| BackgroundPeriodontitis is a chronic disease that affects the integrity of the periodontal system and leads to damage of the periodontal tissues and, ultimately, tooth loss .There are thousands of researches done to explore the solutions of periodontitis from the autogenous bone graft materials to the guide tissue/bone regeneration (GTR/GBR).But the best materials for the regeneration of the lost periodontal tissues are still not found or made now. Autogeneous bone obtained is limited and the allograft and xenograft material for the recipient are easily to cause the immunological rejection. The materials of membranes as a barrier for the GTR/GBR commercially available are mainly collagen. Collagen owns the well biocompatibility, due to one main component of ECM. However, the fast resorption rate and poor mechanical strength lead to a scruple, when the collagen membrane is used.Poly(lactic-co-glycolic acid)(PLGA), is one of the Food and Drug Administration(FDA) approved synthetic polymers and one of the most extensively applied in Pharmaceutics and tissue engineering. PLGA owns excellent biocompatibility, tunable mechanical property and controllable degradation rate which can be readily achieved by varying the copolymer ratio of lactic to glycolic. Chitosan (CS), a natural cationic polymer, is the alkaline decetylated product of chitin and similar in structure to glycosaminoglycan in ECM. CS is widely employed in biomedicine and industry fields, owing to its favorable biological characteristics, such as biodegradability, biocompatibility, non-toxicity, relative hydrophilicity, haemostatic and antibacterial activity.CS is approved by the FDA for use in wound dressings because of the aforementioned remarkable properties.The material that perfectly meets the needs of periodontal tissue regeneration may be not existent, so many research groups have tried to design and develop GTR/GBR periodontal membranes with the requisite features and properties by mixing different materials characterized by different properties,such as combining natural polymers with inorganic materials or synthetic polymers. In general, synthetic polymers provide suitable mechanical and biological properties but lack biocompatibility. Nature polymers offer favorable hydrophilicity and biocompatibility but lack necessary mechanical characteristics. So the studies had put emphasis on the combination of synthetic and nature polymers in the formation of scaffolds. Meng et.al and Li et.al confirmed that the hydrophilicity of scaffold was enhanced with the addition of chitosan. The water contact angle of electrospun PLGA/chitosan nanofibrous scaffold decreased 45.5°compared with the PLGA scaffold. The electrospun chitosan-graft-PLGA nanofibres fabricated by Li et.al not only possessed the improved hydrophilicity, but also maintained the good mechanical property.Dental plaque biofilm is the initial factor of the cause of periodontal disease. The key of periodontal disease successful therapy is good control of the dental plaque. The antibiotics can play an important role in inhibit the bacteria, but they also lead to the emergence of drug-resistance bacteria. Nano-Ag(nAg) is one of the most extensively applied antibacterial materials, due to its broad spectrum of antibacterial activity. Besides antibacterial property, nAg also possesses anti-fungal activity, anti-inflammatory effect, anti-viral activity and anti-cancer effect. Owing to its unique characteristics, nAg has been widely used in medicine dressing, implants, dental materials, surgical masks, water filtration, personal care items, and so on Sofi et.al reported that nAg as an antibacterial agent for endodontic infections could significantly reduce the adherence of Enterococcus faecalis to nAg-treated dentin. The materials must mimic the ultrastructure of the defective tissues as realistic as possible in order to enhance the success rate of therapy. Nanomaterials are able to better mimic the nanostructure in extracellular matrix . PLGA and CS can be easily made into nanoparticles by various methods. Due to their unique physicochemical, nPLGA and nCS have been widely used as drug delivery system to reach release control the drug or not be absorbed in Pharmaceutics and medicine.ObjectiveThe present study aims to prepare the PLGA nanoparticles (nPLGA) and CS nanoparticles (nCS) by an emulsion solvent evaporation method and ionic gelation method, respectively.The cytotoxicity