Comparison of Different Periodontal Healing of Critical Size Noncontained and Contained Intrabony Defects in Beagles.

BACKGROUND: Regenerative techniques help promote the formation of new attachment and bone filling in periodontal defects. However, the dimensions of intraosseous defects are a key determinant of periodontal regeneration outcomes. In this study, we evaluated the efficacy of use of anorganic bovine bone (ABB) graft in combination with collagen membrane (CM), to facilitate healing of noncontained (1-wall) and contained (3-wall) critical size periodontal defects. METHODS: The study began on March 2013, and was completed on May 2014. One-wall (7 mm × 4 mm) and 3-wall (5 mm × 4 mm) intrabony periodontal defects were surgically created bilaterally in the mandibular third premolars and first molars in eight beagles. The defects were treated with ABB in combination with CM (ABB + CM group) or open flap debridement (OFD group). The animals were euthanized at 8-week postsurgery for histological analysis. Two independent Student's t-tests (1-wall [ABB + CM] vs. 1-wall [OFD] and 3-wall [ABB + CM] vs. 3-wall [OFD]) were used to assess between-group differences. RESULTS: The mean new bone height in both 1- and 3-wall intrabony defects in the ABB + CM group was significantly greater than that in the OFD group (1-wall: 4.99 ± 0.70 mm vs. 3.01 ± 0.37 mm, P < 0.05; 3-wall: 3.11 ± 0.59 mm vs. 2.08 ± 0.24 mm, P < 0.05). The mean new cementum in 1-wall intrabony defects in the ABB + CM group was significantly greater than that in their counterparts in the OFD group (5.08 ± 0.68 mm vs. 1.16 ± 0.38 mm; P < 0.05). Likewise, only the 1-wall intrabony defect model showed a significant difference with respect to junctional epithelium between ABB + CM and OFD groups (0.67 ± 0.23 mm vs. 1.12 ± 0.28 mm, P < 0.05). CONCLUSIONS: One-wall intrabony defects treated with ABB and CM did not show less periodontal regeneration than that in 3-wall intrabony defect. The noncontained 1-wall intrabony defect might be a more discriminative defect model for further research into periodontal regeneration.

[Effect of periodontal initial therapy on teeth with chronic periodontitis and secondary occlusal trauma].

OBJECTIVE: To evaluate the effect of subgingival scaling/root planning (SRP) and occlusal adjustment on clinical and occlusal parameters in teeth with chronic periodontitis and secondary occlusal trauma. METHODS: Eighteen patients with chronic periodontitis and occlusal trauma were included and randomly divided into group A and group B. On day 0, group A was treated by full-mouth subgingival scaling and root planning, and group B was treated by occlusal adjustment in occlusal trauma site. On day 28, group A was treated by occlusal adjustment in occlusal trauma site, and group B was treated by full-mouth subgingival scaling and root planning. Probing depth (PD), attachment loss (AL), bleeding index (BI) were evaluated on 0, 28 and 56 d, and the occlusal time (OT) and the percentage of occlusal force were evaluated on 0, 28 and 56 d in occlusal trauma site. The data was statistically analyzed. RESULTS: In baseline, the PD[(4.42 ± 1.41) mm vs (4.36 ± 1.38) mm], AL [(2.75 ± 1.32) mm vs (2.63 ± 1.37) mm] and BI [(2.20 ± 0.81) vs (2.24 ± 0.89)] of the full-mouth showed no significant difference between the two groups (P > 0.05). There was no significant difference in PD [(5.21 ± 1.21) mm vs (5.08 ± 1.12) mm], AL [(4.94 ± 1.47) mm vs (4.89 ± 1.32) mm], BI [(2.61 ± 0.92) vs 2.50 ± 0.79)], OT [(1.29 ± 0.39) s vs (1.34 ± 0.35) s] and the percentage of occlusal force [(6.8 ± 2.1)% vs (7.4 ± 1.7)%] in occlusal trauma site between the two groups(P > 0.05). After SRP therapy, the PD,AL,BI and OT were significantly decreased (P < 0.05).The clinical parameters exhibited no significant difference after only occlusal adjustment(P > 0.05).On 56 d, the reduction in clinical parameters was not significantly different between the two groups(P > 0.05),however the reduction of OT and the change of the percentage of occlusal force in group A [(0.85 ± 0.41) s, (2.2 ± 2.2)%] were more significant than those in group B [(0.70 ± 0.38) s; (1.5 ± 1.6)%] (P < 0.05). After occlusal adjustment, the increase of OT in group A [(0.21 ± 0.11) s] was lower than that in group B [(0.67 ± 0.37) s]through the 28-day observation period (P < 0.05). CONCLUSIONS: Occlusal adjustment alone is inadequate for control and management of periodontitis.SRP therapy can eliminate the inflammation and decrease the OT of tooth with occlusal trauma.The combination of SRP and occlusal adjustment may achieve more stable results.

