| With the rapid pace of life increasing, people are also accelerating their speed of life, expandingthe scope of social activities. Travelling is more and more dependent on traffic tools. Thus there aremore traffic accident and the types of trauma are various and complex. Among them is open jointinjury. At the same time in the fierce confrontation on sports field, in busy construction site, and inroaring factories such injuries are not rare. The following articular cartilage injury and joint infectionare a huge problems for a lot of trauma surgeons and joint surgeons. Especially when pathogeninfection occurs with the existing large range of centralized or scattered articular cartilage injury or alarge area of cartilage surface stripping, which is like one disaster after another. Due to the joint cavityis a sealed cavity, appendant structures like articular cartilage and synovial membrane. And ligamentis the main component of the joint cavity. The internal blood circulation is weak, thus through theintravenous route, antibiotic concentration in the joint cavity has a very poor effect, which lead to alow potency ratiov, poor treatment effect, and the extra side effects of systemic administration. Thelocal repeated injection of antibiotics may lead to exogenous infection and bacterial resistance. At thesame time patients may fear the repeatability of the injection pain. No innervation, poor blood supplyand cartilage cell regeneration ability and other histological features make the repair of incartilagetissue difficult. Continuous destroy of pathogenic bacteria infection will cause failure of any form ofrepair, thus the control of infection is the prerequisite to the repair of cartilage defect. In our study,through the construction of BMSCs–gentamicin-alginate three-dimensional scaffolds, we transplantthem into rabbit knee articular cartilage defects with the infection of Staphylococcus aureus. Throughthe measurement of blood inflammatory index as rectal temperature, blood routine, ESR, CRP,bacterial culture, gross observation, H-E staining, detection methods as Masson staining, toluidineblue staining, alcian blue staining, type II collagen staining, we expect the understanding of itsantibacterial ability, infection control and assessment of cartilage repair defect of the3D scaffoldsgentamicin, providing a reference for the feasibility of clinical application.Objective:We construct BMSCs–gentamicin-alginate three-dimensional scaffolds and transplant theminto rabbit knee articular cartilage defects with the infection of Staphylococcus aureus. Then we detectthe antibacterial ability and the control of infection of the3D scaffolds, and evaluate the repair ofcartilage defect. Methods:1. By adherent screening method, we separated and purified bone marrow mesenchymal stemcells (BMSCs) from rabbit bone marrowblood, then they were incubator proliferation culture passaged.With the application of H-E staining and CD34, CD44,CD90, CD105antibody immunohistochemicalstaining method,the cell phenotype passage and purity will be identified.2. We construct BMSCs-gentamicin-alginate three-dimensional scaffolds, then we culture themin vitro. By cell counting, growth curve we expect to understand the cellular proliferation,transformation from BMSCs to chondrocytes. Through the support of H-E staining, toluidine bluestaining and type II collagen staining, we preliminary had an judgment of gentamicin’s effect on theproliferation and differentiation of BMSCs cells and chondrocytes.3. We Construct non infected rabbit knee articular cartilage defect model A and Staphylococcusaureus infection of rabbit articular cartilage defect model (B, C, D), Injected the standard strains ofStaphylococcus aureus (ATCC25923)1x107cfu/ml,1x106cfu/ml,1x105cfu/ml0.5ml each intothe joints with cartilage defects, and then we acquire the rabbit articular cartilage defect animal modelinfected with Staphylococcus aureus (B, C, D). By measuring the blood inflammatory index as rectaltemperature, blood routine, ESR, CRP and bacterial culture, we can evaluate whether the model canachieve the desired effect and its stability and repeatability. We Choose groups of the right dose as ananimal model of the follow-up experiment.4. The calcium alginate group (scaffold I), BMSCs-calcium alginate group (scaffold II),BMSCs-gentamicin-calcium alginate (scaffold group Ⅲ), blank control group (blank) were grown inthe noninfected rabbit knee articular cartilage defect mode.By measuring the rectal temperature, bloodtest, bacterial culture, gross observation, H-E staining, Masson staining, toluidine blue staining, alcianblue staining, type II collagen staining and other detection method, we can compare the situation ofcartilage repair in each group.5. The calcium alginate group (scaffold I), BMSCs-calcium alginate group (scaffold II), BMSCs-gentamicin-calcium alginate group (scaffold Ⅲ), gentamicin-calcium alginate group(scaffold Ⅳ),blank control group(blank) were grown in the rabbit knee joint cartilage defect model ofStaphylococcus aureus infection.Through measuring rectal temperature, blood routine, ESR, CRPblood inflammatory index test, bacterial culture, gross observation, H-E staining, Masson staining,toluidine blue staining, alcian blue staining, type IIcollagen staining and other detection methods, wecan compare the control of bacterial infection and the repair of cartilage.Results：1. The acquired BMSCs cells adhered rapidly, and the sizes were similar, and the shapes were fusiform or spindle; by flow cytometry we found that the cultured second generations of cell surfaceantigen CD44, CD90, CD105were positive, no expression of CD34; type II collagenimmunohistochemical staining was negative.2. BMSCs had a good biological activity in3D scaffolds; Determination of BMSCs CCK-8cellcount showed a good proliferation, cultured more than two weeks, type II collagen scaffold cellstaining, toluidine blue staining were positive and showed phenotype of chondrocytes.3. Non infected rabbit articular cartilage defect model A was successful. Staphylococcus aureusinfection of rabbit articular cartilage defect model B (ATCC259230.5×107cfu), preoperative andpostoperative measurement of blood inflammatory index as rectal temperature, blood routine, ESR,CRP, and bacterial culture indicated that all group rabbits knee infection were successful. Comparedwith the group