Evaluation of periodontal status and cytokine response in children with familial Mediterranean fever or systemic juvenile idiopathic arthritis.

OBJECTIVES: Familial Mediterranean fever (FMF) and systemic juvenile idiopathic arthritis (sJIA) are chronic inflammatory diseases and anti-inflammatory agents are used in their treatment. This study evaluates the periodontal status and cytokine response in pediatric patients with FMF or sJIA. MATERIALS AND METHODS: Forty-eight FMF/sJIA patients were under treatment/control and in attack-free period; 20 systemically healthy children participated in the study. FMF/sJIA patients were divided into two subgroups based on the treatment they received: receiving anti-IL-1 therapy (anti-IL-1 ( +)) and not receiving anti-IL-1 therapy (anti-IL-1 ( -)). The clinical periodontal indices were recorded. Gingival crevicular fluid (GCF) and serum samples were collected. Cytokine levels (IL-1ß, IL-1α, TNF-α, IL-6, IL-8, IL-10, IL-17, IL-33) in GCF and serum were measured using ELISA kits. RESULTS: There was no significant difference between the groups in terms of GCF IL-1ß and IL-1α levels although, BoP and GI were significantly lower in the anti-IL-1 ( +) group compared to the control group. GCF IL-10 level was higher in the anti-IL-1 ( -) group than in the control group; GCF IL-8 levels were lower in both FMF/sJIA subgroups versus controls. There was no significant difference between serum cytokine levels of FMF/sJIA subgroups. CONCLUSIONS: Considering the significant decrease in GI, BoP, and GCF IL-8 levels in the anti-IL-1 ( +) group, it can be concluded that anti-IL-1 medications may suppress periodontal inflammation clinically and immunologically. CLINICAL RELEVANCE: Anti-IL agents are not currently used in periodontal therapy. However, this study demonstrated the positive effect of anti-IL-1 medications on periodontal inflammation in pediatric patients with FMF or sJIA.

Sex-specific analysis in Behçet's disease reveals higher genetic risk in male patients.

OBJECTIVES: Behçet's disease tends to be more severe in men than women. This study was undertaken to investigate sex-specific genetic effects in Behçet's disease. METHODS: A total of 1762 male and 1216 female patients with Behçet's disease from six diverse populations were studied, with the majority of patients of Turkish origin. Genotyping was performed using an Infinium ImmunoArray-24 BeadChip, or extracted from available genotyping data. Following imputation and extensive quality control measures, genome-wide association analysis was performed comparing male to female patients in the Turkish cohort, followed by a meta-analysis of significant results in all six populations. In addition, a weighted genetic risk score for Behçet's disease was calculated and compared between male and female patients. RESULTS: Genetic association analysis comparing male to female patients with Behçet's disease from Turkey revealed an association with male sex in HLA-B/MICA within the HLA region with a GWAS level of significance (rs2848712, OR = 1.46, P = 1.22 × 10-8). Meta-analysis of the effect in rs2848712 across six populations confirmed these results. Genetic risk score for Behçet's disease was significantly higher in male compared to female patients from Turkey. Higher genetic risk for Behçet's disease was observed in male patients in HLA-B/MICA (rs116799036, OR = 1.45, P = 1.95 × 10-8), HLA-C (rs12525170, OR = 1.46, P = 5.66 × 10-7), and KLRC4 (rs2617170, OR = 1.20, P = 0.019). In contrast, IFNGR1 (rs4896243, OR = 0.86, P = 0.011) was shown to confer higher genetic risk in female patients. CONCLUSIONS: Male patients with Behçet's disease are characterized by higher genetic risk compared to female patients. This genetic difference, primarily derived from our Turkish cohort, is largely explained by risk within the HLA region. These data suggest that genetic factors might contribute to differences in disease presentation between men and women with Behçet's disease.

In vivo efficacy of low-level laser therapy on bone regeneration.

