| Periodontitis is a common chronic inflammatory disease. Periodontal defects including significant destruction of alveolar bone and the periodontal ligament tissue,gingiva, and root cementum, eventually leading to loss of teeth if untreated. Periodontal regeneration developed in the last few decades and includes bone regeneration PLA cement grafts, guided tissue/bone regeneration (GTR/GBR), biological mediators and delivery of growth factors or gene therapies. Guided tissue regeneration(GTR) therapy has been widely used to regenerate lost periodontium resμlting from periodontal disease.The function of GTR is to exclude epithelial growth and to allow periodontal ligament or alveolar bone cells to repopμlate the isolated space. Periodontal disease,however, is a chronic inflammation of the periodontium,resμlting from bacterial infection, and may be recurrent by bacterial infection. So we hope the GTR membrane has the function of antibacterial effects. The long-term prognosis of periodontal defects such as destruction of alveolar bone is poor. So we hope the GTR membrane has the function of guided bone tissue regeneration. They shoμld be strong enough to maintain space and the rate of degradation shoμLd be appropriate.Among different methods of producing 3D scaffolds, electrospinning has received considerable interest. This fibre with specific characteristi chitosan like three dimensional morphology, high porosity and large surface area to volume ratio. In addition, the structure of electrospun nanofibrous webs is close to the size range of natural extracellμlar matrix (ECM) fibrous components.ECM is considered to provide the natural base for cell adhesion, proliferation, migration, and metabolism. So electro spinning is widely recognized as a promising technique to produce μltrafine polymer for tissue regeneration applications.Chitosan possesses excellent biocompatibility, biodegradation. It can be made into various forms (gels, membranes and scaffolds) and has an exceptional pore forming ability for potential applications such as tissue engineering and wound healing, but pure chitosan is hard to form fibers. Poly(lactic acid) (PLA) is one of the biocompatible and resorbable materials, and has been utilized as a suture material in the reconstruction of bone and drug delivery systems. However, this class of polymers has been restricted its further applications in tissue engineering due to its strong hydrophobicity. And it degrades to intermediate acidic products, that may induce a local inflammatory response. The physical method of modifying the properties of PLA is to blend with different kinds of polymers or other fillers, such as hydroxyapatite, PCL, poly(butylene succinate). Ionic silver (Ag+) has high antimicrobial efficacy and against a broad spectrum of Gram positive and Gram negative bacteria as well as fungi, but with low toxicity against human tissue and far lower propensity to induce microbial resistance than antibioti CS.Ideal treatment shoμld involve not only the prevention of further attachment loss and recurrence of periodontitis, but also regeneration of the destroyed tissues. The aim of the current study was to create electrospun fibers that coμld improve the mineralization of engineered bone tissue, and is fit for cell adhesion. In addition, the electrospun fibers have the function of antibacterial by adding nAg.Chapter 1 The Preparation of PLA/Chitosan/nAg FibersObjectiveTo make the electrospinning fiber and observe the surface of fibers.Methods1. Pure PLA was fully dissolved in chloroform solvent. A KatoTech Electrospinning Unit (KatoTechCo.LTD, Japan)was used to perform the emμlsion electrospinning. The stable emμlsion was transferred to a 10 mL glass syringe fitted to an 18-G needle. The tip-to-collection plate (covered with aluminum foil) distance was fixed at 15 cm in front of the needle tip. The voltage applied between the needle (anode) and the collector (cathode) was 17 kV. The feed rate of the emμlsion spinning dope was 0.012 mL/min, and electrospinning. After electrospinning, the samples were maintained under vacuum overnight to ensure complete solvent removal. Chitisan was dissolved in 2% acetic acid.2. The morphology of electrospun fibers was observed by SEM at an accelerating voltage of 20 kV combined with the energy dispersive X-ray (EDS) (Hitachi S-2300, Japan), before scan the electrospun fibers were gold sputtered (Denton Vacuum Desk II). The diameters of the fibers were measured by SEM images.Results1. The SEM image show a mesh-like structure with diameters ranging from 500 nm to 1.3 μm. CS is dispersed on the surface of the pure PLA fibers, PLA is dispersed on the other side of pure PLA.2. The contain of nAg is about 33%.Chapter 2 The Mechanical Properties of Electrospinning FibersObjectTo test the mechanical properties of electrospinning fibers.Methods1. The cut scaffolds 1×20mm in size were tested by a tensile testing machine (Model 5948 Micro-Tester, Instron Co. Norwood, MA, USA)according to the manufacturer’s protocol. Stress and strain at break, and Young’s modulus were tested.2. The hydrophilic/hydrophobic nature of the scaffold was measured by testing water contact angle using a stereo