| Objectives:Isolate,culture and identify adipose-derived Stem Cells(ADSCs).Evaluate the effect of CS-HA-IGF-1C hydrogels,Chitosan(CS)-hyaluronic acid(HA)embellished by Insulin-like growth factor 1(IGF-1)on the biological characteristics of ADSCs in vitro and the survival of ADSCs in vivo.Establish the model of diabetic lower limb ischemia.Transplantation the pretreatment stem cells into the ischemic limbs to evaluate whether the CS-HA-IGF-1C can enhance the ADSCs paracrine role in order to improve its survival ability in the body,thereby promoting angiogenesis,improving blood perfusion and muscle regeneration,and reducing the rate of amputation of limbs.Discuss the mechanism of this biomaterials enhancing the therapeutic effect of cells.Meanwhile provide a new theoretical basis for the treatment of diabetes with lower limb ischemia by using tissue engineering means.Method:1.ADSCs were isolated from adipose tissue of 8-week-old healthy BALB/c male mice for in vitro culture and identification.2.The biocompatibility of the hydrogel was evaluated by in vitro culture of ADSCs with CS-HA-IGF-1C hydrogel.The conditioned medium of ADSCs cultured in hydrogel was used to conduct endothelial cell migration and tube formation experiments.The effect of the hydrogel on paracrine levels of ADSCs was observed.3.Evaluation of survival and resident of transplanted cells:AIE nanoparticles were used to label ADSCs and three-dimensional co-culture with hydrogels.21 BALB/c male nude mice were used to establish lower limb ischemia models and divided into 3 groups(7 in each group),including ADSCs group(106 ADSCs of lower extremity ischemia+intramuscular injection of 100 μL normal saline),ADSCs/CS-HA group(lower limb ischemia+intramuscular injection of 106 μL CS-HA hydrogel 106ADSCs),ADSCs/CS-HA-IGF-1C group(106ADSCs with lower limb ischemia+intramuscular injection of 100 μL CS-HA-IGF-1C hydrogel).All mice were monitored at 0,1,4,7,10,14 and 21 days after treatment using a CRi Maestro EX animal imaging system for dynamic in vivo survival monitoring of transplanted ADSCs.Establishment of diabetic lower limb ischemia model:70 male BALB/c mice were injected intraperitoneally with small dose(1%,50mg/kg)streptozotocin(STZ)to establish a mouse model of type 1 diabetes and the mice were tested.The tail tip blood glucose level determines the rate of molding.Four weeks after modeling,type 1 diabetic mice underwent lower limb ischemia and established diabetic lower limb ischemia models.All animals were divided into 7 groups(10 per group)according to the type of animal receiving treatment,including:Sham group(sham-operated group,diabetic mice were only vascularized 4 weeks after model establishment),Saline group(diabetic mice were modeled After 4 weeks+lower limb ischemia+intramuscular injection of 100μL normal saline),CS-HA group(4 weeks after modelling of diabetes,lower limb ischemia+intramuscular injection of 100 μL CS-HA hydrogel),CS-HA-IGF-1C group(diabetic mice 4 weeks after modelling+lower limb ischemia+intramuscular injection of 100 μL CS-HA-IGF-1C hydrogel),ADSCs group(diabetic mice 4 weeks after model establishment+lower limb ischemia 106ADSCs intramuscularly injected with 100 μL normal saline,ADSCs/CS-HA group(4 weeks after modelling of diabetes+lower limb ischemia+intramuscular injection of 100 μL CS-HA hydrogel 106 ADSCs),ADSCs/CS-HA-IGF-1C group(diabetic mice 4 weeks after modeling+lower limb ischemia+intramuscular injection of 100 μL CS-HA-IGF-1C hydrogel 106 ADSCs).The cell treatment group was a three-dimensional co-culture of ADSCs and hydrogel,and the cells were mixed with saline,CS-HA hydrogel liquid or CS-HA-IGF-1C hydrogel liquid(106ADSCs/100 μL).An insulin syringe was used to suck and inject into the injured muscle.The blood perfusion of the lower extremities was monitored by Doppler blood flow monitor at 0,7 and 21 days after cell therapy,and the PeriCam Psupporting system was used to analyze and obtain the average blood volume.Perfusion rate values;The condition of promoting angiogenesis,accelerating intramuscular cell proliferation,inflammatory cell infiltration levels and controlling the apoptosis ADSCs were evaluated in the early stage(7 days)after injured.The expression of interstitial collagen deposition,the deposition of muscle tissue fibrosis and proangiogenic factor expression was detected in late-stage injuried(21 days).Results:1.ADSCs has a strong ability to proliferate in vitro culture.2.After three-dimensional culture of stem cells and hydrogel materials in vitro,the CS-HA-IGF-1C hydrogel was found to promote cell proliferation and promote ADSCs migration and tube formation.3.Transplantation of AIE-labeled ADSCs into the ischemic lower extremities revealed that the CS-HA-IGF-1C hydrogel pretreated ADSCs could improve the survival of ADSCs in vivo 4.Doppler scanners were used to observe the blood perfusion in the lower extremities of mice.It was found that CS-HA-IGF-1C hydrogel combined with ADSCs transplantation significantly promoted blood flow recovery after ischemia in the lower limbs of mice.and it could promote muscle cell proliferation,reduce inflammatory cell infiltration and promote angiogenesis at early stage after injuried.Meanwhile it can significantly reduce intramuscular collagen deposition and apoptosis,and significantly inhibit muscle fibrosis at the late postoperative,thereby promoting ischemic lower limb histological and functional repair.Ultimately increase the limb salvage rate of damaged lower limbs.Conclusion:1.The diabetic lower limb ischemia model established by ischemic surgery of the lower limbs after T1DM model.There is a simple and effective method can be used.2.In vitro experiments,CS-HA-IGF-1C hydrogel can promote the proliferation of ADSCs,enhance the paracrine function of ADSCs,and have good biocompatibility.3.CS-HA-IGF-1C hydrogel can significantly improve the survival and residence of ADSCs in vivo;4.The ADSCs pretreatmented by CS-HA-IGF-1C hydrogel through improve the ADSCs survival in vivo and enhance its paracrine to promote angiogenesis and tissue repair after injection into damaged muscles. |
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