PURPOSE: In clinical use of low-level laser therapy for bone regeneration (LLLT), application protocol (dose, duration, and repetitions) has not been established. This study aimed to depict a reliable dosage of LLLT by evaluating the efficacy of different dosing of LLLT (diode) on the healing of rabbit cranial defects. METHODS: Critical size defects were prepared in calvarias of 26 New Zealand White Rabbits in such each animal containing both test and control groups. Test groups were irradiated with 4 Joule/cm2 (j/cm2), 6 j/cm2, and 8 j/cm2. The rabbits were subjected to six times of laser treatments in 10 days. At the end of the second week, 5 rabbits were sacrificed for histopathological and immunohistochemical analyses. At the 4th and 8th weeks, 20 rabbits (10 each) were sacrificed for micro-CT and histopathological analyses. RESULTS: Micro-CT evaluation revealed improved new bone formation in all test groups compared to the control group. 6 j/cm2 group demonstrated the highest bone formation. The highest bone morphogenic protein -2 levels were found in the 4 j/cm2 group. Osteocalcin expression was significantly higher in 4 j/cm2 group. CONCLUSIONS: Our findings indicate that LLLT have a positive effect on new bone formation. The high efficacy of doses of 4 j/cm2 and 6 j/cm2 is promising to promote early bone healing.

Evaluation of GCF IL-17, IL-10, TWEAK, and sclerostin levels after scaling and root planing and adjunctive use of diode laser application in patients with periodontitis.

BACKGROUND: The aim of the present study was to evaluate the clinical efficacy of the diode laser as an adjunct to scaling and root planing (SRP) and also determine the biochemical profile by evaluating the gingival crevicular fluid (GCF) levels of interleukin (IL)-17, IL-10, tumor necrosis factor-related weak inducer of apoptosis (TWEAK), and sclerostin. METHODS: A total of 40 systemically healthy, patients with Stage III periodontitis were included in this randomized controlled study. Participants were randomly divided into two groups as SRP + diode laser (L) (0.80W power, 940 nm wavelength and 0.80J/s energy level) and only SRP group. Recording of periodontal parameters and collecting GCF samples were performed at baseline, first and 3rd months. Biomarker levels in GCF were measured with ELISA RESULTS: At baseline, no significant difference was detected between groups in terms of both clinical and biochemical parameters. All biochemical parameters (except for IL-10 in control group), presented a statistically significant difference for 3 months study period in both groups. When laser and control groups were compared, significant differences were not observed, except the lower GCF IL-17 levels (P = 0.025), bleeding on probing (P = 0.028), and clinical attachment level (CAL) (P = 0.0002) values in laser group at third, first, and third months, respectively. Statistically significant correlations were also noted between biochemical parameters and clinical parameters. CONCLUSIONS: The GCF IL-17, TWEAK, and sclerostin levels may be useful for monitoring response to SRP+L therapy. However, long-term studies on higher populations are needed to evaluate the effectiveness of adjunctive use of diode laser application to SRP.

Epigenetic adaptations of the masticatory mucosa to periodontal inflammation.

BACKGROUND: In mucosal barrier interfaces, flexible responses of gene expression to long-term environmental changes allow adaptation and fine-tuning for the balance of host defense and uncontrolled not-resolving inflammation. Epigenetic modifications of the chromatin confer plasticity to the genetic information and give insight into how tissues use the genetic information to adapt to environmental factors. The oral mucosa is particularly exposed to environmental stressors such as a variable microbiota. Likewise, persistent oral inflammation is the most important intrinsic risk factor for the oral inflammatory disease periodontitis and has strong potential to alter DNA-methylation patterns. The aim of the current study was to identify epigenetic changes of the oral masticatory mucosa in response to long-term inflammation that resulted in periodontitis. METHODS AND RESULTS: Genome-wide CpG methylation of both inflamed and clinically uninflamed solid gingival tissue biopsies of 60 periodontitis cases was analyzed using the Infinium MethylationEPIC BeadChip. We validated and performed cell-type deconvolution for infiltrated immune cells using the EpiDish algorithm. Effect sizes of DMPs in gingival epithelial and fibroblast cells were estimated and adjusted for confounding factors using our recently developed "intercept-method". In the current EWAS, we identified various genes that showed significantly different methylation between periodontitis-inflamed and uninflamed oral mucosa in periodontitis patients. The strongest differences were observed for genes with roles in wound healing (ROBO2, PTP4A3), cell adhesion (LPXN) and innate immune response (CCL26, DNAJC1, BPI). Enrichment analyses implied a role of epigenetic changes for vesicle trafficking gene sets. CONCLUSIONS: Our results imply specific adaptations of the oral mucosa to a persistent inflammatory environment that involve wound repair, barrier integrity, and innate immune defense.