and proliferation of nanoparticles (nPLGA, nCS and nAg) for human periodontal ligament cells (hPDLCs) are measured by MTT assay. Alizarin Red S staining and real-time quantitative PCR (RT-qPCR) analysis are performed for the evaluation of osteogenic differentiation. The antibacterial activity is conducted to make sure whether nPLGA will affect the antibacterial effect of nAg.Materials and MethodsChapter I Preparation and characteration of nPLGA and nCSMethods1. PLGA nanoparticles preparation:PLGA nanoparticles(nPLGA) were prepared by a modified oil-in-water emulsion solvent evaporation method.100mg PLGA were dissolved in 10ml acetone,then slowly poured into 40ml 2% polyvinyl alcohol (PVA) stirred on a magnetic stirrer at room temperature. The mixture was continuously stirred at 800rpm for 8hours and the organic solvent was evaporated by stirring.The nanoparticles were obtained by centrifugation at 15,00rpm for 20minutes,wased three times by deionized water and dried naturally.The nPLGA were sterilized by γ-rays and stored before further use.2. Chitosan nanoparticles preparation:Chitosan nanoparticles(nCS) were prerared by ionic gelation method via the interaction with sodium tripolyphosphate (TPP) polyanion. 40mg chitosan powder was dissolved in 40ml of 1% glacial acetic acid under the stirring.Then 10M-NaOH was added into the solution to adjust the pH value to 4.6-6.0.10ml of 0.1%TPP solution was dropped into the chitosan solution one drop about 15s stirred at 1,000rpm by the magnetic stirrer,and the nanoparticles were formed spontaneously. The nanoparticles were collected by centrifugation at 15,000rpm for 20 minutes.The supernatant was discarded, and the nCS were rinsed with deionized water three times and then dried naturally. The nCS were sterilized by γ-rays and stored before further use.3.Characterization:Particles size, polydispersity index(PDI) and zeta potential were measured by a Malvern Zetasizer 3000HS.After the nanoparticles were formed,10ml mixture was moved into a glass bottle. The samples were diluted to a suitable density when measured. The morphology of nanoparticles was analyzed by transmission electron microscopy(TEM).A drop of nanoparticle suspension was placed on a copper grid, the excess liquid was sop up by filter paper, and the grid was dried at ambient temperature.Chapter Ⅱ Biological characteristics of nPLGA、nCS and nAgMethods1.The isolation and culture of hPDLCs:Human Periodontal ligament cells(hPDLCs) were obtained from healthy teeth (11-24 years old) extracted because of impaction or the need for orthodontics at the Department of Oral and Maxillofacial Surgery, Nanfang Hospital, Guangzhou, China, following a protocol approved by the Medical Ethics Committee of Nanfang Hospital. After extraction the teeth were washed at least three times in PBS with penicillin/streptomycin. Periodontal ligament tissue was scraped from the middle third of the root with an aseptic scalpel blade. Tissue transferred to al.5ml eppendorf (EP) tube was minced and washed three times with PBS, and then digested in a solution of 3 mg/ml collagenase type I for 10 minutes at 37℃. The supernatant was discarded, lml complete medium was added which was filled with Dulbecco modified Eagle medium (DMEM) containing 10%fetal bovine serum(FBS),100U/ml penicillin,100 mg/ml streptomycin, and 50mg/ml ascorbic acid. A 24mm X 24 mm coverslip was covered on the tissues flattened on a 35mm petri dish.1.5ml complete medium was added to the petri dish. Cells were cultured at 37℃ with 5% CO2 and the medium was replaced every 3days.Upon reaching 80% confluency the cells were passaged using trypsin/EDTA(0.25%w/v crude trypsin and 1mM EDTA) and passage 3-5 was used in this experiment.2. Cytotoxicity and proliferation of nPLGA, nCS and nAg for hPDLCs:The suitable medium was added into 4ml tube containing PLGA, nPLGA, CS, nCS and nAg to respectively prepare lmg/ml PLGA,lmg/ml and 2mg/ml nPLGA, lmg/mlCS, lmg/ml and 2mg/ml nCS and 100μg/ml nAg suspensions. nPLGA group and nCS group were added 100μl material suspension into corresponding wells and the final concentration of nAg group in wells was adjusted to2μg/ml,10μg/ml,20μg/ml, 30μg/ml,40μg/ml,50μg/ml. HPDLCs were seeded into 96-well plates at a density of 3-5 ×103 cells/well and treated with aforementioned suspensions. The cytotoxicity and proliferation assay was evaluated with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT). The cytotoxicity and