[Effects of basic fibroblast growth factor and vascular endothelial growth factor on the proliferation, migration and adhesion of human periodontal ligament stem cells in vitro].

OBJECTIVE: To evaluate the effects of basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF) on the proliferation, migration, and adhesion of human periodontal ligament stem cells (PDLSC) in vitro. METHODS: Human PDLSC were cultured in vitro using tissue culture method.The cells were cultured and incubated with various concentrations of FGF-2 and VEGF [A:α-MEM with 2% fetal bovine serum (FBS) (control 1); B:A supplemented with 20 µg/L FGF-2; C:A supplemented with 10 µg/L VEGF; D:A supplemented with 20 µg/L FGF-2 and 10 µg/L VEGF; E:α-MEM with 10% FBS (control 2); F:E supplemented with 20 µg/L FGF-2; G:E supplemented with 10 µg/L VEGF; H:E supplemented with 20 µg/L FGF-2 and 10 µg/L VEGF]. Soluble tetrazolium salts assay was used to evaluate the proliferative capacity on the 1st, 3rd, 5th and 7th d. Then the groups were changed according to result of the proliferation assay (control:α-MEM with 2% FBS; FGF-2 group:control supplemented with 20 µg/L FGF-2; VEGF:control supplemented with 10 µg/L VEGF; Combination group:control supplemented with 20 µg/L FGF-2 and 10 µg/L VEGF). The cell cycle, migration and adhesion capacities were evaluated using flow cytometer, soluble tetrazolium salts assay, cell adhesion assay and scratch wound-healing motility assay. RESULTS: In 2% volume fraction serum containing medium, FGF-2 and VEGF did not stimulate the cell proliferation. However, in 10% serum condition, in groups treated with FGF-2 for 3,5 or 7 d, the A value was (1.22 ± 0.17, 2.15 ± 0.19, 2.72 ± 0.11) respectively, which were significantly higher than that in the control group (0.76 ± 0.16, 1.25 ± 0.06, 1.64 ± 0.09) (P < 0.01) while lower than that in the group treated with FGF-2 and VEGF in combination on the 5 th and 7 th d (2.46 ± 0.17, 3.18 ± 0.27) ( P < 0.05). The A value in the VEGF group on the 5 th and 7 th d is higher than the control group while lower than the FGF-2 group (1.66 ± 0.05, 2.13 ± 0.13) (P < 0.05). Flow cytometer showed that the proliferation index in VEGF group [(34.3 ± 2.0)% ] were significantly lower than those in FGF-2 [(46.8 ± 3.2)%] group and (FGF-2+ VEGF) group [(45.0 ± 4.0)%] but higher than in the control group [(14.5 ± 1.7)%] (P < 0.01). The cell migration assay indicated that the group stimulated with FGF-2 showed no migration promoted effect. Cell adhesion assay showed that the ratio of the adhesive cells number to the original cells number is greater in the FGF-2 group (79 ± 4) than in the VEGF group (62 ± 4) (P < 0.05). Light microscope identified a better cellular morphology on the adhesive surface in the group with FGF-2 than groups without FGF-2. CONCLUSIONS: Both FGF-2 and VEGF could simulate the proliferation of PDLSC in a dose dependent manner, and showed an synergistic effect. FGF-2 was more effective to promote the adhesive capacity of PDLSC compared with VEGF. VEGF could facilitate the migration of PDLSC to the wound side.

MicroRNA-137 promoter methylation in oral lichen planus and oral squamous cell carcinoma.

