The Rat Decellularized Gracilis Bio-scaffold Was Prepared By Perfusion Method

Objective: To establish an experimental model of gracilis muscle flap in rats for microsurgery,ischemia-reperfusion and regenerative medicine through the detailed vessel anatomical observation of gracilis muscle flap and summarized surgical techniques.Methods: Six SD rats(280-300g)were perfused with Microfil dye and anatomical observation of gracilis muscle flap and micro CT scan.Six Lewis rats(250-280g)were observed of the vessel of gracilis muscle flap through the surgery.Nine Nude rats(150-300 g)were observed through the surgery.Six fresh frozen gracilis muscle flap were perfused with Indian ink in 3 groups at 0.2ml / min,0.5ml / min and 1.0ml /min.Twelve fresh gracilis muscle flap were perfused with Indian ink at 0.5 ml /min.These flaps were evaluated by gross morphology,histology,integrity of the microcirculatory networks,and DNA quantification.Results: The vascular anatomy of different species and different weight rats and femoral muscle were similar.It was easy to distinguish between gracilis and semitendinosus muscle gap in limb abduction position.The Femoral arteryand femoral vein were accompanied by the femoral nerve.The femoral artery has three main lateral branches and two medial branches which are to anterior and posterior to the gracilis muscle.Gracilis muscle derives from medial sacral and inserts in medial femur.The obturator nerve had anterior branch and deep branch with blood vessels which are to anterior and posterior to the gracilis muscle.The femoral artery of gracilis muscle was inserted into the catheter of the diameter of 0.75 mm.The femoral vein gracilis muscle flap was opened and fluid returned after perfusion of the femoral artery with heparin saline.There is no obvious no swelling and fluid leaking in whole gracilis flap.The femoral vein gracilis muscle flap did not have fluid returned in 3 frozen Nude rats with swollen muscle.After perfusion of the femoral artery with India ink at different flow rate,there was a leaking of India ink out of the gracilis muscle.The gracilis flap at 0.5ml / min perfusion was observed to be a little swollen while the vascular of muscle of the perfusion of ink was uniformly distributed.Histology of hematoxylin and eosin stain showed the ink in the muscle capillary network in 12 flesh gracilis muscle of the perfusion of ink at 0.5ml/min.Conclusion:SD,Lewis and Nude rats,weighing from 150 to 300 g,were easier to harvest the gracilis muscle with femoral pedicle according to our surgery method.The gracilis muscle can obtain a longer vascular pedicle and variety.The femoral artery can be inserted into the catheter of the diameter of0.75 mm.The first anatomy foundation is the nutrition vascular integrity of gracilis muscle flap.The nutrition vessels of gracilis muscle flap are from the femoral artery muscle vessels,also from the vessels accompanied by adductor muscle branch nerve.The capillary of gracilis muscle flap existed visible ink at0.5 ml / min perfusion rate of India ink.The outcome will provided the evidenceof the appropriate flow rate for the perfusion method of decellularized gracilis muscle flap in ratsObjective: To investigate the effect of the artery perfusion method to construct a rat decellularized gracilis muscle flap scaffold.Methods: Twenty gracilis muscle flaps were harvested under the microscope.The femoral artery was perfused with detergent at 0.5ml / min through the inserted catheter of the diameter of 0.75 mm of the femoral artery.The decellularized gracilis muscle flaps were evaluated by gross morphologic examination,vascular perfusion observation,histological examination,confocal microscopy,transmission electron microscopy and DNA quantification.Results: Twenty gracilis muscle flaps were harvested successfully.The size of gracilis muscle flap is about 2.5cm X2.0cm,weight 268±42mg.Fourteen decellularized rat gracilis muscle flaps were perfused for decellularized scaffold.The decellularized gracilis muscles flaps are about 2.2cm X1.8cm,weight160±30mg.The appearance of scaffolds were translucent.The size of scaffolds was smaller than normal gracilis muscle flap.The decellularization effect of one gracilis muscle flap was not good.The vascular perfusion in the decellularized gracilis muscle showed the integrity of arterial and venous network,but the femoral vein cannot reflux arterial infusion fluid.Optical inverted microscope can observe muscle translucent and intramuscular vascular network.The alternate perfusion of PBS and DMEM was uniformly distributed in the decellularized gracilis muscle.Hematoxylin and eosin stain of normal muscle showed the multiple nuclei and fibers arranged,while the decellularized gracilis muscle flaps showed extracellular structure and no nuclei.Hematoxylin and eosin stain of normal nerves showed the nerve bundle membrane clearly and densely,with blue-stained nuclei,while the decellularized femoral nerve and adductor nerve showed nerve bundle membrane loose,with no blue-stained nuclei.Hematoxylin and eosin stain of femoral artery and vein show the bluestained nuclei of endothethiel cell in the vessel while the decellularized femoral artery and vein showed no blue-stained nuclei and extracellular matrix.Dapi stain of the normal muscle showed blue-stained nuclei under the confocal microscopy observation while the decellurizaed muscle showed no blue-stained nuclei imaging.Transmission electron microscopy of the normal muscle showed complete sarcoplasmic,plastosome and myofibrils while the decellurizaed muscle retains muscle ultrastructure,such as collagen and elastic fibers.The decellurizaed muscle showed no sarcoplasmic and no nuclei of muscle cell.Transmission electron microscopy of the flesh nerve showed the integrity structure of myelin and axons.While the decellularized nerve showed no myelin and no axons,showing collagen,elastic fibers and complete basement membrane.The DNA content of the normal dry weight muscle was 4896 ±1303ng/mg,while the decellularized dry weigh muscle was 214 ± 37 ng/mg (P=0.0001).The decellularized muscle was removed 95.7% DNA content of normal muscle.Conclusions: The perfusion method at 0.5 ml/min is effective for rat decellurlarized gracilis muscle flap.The scaffold of vascular pedicle flap had intact arteries and vascular network structure.