PD, clinical attachment level (CAL), bleeding on probing (BOP), plaque index and gingival index (GI) were performed at baseline and 4 weeks after the initial periodontal therapy. The subgingival microflora were assessed by terminal restriction fragment-length polymorphism analysis. Relative to baseline values, the mean scores for PD-, CAL-, BOP-, and GI-sampled sites were significantly decreased post treatment in both groups (p < 0.01). The intra-comparisons showed a significant decrease in the counts of the genera Eubacterium, Parvimonas, Filifactor, Veillonella, Fusobacterium, Porphyromonas, Prevotella, and unknown species in the SRP + MO group (p < 0.05). Inter-comparisons indicated a significant decrease in the genera Veillonella in the SRP + MO group (p = 0.01). Combination therapy of SRP and local MO induced a change in the subgingival microbial community: particularly, the number of Veillonella spp. was markedly reduced.

Periodontal Tissue Healing After Autologous Tooth Transplantation: A Retrospective Analysis of Case Series.

PURPOSE: Tooth transplantation is a treatment that uses non-functional teeth to compensate for defects caused by tooth extractions. Surgical procedures have yielded high success rates in autologous tooth transplantation using a tooth with a complete root. This study aimed to evaluate periodontal tissue healing after transplantation of 14 molar teeth. MATERIALS AND METHODS: Fourteen individuals aged 28-53 years who underwent autologous transplantation of third molars with completely developed roots between December 2010 and March 2017 were included in the study. The donor tooth was carefully extracted, placed into the prepared transplant site, and stabilised with an orthodontic wire and 4-0 silk sutures for a few weeks. Endodontic treatment was performed after 3-4 weeks. To evaluate the periodontal tissue healing, clinical measurements of the probing pocket depth (PPD), clinical attachment level (CAL), and keratinised gingival width (KGW) were performed, along with radiographic examinations of bone defect fill (BDF) at baseline and at 6 and 12 months after surgery. Statistical analysis was performed using the Mann-Whitney U test. RESULTS: The changes in PPD and CAL at baseline, 6, and 12 months were statistically significant (P <0.05). KGW did not show a statistically significant decrease. The postoperative-BDF amount at 6 and 12 months was 2.2 ± 1.4 and 4.2 ± 1.4 mm, respectively. CONCLUSION: Periodontal tissue healing may occur in tooth autotransplantation even in the presence of complete root development in the donor tooth.

Angiogenic Effects of Secreted Factors from Periodontal Ligament Stem Cells.

Periodontal disease is a chronic inflammation of tooth-supporting tissues, and the destruction of these tissues results in tooth loss. Regeneration of periodontal tissues is the ultimate goal of periodontal treatment. We previously reported that transplantation of conditioned medium (CM) of periodontal ligament stem cells (PDLSCs) demonstrated the enhancement of periodontal tissue regeneration, compared to CM from fibroblasts (Fibroblast-CM). We hypothesized that the angiogenic effects of PDLSC-CM might participate in the enhanced wound healing of periodontal tissues. The aim of this study was to investigate the effect of PDLSC-CM on the functions of endothelial cells. PDLSCs were cultured from periodontal ligament tissues obtained from healthy volunteers. Human gingival epithelial cells, dermal fibroblasts, osteoblasts, and umbilical vein endothelial cells (HUVECs) were purchased from commercial sources. The functions of endothelial cells were examined using immunostaining of Ki67, observation of nuclear fragmentation and condensation (apoptosis), and network formation on Matrigel. Vascular endothelial cell growth factor (VEGF) level was measured using an ELISA kit. HUVECs demonstrated higher cell viability in PDLSC-CM when compared with those in Fibroblast-CM. HUVECs demonstrated a higher number of Ki67-positive cells and lower apoptosis cells in PDLSC-CM, compared to Fibroblast-CM. Additionally, HUVECs formed more capillary-like structures in PDLSC-CM than Fibroblast-CM. PDLSC-CM contained higher levels of angiogenic growth factor, VEGF, than Fibroblast-CM. Our results showed that PDLSC-CM increased cell viability, proliferation, and capillary formation of HUVECs compared to Fibroblast-CM, suggesting the angiogenic effects of PDLSC-CM, and the effect is a potential regenerative mechanism of periodontal tissues by PDLSC-CM.

Conditioned Medium from Periodontal Ligament Stem Cells Enhances Periodontal Regeneration.