Oral lichen planus (OLP) is a common oral mucosal disease, which is generally considered a potentially malignant lesion. To identify efficiently prognostic biomarker, we investigated the microRNA-137 (miR-137) promoter methylation in OLP and compared with the samples from healthy volunteers and patients with oral squamous cell carcinoma (OSCC). A total of 20 OLP and 12 patients with OSCC as well as 10 healthy subjects were subjected to miR-137 promoter methylation analysis using methylation-specific PCR (MSP). To address the malignancy prediction potential from miR-137 promoter methylation status, methylation of the p16 gene, a well-known tumor suppressor, was investigated in the same samples. The p16 methylation and miR-137 promoter methylation were found to be 25% and 35% in patients with OLP, 50% and 58.3% in patients with OSCC, and 0% and 0% in healthy subjects, respectively. The differences between miR-137 and p16 methylation levels were statistically significant between healthy controls and patients. Methylation levels of the two promoters were also influenced by age, gender, and lesion duration. Interestingly, aberrant promoter methylation of the p16 and miR-137 genes was only found in the epithelium but not in the connective tissue from patients with OLP. This raises the possibility to use miR-137 methylation as a biomarker for malignant prediction in patients with OLP.

Development of a reverse transcription loop-mediated isothermal amplification assay for rapid detection of grass carp reovirus.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a gene amplification method that can amplify the RNA template by isothermal incubation. This paper reports a rapid and sensitive RT-LAMP method which was developed for the detection of grass carp reovirus (GCRV). The present study concluded the optimal conditions for the LAMP reaction of which the Mg(2+) concentrations in the reaction mixtures, the incubation temperature, and the reaction time are at 8mM, 64°C, and 30 min, respectively. The analytical sensitivity of the RT-LAMP method was revealed as low as 7 copies of viral templates and 100-fold more sensitive than the published RT-PCR method. A visual inspection of in-tube LAMP products stained with a DNA fluorescent dye demonstrated that the positive and negative reactions exhibit distinct and different colors in daylight, which means that gel electrophoresis is not necessary to judge the positive or negative results. As the application of the method is rapid, easy, and no complicated instrument required, the GCRV-RT-LAMP method established in this study has great potential for the detection of GCRV in both the laboratory and the farm.

Multiple biological complex of alkaline extract of the leaves of Sasa senanensis Rehder.

Previous studies have shown anti-inflammatory potential of alkaline extract of the leaves of Sasa senanensis Rehder (SE). The aim of the present study was to clarity the molecular entity of SE, using various fractionation methods. SE inhibited the production of nitric oxide (NO), but not tumour necrosis factor-α by lipopolysaccharide (LPS)-stimulated mouse macrophage-like cells. Lignin carbohydrate complex prepared from SE inhibited the NO production to a comparable extent with SE, whereas chlorophyllin was more active. On successive extraction with organic solvents, nearly 90% of SE components, including chlorophyllin, were recovered from the aqueous layer. Anti-HIV activity of SE was comparable with that of lignin-carbohydrate complex, and much higher than that of chlorophyllin and n-butanol extract fractions. The CYP3A inhibitory activity of SE was significantly lower than that of grapefruit juice and chlorophyllin. Oral administration of SE slightly reduced the number of oral bacteria. When SE was applied to HPLC, nearly 70% of SE components were eluted as a single peak. These data suggest that multiple components of SE may be associated with each other in the native state or after extraction with alkaline solution.

[Influence of transfection with human transforming growth factor-beta1 gene on the osteogenic potential of the cultured human gingival fibroblast].

OBJECTIVE: To study the influence of transfection with human transforming growth factor-beta1 (hTGF-beta1) gene on the osteogenic potential and differentiation of the cultured human gingival fibroblast (GF). METHODS: Enzyme kinetics method was used to measure the effects of the transfection on the alkaline phosphatase (ALP) activity. Immunohistochemistry stain and image analysis were applied to evaluate the alteration of the content of osteopontin (OPN), bone sialoprotein (BSP), osteonectin (ON), osteocalcin (OC) in the GF with transfection. Mineralization nodules formation in vitro was also used. RESULTS: The ALP activity of the GF after transfection was higher than the GF without transfection significantly (P<0.05), and was close to that of the PDLCs (P>0.05). The content of OC in GF was not improved after transfection, was similar with that of PDLCs (P>0.05). Under immunohistochemistry stain, the contents of OPN, ON, BSP expressed in GF with transfection were higher than those of GF without transfection (P<0.05), but similar to those of PDLCs (P>0.05). In the mineralized cultured medium, the nodules were observed in the GF with transfection and PDLCs after 21 days and 24 days alternatively. After von Kossa stain, purple mineralization nodules were observed. CONCLUSION: The GF transfected with pcDNA3-hTGF-beta1 could express some osteogenic characters, though these characters are restricted.