Genetic Association of a Gain-of-Function IFNGR1 Polymorphism and the Intergenic Region LNCAROD/DKK1 With Behçet's Disease.

OBJECTIVE: Behçet's disease is a complex systemic inflammatory vasculitis of incompletely understood etiology. This study was undertaken to investigate genetic associations with Behçet's disease in a diverse multiethnic population. METHODS: A total of 9,444 patients and controls from 7 different populations were included in this study. Genotyping was performed using an Infinium ImmunoArray-24 v.1.0 or v.2.0 BeadChip. Analysis of expression data from stimulated monocytes, and epigenetic and chromatin interaction analyses were performed. RESULTS: We identified 2 novel genetic susceptibility loci for Behçet's disease, including a risk locus in IFNGR1 (rs4896243) (odds ratio [OR] 1.25; P = 2.42 × 10-9 ) and within the intergenic region LNCAROD/DKK1 (rs1660760) (OR 0.78; P = 2.75 × 10-8 ). The risk variants in IFNGR1 significantly increased IFNGR1 messenger RNA expression in lipopolysaccharide-stimulated monocytes. In addition, our results replicated the association (P < 5 × 10-8 ) of 6 previously identified susceptibility loci in Behçet's disease: IL10, IL23R, IL12A-AS1, CCR3, ADO, and LACC1, reinforcing the notion that these loci are strong genetic factors in Behçet's disease shared across ancestries. We also identified >30 genetic susceptibility loci with a suggestive level of association (P < 5 × 10-5 ), which will require replication. Finally, functional annotation of genetic susceptibility loci in Behçet's disease revealed their possible regulatory roles and suggested potential causal genes and molecular mechanisms that could be further investigated. CONCLUSION: We performed the largest genetic association study in Behçet's disease to date. Our findings reveal novel putative functional variants associated with the disease and replicate and extend the genetic associations in other loci across multiple ancestries.

The tendency of reduced periodontal destruction in acromegalic patients showing similar inflammatory status with periodontitis patients.

PURPOSE: Evaluate periodontal status of acromegalics through clinical and biochemical variables. METHODS: Demographics, hormone and metabolic variables, periodontal variables, gingival crevicular fluid (GCF) volume, and content data were collected from 30 patients with acromegaly, 30 patients with periodontitis, and 20 healthy subjects and comparatively analyzed. RESULTS: GH differences between acromegaly (2.56 ± 4.86) and periodontitis (0.53 ± 0.95) (p < 0.001) were statistically significant. IGF-1 was lowest at periodontitis (113.31 ± 45.01) and lower (152.11 ± 45.56) at healthy group compared with acromegalics (220.38 ± 167.62) (p < 0.05). GH and IGF-1 had positive correlation (p < 0.05). IGF-1 and CAL had negative (p < 0.01) correlation except healthy group that showed the same correlation at the opposite direction (p < 0.05). Besides similar plaque and gingival indices with periodontitis, acromegalics showed relatively less CAL and GCF volume but except CAL, all their periodontal variables were higher than healthy subjects. GCF GH and prolactin showed higher values in acromegalics while healthy subjects showed relatively high interleukin-1, -10 and carboxyterminal telopeptide of type I collagen compared with others. CONCLUSION: Acromegalics have a tendency of slowed periodontal destruction with an influence of GH and IGF-1 to the inflammation- and collage metabolism-related mechanisms rather than bone-associated ones. However, this information must be confirmed with further studies exploring the mechanisms possibly bonded to others.