Periodontal disease is one of the most common infectious diseases in adults and is characterized by the destruction of tooth-supporting tissues. Mesenchymal stem cells (MSCs) comprise the mesoderm-originating stem cell population, which has been studied and used for cell therapy. However, because of the lower rate of cell survival after MSC transplantation in various disease models, paracrine functions of MSCs have been receiving increased attention as a regenerative mechanism. The aim of this study was to investigate the regenerative potential of transplanted conditioned medium (CM) obtained from cultured periodontal ligament stem cells (PDLSCs), the adult stem cell population in tooth-supporting tissues, using a rat periodontal defect model. Cell-free CM was collected from PDLSCs and fibroblasts, using ultrafiltration and transplanted into surgically created periodontal defects. Protein content of CM was examined by antibody arrays. Formation of new periodontal tissues was analyzed using microcomputed tomography and histological sections. PDLSC-CM transplantation enhanced periodontal tissue regeneration in a concentration-dependent manner, whereas fibroblast-CM did not show any regenerative function. Proteomic analysis revealed that extracellular matrix proteins, enzymes, angiogenic factors, growth factors and cytokines were contained in PDLSC-CM. Furthermore, PDLSC-CM transplantation resulted in the decreased mRNA level of tumor necrosis factor-α (TNF-α) in healing periodontal tissues. In addition, we found that PDLSC-CM suppressed the mRNA level of TNF-α in the monocyte/macrophage cell line, RAW cells, stimulated with IFN-γ. Our findings suggested that PDLSC-CM enhanced periodontal regeneration by suppressing the inflammatory response through TNF-α production, and transplantation of PDLSC-CM could be a novel approach for periodontal regenerative therapy.

Recruitment of bone marrow-derived cells to periodontal tissue defects.

Bone marrow-derived cells (BMCs) are considered to be a major source of mesenchymal stem cells (MSCs) in adults and are known to be effective in periodontal tissue regeneration. However, whether endogenous BMCs are involved in periodontal tissue repair process is uncertain. We therefore created periodontal tissue defects in the buccal alveolar bone of mandibular first molars in bone marrow chimeric mice, and immunohistochemically examined the expression of stromal cell derived factor-1 (SDF-1) and the mobilization of BMCs. We found that SDF-1 expression was increased around the defects at as early as 1 week after injury and that BMCs were mobilized to the defects, while GFP+/CD45+ were rarely observed. Fluorescence-activated cell sorting (FACS) analysis demonstrated that the number of platelet-derived growth factor receptor (pdgfr) α+/Sca-1+ (PαS) cells in the bone marrow decreased after injury. Taken together, these results suggest that BMCs are mobilized to the periodontal tissue defects. Recruitment of BMCs, including a subset of MSCs could be a new target of periodontal treatment.

Periodontal regeneration using periodontal ligament stem cell-transferred amnion.

Periodontal disease is characterized by the destruction of tooth supporting tissues. Regeneration of periodontal tissues using ex vivo expanded cells has been introduced and studied, although appropriate methodology has not yet been established. We developed a novel cell transplant method for periodontal regeneration using periodontal ligament stem cell (PDLSC)-transferred amniotic membrane (PDLSC-amnion). The aim of this study was to investigate the regenerative potential of PDLSC-amnion in a rat periodontal defect model. Cultured PDLSCs were transferred onto amniotic membranes using a glass substrate treated with polyethylene glycol and photolithography. The properties of PDLSCs were investigated by flow cytometry and in vitro differentiation. PDLSC-amnion was transplanted into surgically created periodontal defects in rat maxillary molars. Periodontal regeneration was evaluated by microcomputed tomography (micro-CT) and histological analysis. PDLSCs showed mesenchymal stem cell-like characteristics such as cell surface marker expression (CD90, CD44, CD73, CD105, CD146, and STRO-1) and trilineage differentiation ability (i.e., into osteoblasts, adipocytes, and chondrocytes). PDLSC-amnion exhibited a single layer of PDLSCs on the amniotic membrane and stability of the sheet even with movement and deformation caused by surgical instruments. We observed that the PDLSC-amnion enhanced periodontal tissue regeneration as determined by micro-CT and histology by 4 weeks after transplantation. These data suggest that PDLSC-amnion has therapeutic potential as a novel cell-based regenerative periodontal therapy.