Gene delivery for periodontal tissue engineering: current knowledge - future possibilities.

The cellular and molecular events of periodontal healing are coordinated and regulated by an elaborate system of signaling molecules, pointing to a primary strategy for functional periodontal compartment regeneration to replicate components of the natural cellular microenvironment by providing an artificial extracellular matrix (ECM) and by delivering growth factors. However, even with optimal carriers, the localized delivery of growth factors often requires a large amount of protein to stimulate significant effects in vivo, which increases the risk and unwanted side effects. A simple and relatively new approach to bypassing this dilemma involves converting cells into protein producing factories. This is done by a so-called gene delivery method, where therapeutic agents to be delivered are DNA plasmids that include the gene encoding desired growth factors instead of recombinant proteins. As localized depots of genes, novel gene delivery systems have the potential to release their cargo in a sustained and controlled manner and finally provide time- and space- dependent levels of encoded proteins during all stages of tissue regrowth, offering great versatility in their application and prompting new tissue engineering strategy in periodontal regenerative medicine. However, gene therapy in Periodontology is clearly in its infancy. Significant efforts still need to be made in developing safe and effective delivery platforms and clarifying how gene delivery, in combination with tissue engineering, may mimic the critical aspects of natural biological processes occurring in periodontal development and repair. The aim of this review is to trace an outline of the state-of-the-art in the application of gene delivery and tissue engineering strategies for periodontal healing and regeneration.

[Effects of enamel matrix proteins loaded in chitosan thermosensitive hydrogel on bone marrow stromal cells in vitro].

PURPOSE: To investigate the effects of enamel matrix proteins (EMPs ) on proliferation, alkaline phosphate (ALP) activity of bone marrow stromal cells (BMSCs) seeded on the scaffold of chitosan thermosensitive hydrogel. METHODS: Chitosan thermosensitive hydrogel was prepared and its slow-releasing effect of EMPs was checked by coomassie blue staining kit. Rat BMSCs were obtained from rat bone marrow aspiration and cultured in DMEM medium with 10% fetal bovine serum (FBS). Rat BMSCs were exposed to various concentrations of EMPs (0,50,100 and 150 microg/mL) and their proliferation rates were assessed by MTT assay. The proliferation rates and ALP activity of rat BMSCs were examined by MTT assay and ALP kit when BMSCs cultured on the scaffolds of chitosan thermosensitive hydrogel loading with or without 100 microg/mL EMPs .The data was statistically analyzed with SPSS11.0 software package for a parametric one-way analysis of variance (ANOVA) and two-sample t test. RESULTS: The release of EMPs in chitosan thermosensitive hydrogel lasted for more than 3 weeks. In DMEM medium, 50 microg/mL EMPs significantly enhanced BMSCs proliferation from day 3 over the experiment(P<0.01). In chitosan thermosensitive hydrogel scaffolds loading 100 microg/mL EMPs, both the proliferation at day 3 and 5 (P<0.05)and the ALP activity at day 7 (P<0.05) and 9(P<0.01) of BMSCs in the experiment were promoted. CONCLUSION: EMPs loaded on the chitosan thermosensitive hydrogel exhibits significant effects on proliferation and ALP activity of rat BMSCs.

Non-apoptotic cell death induced by nutritional starvation in J774.1 mouse macrophage-like cell line.

The growth and amino acid utilization of a mouse macrophage-like cell line J774.1 was investigated in two different culture media supplemented with 10% fetal bovine serum (FBS). The J774.1 cells grew faster, and consumed glutamine and serine at higher rates in DMEM than in RPMI1640 medium. The consumption of other amino acids was much less, while considerable quantities of alanine, glutamic acid and glycine were produced by the J774.1 cells. When the cells became confluent, serine, but not glutamine, was nearly depleted from the culture medium, followed by cell death characterized by smear DNA fragmentation, slight caspase-3 activation and structural damage of the mitochondria. Serine is required for the growth of mouse macrophage-like cell lines, and DMEM is superior to RPMI1640 for long-term cell culture.

[An animal experiment for the regeneration of periodontal defect by application of the dual-release chitosan thermosensitive hydrogel system].

