| With the increase of social population, due to trauma, osteomyelitis, tumor or other bone diseases caused by bone defect were more and more, so the clinical bone grafting materials demand more and more, a lot of demand driven research and development of artificial bone. Many inorganic bone substitute material has now reported, α-calcium sulfate hemihydrate has good biocompatibility, biodegradable, osteoconductive and other valuable characteristics [1-4], a-calcium sulfate hemihydrate whiskers can form a slightly acidic environment in the degradation process, calcium ion and its degradation resulting from the formation of the high calcium concentration environment to osteoblasts play a stimulating effect and osteogenesis of [5]. Based on calcium sulfate bone cement can be implanted in the free plastic and no stimulation on the body, after curing the matrix left a large number of pores, so its as a drug carrier can effectively to achieve both bone defect filling, but also can effectively prevent bone infection, accelerate bone healing, promote the new bone regeneration purposes.Research shows that, the element strontium (Sr) is a kind of trace elements present in humans, is a dear bony elements. SR 99% above in the human body are present in human bone, cell fluid in addition to 0.65% of strontium ions exists in the form of a human body, make the strontium concentration in a dynamic balance. The concentration of strontium ions will maintain a high process in new bone formation in order to promote new bone formation, after the new bone formation and ion to maintain a certain concentration in the neutralization of bone cells in liquid to maintain the bone of normal function of [6]. Its mechanism of action is to increase the expression of osteogenic related genes and bone marrow mesenchymal stem cells (BMSCs) in alkaline phosphatase activity, and inhibition of BMSCs necessary to osteoclast differentiation of NF-B receptor activated factor [7-8]. In the calcium sulfate hemihydrate can be added with a certain amount of Strontium in order to maintain the concentration of local strontium ions will promote role for bone repair, but encountered in the application of the process in question is:the strontium containing calcium sulfate hemihydrate strontium ion implantation in the human body total, will release rate cannot be too fast, formation and bone the new bone matching. At present in the clinical application of calcium sulfate hemihydrate complete degradation time is 6-9 weeks, much faster than the rate of new bone formation (12 weeks), the rate of degradation rate of material makes faster than new bone formation, can lead to bone defect area appeared empty part, which will be fibrous tissue ingrowth, will affect the treatment effect, is not conducive to the healing of bone defect.Therefore, in order to solve the strontium containing calcium sulfate hemihydrate degradation rate too fast and strontium ion release rate high, the need for strontium containing calcium sulfate hemihydrate improvement. Chitosan is widely used for this study suggested that chitosan also known as chitin, chitosan, also called chitin Tang, chitosan, chitosan, capsid protein,which is similar to the plant fiber six carbon sugar polymer, is the nature of the only edible animal fiber containing free amino alkaline cations, is chitin deacetylation and get a natural cationic polysaccharide. Chitosan belongs to the amino polysaccharide, molecular formula (C8H13NO5), molecular weight is about 106, the theory of the nitrogen content of 6.9%. The structure of chitosan and cellulose molecules of similar molecular shell is a straight chain, very strong, can generate different derivatives, different nature, different uses. It exists in the microbial cell wall, lower plant fungi, algae cells and yeast, arthropod shrimp, crab and insects in the shell and cartilage, mollusks (such as squid, cuttlefish) inner shell and cartilage. The scope of application of chitosan widely, is a kind of natural bioactive molecules,special molecular structure and physicochemical properties of the chitosan has antimicrobial, anti-inflammatory, hemostatie, promote wound healing, nourishing lubrication mucosa and other biological activity and function, applied to artificial organs, wound dressings, hemostatie materials, bone repair material, anti adhesion even the agent, the immune preparation medicine field. At the same time is widely used in food, chemical industry, environmental protection industry.Chitosan is the appearance of white or light yellow translucent solid, soluble in dilute acids such as hydrochloric acid, most of acetic acid, benzoic acid solution, and is soluble in acid, molecular amino group can be combined with a proton, make oneself with positive charge, and chitosan in acid solution of acid catalyzed hydrolysis reaction occurs, the final hydrolysis a monosaccharide and oligosaccharide. However, chitosan is insoluble in water and alkali solution, do not dissolve in sulfuric acid and phosphoric acid. Acetic acid solution of ehitosan dissolved in 1% mass fraction of formed transparent viscous chitosan colloidal solution, which is one of the important properties of. Chitosan is nontoxic and harmless, and has good moisture resistance, wettability, but strong hygroscopicity, water decomposition [9]. In recent years, the domestic and foreign [10] has a large number of reports in the literature using chitosan modified liposomes,microspheres, microcapsules as drug delivery system. The preparation of chitosan microspheres using, can control the release of drugs, improve the easy degradation (such as protein) bioavailability, enhanced the hydrophilic drug permeability through the epithelial layer.The present method for the preparation of chitosan microspheres are:complex coacervation, covalent crosslinking method, ionic gelation method, spray drying method, emulsion crosslinking method.Xie Yu et al to bovine serum albumin (BSA) as a model drug, the study prepared by ionic gelation of chitosan nano microspheres for BSA encapsulation and release effect. The purpose of this study is the use of Chitosan on strontium containing calcium sulfate hemihydrate was coated to form microspheres, to form a shell surface of strontium containing calcium sulfate hemihydrate, effectively solves the problem of hemihydrate calcium sulfate degradation rate too fast and easy loss problems encountered a lot of fluid, also make the strontium can reach a long-term stability release effect.This