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The Experimental Study Of Repairation Of Cartilage Full-thickness Defects By Small Instestinal Submucosa Combined With Chondrocyte.

Posted on:2009-10-19Degree:DoctorType:Dissertation
Country:ChinaCandidate:D WuFull Text:PDF
GTID:1114360245458802Subject:Surgery
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Background:To investigate relatively the result of repair the cartilage full-thickness defects with cultivated the autologous chondrocyte in high density as the bone tissue engineering seed cells, combined it with SIS and chitosan-collagen scaffold to fabricate the tissue engineered cartilage. Thereby discuss the feasibility and efficacy of the cartilage full-thickness defects repairation with different tissue engineered cartilage,then provide experimental evidence for the further clinical using of tissue engineered cartilage.Methods:1.The articular genu cartilage was collected from 2 month old swine,the cartilage were digested by theⅡtype collagenase,then cultivathed in different density for obtaining the cartilage cells,paaaaged and amplificated,then observed the morphous and draw the auxodrome, identified by immunohistochemistry and Mallory stain and TB stain.2.According Abraham method,the Small instesfinal submucosa was made,and reserved after radiosterilization;Then transplanted the cartilage cells which was cultivated to 3rdgeneration into the SIS,this complex was cultivated in 48 hours to establish the chondrocyte- scaffold complex, and then observed by inverted microscope and electron microscope.3.The chitosan-collagen scaffold was made and reserved after radiosterilization for using.Then tested its cytotoxicity,pyrogenic,hemolysis,sensitivity test by leaching liquor.The cartilage cells which was cultivated to 3rdgeneration was transplanted onto chitosan-collagen scaffold,and this complex was cultivated in 48 hours to establish the chondrocyte- scaffold complex,and then observed by inverted microscope and electron microscope.4.the swine articular genu cartilage full-thickness defects at condyles of femur was made,and devided randomly into 4 groupes:1 and 3 groupe(autologous chondrocyte+ SIS scaffold)was at right knee,2 groupe(SIS caffold)at left knee,4groupe((blank control group)was at left knee. After 16 weeks,macroscopic observation,HE,TB,Mallory stain andⅡtype collagenase immunohistochemistry stain observation was taken.And graded as Wakitani grading system.Results:1.The articular genu cartilage was obtained 40-50mg,and the cell volume dose was 1. 5-2.5×105 after digested completely according 'One step method',the activity was 96.4%,the ceils were 3.2×106 after cultivated to 4thgeneration in monolayer high density.The positive reaction was showed in TB,Mallory stain andⅡtype collagenase immunohistochemistry stain. 2.electron microscope showed surface of SIS is rough hard sphere,cellules were distributed in the formatio reticularis,the size of cellules is 100-400pro,the chondrocyte adhered to the scaffold and grew well in the cellules.HE stain showed cells presented monolayer or multilayer grown on surface of the SIS.Immunohistochemistry stain shows there is a successive positive expression strap between the chondrocyte and SIS.3.Electron microscope showed the chitosan-collagen scaffold presented a structure with a rough hard sphere surface and adqulis lamellar cellules,the size of cellules was 50-300μm,the osteal -analog strcture was appeared,chondrocyte with manipulus adhered and grew.Its leaching liquor didn't cause pyrogenic,hemolysis,sensitivity reaction.4.After 16 weeks,cartilage full-thickness defects in the 1 and 3group were repaired well, demonstrated the hyaline cartilage formation,the structure of the cartilage was integrated,and neogenesis tissue syzygia with other structure,demonstrated a hyaline-like syzygium.2 group presented a fibrous tissue or fibrocartilage-like formation,vascular tissue contained.In 4 groupedefects didn't be repaired,and degeneration sign was found.Wakitani grade showed there was no difference with significance(P>0.05)in 1 and 3group,but surpassed 2 and 4 group(P<0.01; MRI showed there was no difference with significance(P>0.05)inl and 3group,but surpassed 2 and4 group(P<0.01)Conclusions:Chondrocyte digested by Single collagenase and cultivated in high density not only obtained sufficient cell population,but also maintained cytodifferentiation phaenotype.Culture chondrocytes by high density could obtain tissue-like cell aggregates.This method is a reliable approach to obtain seed cells of tissue engineered cartilage.Serial subcultivation and dedifferentiation chondrocyte cultivation by high density could restore phaenotype partially. Cultivation by high density at primary cell or after serial subcultivation was a good method for chondrocyte cultivating in vitro.Chitosan-collagen scaffold was a cartier of tissue engineered cartilage,but its preparation and biocompatibility need further improvement.SIS was a good carrier in tissue engineering,and combined it with autologous chondrocyte to fabricate the tissue engineered cartilage,which could repair cartilage full-thickness defects and acquired hyaline-like repair;but transplanted the SIS singly or without any materials couldn't cartilage full-thickness defects.
Keywords/Search Tags:articular cartilage, autologous, tissue engineering, chondrocyte, transplantation, collagen, chitosan, SIS
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