OBJECTIVE: To observe the effect of the self-made chitosan thermosensitive hydrogel system with dual-release bone morphogenetic protein and chlorhexidine on periodontal defects repair. METHODS: The furcation defect model was established on dog premolar. The models were divided into five groups, including three experimental groups, one control group and one blank control group. The hydrogel with the chlorhexidine/3-cyclodextrin inclusion complexes (IC) /rhBMP-2, hydrogel with rhBMP-2, hydrogel with IC, the pure hydrogel were applied to the defects of the four groups, respectively, and the blank control group did not receive any agent. The dogs were sacrificed 8 weeks later and the periodontal regeneration and gingival condition were observed by histological examination. RESULTS: Obvious periodontal tissue regeneration was found in group one and two. The heights of new bone reached 99.2% of the defects in group one, 87.8%, 63.6%, 37.0% and 34.3% in group two, three, four and blank control groups, respectively. The inflammation of the affected gingiva showed less significant in group one and group three than in the other groups. CONCLUSIONS: rhBMP-2 and chlorhexidine played their independent role in repairing periodontal defects and the dual-release chitosan thermosensitive hydrogel system is effective and convenient to use.

[Effects of Porphyromonas gingivalis on inflammatory factor secreted by human umbilical vein endothelial cells].

PURPOSE: To investigate the effect of porphyromonas gingivalis (P.g) on interleukin-18 (IL-18) and CRP secretion in human umbilical vein endothelial cells (HUVECs). METHODS: Anaeropack method was used for P.g cultivation and then HUVECs were co-cultured with P.g followed by assaying the amount of IL-18 and CRP with enzyme-linked immunosorbent assay (ELISA) kit. Linear regression and variance analysis were performed with SPSS11.0 software package. RESULTS: The unstimulated HUVECs expressed small amounts of IL-18 and CRP, P.g dose-dependently increased the production of IL-18 and CRP in HUVECs. Elevated IL-18 and CRP were found at 4-hour, then continued to increase at 8-hour, 12-hour and 24-hour after co-cultivation; under the effect of same concentration, the quantity of CRP was significantly higher than that of IL-18 secreted by HUVECs. There was significant different, P<0.05. CONCLUSION: It is concluded that P.g increased the production of IL-18 and CRP in HUVECs in a dose and time dependent manner, elevated IL-18 and CRP may be involved in the regulation of inflammatory both in periodontal disease and formation of atherosclerotic plaque in cardiovascular disease(CVD).

Epidemiology and preventive direction of periodontology in China.

OBJECTIVE: The aim of this work was to estimate the present periodontal problems of people in China, based on an epidemiological investigation of adults. MATERIAL AND METHODS: The data were collected from the northwest, southwest, northeast and east regions (400 subjects from each region) of China. All subjects were over 25 years of age. About half of the subjects were farmers and about half were urban professionals. Everyone was asked to fill out a questionnaire and to undergo a professional oral examination. Periodontal health status was evaluated by a simplified oral hygiene index (OHI-S), gingival index (GI), bleeding on probing (BOP), probing pocket depth (PD), clinical attachment loss (CAL), and tooth mobility. RESULTS: Of the 1590 subjects enrolled in this investigation, 45.7% were male, 45.5% were farmers, and the remaining were urban professionals, and 27.7% of the subjects were smokers. There was a significant difference in the educational background but not smoking between the rural and urban groups. While 34.9% of the subjects in the urban group brushed only once per day, 56.1% of the subjects in the rural group did so. The prevalence of bleeding during brushing was 71.1%, while about 61.4% of the subjects know nothing about scaling. All periodontal indices were significantly higher in males than in females and higher in the rural group than in the urban group. PD, CAL and tooth mobility increased with age. The percentage of sites with CAL>3 mm in the rural group (49.5%) was significantly higher than that in the urban group (37.5%). Both current and former smokers showed increased CAL than non-smokers. CONCLUSION: Gingivitis and periodontitis are common findings in China. Most Chinese have no knowledge of common periodontal prevention and treatment and very few have regular dental care. The data of this study suggest that age, smoking, and limited education are significantly associated with Chinese adult periodontal attachment loss. Preventive periodontal care and education should be reinforced in the future by establishing relevant oral health projects.

MMP-2, MMP-9 and TIMP-2 gene polymorphisms in Chinese patients with generalized aggressive periodontitis.