proliferation assay was respectively conducted on days 1,2,3 and 1,2,3,5,7. On the day of test, every well was added 20μl MTT solution at the concentration of 5mg/ml and incubated for 4h at 37 ℃ in humid environment with 5% CO2. After the incubation period, the solution was removed gently followed by the addition of 150μl dimethylsulfoxide (DMSO). The plate was vibrated for 10 minutes before measurement.The absorbance was read at 490nm using a SpectraMax M5. Relative growth rate(RGR) was used to represent cytotoxicity(Table 1). The experiment was repeated in triplicate. RGR=×100% where A was the negative control only containing cells; B was the experiment group containing different materials.3. Alizarin Red S staining:HPDLCs were seeded into 6-well plates at a density of 1-5 ×105 cells/well with 1.5ml complete culture medium, lml of the above suspensions, besides nAg, was added into 6-well plates, when the cell confluency reached 80%. After 3days, hPDLCs were induced by mineralized solution supplemented with 10mmol/1β-glycerol phosphate,50μl/ml ascorbic acid, and 10-7mol/l dexamethasone in complete medium for 3 weeks. The induced cells were washed three times in PBS and fixed in methanol for 10 minutes at room temperature. The cells were washed three times with PBS and stained with 1%Alizarin Red S(ARS) for 10minutes. After several washes with PBS, the cells were observed under optical microscope.4. Real-time quantitative PCR (RT-qPCR) analysis for gene expression:At day 21, the total RNA was isolated from hPDLCs using TRIzol according to the manufacturer’s instructions. Equivalent amount of RNA samples was reverse transcribed for first strand cDNA synthesis using the PrimeScript(?) RT reagent kit. RT-qPCR were performed on a Light Cycle 480Ⅱ using SYBR Premix DimerEraser kit. The relative expression of the genes was normalized against the housekeeping gene β-actin. All samples were assayed in triplicate and the experiment was performed three times. The cycle threshold(Ct) value of each target gene was normalized against the Ct value of P-actin. The relative expression of the target gene was calculated using the formula 2-. Primer sequences for osteocalcin(OCN), alkaline phosphatase(ALP), osteopntin(OPN) and p-actin were as follows.5. Antibacterial studies:A certain amount of Luria Broth (LB) was added into 2ml EP tubes containing materials to respectively prepare 5mg/ml, lmg/ml of CS and nCS,lmg/ml of nAg, and 5mg/ml,10mg/ml,15mg/ml and 20mg/ml of PLGA and nPLGA suspensions. When tested, CS and nCS suspensions was diluted to the final concentrations (800μg/ml,lmg/ml,2mg/ml and 5mg/ml). nAg suspensions was diluted to the concentrations of 400μg/ml,600μg/ml,800μg/ml and lmg/ml. Gram negative bacterium Escherichia coli (E.coli) was used for checking the antibacterial properties of the materials and the effectiveness was evaluated by plate colony-counting methods.1 μl E.coli suspension was dropped into 50μl different concentrations of materials suspensions, which was shook for 12h in the constant temperature incubator shaker. The suspensions was appropriately diluted, 1μl bacterial solution was transferred to agar plate and incubated for 24h at 37℃ to count colonies. Every concentration was performed three plates and this experiment repeated five times.Chapter Ⅲ Preparation and biological characterization of the mixture of nPLGA、nCS and nAgMethods1.Cytotoxicity and proliferation of the mixture of nPLGA/nCS for hPDLCs: According to above assay, suitable concentration of each material was chose to conduct this part. nPLGA and nCS were added together to 96-well plate at the ratio of 9:1,8:2,7:3. MTT assay was used to evaluate the cytotoxicity and proliferation of hPDLCs.2. Alizarin Red S staining of the mixture of nPLGA/nCS for hPDLCs lml of nPLGA and nCS was added together to 6-well plate at the ratio of 9:1,8:2,7:3. hPDLCs were induced by mineralized solution 21days.Well without materials was negative control. Every group was repeated three wells. When the time was over, the wells were stained by ARS and observed the developed mineralized nodule under optical microscope.3. Real-time quantitative PCR (RT-qPCR) analysis:The effect of the mixture on hPDLCs osteogenic differentiation was assessed by RT-qPCR to measure the mRNA expression of OCN, ALP, OPN. Total RNA was extracted using TRIzol reagent. Complementary DNA was synthesized from 500ug of total RNA following the manufacturer’s protocol.4. The effect of mixture of nPLGA/nCS/nAg on hPDLCs:According to above assays, the best ratio of nPLGA and nCS and the suitable concentration of nAg were chose. 