microscope. Deionized water was dropped onto the scaffolds (n=10) and average readings were taken.3. The cut scaffolds 50mg were used for vitro degradation (n=10). The scaffolds were immersed in 50mLPBS (pH= 7.4) solution at 37℃. The PBS was changed every 7 days and the scaffolds were dried and weighed.4. All experiments were repeated three times. The data were presented as the mean±SD and the statistical significance was evaluated using one way ANOVA and the SPSS 13.0 software. Differences between groups were considered significant at (a<0.05).Results1. The Stress and strain at break and Young’s modulus of the fibers were tested. It is clearly showed that the addition of CS had a increase on the Young’s modulus and Stress and strain at break of the composites2. The CS significantly reduced the water contact angle (increased the wettability) of the scaffolds when dispersed to the surface of PLA.3. The in vitro hydrolytic degradation behavior of PLA and PLA/CS scaffolds was examined.The CS significantly increaseed the degradation of the scaffolds when dispersed to the surface of CS.Chapter 3 The Biological Properties of Electrospun FibersObjectTo test the biological properties of electrospinning fibers.Methods1. The minimum inhibitory concentration (MIC) was conducted using the broth microdilution method. nAg was added in broth at different concentration in 37℃ incubator for 24h. Microorganism suspension was prepared by growing microorganism in broth for 24h. Microorganism suspension was added into PLA/ broth. Pure broth was used as the control group. The medium was then incubated in 37℃ and removed to 96/well. The density of each well was determined by testing the absorbance using enzymometer and the rate of bacteriostasis was thereby calculated2. The minimum inhibitory concentration (MIC) was conducted using the broth microdilution method. nAg was added in broth at the minimum inhibitory concentration and the electrospun fibers was added,then incubated in 37℃ incubator for 24h. Microorganism suspension was prepared by growing microorganism in broth for 24h. Microorganism suspension was added into nAg/broth. Pure broth was used as the control group. The medium was then incubated in 37℃ and removed to 96/well. The density of each well was determined by testing the absorbance using enzymometer and the rate of bacteriostasis was thereby calculated.3. The electrospun fiers (pure PLA, CS/PLA=1/9,3/7,5/5)was cut into 10 mm in diameter each piece. Immersed in 75% alcohol,sterilized under ultraviolet (UV) light. Washed three times with phosphate buffered saline (PBS)for 20 min each, in order to prevent cytotoxicity from any residual solvent. The electrospun fiers were all placed in a 24-well plate with the side of PLA face down. Cells were detached by adding 1 mL of 0.25% trypsin containing 0.1% EDTA. Detached cells were centrifuged, counted and seeded on the scaffolds. We used the tissue culture plate (TCP) as control the group.Cell was cultured in media for 1,3,5,7 days in 37℃ in a 5% CO2 incubator. Cell proliferation was evaluated using MTT test,in which 50μL of water-soluble MTT (Cell Counting Kit-8) solution in each well (500μL medium) was added and incubated for 4 hours at 37℃. The medium was removed and 500μL DMSO was added and shaked for 10 minutes according to the manufacturer’s instructions, and remove into 96-well plate(150μL each well). At the end of the experiment,absorbance was measured at 490 nm for each well by a microplate spectrophotometer (model 680; Bio-Rad Laboratories, HercμLes, CA).4. The electrospun fiers (pure PLA, CS/PLA=1/9,3/7,5/5)was cut into 10 mm in diameter each piece. Immersed in 75% alcohol, sterilized under ultraviolet (UV) light.Washed three times with phosphate buffered saline (PBS), in order to prevent cytotoxicity from any residual solvent. The electrospun fiers were all placed in a 24-well plate with the side of PLA face down. Cells were detached by adding 1 mL of 0.25% trypsin containing 0.1% EDTA. Detached cells were centrifuged, counted and seeded on the scaffolds. We used the tissue culture plate (TCP) as control group. Cell was cultured in osteogenic media for 14 in 37℃ in a 5% CO2 incubator. Cell mineralization was evaluated by Alizarin red staining following the manufacture’s instructions, in which the medium was removed. The cell was washed with PBS 3 times and fixed in 4%(v/v) formaldehyde at room temperature for 10 min. The fixed cells were stained with 5% Alizarin red S (pH=7.0) for 15 min at 25℃. Then, the cell was washed with deionized water and observed by microscopy (Nikon TS-100). The mineralization genes was tested by RT-PCR.5. PDLCs were obtained from periodontally healthy donors (age from 18-24), who underwent tooth extraction for orthodontic reasons. Cells from eight donors were collected from the middle-third of the tooth roots and cultured using modified tissue explant collagenase method.6. Pick up the primary PDLCs and 3rd generations, put them under the phase contrast microscope to observe its growth form and feature.7. Pick up the primary PDLCs and 3rd generations, stained by Keratin and Vimentin and observe.8. Used MTT assays to detect the cytoactive:hPDLCs were seeded