A, reoperative indicators had no significant difference (P>0.05). Postoperative bodytemperature and blood indexes were higher than those in group A. Compared with group A, bacterialculture were positive and the difference was statistically significant(P<0.05). Model C and D got onlypart of the rabbit knee infection because of the lack of bacterial infection doses.4. Before and after planting, non infection model of rabbit articular cartilage defects in groupscaffoldⅠ, group scaffold Ⅱ, group scaffold III and the blank group the anus temperature, bloodinflammatory markers and bacterial culture results, the pairwise comparison has no significance(P>0.05). H-E staining, Masson staining, toluidine blue staining, alcian blue staining, type II collagenstaining showed: scaffold I group can repair cartilage defects, and the major was hyaline cartilagesurrounded by fibrous cartilage. The repaired cartilage layer was thinner, and the cell arrangement andstructure were in a mess; in group scaffold II and III the repairment of cartilage defect is overallsatisfactory, substantially transparent cartilage. Around the edge there is fiber cartilage. The thicknessof the cartilage repaired flush with the edge around. Cells were arranged, and the hierarchy was fair.There was an abundant extracellular matrix; in blank group cartilage defects was still obvious. Thesurface was mainly fiber cartilage. The repaired cartilage layer was thick and the cells arrangeddisorderly, and structure hierarchical.5.In the model of rabbit articular cartilage defects of staphylococcal infection: before and afterplanting, rectal temperature, blood inflammatory markers and bacterial culture results, the pairwisecomparison had no significance (P>0.05); in group scaffold III, group scaffold IV rectal temperature,the blood of inflammatory indicators and the results of bacterial culture, differences between the twogroups were without significance (P>0.05); there was statistical significance between group scaffoldⅠ, group scaffold Ⅱ, blank group and Ⅲ or Ⅳ groups (P<0.05). H-E staining, Masson staining,toluidine blue staining, alcian blue staining, type II collagen staining showed: in group scaffoldⅠ, group scaffoldⅡ, and the blank group, the cartilage defects were still obvious, even expanded, andthere were osteophytes, joint degeneration, and inflammatory cell filling, confusion of structure; ingroup scaffold III, the repair of the cartilage defect was the overall satisfactory, substantiallytransparent cartilage. around the edge there was fiber cartilage, the thickness of the cartilage repairedflush with the edge around. Cells were arranged, and the hierarchy was fair. There was an abundantextracellular matrix; in group scaffold IV cartilage defects could be repair. The subject was hyalinecartilage, surrounded by fibrous cartilage. The repaired cartilage layer was thinner. Cells’ arrangementand hierarchical structure were in disorder.Conclusion:1. The purification of rabbit BMSCs screening adherent method, separation, detection by flowcytometry showed CD34negative, CD44, CD90, CD105antibody positive, and group type II collagenimmunohistochemical staining negative, consistent with the characteristics of BMSCs, proving thatthough this method can isolate and culture the undifferentiated BMSCs with high purity.2. BMSCs maintain the biological activity in sustained release of gentamicin in gentamycin-calcium alginate scaffold in the case, showing a three-dimensional growth and proliferation. In1weeks-3weeks of culture, cell proliferation was active, and group type II collagenimmunohistochemical staining and toluidine blue staining indicated that BMSCs eventuallydifferentiated into chondrocytes.3. The result of non infected rabbit articular cartilage defect model A was ideal, which could bean animal model for the late planting scaffold; The result of Staphylococcus aureus infection of rabbitarticular cartilage defect model B was ideal, with bacterial infection rate of100%, which can be usedas animal model of late planting scaffold; in model of C and D due to the insufficient dose of bacterialinfection, the bacterial infection rates were66.7%and33.3%, which could not reach the standardanimal model of presupposition, thus they cannot be as animal model for the latter test.4. Non infection model of rabbit articular cartilage defects in group scaffoldⅠ, group scaffoldⅡ,group scaffold III and blank group, there were no differences in inflammatory markers before andafter planting. Through a comprehensive analysis of the various staining method we could see: groupscaffold II, Ⅲ were most ideal scaffold for the repair of cartilage defect, close to hyaline cartilage,and flushed with the area around; and the repair ability of scaffoldⅠ was slightly worse, most wererepaired into hyaline cartilage, the area between the defect and the scaffold were fiber cartilage repair,and there was uneven part in the region; the blank group cartilage defects became fiber cartilage, andthe repair was not smooth. 5. In the model of rabbit articular cartilage defects of staphylococcal infection: in group scaffoldIII, group scaffold IV group the anus temperature, blood inflammatory index decreased rapidly afterplanting, joint fluid germi culture was negative,suggesting that the joint control of bacteria infectionwas ideal, and bacteria was killed; in group scaffold I、group scaffold II and blank group the rectaltemperature, all kinds of inflammatory markers, and joint fluid bacterial culture showed no significantchanges after planting, suggesting that bacterial infections of joints had not been effectively controlled,with inflammation persistent. Through a comprehensive analysis of the variety of staining we couldconclude that scaffold III was the most ideal scaffold for cartilage repair: close to the hyaline cartilagedefect, and flush with the area around and with less inflammatory cells; and the repair ability of groupscaffold IV was slightly worse, most repair was hyaline cartilage, the area between the defect and thescaffold were fiber cartilage repair, and there was uneven part in the region, and there were lessinflammatory cells; group scaffold Ⅰ, group scaffoldⅡ and blank group repaired in failurebecause of nonadequate control of infection, obvious damage of cartilage defect region, andinflammatory cell infiltration. |
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