Influence of transfection with human transforming growth factor-beta1 gene on the osteogenic potential of the cultured human gingival fibroblast / 华西口腔医学杂志

<p><b>OBJECTIVE</b>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).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>The GF transfected with pcDNA3-hTGF-beta1 could express some osteogenic characters, though these characters are restricted.</p>

Effects of gingival fibroblasts transfected with human transform growth factor-beta1 gene on improving the periodontal tissue regeneration / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To evaluate the effects of gingival fibroblasts (GF) transfected with hTGF-beta1 gene on improving the periodontal tissue regeneration for the repair of degree II artificial furcation defects.</p><p><b>METHODS</b>The gingival fibroblasts transfected with hTGF-beta1 gene was compounded to the cuttlebone-transformed nanometer hydroxyapatite (CBHA) material from the cuttlefish in vitro, the degree II furcation defects on the premolars of dogs were produced surgically, and the compound was to implanted into the defect (transfected group), and compared with the compound of periodontal ligament cells (PDLC) with nanometer HA material and the compound of untransfected GF with HA. The results were examined histologically 8 weeks after operation.</p><p><b>RESULTS</b>In the transfected group and the positive control group, more new attachment was found compared with the negative control (P < 0.01), and the NC, NB and NC of the transfected group and the positive control group were: (2.97 +/- 0.50), (4.29 +/- 0.26) and (4.73 +/- 0.06) mm; (3.09 +/- 0.26), (4.46 +/- 0.25) and (4.69 +/- 0.10) mm, respectively. There was no significant difference between the two groups (P > 0.05). Although the alveolar bone regeneration was found in the untransfected group [NB = (3.46 +/- 0.32) mm], the root resorption was observed. The tracing experiment showed that the transfected GF were found in the new alveolar bone and the periodontal membrane.</p><p><b>CONCLUSIONS</b>GF transfected with hTGF-beta1 gene can significantly improve the periodontal tissue regeneration in treatment of degree II furcation defects and is involved in the formation of the new alveolar bone and the new periodontal membrane.</p>

A study on the effect of the chitosan thermosensitive hydrogel loading recombinant human bone morphogenetic protein-2 on repairing periodontal defects / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To observe the effect of the chitosan thermosensitive hydrogel loading recombinant human bone morphogenetic protein-2 (rhBMP-2) on repairing periodontal defects.</p><p><b>METHODS</b>To prepare artificial furcation defects model in the posterior area in 3 healthy male dogs, and then to inject chitosan thermosensitive hydrogel loading of rhBMP-2 after fast suturing tissue flap. The groups filled with nothing or filled only with chitosan thermosensitive hydrogel were the controls. The dogs were sacrificed after 5 weeks and the periodontal regeneration was observed histologically.</p><p><b>RESULTS</b>The histological observation showed that the chitosan thermosensitive hydrogel loading rhBMP-2 group achieved apparent periodontal tissue regeneration occupying the majority of the defects and the control groups got only a small amount of periodontal tissue regeneration.</p><p><b>CONCLUSION</b>The chitosan thermosensitive hydrogel loading rhBMP-2 can effectively promote the periodontal tissue regeneration, while simplifying the surgical operation. It might be a potential means for periodontal regeneration.</p>

An animal experiment for the regeneration of periodontal defect by application of the dual-release chitosan thermosensitive hydrogel system / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To observe the effect of the self-made chitosan thermosensitive hydrogel system with dual-release bone morphogenetic protein and chlorhexidine on periodontal defects repair.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Preparation of functional chitosan thermosensitive hydrogel for slow release both rhBMP-2 and chlorhexidine / 生物工程学报