Evaluation of gingival crevicular fluid and peri-implant crevicular fluid levels of sclerostin, TWEAK, RANKL and OPG.

BACKGROUND: The combination of local and systemic factors play role in the pathogenesis of periodontal and peri-implant diseases. Host-derived enzymes, cytokines and other proinflammatory mediators play an integral role in this destruction. The aim of this study is to evaluate gingival crevicular fluid (GCF) and peri-implant crevicular (PICF) fluid levels of sclerostin, TNF-related weak inducer of apoptosis (TWEAK), receptor activator of nuclear factor kappa-beta ligand (RANKL) and osteoprotegerin OPG in periodontal and peri-implant tissues in disease and health conditions and also to assess the potential for use as biomarkers. MATERIALS AND METHODS: The study population was consisted of 50 women and 41 men, in the total of 91 individuals, with a mean age of 51.84 ±â€¯14.05. Periodontitis (n = 22), periodontal health (n = 17), peri-implantitis (n = 27) and peri-implant health (n = 25) groups were established according to clinical and radiographic examination results of 39 teeth and 52 implants restored with fixed prosthetic restorations. In all groups, periodontal and peri-implant parameters (probing depth, gingival recession, gingival bleeding time index, gingival index, and plaque index) were recorded and GCF and PICF samples were also collected. Sclerostin, TWEAK, RANKL and OPG levels in GCF and PICF were measured with ELISA tests. RESULTS: Peri-implantitis group presented significantly higher levels of Sclerostin (p = 0.002), TWEAK(p < 0.0001), RANKL(p < 0.0001), and OPG (p = 0.037) compared to peri-implant health group. Similarly, significantly higher levels of TWEAK (p = 0.001), RANKL(p < 0.0001), and OPG(p = 0.025) were detected in periodontitis group when compared to periodontal health group. Statistically significant correlations were also noted between biochemical parameters and clinical parameters. CONCLUSION: Findings of this study evaluating four different bone metabolism related proteins at the same time, suggests levels of sclerostin may be a biomarker for peri-implant disease presenting significantly higher levels in the peri-implantitis group than in the peri-implant health group. Moreover, levels of TWEAK can be a good indicator for both periodontal and peri-implant disease, due to the correlations with periodontal clinical parameters and the higher levels of TWEAK in diseased sites compared to the healthy sites for both dental implants and teeth.

Evaluation of gingival crevicular fluid and peri-implant sulcus fluid levels of periostin: A preliminary report.

BACKGROUND: Periostin is a protein present in alveolar bone and periodontal ligament whose function is related to response to external forces. The aims of this study are to detect levels of periostin in peri-implant sulcular fluid (PISF) and gingival crevicular fluid (GCF) and to evaluate the relationship between periostin, pyridinoline cross-linked carboxyterminal telopeptide of Type I collagen (ICTP), and C-terminal cross-linked telopeptide of Type I collagen (CTX) levels and clinical inflammatory symptoms and duration of functional loading. METHODS: The study population comprised nine women and four men with mean age 43.23 ± 12.48. Twenty "bone-level designed" dental implants (DIs) placed in molar or premolar sites, without any signs of peri-implant bone loss and with a restoration in function for at least 12 months, were included in the study with 20 contralateral natural teeth (NT) as controls. Clinical parameters and restoration dates of the implants were recorded. PISF, GCF, ICTP, CTX, and periostin levels were evaluated using enzyme-linked immunosorbent assay. RESULTS: ICTP, CTX, and periostin levels were similar between DI and NT groups. There were no statistically significant differences between PISF and GCF values. When implants were grouped as healthy (gingival index [GI] = 0) and inflamed (GI ≥0), ICTP levels and PISF volume were lower in healthy implants compared with the inflamed group. Both periostin and CTX levels were negatively correlated with functioning time, suggesting less bone remodeling around DIs at later stages of functioning. CONCLUSION: Findings of this study suggest collagen breakdown products may be used as markers to evaluate peri-implant metabolism.

Sequential IGF-1 and BMP-6 releasing chitosan/alginate/PLGA hybrid scaffolds for periodontal regeneration.

