| Joint infection is a problem needed to solve by surgeons. Severe joint infection usually complicates with large articular cartilage damage or large areas of cartilage stripping. The difficulty of articular cartilage injury and infection treatment lies in that joint cavity is an enclosed chamber with low blood circulation, systemic application of antibiotics can only reach very low concentration in joint cavity, what is more, local injection of antibiotics may lead to of exogenous infection and the possibility of bacterial resistance, and patients are not tolerate of the pain brought by repeated injection. As cartilage has no nerves and lacks of blood supply, the chondrocytes are of low proliferation and migration features, so articular cartilage is difficult to repair itself after injury. In our research, we built a BMSCs - gentamicin - alginate three dimensional co-culture system, through general observation, HE stain, toluidine blue staining,â…¡collagen staining, microbiological assay and other detection methods to observe the release of three-dimensional coagulated gentamicin beads and the BMSCs in cell morphology, proliferation and phenotypic features, thus evaluated the function of gentamicin on BMSCs and chondrocytes in their proliferation and differentiation.Objective:Constructing BMSCs - gentamicin - alginate three dimensional co-culture system, detecting the release of three-dimensional coagulated gentamicin beads, observing cell morphology, proliferation and phenotypic changes of BMSCs, and evaluating the function of gentamicin on BMSCs and chondrocytes in their proliferation and differentiation.Methods:1. Adherence screening method was used to isolate and purify BMSCs from rabbit bone marrow. BMSCs were cultured and amplifed in vitro in flat plates. HE staining, immunohistochemistry of CD34, CD44, CD105 to identify cell phenotype and purity.2. BMSCs - gentamicin - alginate three dimensional co-culture system was constructed. Cell counting, growth curve, HE stain, toluidine blue staining and typeâ…¡collagen immunohistochemical staining were applied to observe biological characteristics of cells in the three-dimensional alginate gel.3. Three groups of BMSCs - gentamicin - alginate three dimensional co-culture system were estibalished: U, S, T group. The concentration and duration of drug release were detected by UV spectrophotometry and microbial assay, to calculate the cumulative release of drug at different time points, comparing with bone cement containing gentamicin (Y group) to find out the optimal concentration ratio and a more stable and lasting release of gentamicin.4. Constructing the BMSCs - gentamicin - alginate three dimensional co-culture system to evaluate cell morphology, proliferation and phenotypic features of BMSCs by cell counting, growth curve, HE stain, toluidine blue staining and typeâ…¡collagen immunohistochemical staining, and evaluated the function of gentamicin on BMSCs and chondrocytes in their proliferation and differentiation.Results:1.BMSCs are strongly adherent in culture,and the morphology of cells are spindle -shaped and similar size. Immunocytochemical revealed 1-3 cultured cells surface antigens CD44, CD105 positive expression, but CD34,â…¡collagen negative.So it is indicated that the cultured cells are undifferentiated BMSCs.BMSCs have good biological activity in alginate which in a certain degree of flexibility ,strength and well biocompatibility.2.After 2 weeks of culture in alginate beads in three-dimensional environment,the immunohistochemical of planted cells are collagenâ…¡positive expression.Toluidine blue staining was also significative changes.Shows cultured in alginate beads in three -dimensional environment,BMSCs can differentiate into chondrocytes.3.The cumulative release of gentamicin in three-dimensional alginate group(u group) was no significant difference with bone cement group(Y group),and greatly higher than S,T group.But the difference of S,T group was not detected.The gentamicin release of U group at each time point were significantly different with the Y ,S and T group.Between S, T group there were no significant differences.According to curve relationship of release and timepoint,the U group was similar with Y group,and at the time point of forward 24 hours the U group was higher than the Y ,S and T group.The drug release of all groups were detected in the 30 day period,which all higher than the minimum inhibitory concentration.4.BMSCs have good biological activity in the coculture system of BMSCs - gentamicin - three - dimensional alginate.Gross observation of microscope,the growth of BMSCs was in good condition,and the cell growth curve is normal.After longer than 2 weeks of culture in three-dimensional alginate,immunohistochemical of planted cells are collagenâ…¡positive expression,Toluidine blue staining was also significative changes.It is indicated that cells show chondrocyte phenotype.Conclusion:1.Experiments used adherence method isolated and purified the rabbit bone marrow mesenchymal stem cells.The immunohistochemical show CD34, CD44, CD105 antibody staining positive,â…¡collagen antibody staining negative that consistent with the characteristics of BMSCs.So the methods can successfully harvest the high purity, undifferentiated mesenchymal stem cells.2.After construct the coculture system of BMSCs-gentamicin-three-dimensional alginate,the gross observation,microscope observation and cell growth curve show that cell growth well in this coculture system.The 7-21 days of culture,cell proliferation significantly.The amount of cells in 3-D culture system is more than in normal system.BMSCs can maintain good biological characterization and then differentiated to chondrocyte state after 2 weeks.Immunohistochemical of collagenâ…¡and toluidine blue staining was positive.3.Gentamicin-three-dimensional alginate system could release gentamicin extendedly and persistently,and got the minimum inhibitory concentration in the local.In the 30-day test period, the encapsulation rate of calcium alginate condensate beads which prepared by U group program is 53.99%.The velocity and total release of gentamicin were perfect, that more than the minimum inhibitory concentration,same with the gentamicin release of bone cement.Because of the biodegradable,the early release of gentamicin in calcium alginate was more than in bone cement.4.BMSCs-gentamicin - three-dimensional alginate system could release gentamicin persistently and also maintain the biological characterization of BMSCs.Cell growth well in this system,in the 7-21 days of culture,cell proliferation significantly and differentiated to chondrocyte state.Immunohistochemical of collagenâ…¡and toluidine blue staining of those cells was positive. |
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