The chitosan thermosensitive hydrogel is liquid at room temperature but gels rapidly when heated to body temperature. This hydrogel are wildly used for cell encapsulation, drug delivery or tissue-engineered scaffolds. The system can sustain the release of macromolecules over a period of several hours to a few days. However, with low-molecular-weight compounds, the release is generally completed within 24 h. To prepare a functional chitosan thermosensitive hydrogel for slow release both broad-spectrum antibiotic chlorhexidine and growth factor recombined human bone morphogenetic protein-2 (rhBMP-2), The beta-cyclodextrin was used to prepare an inclusion complex with chlorhexidine, and then the latter was incorporated into the chitosan thermosensitive hydrogel system. Simultaneously, rhBMP-2 was added into the hydrogel system. By HAAKE viscosity measuring instrument, we contrasted the viscoelastic properties of system with or without objective factors. And the in vitro release kinetics of chlorhexidine and rhBMP-2 was investigated by HPLC (high performance liquid chromatography) and ELISA (enzyme-linked immunosorbent assay) respectively. The results showed that the addition of chlorhexidine/beta-cyclodextrin inclusion complex to the thermosensitive solution did not change the gelling behavior of the thermosensitive system. Further, the in vitro release profiles demonstrated that the release rate of chlorhexidine and rhBMP-2 from hydrogel became slower, controlled delivery over at least 1 month. By first preparing chlorhexidine/beta-cyclodextrin inclusion complex, and then mixing the IC and rhBMP-2 into the chitosan thermosensitive hydrogel, a functional chitosan thermosensitive hydrogel system with ability of slow release both rhBMP-2 and chlorhexidine is successfully made.

Experimental study on dog's bone marrow stem cells transfected by pIRES2-EGFP-IGF-1 gene / 中华口腔医学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Preparation of enamel matrix proteins controlled release microspheres and their biological effects on the proliferation and differentiation of human periodontal ligament cells in vitro / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To prepare enamel matrix proteins (EMPs) loaded dextran-based hydrogel microspheres (EMPs-dex-MPs), and to evaluate their EMPs controlled release property and their biological effects on the proliferation and differentiation of human periodontal ligament cells (PDLCs) in vitro.</p><p><b>METHODS</b>Using dimethylbenzene as the oil phase, EMPs-dex-MPs were achieved by emulsion-chemical crosslinking technique. The process of the recombination preparation was optimized by orthogonal factorization method. The configuration and size of EMPs-dex-MPs were determined by scanning electron microscope. The EMPs loading content and encapsulation rate of EMPs-dex-MPs, and their biodegradation characteristic were studied by routine analysis methods. Dynamic dialysis method was used to determine the release characteristic of EMPs-dex-MPs in vitro and its influencing factors. The proliferation of cultured PDLCs was measured by MTF method and the differentiation of PDLCs was measured by their alkaline phosphatase (ALP) activities.</p><p><b>RESULTS</b>The results showed that EMPs-dex-MPs were homogenous and stable with the average diameter 25 microm, and the EMPs loading content was (32.8 +/- 1.2)%, the encapsulation rate was (78.9 +/- 1.0)%. Under 9% physiological saline solution contained a very thimbleful quantity of dextranase EMPs-dex-MPs could be biodegraded completely during about 40 days. The in vitro experiments showed that about 80% of EMPs could be released out in 20 days. Using EMPs-dex-MPs could enhance the proliferation responses and ALP activities of PDLCs more than 12 days.</p><p><b>CONCLUSION</b>As a new sustained release system of growth factors, the dex-MPs is stable, workable and biodegradable. EMPs-dex-MPs, whose drug release can be controlled by preparation technique, may be more effective in promoting periodontal tissue regeneration.</p>

Preliminary study on anti-periodontitis immunization with DNA vaccine / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To observe the protection against periodontal bone loss in the Sprague-Dawley (SD) rats periodontitis model, with the recombined plasmid pcDNA3.1+/kgpcd as DNA gene vaccine.</p><p><b>METHODS</b>PcDNA3.1+/kgpcd was delivered into rats by submandibular gland-targeted injection. The anti-KGPcd sIgA in saliva was measured by indirect ELISA method. Immunohistochemistry staining was used to assess the protection in the animal model.</p><p><b>RESULTS</b>The level of specific anti-KGPcd sIgA in saliva of the experimental group was significantly higher than that of control group. HE staining showed that immunization with recombined plasmid pcDNA3.1+/kgpcd could protect or minimize tissue destruction caused by subsequent P. gingivalis challenge in the rat model.</p><p><b>CONCLUSIONS</b>The results indicate that pcDNA3.1+/kgpcd was a good candidate for anti-periodontitis gene vaccine and could provide protection against Porphyromonas gingivalis-caused periodontitis in rat lesion model.</p>

