| Periodontitis is currently one of the major causes of tooth loss in adults.The ultimate goal of periodontal treatment is to obtain structural and functional regeneration and reconstruction of the periodontal support tissues,including alveolar bone,periodontal ligament and cementum.To achieve this purpose,biological graft materials are always needed to be placed in the bone defect.In recent years,synthetic biomaterials for bone defect repairing has drown more and more attention.The latest study confirmed Nano Hydroxyapatite/Chitosan Composite(n HA/CS)had a good biocompatibility,osteoconductive ability and injectable ability.Many scholars treated it as a drug carrier loading Chinese Medicine,protein drugs,antibiotics,etc.,to achieve the sustained release of drugs.Oral cavity had a bacterium environment.In the early period of implant bone repairation,bacteria were needed to control and infection should be prevented.In the long-term bone resorption caused by periodontitis should be prevented.In this case,traditional systemic use of antibiotics could lead to poor results,and can cause a variety of side effects and cumulative drug toxicity in vivo.Therefore,this study took n HA / CS as a drug carrier,loaded the minocycline hydrochloride which was commonly used in clinical treatment of periodontitis,to construct Minocycline-Nano Hydroxyapatite/Chitosan(M-n HA/CS).The n HA/CS and M-n HA/CS were foamed and made hole via chemical reaction and can be filled by a doctor needed to reconcile the powder-liquid,which avoided the shortcoming that traditional bone filling materials could not fit closely with the host bone due to prefabricated moldings.The physical and chemical properties,drug release,antimicrobial resistance and biocompatibility of the newly constructing M-n HA/CS were evaluated to provide experimental basis for clinical application of bone defect.Part I Physical and chemical property investigations of Minocycline-Nano Hydroxyapatite / Chitosan(M-n HA/CS)Objective: Through test of the porosity,scanning electron microscopy,the curing time,compressive strength and p H value of the bone cement before and after drug loading,to investigate the effects on physical and chemical properties of n HA/CS caused by minocycline.Methods: The n HA/CS and M-n HA/CS were prepared by using co-precipitation method.Porosities of the bone cements were tested by The Archimedes Principle.Initial setting time and final setting time of the two bone cement were measured by Vicat.Pore sizes and distributions were scanned by an electron microscopy.The compressive strength was measured by the universal testing machine.The acidities within one week of the two cement extract were tested by a p H meter.Results: Bone cement initial setting time and final setting time after loading Minocycline were extended with statistically significant difference(P<0.05).Compressive strength significantly downward trend,n HA/CS compressive strength 37.65 MPa,M-n HA/CS compressive strength of only 21.7 MPa,the difference was statistically significant(P<0.05).M-n HA/CS and n HA/CS interior material was porous structure,with better penetration and different pore shapes and sizes.The average porosity was 43.68% of n HA/CS group and 53.99% of M-n HA/CS group.M-n HA/CS group had largher number of holes than that of n HA/CS group with statistically significant difference(P<0.05).M-n HA/CS group had large aperture than that of n HA/CS group,ranging from 20 to 200?m.After the fourth day,two materials' p H value all reached 7.3 and kept stable.Throughout the experiment p H values are in the range of 6.9-7.4 and kept neutral,which did not show relatively strong acidity.There was no statistically significant of the daily p H values in the two groups of materials(P>0.05).Conclusion: 1.The bone cement curing time can be extended by loading Minocycline.2.The load of the compressive strength of bone cement can be reduced by Minocycline.3.Minocycline is loaded so that the bone cement pore size increases,the porosity increased.4.The p H value of the bone cement before and after loading Minocycline does not change significantly or show strong acid or alkaline.Part II The in vitro release and inhibiting effect research of Minocycline-Nano Hydroxyapatite / Chitosan(M-n HA/CS)Objectives: Observe the in vitro release of the drug and its inhibiting effect on Porphyromonas gingivalis and Staphylococcus aureus.Methods: Two sets of the composite were soaked into the stimulated saliva separately,and the high performance liquid chromatography(HPLC)was used to measure the release of minocycline everyday.The bacteria-inhibiting ring method was used to test the inhibiting effect on Porphyromonas gingivalis and Staphylococcus aureus.Results: The daily release of minocycline reached peak value of 3.77?g,and decreased day by day.After a week,the daily release remained stable with 0.5-1?g/d.On the 14 th day,the release was 0.83?g/d.M-n HA/CS released drug early by bone dissolution and diffusion after.The release best conformed to the Higuchi model at the first four days,and from the 5th to 14 th day,it conformed to zero-order or first-order release pattern.After a week,20 mm inhibition zone of the Staphylococcus aureus and 24 mm inhibition zone of the Porphyromonas gingivalis still could be observed around M-n HA/CS.M-n HA/CS had a good inhibiting effects on the Staphylococcus aureus and Porphyromonas gingivalis.Conclusions: 1.The release of M-n HA/CS best conformed to the Higuchi model at the first four days,and from the 5th to 14 th day,it conformed to zero-order or first-order release pattern.It released drug early by bone dissolution and diffusion after.2.Using HPLC and bacteria-inhibiting ring method can get a result that M-n HA/CS can provide a long-term,slow release of minocycline with effective inhibitory concentration.Part III Biocompatible detection of minocycline nano-hydroxyapatite / chitosan compositeObjective: Observe the proliferation and adhesion of the periodontal ligament stem cells on the surface and in the leach liquor of M-n HA/CS and n HA/CS,and also test the osteogenic differentiation potential of periodontal ligament stem cells cultured in the leach liquor of M-n HA/CS and n HA/CS.Methods: Primary cultured periodontal ligament stem cells(PDLSCs)with tissue block method and prepared the leach liquor of M-n HA / CS and n HA/CS.Measured the proliferation of PDLSCs in two groups of leach liquor by CCK-8 kit,OD values of each group were measured by a microplate reader.PDLSCs were seeded on the surface of M-n HA/CS and n HA/CS.Fifteen days after,both groups were fixed,dehydrated,dried with critical point drier,sputter-coated with gold and examination under SEM.ALP,COL-1,OCN,OPN and OPG osteogenic markers of the periodontal ligament stem cells which were cultured in two groups of leach liquor were tested by real-time quantitative PCR method.Result: OD values of two composite groups on the third day were higher than that of the control group.OD values of M-n HA/CS group was the highest of three.The two groups of leach liquor after culturing PDLSCs for 72 h,OD values showed significant different from that of the control group(P<0.05).Compared with control group,the relative growth rate of n HA/CS was 98% in the previous two days,cytotoxicity grade level one,the third day rised to more than 100%,grade zero cytotoxicity level.Relative growth rate of M-n HA/CS group is more than 100% within three days,grade zero cytotoxicity level.Scanning electron microscopy showed that compared to n HA/CS,the cells on the surface of M-n HA/CS was denser,they overlapped and covered each other.PCR showed that the expression level of ALP in M-n HA/CS group was slightly lower compared with n H/CS group without statistically significant(P>0.05).Compared with n HA/CS group,each osteogenesis-related genes(COL-1,OCN,OPN,OPG)expression level of M-n HA / CS was higher with statistically significant(P<0.05),Which could be considered that compared to n HA/CS,M-n HA/CS had more ability to promote periodontal ligament stem cells into osteogenic differentiation.Conclusion : 1.M-n HA/CS and n HA/CS exhibites good cytocompatibility.2.M-n HA/CS can better promote the adhesion and proliferation of PDLSCs on the surface of the composite in vitro.3.M-n HA/CS can promote the osteogenic differentiation of periodontal ligament stem cell. |
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