The goal of periodontal tissue engineering is to repair or regenerate the destructed or lost periodontium by improving functions of cells in the remaining tissue. For continuty of cell growth process, two group of growth factors, i.e. competence factors and progression factors, are needed to act together. However, the short biological half-life of these factors limits their effects on cells and their clinical efficacy. The purpose of this study is to develop different microparticles-loaded chitosan carriers/scaffolds for controlled and sequential delivery of a competence factor, insulin-like growth factor (IGF-1), and progression factor, bone morphogenetic factor-6 (BMP-6). Alginate and poly (lactic-co-glycolic acid) (PLGA) microparticles provided release of IGF-1 and BMP-6 for early short period and for long period, respectively. The cell culture studies showed that, chitosan/alginate/PLGA hybrid scaffolds induced proliferation and osteoblastic differentiation of cementoblasts when compared with IGF-1 and BMP-6 free chitosan scaffold.

A genome-wide association study identifies nucleotide variants at SIGLEC5 and DEFA1A3 as risk loci for periodontitis.

Periodontitis is one of the most common inflammatory diseases, with a prevalence of 11% worldwide for the severe forms and an estimated heritability of 50%. The disease is characterized by destruction of the alveolar bone due to an aberrant host inflammatory response to a dysbiotic oral microbiome. Previous genome-wide association studies (GWAS) have reported several suggestive susceptibility loci. Here, we conducted a GWAS using a German and Dutch case-control sample of aggressive periodontitis (AgP, 896 cases, 7,104 controls), a rare but highly severe and early-onset form of periodontitis, validated the associations in a German sample of severe forms of the more moderate phenotype chronic periodontitis (CP) (993 cases, 1,419 controls). Positive findings were replicated in a Turkish sample of AgP (223 cases, 564 controls). A locus at SIGLEC5 (sialic acid binding Ig-like lectin 5) and a chromosomal region downstream of the DEFA1A3 locus (defensin alpha 1-3) showed association with both disease phenotypes and were associated with periodontitis at a genome-wide significance level in the pooled samples, with P = 1.09E-08 (rs4284742,-G; OR = 1.34, 95% CI = 1.21-1.48) and P = 5.48E-10 (rs2738058,-T; OR = 1.28, 95% CI = 1.18-1.38), respectively. SIGLEC5 is expressed in various myeloid immune cells and classified as an inhibitory receptor with the potential to mediate tyrosine phosphatases SHP-1/-2 dependent signaling. Alpha defensins are antimicrobial peptides with expression in neutrophils and mucosal surfaces and a role in phagocyte-mediated host defense. This study identifies the first shared genetic risk loci of AgP and CP with genome-wide significance and highlights the role of innate and adaptive immunity in the etiology of periodontitis.

Platelet-rich plasma-loaded chitosan scaffolds: preparation and growth factor release kinetics.

The aim of this study is to compare the effects of different platelet-rich plasma (PRP) preparation methods on platelet activity and to investigate the growth factor (GF) release kinetics from PRP-loaded chitosan scaffolds for tissue engineering applications. Flow cytometry analysis showed that centrifugation processes used for PRP preparation did not cause significant effect on platelet activation levels by means of markers investigated. Two different methods were used to prepare PRP-loaded chitosan scaffolds: (i) PRP was added to chitosan gel before freeze-drying to prepare scaffolds called as "GEL" and (ii) PRP was embedded to freeze-dried chitosan scaffolds to prepare scaffolds called as "SPONGE." In addition, nonactivated PRP and PRP activated with type-I collagen were used as control groups. Scanning electron microscopy images demonstrated that, in GEL group, there is no deterioration on the scaffolds porous, 3D, and interconnected structure. GF release kinetics was determined by enzyme-linked immunosorbent assay for platelet-derived GF-BB, transforming GF-ß1, and insulin-like GF-1. A sustained release of GFs was achieved in GEL group while a sharp burst release was observed for all the GFs from the SPONGE groups. Moreover, platelet-derived GF-BB, insulin-like GF-1, and transforming GF-ß1 releases were prolonged to 20 days in GEL groups, and the biological activities of all GFs released from GEL and SPONGE scaffolds were preserved. This study demonstrated that chitosan scaffold that was called GEL could be an appropriate carrier for PRP applications by providing sustained release of GFs.