BACKGROUND: Aggressive periodontitis (AgP) has a genetic basis. It has been reported that the functional gene polymorphisms of matrix metalloproteinase (MMP)-2, MMP-9 and tissue inhibitor of metalloproteinase-2 (TIMP-2) alter their expressions in transcriptional level and they are involved in the tissue destruction of periodontitis. The study was carried out to analyse the association of functional polymorphisms in MMP-2, MMP-9 and TIMP-2 with generalized AgP (G-AgP) in a Chinese population. MATERIAL AND METHODS: The study population consisted of 79 Chinese patients with G-AgP and 128 healthy controls. DNA was obtained from oral mucosa swab samples. MMP-2 genotypes were determined by PCR-based denaturing high-performance liquid chromatography analysis while MMP-9 and TIMP-2 genotypes were identified by a PCR-based restriction fragment length polymorphism. Chi2 test after Yates' correction was used to investigate the possible association of the genotypes with the G-AgP. RESULTS: Although gene polymorphisms for MMP-2 and MMP-9 did not show any association with the G-AgP, the analysis of the TIMP-2 -418G to C gene polymorphism revealed significant differences between the patients and controls. Compared with controls, a significant increasing trend of TIMP-2 -418C carrier in the G-AgP patients occurred (p=0.013). CONCLUSION: It is suggested that the TIMP2 -418G to C gene polymorphism is associated with G-AgP in the Chinese subjects.

Novel glycidyl methacrylated dextran (Dex-GMA)/gelatin hydrogel scaffolds containing microspheres loaded with bone morphogenetic proteins: formulation and characteristics.

Novel thermomechanical hydrogel scaffolds containing our previously prepared microspheres loaded with bone morphogenetic proteins (BMP) were successfully generated by radical crosslinking and low dose gamma-irradiation from combination of two kind of biomaterials: glycidyl methacrylated dextran (Dex-GMA) and gelatin. The structure of those resulting smart hybrid hydrogels was evaluated by mercury intrusion porosimetry (MIP) and scanning electron microscopy (SEM) analyses, and as a function of the degree of Dex-GMA's substitution (DS), the proportion between Dex-GMA and gelatin, and the initial polyethyleneglycol (PEG) concentration used in the preparation of the hydrogels. The swelling and degradation properties and the temperature-sensitive drug release manner were determined by dynamic evaluation methods in vitro, and the gel content was also calculated. MIP analysis showed that by systematically altering the preparation parameters, the overall networks were clearly macroporous with pore sizes ranging from 5.6+/-4.2 to 37.7+/-13.7 microm. As expected, the pore size decreased as DS and initial PEG concentration increased, whereas the opposite was found for the gel content. Moreover, the porosity values ranged from 73.7+/-12.4% up to 89.6+/-6.3%. The SEM results also showed the inter-connective pores as well as microspheres encased into their porous structure of those hydrogels. The swelling and degradation properties of the resultant hydrogels varied according to the DS of Dex-GMA and initial PEG concentration, while the proportion between Dex-GMA and gelatin had no significant influence on those characterizations. By changing the composition ratio of the two precursors, the phase transition temperature (lower critical solution temperature, LSCT) of the hydrogel scaffolds could also be adjusted to be or near the body temperature, so BMP release from microsphere-hydrogel compounds could be accordingly controlled and the release period could be varied from 18 to more than 28 days. These results demonstrated that a novel temperature-sensitive and biodegradable Dex-GMA/gelatin scaffold containing microspheres loaded with BMP could be successfully developed from both dextran- and gelatin-based biomaterials, which could promisingly satisfy the need, desire, and expectation of both self-regulated drug delivery and tissue-engineering applications.

Enhancement of periodontal tissue regeneration by locally controlled delivery of insulin-like growth factor-I from dextran-co-gelatin microspheres.