Construction of eukaryotic expression vector for KGPcd gene from Porphyromonas gingivalis and expression in mammalian cells / 华西口腔医学杂志

<p><b>OBJECTIVE</b>This study aimed at constructing secretory eukaryotic expression vector of KGPcd gene encoding whole amino acid residues of mature KGPcd from Porphyromonas gingivalis and investigating the transcription and expression of recombined plasmid VR1020/KGPcd in mammalian cells.</p><p><b>METHODS</b>Eukaryotic expression plasmid VR1020/KCPcd was constructed by using molecular cloning methods. Then, the VR1020/KGPcd was transfected into mammalian cell COS7 with Lipofectamine 2000 according to the manufacturer's instruction. The transcription of VR1020/KGPcd was assayed by reverse transcription polymerase chain reaction (RT-PCR). The expression product of VR1020/KGPcd was analyzed by using indirect immunofluorescence. The protein secretion in cultural medium was detected by ELISA method.</p><p><b>RESULTS</b>It proved that the VR1020/KGPcd could be transcribed and translated into transfected COS7 cells. The expressed targeted protein could be secreted into cultural supernatant and could be detected by ELISA.</p><p><b>CONCLUSION</b>The eukaryotic expression plasmid of VR1020/KGPcd was constructed successfully and its product can be expressed in mammalian cells. The results indicated that the recombinant plasmid has antigenicity and may be acted as candidate gene vaccine. This laid a basis for its use as gene vaccine candidates in the development of anti-periodontitis and paved the way for further study.</p>

A study on the effects of cells and scaffolds tissue engineering on the periodontal regeneration / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To observe the effects of cells and scaffolds tissue engineering on the periodontal regeneration, and to evaluate the feasibility of nano-Hap-collagen (nHAC) as the scaffold material for periodontal tissue engineering.</p><p><b>METHODS</b>Dog autogenous periodontal ligament cells (PDLCs) cultured in vitro were collected and seeded on the three-dimensional framework of nHAC. The cell growth in the scaffolds was observed by scanning electron microscope. And then the PDLCs-nHAC composites were transplanted into man-made periodontal defects, and the groups filled with nothing or filled only with nHAC were the controls. The dogs were sacrificed after 8 weeks and the periodontal regeneration was observed histologically.</p><p><b>RESULTS</b>Scanning electron microscope showed the porous structure of nHAC and the eugonic growth of cells in the nHAC scaffolds. The histological observation showed that the PDLCs-nHAC groups exhibited more new bone, new periodontal ligament and new cementum occupying the majority of the defects than the control groups, and the epitheliums were not observed.</p><p><b>CONCLUSIONS</b>Periodontal regeneration could be enhanced by the cells and scaffolds tissue engineering, and the PDLCs and nHAC could be used as the seed cell and the scaffold material for periodontal tissue engineering.</p>

CLONING AND SEQUENCING ANALYSIS OF GINGIPAIN K OF PORPHYROMONAS GINGIVALIS / 微生物学通报

The desired DNA product of KGPcd and KGP-hag was obtained from the total DNA of Porphyromonas gingivalis by PCR with two pairs of gene specific primers. The segment of KGPcd and KGP-hag (about 1.5kb and 1.6kb) was inserted into pGEM-T easy Vector. The double-stranded DNA of the postitive clone was analyzed by restriction endonuclease mapping and DNA sequenceing. The sequences of KGPcd and KGP-hag were consistent with those of the references appeared. The proteins of KGPcd and KGP-hag will be obtained for further study.