| With the social progress and with the development of science and technology,people's quality life and life expectancy continues to increase, and the human has steppedinto an aging society. The aging society brings us a series of challenges, including wearand generation of articular cartilage, even stripping, defect. The lesion of articularcarticulage is a hot problem in sport medicine. In addition, the industrial revolutionmakes our life more convenient, all kind of traffic accident and other high energy injuryalso increases increasingly. In clinical, articular cartilage defect of hip and knee with alarge area, even with open injury of joint surgery, are common and bring the opportunityof development.No nerves, lacks of blood supply, low proliferation and migration features, afterinjury, articular cartilage is difficult to be repaired by itself. In addition, large areas ofarticular cartilage damage or stripping complicates with joint infection, easy. Joint cavityis an enclosed chamber, lack of blood circulation, very low concentration of antibioticscan reach joint cavity with systemic application of antibiotics, what is more, localinjection of antibiotics may lead to of exogenous infection and pain brought by repeatedinjection. So, articular catilage lesioni is a most difficult problem for surgrons,and is adisaster disease with infection.In our research, we built a BMSCs-gentamicin-alginate three dimensionalco-culture system to observe the release of three-dimensional coagulated gentamicinbeads and the BMSCs in cell morphology, proliferation and phenotypic features, thusevaluated the function of gentamicin on BMSCs and chondrocytes in their proliferationand differentiation, and provided a basic study for the clinical treatment of aticular lesion,and for open or infectious aticular lesion.Objective:1. To master the techniques of BMSCs-gentamicin-alginate three dimensionalco-culture system in vitro. To master the establishment of the animal model of articularfull-thickness defect and the operational procedure of tissue engineering plus auto-periostcovering.2. To detecting the release of three-dimensional coagulated gentamicin beads,observing cell morphology, proliferation and phenotypic changes of BMSCs. 3. To explore the repaire effects of articular full-thickness defect with the procedureof the tissue engineering plus auto-periost covering in rabbits.4. To provide a basic study for the clinical treatment of aticular lesion, and for openor infectious aticular lesion.Methods:1. The source of the BMSCs Bone marrow was extracted from the posterior superiorsine of ilim in rabbits. BMSCs were isolate and purify with density gradientcentrifugation and adherence screening method, and amplifed in flat plates, and identifiedby the cell phenotype and purity.2. To manufacture gentamicin alginate controlled-release bead Gentamyc3.0g wasdissolved in10ml ddH2O, titrated to PH7.4, then HEPES10μL,0.0875gNaCl, mixedfully, then added into20ml triple standard concentration sodium algilate. This mixturecontains Gentamycin3.0g and standard concentration sodium algilate. Different groupsof gentamicin alginate sodium was added into40ml CaCl2solution (5mmol/l, HEPES,PH7.4), and produced some beads, which showed milky white or translucent primroseyellow. The test samples in the bags were put into the containers that have100ml0.9%sodium chloride, then exerted in a bed of the machine. At the estabished different period,accurately drawn as samples for10ml level from the container, the samples were sent tothe bacteric test for the calculation of the cumulative release rate and duration of durgrelease. Then0.9%additional10ml sodiom chloride was got into the container to persistno change in volume.3. To build the BMSCs-gentamicin-alginate three dimensional co-culture systemGentamycin3.0g was dissolved in10ml ddH2O, titrated to PH7.4, then HEPES10μL,0.0875g NaCl, mixed fully, then added into20ml triple standard concentration sodiumalgilate. Then0.5ml additional liquid of the second generation of BMSCs was mixed with2ml solution of gentamicin alginate sodium, fully. Then the BMSCs-gentamicin-alginate three dimensional co-culture system were compared with the BMSCs-alginatethree dimensional co-culture system.4. The transplant of the BMSCs-gentamicin-alginate three dimensional co-culturesystem BMSCs-gentamicin-alginate three dimensional co-culture system is stuffed intothe area of articular defect with the auto-periost.5. To establish animal model and design the groups of the experiment Twocylindrical, full-thick articular defect were drilled in the double knee jionts of12rabbits, 5.0in diameter and depth. According to the design of this experiment, these models weredivided into four groups: experimental group (BMSCs-gentamicin-alginate bead);experimental control group (BMSCs-alginate bead); simple auto-periost flap; the blankcontrol group (without both bead and periost). In12weeks, the distal femurs of theserabbits were cut for the macroscopic observation, H-Estain and Immunocyto-chemicalmethods.Results:1. In culture, BMSCs are strongly adherent, and the morphology of cells are spindle-shaped and similar size. Immunocytochemical methods of the1-3rd cultured cellsrevealed surface antigens CD44, CD105positive expression, CD34negative. It isindicated that the cultured cells are undifferentiated BMSCs.2. There was a significant statistical difference among the four groups (P<0.01) inthe release of Gentamycin. The cumulative release of gentamicin in three-dimensionalalginate group(U group) was no significant difference with bone cement group(Y group),and greatly higher than S,T group. The gentamycin concentrations in all of four groupsof delivery release beads, could achieve minimum inhibitory concentration (MIC)>2μg/ml of Staphylococcus aureus in the inhibition assays conducted.3. In the co-culture system of BMSCs-gentamicin-three-dimensional alginate,BMSCs have good biological activity. Gross microscopic observation revealed asatisfactory growth of BMSCs and a normal cell growth curve. Afte more than2weeksof culture, immunohistochemical analyses of planted cells exhibit collagen Ⅱpositiveexpression, and shows significant changes with Toluidine blue staining, these denotefully that cells show chondrocyte phenotypes.4. Successfully established the modeles of full-thick articular defect,12rabbits wereall survival till to12weeks after operations. The surface of carticular defect inexperimental groups filled by milky white tissue, similar color, and clear boundaries withthe surrounding tissue, no significant gap. Tissue sections showed that the fields ofcarticular defect have been contacted with the normal carticulage. There were no residuesof the delivery material. The cells were round, surrounding the formation of cartilagelacunae, but the numbers of cell were more than normal tissue, cellular derangement.Immunohistochemical detection of typeⅡcollagen antibody were positive. The results ofthe experimental groups were consistent with the experimental control groups. The repair tissue of simple periosteal flap groups were white, the surface depression, and clear gapwith the surrounding tissue. Tissue sections showed that the fields of carticular defectwere fiber cell-like rough surface, depression, poor healing, and a small aomount of boneformation at the bottomm. Collagen typeⅡimmunohistochemical staining were negative.Blank control group were obvious depression, pink granulation tissue at the bottom, andno siginificant tissue filling.Conclusion:1.In the Experiment, theBMSCs of rabbits were isolated and purified by gradientcentrifugation with adherence method. The immunohistochemical tests show CD34,CD44, CD105antibody staining positive, Ⅱcollagen antibody staining negative, these isconsistent with the biological characterization of BMSCs. So these methods can harvestthe high purity, undifferentiated mesenchymal stem cells, successfully.2. In the30-day test periods, the Gentamycin concentrations in all of four groups ofthe delivery release beads, could achieve minimum inhibitory concentration(MIC)>2μg/ml of Staphylococcus aureus in the inhibition assays conducted. Theencapsulation rate of calcium alginate condensate beads which prepared by U groupprogram is53.99%. The velocity and total release of gentamicin were perfect, that morethan the minimum inhibitory concentration, same with the gentamicin release of bonecement. Because of the biodegradable, the early release of gentamicin in calciumalginate was more than in bone cement.3. Gentamicin can be released from the BMSCs-gentamicin-three-dimensionalalginate system, persistently. the biological characterization of BMSCs can also bemaintained. In the7-21days of culture, cells grow up well proliferate significantly anddifferentiate to chondrocyte in these three-dimensiona system. Immunohistochemical ofcollagenⅡ and toluidine blue staining of those cells was positive expression. Thesecharacterizations are consistent with the three-dimensional BMSCs alginate system. Thisis to say, BMSCs-gentamicin-three-dimensional alginate system have goodhistocompatibility.4. In vivo, the repair effects of articular full-thickness defect of theBMSCs-gentamicin-alginate three dimensional co-culture system is consistent with thegentamicin alginate beads, and is good in short time. BMSCs-gentamicin-three-dimensional alginate system has good histocompatibility. It confirmed that BMSCs were active in cell division and proliferation in alginate three-dimensional culturesystems, and can be used in tissue engineering research.
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