Periodontal ligament cell behavior on different titanium surfaces.

AIM: The purpose of this study was to investigate proliferation, morphology, mineralization and mRNA expressions of mineralized tissue associated proteins of PDL cells on smooth (S), sandblasted small-grit (SSG), sandblasted large-grit (SLG) and sodium titanate (NaTi) coated titanium alloys, in vitro. METHODS AND MATERIALS: PDL cells were cultured with DMEM media containing 10% FBS on the S, SSG, SLG and NaTi titanium surfaces. PDL cell proliferation, mineralization and immunohistochemistry experiments for Bone Sialoprotein (BSP) were performed. The morphology of the PDL cells was examined using confocal and scanning electron microscopy (SEM). Gene expression profiles of cells were evaluated using a quantitative-polymerase chain reaction (Q-PCR) for type I collagen (COL I), Osteocalcin (OCN), osteopontin (OPN) and Runt-related transcription factor-2 (Runx2) on days 7 and 14. RESULTS: Proliferation results on days 6 and 10 were similar in groups, while those of day 13 revealed a decrease in the NaTi group when compared to the S group. NaTi surface induced BSP mRNA expression which was correlated with mineralization tests and BSP immunostaining results. Increased Runx2 mRNA expression was also noted in the NaTi surface when compared to other surfaces. CONCLUSIONS: This study considers the NaTi surface as a potential alternative to SSG and SLG surfaces. This surface might provide a promising environment for PDL ligament-anchored implants.

Bone morphogenetic protein-7 enhances cementoblast function in vitro.

BACKGROUND: Bone morphogenetic protein (BMP)-7 is a potent bone-inducing factor and was shown to promote periodontal regeneration in vivo and in vitro; however, to our knowledge, the specific effect of BMP-7 on cementoblasts has not been defined. We aimed to investigate the effects of BMP-7 on cementoblasts, which are cells responsible for tooth root-cementum formation. We hypothesized that BMP-7 would regulate mineralized tissue-associated genes in cementoblasts and influence the expression profile of genes associated with cementoblast extracellular matrix (ECM) and cell adhesion molecules (CAMs). METHODS: A murine immortalized cementoblast cell line (OCCM.30) was cultured with and without 50 ng/ml BMP-7. After 72 hours, total RNA was isolated, and mRNA levels for bone/cementum markers, including bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN), and runt-related transcription factor-2 (Runx2), were investigated by real-time quantitative reverse transcription-polymerase chain reaction (Q-PCR). In vitro mineral nodule formation was assayed on day 8 using von Kossa staining. A pathway-specific gene-expression array was used to determine BMP-7-responsive ECM and CAM genes in cementoblasts. RESULTS: Mineralized tissue markers were strongly regulated by BMP-7, with an almost three-fold increase in BSP and OCN transcripts and significant increases in OPN and Runx2 mRNA expressions. BMP-7 treatment markedly stimulated cementoblast-mediated biomineralization in vitro compared to untreated cells at day 8. BMP-7 treatment altered the OCCM.30 expression profile for ECM and CAM functional gene groups. BMP-7 tended to increase the expression of collagens and matrix metalloproteinases (MMPs), mildly decreased tissue inhibitors of MMPs (TIMPs), and had mixed regulatory effects on integrins. Using Q-PCR, selected array results were confirmed, including a significant BMP-7-induced increase in MMP-3 and a decrease in TIMP-2 mRNA expression. CONCLUSION: These results support the promising applications of BMP-7 in therapies aimed at regenerating periodontal tissues lost as a consequence of disease.

Bone morphogenetic protein-6-loaded chitosan scaffolds enhance the osteoblastic characteristics of MC3T3-E1 cells.