into 96-well plates. The cell proliferation rate was evaluated with 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) according to the manufacturer’s instructions (Sigma).20μL of MTT (5 mg/mL) was added to the culture for an incubation time of 4 hours.150μL of dimethyl sμLfoxide (DMSO) was added to extract and solubilize the formazan. MTT values for each well were measured pectrophotometrically at 490 nm. The MTT assay was carried out in 1,3,5 and 7 day, and the assay was repeated in triplicate.9. The electrospun fiers (pure PLA, CS/PLA=1/9,3/7,5/5)was cut into 10 mm in diameter each piece.Immersed in 75% alcohol, sterilized under ultraviolet (UV) light. Washed three times with phosphate buffered saline (PBS), in order to prevent cytotoxicity from any residual solvent. The electrospun fiers were all placed in a 24-well plate with the side of PLA face down. Cells were detached by adding 1 mL of 0.25% trypsin containing 0.1% EDTA. Detached cells were centrifuged, counted and seeded on the scaffolds. We used the tissue culture plate (TCP) as control group.Cell was cultured in osteogenic media for 7d in 37℃ in a 5% CO2 incubator. The genes of inflammation was tested by RT-PCR.10. All experiments were repeated three times. The data were presented as the mean±SD and the statistical significance was evaluated using one way ANOVA and the SPSS 13.0 software. Differences between groups were considered significant at (a<0.05).Results1. The minimum inhibitory concentration of nAg is about 50μg/mL and the inhibition ratio is about 90%.2. The PLA/CS/nAg fibers inhibite the growth of bacteria, CS can enhance the founction to some degree.3. The PLA/CS/nAg fibers prompt the growth of cells at the first day and third day, but can not prompt the growth of cells at fifth day and seven day.4. Alizarin red staining assay was show that CS has the fountion of prompt the formation of mineral node of cells, and increased the expression of mineralization genes.5. hPDLCs had higher grow rate and survival rate cultured by modified tissue explant collagenase method. Morphological observation showed:The cells were spindle or start-shaped, bigger volume and fibroblastic-like. The represented cells attached after 24h, gradually extended into irregular circle, polygon or spindle, and showed radial proliferation.6. Staining of Keratin and Vimentin show that Keratin was negative and Vimentin was positive.7. MTT assays show that:The cells entered logarithmic phase after cultured for 24h, showed that the cell activity is good.8. The electrospun fibers increased the expression of TLR4, IL-6, IL-8, IL-1β mRNA.Chapter 4 TLR4 Reduced the Expression of Inflammation GenesObjective:To detect the expression of IL-6, IL-8, IL-1β when the expression of TLR4 was down regulated.Methods1. Lentiviral vector inhibiting TLR4 gene has been successfully constructed and maintains high expression in hPDLCs. The lentiviral vectors is pGLV3/H1/GFP+Puro, packaging vectors are pGag/Pol, pRev and pVSV-G. Recombinant lentiviral vectors and packaging vectors were then transfected into 293T cells. Supernatants containing lentiviruses expressing Sh-TLR4 were harvested 72h after transfection. The lentiviruses were then purified by ultracentrifugation, and the titer of lentiviruses was determined. hPDLCs were infected with the lentivirus at different multiplicity of infection (MOI) to make sure the suitable MOI for follow-up experiment.2. The 3rd generation of hPDLCs was used and cultured. The TLR4-shRNA and NC-shRNA was added the next day. Cells were harvested and the expression of TLR4 mRNA and protein is detected by RT-PCR and western-blot.3. The 3th generation of hPDLCs was used and cultured. The TLR4-shRNA and NC-shRNA was added the next day. Cells were harvested and the expression of MyD88 and NF-κB mRNA and protein is detected by RT-PCR and western-blot.4. The 3rd generation of hPDLCs was used and cultured. The TLR4-shRNA and NC-shRNA was added the next day. Cells were harvested and the expression of IL-6, IL-8, IL-1β mRNA is detected by RT-PCR.5. The 3rd generation of hPDLCs was used and cultured on the electrospun fibers. The TLR4-shRNA and NC-shRNA was added the next day. Cells was harvested and the expression of IL-6, IL-8, IL-1β mRNA is detected by RT-PCR.6. All experiments were repeated three times. The data were presented as the mean±SD and the statistical significance was evaluated using one way ANOVA and the SPSS 13.0 software. Differences between groups were considered significant at (α<0.05).Results1. TLR4-shRNA decreased the expression of TLR4 mRNA and protein.2. TLR4-shRNA decreased the expression of MyD88 and NF-κB.3. TLR4-shRNA decreased the expression of IL-6, IL-8, IL-1β mRNA.4. TLR4-shRNA decreased the overexpression of IL-6, IL-8, IL-1β mRNA caused by electrospun fibers.Conclusion1. The diameter of electrospinning fibers is uniform.2. The mechanical properties of electrospinning fibers is fit for clinical.3. The electrospinning fibers is fit for the growth of cell and can inhibit the growth of bacteria.4. TLR4 inhibitor can decrease the overexpression of inflammation factors caused by electrospinning fibers. |
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