100ul of the mixture of nPLGA and nCS was added. The suitable volume of nAg was added to reach the chosen concentration. After 1,2,3,5 and 7 days of the materials suspensions added, MTT assay was conducted. When the cells were cultured for 21 days by mineral solution, the cells were stained by ARS and the total RNA was extracted.5.The antibacterial activity of the mixture of nPLGA/nCS/nAg:To evaluate the influence of nPLGA for the antibacterial of nCS and nAg,nPLGA was mixed with the same amount of nCS and nAg. The concentration was set 800μg/ml. The experiment was divided into negative control, nCS, nAg, nAg/nCS, nAg/nPLGA, nCS/nPLGA, nAg/nCS/nPLGA groups. Other operations was the same with previous processes.Statistical analysisAll quantitative data were expressed as the mean ±standard deviation. Statistical analysis was performed using one-way ANOVA. All statistical tests were analyzed with SPSS 20.0. A value of p<0.05 was considered statistically significant for this study.Result1.Characterization of the nPLGA, nCS:The mean diameter of PLGA nanoparticles we obtained is 112.4±8.33nm and the PDI is 0.13±0.05. Zeta potential values of the nPLGA were found to be-25.4±2.6mv.TEM showed that nPLGA is spherical and regular shape.The mean diameter and PDI of nCS are 180.3±11.2nm and 0.37±0.07 measured by Malvern Zetasizer. Zeta potential values for nCS were found to be +34.0±0.8mV. The nCS is aspheric and the particles edge is clear.2. Cytotoxicity and proliferation of nPLGA, nCS and nAg for hPDLCs:The results of toxicity grading are 0 or 1,which can be considered to be nontoxicity according to ISO 10993-5. The toxicity grading shows different structures or different concentration of a kind of structure or material are all nontoxicity in the experiment. nPLGA or nCS possess the same cytocompatibility with PLGA and CS. With the concentration increased, nanoparticles still keep favorable cytocompatibility. nAg also maintains the good cytocompatibility with theconcentration and the time increased.The cell numbers were increased with the culture periods extended for the three nanoparticles. The proliferation of cells is not affected by the structure and the concentration of materials. The proliferation of cells for nAg group is not different from that in negative control group three days before.The proliferation of cells for 40μg/ml and 50μg/ml nAg is much slower than the control group on the fifth day and seventh day.3. Alizarin Red S staining:More minerlized nodules were observed on the wells with materials than that on negative control group. The number of nodules was not significantly different for the different structure or concentration of the same material.4. Real-time quantitative PCR (RT-qPCR) analysis:The mRNA relative expression of OCN and OPN for lmg/ml nPLGA and 2mg/ml nPLGA was not significantly different from the negative control group(p>0.05), while that of ALP was significantly difference with the negative control group (p<0.05). The relative expression of ALP in lmg/ml nPLGA and 2mg/ml nPLGA groups was much higher than the control group. The mRNA relative expression of OCN and OPN in hPDLCs with lmg/ml PLGA was higher than the control group (p<0.05), and the expression of OCN in PLGA group was also different from the InPLGA and 2nPLGA group (p<0.05). The expression of OPN in PLGA group was significantly difference with lmg/ml nPLGA group. For the relative expression of ALP, PLGA group was much less than lmg/ml nPLGA group. For the expression of OCN, ALP and OPN, there was not significantly difference between lmg/ml nPLGA and 2mg/ml nPLGA groups.Cells cultured with lmg/ml CS was the highest in the expression of OCN and OPN, and was significantly difference with the control group(p<0.05). For the expression of OCN,2mg/ml nCS was less than CS and there was distinction between the two groups(p<0.05). The CS group was statistically difference with lmg/ml nCS and 2mg/ml nCS in the relative expression of OPN(p<0.05).5. Antibacterial studies:The antibacterial ratio was gradually increased with the increase of the concentration of materials. The inhibitory rate of nAg at 400μg/ml was 64.1±10.0%. When the concentration was up to lmg/ml, the inhibitory rate reached 91.9±10.2%. The antibacterial ratio of nCS was higher than CS at the