The present work focused on the design of novel hydrogel microspheres based on both dextran- and gelatin-derived biomaterials, and discussed whether locally controlled delivery of IGF-I from dextran-co-gelatin hydrogel microspheres (DG-MP) was useful for periodontal regeneration enhancement. Microspheres were synthesized when gelatin was cooperating with glycidyl methacrylate (GMA) derivatized dextrans (Dex-GMA) and the resultant DG-MP with a hydrogel character of which the cross-linking density could be controlled by the degree of substitution (DS, the number of methacrylates per 100 glucopyranose residues) of Dex-GMA. In this study, three types of DG-MP (DG-MP4.7, DG-MP6.3 and DG-MP7.8) obtained from gelatin and Dex-GMA (differing in DS: 4.7, 6.3 and 7.8 respectively) were prepared and characterized by swelling and degradation properties, drug release kinetics and biological capability in promoting tissue regeneration. By swelling in aqueous positively charged IGF-I solutions, the protein could be encapsulated in DG-MP by polyionic complexation with negatively charged acidic gelatin. No obvious influence of Dex-GMA's DS on DG-MP's configuration and size was observed, and the release and degraded properties showed no significant difference between three types of DG-MP in PBS buffer either. However, high DS of Dex-GMA could lower microsphere's swelling, prolong its degraded time and minimize IGF-I burst release markedly in dextranase-containing PBS, where IGF-I release from a slow release type of microspheres (DG-MP7.8) could be maintained more than 28 days, and an effective protein release kinetics without a significant burst but a relevantly constant release after the initial burst was achieved. IGF-I in DG-MP resulted in more new bone formation in the periodontal defects within 4 or 8 weeks than IGF-I in blood clot directly did (P < 0.01). The observed newly formation of periodontal tissues including the height and percentage of new bone and new cementum on the denuded root surfaces of the furcation area in DG-MP7.8 group were more than that in other groups (P < 0.05). The adequate width of regenerative periodontal ligament (PDL), regular Sharpey's fibers and alveolar bone reconstruction could be observed only in DG-MP7.8 group. These combined results demonstrate that effective release kinetics can be realized by adjusting the DS of Dex-GMA and followed cross-linking density of DG-MP, and that locally controlled delivery of IGF-I from slow release type of DG-MP may serve as a novel therapeutic strategy for periodontal tissue regeneration.

Influence of baicalin on the expression of receptor activator of nuclear factor-kappaB ligand in cultured human periodontal ligament cells.

BACKGROUND: Baicalin is a flavonoid purified from the medicinal plant Scutellaria baicalensis Georgi. It has been reported that baicalin exhibits antibacterial, anti-inflammatory and analgesic effects and can inhibit nuclear factor-kappaB activation. Periodontal disease is a chronic infective disease of the periodontium caused by bacteria present in dental plaque inducing alveolar bone resorption until teeth are lost. Human periodontal ligament (HPDL) is the connective tissue between alveolar bone and tooth. Receptor activator of nuclear factor-kappaB ligand (RANKL), a member of the tumor necrosis factor (TNF) ligand family, plays an important role in osteoclastogenesis from osteoclast precursors to mature osteoclasts. In this study we investigate the effects of baicalin on RANKL protein production and messenger RNA (mRNA) expression induced by IL-1beta in cultured HPDL cells. METHODS: To induce RANKL expression, IL-1beta was added to serum-free medium HPDL cells and incubated. Various concentrations of baicalin (0, 0.001, 0.01 and 0.1 microg/ml) were added to the medium and the cells were treated for 0, 12, 24, 48 and 72 h, respectively. RANKL in the cells was detected using immunocytochemistry. The mRNA of RANKL, osteoprotegerin (OPG) and cyclooxygenase-2 (COX-2) were measured by semiquantitative reverse transcription-polymerase chain reaction. RESULTS: The expression of RANKL at mRNA and protein levels in HPDL cells was stimulated by IL-1beta. Baicalin suppressed IL-1beta-induced RANKL and COX-2 production at a concentration of 0.01 microg/ml. The most prominent effect was observed with 48 h of baicalin treatment. The inhibition of baicalin on the rhIL-1beta-stimulated OPG expression was first apparent at 24 h after the start of treatment, however it did not reach significant differences. CONCLUSIONS: The data suggest that baicalin may inhibit RANKL mRNA expression via the suppression of COX-2 expression induced by IL-1beta. In addition to its antibacterial and anti-inflammatory properties, baicalin was shown to be effective in periodontitis and alveolar bone resorption.

Release of bioactive BMP from dextran-derived microspheres: a novel delivery concept.

