Crosslinking Of Decellularized Kidney Scaffold By Glutaraldehyde And Heparin

Objiective :Glutaraldehyde and heparin crosslinking modificaiton of the decellularization of intact kidney by perfusion,try to enhance indexes including the time of degradation in in vitro and vivo?biomechanical property?anticoagulation and so on,in order to closer to clinical needs.Methods:1.Experimental group160 SD rats,randomly divided into 4 groups,each group 40,only the first group(group D):decellularization kidney scaffold;The second group(group G):Decellularization kidney scaffold crosslinking glutaraldehyde;The third group(group H):decellularization kidney scaffold crosslinking heparin;The fourth group(group G+H):decellularization kidney scaffold crosslinking glutaraldehyde and crosslinking heparin.2.Preparation of each group decellularization kidney scaffold Putting indwelling needle in SD rat's aorta abdominalis,by below of the abdominal aorta putted 2# needle and fixed,cut broken upper and inferior vena cava blood flow out as soon as possible,connecting the peristaltic pump,each kidney is in turn into corresponding 0.01% heparin solution,1% Triton X-100,0.8% sodium dodecyl sulfate(SDS),deionized water,0.625% glutaraldehyde(GA),0.002% heparin sodium solution and containing double anti-(penicillin streptomycin)in deionized water,each group decellularization kidney scaffold are soaked in physiological saline in 4 ? refrigerator storage for later use.3.Pathological observation1)General observation kidney shape and color difference;2)HE staining?masson staining and scanning electron microscopy(SEM)observation each group collagen morphological microstructure of decellularization kidney scaffold;4.Biomechanical structure stability observation1)Through the in vitro degradation rate,bibulous rate to evaluate the internal structure of stability;2)Through biomechanical testing,including: ultimate tensile strength(Mpa),elongati-on at break(%)and modulus of elasticity.5.Anticoagulant observation Toluidine blue staining to observe whether successful crosslinking heparin1)The platelet adhesion test Cut each group decellularization kidney scaffold into0.5 cmx0.3 cm size tissue block,putted into the rich platelet plasma oscillation within6 h,in turn,by deionized water immersion,0.5% glutaraldehyde fixation,gradient dehydration,drying,gold-plated,finally by the Scanning electron microscope observe samples and gather image.6.Tissue compatibility testing Rats back on both sides of the midline incision through separation to take 1 cm size deep subcutaneous fascia layer to build four pouch,were implanted into four groups of specimens(1/4single kidney),in 3 d,1 w,2 w,4 w and 8 w remove subcutaneous implant stents,HE dyeing observation stent internal inflammation and angiogenesis.7.Cellular compatibility(1)CCK-8 cell proliferation-toxicity test Step 1: making standard curve cell count plate count the number of cells in the preparation of cell suspension,the cell suspension geometric dilution into 3-5 cell concentration gradient(each group to do four complex hole)vaccination in 96-well plates,37 ? 5% CO2 incubator 2 h incubation see cell wall,join the CCK 8(10 ul)2 h,enzyme standard instrument measuring 450 nm absorbance(OD value),making standard curve,among them,the X axis:cells;Y:OD value.Step 2: the determination of samples and test each renal support line of frozen section(10um thick)attached with six orifice climb standby,vascular endothelial cell count after(average of 7-10X104 / ml)from cell suspension,vaccination respectively in standbycrawl and blank piece and in 37 ? 5% CO2 incubator incubation 7 d,5 d,3 d,2 d,1 d,each hole to join 200 ul CCK 8 solution,into the incubator 2 h incubation,pipetting gun into 96-well plates,enzyme standard instrument measuring450 nm absorbance,according to the standard curve to calculate the cellsurvival rate,the inhibition rate.(2)Immunofluorescence staining Each groups of decellularization kidney scaffold by frozen section(10 um thick)attached with six orifice climb standby,vascular endothelial cell count after(an avera-ge of 7-10X104/ml)drop to climb cocultivation,three days after take out and Immunofluorescence staining observation,PBS cleaning residue medium,formaldehyde fixed,serum closed,add a resistance(Collagen IV antibodies anti rat,sma rabbit)resistance in hatch cassette in 4 ? refrigerator overnight,the next day,plus two drops resistance(755 green light anti-rat,694 red light anti-rabbit)and DAPI by fluorescence microscope observation.Results1.General observation Each group observation preserve decellularization kidney scaffold original shape,group D is translucent white,slightly collapse,the remaining three groups to renal scaffold morphology more solid than before crosslinking,crosslinking glutaraldehyde to cells after kidney is pale yellow in colour.2.Pathology observation HE staining and Masson staining to observe the each group of extracellular matrix(ECM)composition is complete,no fracture of collagen,is clearly visible clearly similar glomerular,renal tubules,outline of vascular structures,institutions such as continuous fiber and basement membrane integrity,renal glomerulus and there were no cells remain in small tube structure;Scanning electron microscopy(SEM)is clearly visible collagen continuous connection,no fracture,crosslinking group attached to the corresponding drug particles.3.The biomechanical stability and organization observation G+H group and G group renal stents degradation rate is far lower than the group D(P <0.05);Group D bibulous rate(94.88%)than(88.14%)in group G and G + H group(88.72%)(P < 0.05);Glutaraldehyde crosslinking increased after the mechanical performance of the decellularization kidney scaffold,including: the ultimate tensile strength and modulus ofelasticity(P < 0.05).4.Observation of heparin anticoagulant Crosslinking group(group H and G + H)of decellularization kidney scaffold not seen obvious blood platelet,but no heparin crosslinking group(group D and G)renal stent is full of platelets.5.Histocompatibility observation Back subcutaneous embedding the results showed that the kidney stent of non-crosslinking began to degrade after in the embedding two weeks,through GA crosslinked stent degradation phenomenon observed in 4 weeks,both groups of embedding kidney internal stent new blood vessels.6.Cellular compatibility Between groups of CCK 8 research suggest that the decellularization kidney scaffold and vascular endothelial cells after the co-culture,did not produce poisonous to the cells,compared with the blank group at the same time,show a certain role in promoting cell proliferation.The experimental group and control group of the decellularization kidney scaffold and renal vascular endothelial cell co-culture 3 days after immunofluorescence staining showed that cells can grow well attached to the bracket,showed good cell compatibility.Conclusion1.Compared with the pure decellularization kidney scaffold,through perfusion of glutaraldehyde and heparin crosslinking of the decellularization kidney scaffold with more stereo shape at the same time,a long time degradation in vivo and in vitro and the biological mechanics performance is more stable;2.Compared with the pure decellularization kidney scaffold,heparin after crosslinking have a certain anticoagulant properties;3.the decellularization kidney scaffold of crosslinked by glutaraldehyde and heparin can still keep the good histocompatibility and cell compatibility.