same concentration. The antibacterial ratio of nCS at 800μg/ml was 87.7 ±2.5%, while that of CS was 49.1±18.3%. When the concentration is lower, the inhibitory rate between nCS and CS is significantly different. When the concentration was 5mg/ml, the antibacterial ratio of nCS and CS was respectively 90.7±14.0% and 100.0±0.1%. PLGA and nPLGA from 5mg/ml to 20mg/ml were found no antibacterial activity.The adding of nPLGA brings no influence on the antibacterial ratio of nCS and nAg against E.coli (p>0.05).6. Cytotoxicity and proliferation of the mixture of nPLGA/nCS for hPDLCs:The mixture in different ratio owns preferable cytocompatibility. RGR values were all more than 90% in the three days. According to ISO 10993-5, the mixture of different ratio of nPLGA and nCS was not toxic.The proliferation of hPDLCs cultured with different ratio mixture was not different from negative control group(p>0.05) and no difference found among the different ratio mixture(p>0.05).7. Alizarin Red S staining of the mixture of nPLGA/nCS for hPDLCs:hPDLCs with the mixture of nPLGA and nCS induced by mineralized solution were stained by ARS on the twenty-first day. The obviously difference cannot be found.8. Real-time quantitative PCR (RT-qPCR) analysis:The relative expression of the above-mentioned genes was gradually increased with the increase of nCS. When the ratio is 9:1, the expression of OCN and ALP was significantly different from negative control group(p<0.01). For the expression of ALP, there was no difference found between the control group and 8:2 group,7:3group. For the 8:2 group, the expression of OCN was not found different from control group (p>0.05), and the expression of OPN was difference with control group (p<0.05).The 7:3 group was the highest for the expression of OCN and OPN. The 7:3 group was higher and significantly different from the control group for the expression of OCN (p<0.05).The expression of OPN in the 7:3 group was about 1.5 times that in the control group (p<0.01).9. The effect of mixture of nPLGA/nCS/nAg on hPDLCs:The RGR values of nPLGA/nCS/nAg and PLGA/CS/nAg for hPDLCs were all more than 90%. nPLGA/nCS/nAg and PLGA/CS/nAg were non-toxic for hPDLCs, according to ISO10993-5. The number of hPDLCs in three groups was more and more with the culture time increased.The proliferation of hPDLCs treated with nPLGA/nCS/nAg and PLGA/CS/nAg was not different from the negative control group(p>0.05). There was also no difference between nPLGA/nCS/nAg group and PLGA/CS/nAg group (p>0.05).The colour and number of mineralized nodule of Alizarin Red S staining for nPLGA/nCS/nAg and PLGA/CS/nAg was deeper and more than the negative control group.There was no significantly difference between nPLGA/nCS/nAg group and PLGA/CS/nAg group.The relative expression of OCN and OPN in nPLGA/nCS/nAg group and PLGA/CS/nAg group was evidently higher than the negative control (p<0.05).The expression of ALP in PLGA/CS/nAg group was slightly higher than the negative control group,but there was no difference between groups(p>0.05). The expression of OCN、ALP and OPN between nPLGA/nCS/nAg group and PLGA/CS/nAg group was no significantly difference(p>0.05).The antibacterial test shows that nPLGA was no influence on the antibacterial activity of nCS and nAg for E.coli. There was no found synergistic effect between nCS and nAg, when the antibacterial concentration was 800μg/ml.Conclution1. nPLGA and nCS were successfully obtained.The nanoparticles were regular in shape,uniform in size and clear.2. Like PLGA and CS, nPLGA and nCS had good biocompatibility.nPLGA and nCS were no toxic for hPDLCs and had no effect on the proliferation of hPDLCs.nAg was also nontoxic for hPDLCs.nAg was no effect on the proliferation of hPDLCs when the concentration was not more than 30μg/ml.3.nCS and CS owned strong antibacterial activity for E.coli.The antibacterial rate of nCS was much stronger than CS,when the concentration was lower.The antibacterial ratio of nAg for E.coli was also strong.When the concentration was 400μg/ml,the antibacterial ratio of nAg exceeded 50%. nPLGA and PLGA had no antibacterial activity and nPLGA had no effect on the antibacterial activity of nCS and nAg.4.PLGA、CS and nPLGA、nCS could improve the mineralization ability of hPDLCs on some extent.Likewise,nPLGA/nCS/nAg and PLGA/CS/nAg also could improve the mineralization ability of hPDLCs on some extent. |
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