Recent developments of biotechnology have produced a great variety of protein and bioactive drugs. For these drugs to be used therapeutically, suitable drug delivery systems have become increasingly essential. Dextran-derived biomaterials have been considered to be compatible matrices for protein and bioactive drugs because of their hydrophilic properties and ability to control drug dissolution and permeability. A novel class of dextran-glycidylmethacrylate (Dex-GMA)/poly(ethylene glycol) (PEG) microspheres were designed and synthesized by polymerization of Dex-GMA emulsified in an aqueous PEG solution. Dex-GMA was prepared by substituting the hydroxyl groups in Dex by GMA. The drug loading and in vitro drug release was evaluated by routine procedure and the biological activity of BMP-loaded microspheres was studied by experimental cytology methods. Recombinant human bone morphogenetic protein-2 (rhBMP-2) were entrapped in dextran-derived microspheres quantitatively and with full preservation of their biological activity. In vitro release kinetics indicated that dextran-derived microspheres could retain rhBMP-2 in a variable manner depending on the preparation and degradation of the microspheres. The release profiles of rhBMP-2 from microspheres as a function of time showed that rhBMP-2 releasing kinetics in vitro fitted to first-order and Higuchi equations. The release profile in vitro was in accord with two phases kinetics law and more than 60% drug were released during 20 days. Cytology studies showed rhBMP-2 microspheres have good biological effects on cultured periodontal ligament cells, and could achieve a longer action time than concentration of rhBMP-2 solution. These properties make those microspheres interesting osteo-conductive BMP carriers, allowing to decrease the amount of implanted factor required for tissue regeneration.

[Experimental study on dog's bone marrow stem cells transfected by pIRES2-EGFP-IGF-1 gene].

OBJECTIVE: To establish the bone marrow stem cells (MSC) model which could highly express the insulin-like growth factor 1 (IGF-1) transfected by dog's IGF-1 gene. METHODS: pIRES2-EGFP-IGF-1 was transfected into MSC by lipofectamine. Positive clones were selected with G418. The expression of IGF-1 protein in the MSC was determined by immunohistochemistry and Western blot analysis. The IGF-1 in the supernatant of the transfected MSC was detected by sandwich-in ELISA. The periodontal ligament cells (PDLC) were cultured in the supernatant of the transfected MSC. The changes of PDLC' proliferation were observed by MTT. RESULTS: IGF-1-transfected MSC could apparently express IGF-1. The IGF-1 protein in the supernatant of the transfected MSC was confirmed by sandwich-in ELISA. IGF-1 could promote the PDLC' proliferation. CONCLUSIONS: The MSC transfected by dog's IGF-1 gene can highly express IGF-1, which may lay the foundation for further study on periodontal regeneration.

[Preparation and property of recombinant human bone morphogenetic protein-2 loaded hydrogel nanospheres and their biological effects on the proliferation and differentiation of bone mesenchymal stem cells].

PURPOSE: To prepare and study the recombinant human bone morphogenetic protein-2 loaded dextran-based hydrogel nanospheres (rhBMP(2)-dex-NPs) sustained release system, and to evaluate its biological effects on cultured rabbit bone mesenchymal stem cells(BMSCs). METHODS: The rhBMP(2)-dex-NPs were prepared by improved emulsion polymerization method. Their morphology, size and size distribution, encapsulated ratio and stability were assessed by routine procedure. Dynamic dialysis method was used to determine the release characteristics of rhBMP(2)-dex-NPs in vitro. Cell culture technique and MTT colorimetric assay were used to evaluate the proliferation and differentiation of the BMSCs, ALP kit was used to evaluate the ALP activity of the BMSCs so as to show the differentiation of the cells by adding the rhBMP(2)-dex-NPs to the DMEM culture medium (B group) or rhBMP2 only (A group). Adding dex-NPs without rhBMP2 (C group) and adding nothing (D group) were taken as the controls. The results were analyzed by statistical analysis software (SPSS10.0). RESULTS: The shape of rhBMP(2)-dex-NPs was spherical, with a size distribution of 20 nm. The encapsulated ratio was 83% and rhBMP(2)-dex-NPs could be kept more than 6 months under 4 degrees C without decomposition , destruction or deposition. The release profile in vitro was in accordance with two phases kinetics law, and more than 80% of the encapsulated rhBMP(2) can be released during 12 days. Statistical analysis showed that rhBMP(2)-dex-NPs had biological activity, and could enhance both proliferation and differentiation of rabbit BMSCs significantly, the effect of the rhBMP(2)-dex-NPs was significantly higher than that of rhBMP(2) (P<0.01). During the first 3 days, the proliferation and differentiation of BMSCs between group A and B had no significance (P>0.05), but much faster than group C and D. After 5 to 7 days, rhBMP(2)-dex-NPs could enhance BMSCs proliferation and differentiation continually, but rhBMP2 had no enhancement any more. 7 days later, the difference between group A and B become much more significant (P<0.001). CONCLUSIONS: The rhBMP(2)-dex-NPs can release rhBMP2 more than 12 days and have long-drawn biological effects. To encapsulate rhBMP2 into dextran-based hydrogel nanospheres may be an effective way of growth factor controlled release in tissue engineering.