The purpose of this study is to investigate the convenience of bone morphogenetic protein-6 (BMP-6)-loaded chitosan scaffolds with preosteoblastic cells for bone tissue engineering. MC3T3-E1 cells were seeded into three different groups: chitosan scaffolds, BMP-6-loaded chitosan scaffolds, and chitosan scaffolds with free BMP-6 in culture medium. Tissue-engineered constructs were characterized by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide assay, scanning electron microscopy (SEM), mineralization assay (von Kossa), alkaline phosphatase (ALP) activity, and osteocalcin (OCN) assays. BMP-6-loaded chitosan scaffolds supported proliferation of the MC3T3-E1 mouse osteogenic cells in a similar pattern as the unloaded chitosan scaffolds group and as the chitosan scaffolds with free BMP-6 group. SEM images of the cell-seeded scaffolds revealed significant acceleration of extracellular matrix synthesis in BMP-6-loaded chitosan scaffolds. Both levels of ALP and OCN were higher in BMP-6-loaded chitosan scaffold group compared with the other two groups. In addition, BMP-6-loaded scaffolds showed strong staining in mineralization assays. These findings suggest that BMP-6-loaded chitosan scaffold supports cellular functions of the osteoblastic cells; therefore, this scaffold is considered as a new promising vehicle for bone tissue engineering applications.

bFGF-loaded HA-chitosan: a promising scaffold for periodontal tissue engineering.

A scaffold containing growth factors promoting regeneration may be a useful device to maintain periodontal regeneration when applied with appropriate cells. The aim of this study is to evaluate the convenience of chitosan and hydroxyapatite (HA)-chitosan scaffolds loaded with basic fibroblast growth factor (bFGF) for periodontal tissue engineering applications. Scaffolds were fabricated by freeze-drying technique using 2 and 3% chitosan gel in the absence or presence of HA particles. Addition of HA beads to chitosan gels produced a novel scaffold in which the pore sizes and interconnectivity were preserved. The scaffolds were loaded with 100 ng bFGF by embedding technique. HA-chitosan scaffolds provide better controlled release kinetics for bFGF compared with chitosan scaffolds and total release continued up to 168 h. Cell culture studies were carried out with periodontal ligament (PDL) cells and cementoblasts. Both 3-[4,5-dimethylthiazol-2-yl]-diphenyltetrazolium bromide (MTT) assay and confocal laser scanning microscope analysis revealed cells proliferating inside the scaffolds. The results demonstrated that bFGF-loaded HA-chitosan scaffolds provide a suitable three-dimensional environment supporting the cellular structure, proliferation, and mineralization.

In vitro release of dexamethasone or bFGF from chitosan/hydroxyapatite scaffolds.

Chitosan scaffolds containing dexamethasone (Dex) or basic fibroblast growth factor (bFGF) were developed to create alternative drug-delivery systems for possible tissue-engineering applications such as periodontal bone regeneration. Chitosan solutions (2% and 3% (w/v) in acetic acid) were prepared from chitosan flakes with high deacetylation degree (>85%), then these solutions were freeze-dried at -80 degrees C to obtain scaffolds with interconnected pore structures. Dex and bFGF were incorporated into scaffolds by embedding method (solvent sorption method). The initial loading amounts were varied as 300, 600 and 900 ng Dex per dry scaffold (average dry weight is 3 mg) and 50 or 100 ng bFGF per dry scaffold to a range of deliverable doses. Release studies which were conducted in Dulbecco's phosphate-buffered saline (DPBS) showed that 900 ng Dex loaded chitosan scaffolds in both compositions released total Dex during a 5-day period at a nearly constant rate after the initial burst. However, bFGF release from all scaffolds with both loading amounts (50 ng or 100 ng) was completed in 10 or 20 h. In order to prolong the release period of bFGF, composite scaffolds were fabricated in the presence of hydroxyapatite (HA) beads with average particle size of 40 mum. Sustained release of bFGF up to 7 days was achieved due to the electrostatic interactions between HA and bFGF molecules. These results suggested that chitosan scaffolds can be suitable for Dex release; however, the presence of HA in the chitosan scaffold is necessary to achieve the desired release period for bFGF.