experiment includes two parts, the first part is the chitosan coated strontium containing alpha calcium sulfate hemihydrate microsphere compound preparation and the materials were detected, verify that it is for the product; the second part of the basis of medical biological materials (implant) relevant standard safety evaluation (people’s Republic of China national standard GB/T16886), complete the required to:(1) cell toxicity test (2) genetic toxicity test (3) acute toxicity test (4) in subacute toxicity test, skin irritation test (5) (6) of intradermal skin sensitization test (7) subcutaneous implant test, required to complete the requirements of national standards to preliminary biological material evaluation of chitosan coated strontium containing alpha calcium sulfate hemihydrate composite microspheres provide biocompatibility, biological safety data for the application of the material.The first part of chitosan coated strontium containing alpha calcium sulfate hemihydrate microsphere compound preparationObjective:To study the preparation of chitosan coated strontium containing alpha calcium sulfate hemihydrate microsphere compound and to detect whether the productverification.Materials and methods:first, the strontium containing two water calcium sulfate were prepared by the method of CO crystallization of the system, and then use the atmospheric salt solution was prepared by strontium containing calcium sulfate hemihydrate, electrostatic attraction between the last under PH=5 conditions using chitosan solution and strontium containing calcium sulfate hemihydrate in high-speed stirring conditions preparation the chitosan coated strontium containing calcium sulfate hemihydrate microspheresResults:the successful preparation of chitosan coated strontium containing alpha calcium sulfate hemihydrate microsphere complexes, the XRD results showed that, the XRD analysis results showed that the 2 theta=14.75,25.71,29.76,31.91 respectively corresponding to a typical alpha of the -CSH crystal (110), (310), (220), (-114) crystal surface 9, said there was no impact of strontium ions joining the crystallization behavior of CSH, CSH has excellent characteristics of its good crystallization performance and its. In addition, CS-C-CSH (Sr) and the intensity of CSH peak (Sr) is weaker than, the possible reason was coated on the surface of a layer of chitosan, thus affecting the strength of the signal; the SEM results show that from the left, we can see that the CSH (Sr) is a long rod like structure of 10, and its length is even, the average length of approximately equal to 50um. On the right is CS-C-CSH (Sr) microspheres, see after chitosan coating can be from the graph,CSH (Sr) were spherical in shape, this is because the CSH (Sr) is a rod structure, chitosan is difficult to be wrapped into spherical rules, and also can be seen from the chart, the diameter of about 60um in, and the particle size is consistent, which contributes to the later uniform release strontium ion.Conclusion:the preparation of the experiment system of chitosan coated strontium containing alpha calcium sulfate hemihydrate microspheres with uniform diameter, and in the reaction process without any organic solvent, ensure safety of microspheres.Part Two:chitosan coated strontium containing alpha compatibility of hemihydrate calcium sulfate composite microspheres of biologicalObjective:to preliminarily evaluate the chitosan coated strontium containing alpha calcium sulfate hemihydrate composite microspheres provide biocompatibility, biological safety data for the application of the material. Materials and methods:(1) cell toxicity assessment material extract fibroblast morphology and influence the relative proliferation rate of L-929 mice. (2) the skin stimulation test (3) of mouse bone marrow polychromatic erythrocyte micronucleus test (4) implantation in vivo test to assess their response to local toxicity of living tissue. (5) the subcutaneous sensitization test (6) acute toxicity test (7) subacute toxicity testResults:(1) experimental group, negative control group at different time points of toxicity was grade 0-1, no cell toxicity; positive control group toxicity was grade 111, cytotoxicity; micronucleus rate (2) were reported in the literature in the normal range, that extracts did not make mice bone marrow micronucleus rate change, each dose group PCE/NCE of not less than 20% in the control group, normal range; (3) implantation in vivo results show that the material and the body reaches a steady state. (4) intradermally sensitized experiment, experimental observation on rat injection of local and surrounding skin tissue reaction, erythema, edema and necrosis were not B point area of skin test side and control side, shows that the material extract had no irritation to the skin, are in line with national standards for intradermal skin sensitization test. (5) extract group and negative control group unanimously skin stimulation test, no skin edema, skin prickle cell layer, perivascular edema, diffuse dermal and epidermal infiltration of mononuclear cells, see scattered in a small amount of basophilic cells. (6) acute toxicity test, the test group and the control group animal appeared adverse reaction. Experimental animal group than in the control group without a large animal reaction,Mice with good activity and eating, breathing normally, no paralysis, convulsions and death occurs, all aspects of performance and the same as the experimental group. Mouse tail vein injection weight stable, a slight increase in part. (7) the subacute toxicity test, the observation period the test group and the control group rats were active, quick reaction, hair color, eating normal. Loss of appetite obvious to suit all did not appear animal, the rats were also seen hypokinesis, difficulty in breathing, diarrhea and other symptoms of toxicity, no animal death phenomenon. The experimental group and the control group were lower than those after injection than before the injection weight slightly increased, little change in body weight, relatively stable. An important organ pathological biopsy was experimental and control group animal heart, liver, spleen, kidney and other congestion,hemorrhage, edema or other pathological changes.Conclusion:Preliminary biocompatibility tests confirmed that the novel chitosan parcels containing strontium calcium sulfate hemihydrate microsphere composite has good biocompatibility. |
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