Identification of the difference in extracellular matrix and adhesion molecules of cultured human gingival fibroblasts versus juvenile hyaline fibromatosis gingival fibroblasts using cDNA microarray analysis.

BACKGROUND: A difference from the normal range in collagen profile and perivascular hyaline deposition in the dermis and gingiva has been demonstrated histopathologically in juvenile hyaline fibromatosis (JHF), which is an autosomal recessive disease. The aim of this study was to understand the mechanism of gingival overgrowth in JHF, and to observe differences in the expression of genes regulating extracellular matrix organization. METHODS: Human gingival fibroblasts (GF) were obtained from individuals who have clinically healthy gingival tissue. JHF-GF were obtained from a patient who underwent a gingivectomy. Cultured fibroblast cells were examined visually using a phase contrast microscope. Total RNA from both cell types was isolated, and after biotin-deoxyuridine triphosphate (dUTP) labeling of cDNA, hybridization was performed with a pathway-specific gene expression profiling array membrane. Extracellular matrix (ECM) and adhesion molecule (AM) mRNA expressions in GF and JHF-GF were analyzed, and microarray data on genes modulating ECM remodeling were confirmed with reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Cell morphology differences were observed between fibroblast types. Although type I collagen gene expression levels were almost the same, decreased type IV collagen expression was noted in JHF-GF versus GF. Decreased matrix metalloproteinase (MMP) and increased tissue inhibitor of matrix metalloproteinase (TIMP) transcripts were noted in JHF-GF versus GF. Increased fibronectin and decreased laminin mRNA expression were observed in JHF-GF when compared to GF. The present findings suggest that GF and JHF-GF differ not only morphologically but also in the expression level of ECM and AM genes involving connective tissue turnover and remodeling. CONCLUSIONS: Results from these analyses may be helpful to clarify the nature of overgrowth mechanisms, especially regarding enzymes and their inhibitors. This information is important in understanding the remodeling of ECM. The gingival overgrowth that is observed in JHF patients may be explained by a decreased level of MMPs and increased blockage of MMPs with TIMPs.

Dexamethasone and basic-fibroblast growth factor regulate markers of mineralization in cementoblasts in vitro.

BACKGROUND: The aim of this study was to determine the effects of basic-fibroblast growth factor (b-FGF) and/or dexamethasone (Dex) on cementoblasts in vitro. METHODS: Murine cementoblasts were treated as follows: 1) 5% FBS (fetal bovine serum) + ascorbic acid (AA, 50 microg/ml, control); 2) 5% FBS + Dex (10(7)M) + AA; 3) 5% FBS + b-FGF (50 ng/ml)+AA; or 4) 5% FBS + Dex (10(7) M) + b-FGF (50 ng/ml)+AA and then evaluated by Northern analysis for changes in specific genes and by von Kossa stain for changes in mineral nodule formation. RESULTS: Mitotic activity: b-FGF stimulated DNA synthesis significantly versus negative control. Gene expression: osteocalcin (OCN): Dex or b-FGF or the combination resulted in a decrease in expression versus control. Bone sialoprotein (BSP): Dex increased expression of BSP mRNA levels, b-FGF decreased transcript for BSP at 6 and 24 hours. Long-term (8 days) Dex, b-FGF, or Dex plus b-FGF caused a decrease in BSP expression versus control; osteopontin (OPN): both Dex and b-FGF increased transcripts for OPN seen by 6 hours, with a greater increase noted with b-FGF versus Dex. No apparent additive effect of Dex with b-FGF was noted; matrix gamma-carboxyglutamic acid protein (MGP): b-FGF induced transcripts for MGP and addition of Dex increased this effect, while Dex alone had no effect on expression. Biomineralization: Dex increased cementoblast- mediated biomineralization, while b-FGF blocked this activity, and addition of Dex to b-FGF did not alter FGF associated inhibition. CONCLUSION: Dex and FGF alone and in combination alter cementoblast behavior, but additional studies are required to determine whether these factors have beneficial effects at the clinical level.