| Objective:1.After mixing human adipose tissue and normal saline in a specific proportion,human adipose tissue homogenate was prepared by the self-developed temperature controlled high-speed soft tissue homogenate equipment.The optimal preparation conditions of the homogenate were explored based on the fact that the homogenate could be passed through a 27 G needle(inner diameter 0.21mm).2.The tissue structure of homogenate and whether it contained ADSCs were observed.3.To observe the filling effect and degradability of homogenate injected into the back subcutaneously of Wistar rats,and to explore the feasibility of human adipose tissue homogenate as a new injectable soft tissue filling material.Methods:1.The purification of the fat particles and normal saline mixed according to 1:1,1:2 and 1:3,the homogenate equipment speeded to 3000 rpm,4000 rpm,5000 rpm,homogenate time seted to 30 seconds,60 seconds,90 seconds,120 seconds,150 seconds,180 seconds,in each of the parameters under the condition of the preparation of adipose tissue homogenate,homogenate through 80 mesh sieve(0.2mm diameter)to remove the thick fibrous connective tissue,there was no resistance in homogenate could through 27 G needles for the standard,the optimum volume of fat tissue and normal saline,optimum speed and time of homogenizer.2.The color and texture of human adipose tissue homogenate were observed by naked eyes;The histological structure of HE,Masson and DAPI staining were observed by microscope.The particle size was observed by scanning electron microscope.To observe whether adipose tissue-derived stromal cells were contained in human adipose tissue homogenate by cell culture.3.Fifteen wistar rats were selected,and 1m L human adipose tissue homogenate was injected subcutaneously into the back of both sides of the rats.The control group was injected with 1m L nanofat 2.0 and 1m L granulated adipose.Three rats were randomly selected at 1,2,4,6 and 8 weeks after surgery,and subcutaneously grafts were removed from the back of the rats.The color,texture,liquefaction,cyst,necrosis and calcification of the graft were observed.Weighing the weight of the graft,measuring the volume of the graft and calculating the retention rate of graft volume;The grafts were stained with HE and Masson to observe the structural changes and fibrosis degree of the grafts.Immunohistochemical staining of CD31 was used to observe the angiogenesis of the grafts.Immunohistochemical staining of CD68 was used to observe the infiltration degree of macrophages.Results:1.The homogenate equipment could prepare human adipose tissue homogenate that could pass through a 27 G needle.The optimal parameters were as follows: the optimal volume ratio of adipose tissue and normal saline was 1:1,the optimal rotation speed of the homogenizer was 4000 rpm,and the optimal homogenizing time was 120 seconds.2.The macroscopic observation in homogeneous emulsion state homogenate,yellowish-white appearance.HE staining showed that the structural integrity of adipocytes was destroyed and some adipocytes were preserved.Masson staining showed that most of the structure of the adipose extracellular matrix was intact.DAPI staining shows that some of the living adipose nuclei have been stained blue.Scanning electron microscopy showed that the size of homogenate particles was approximately uniform,and the diameter of homogenate particles was(27.00±4.76)μm.On the 10 th day of homogenate cell culture,there was no obvious adipose tissue-derived stromal cells.After repeated several times of cell culture,there was still no obvious adipose tissue-derived stromal cells.3.Human adipose tissue homogenate,nanofat 2.0 and granular fat were injected subcutaneously into the back of Wistar rats.(1)Graft gross observation: At the first week,the graft was forming a complete capsule,with the surrounding tissue boundaries clear,the graft was circular,graft groups in terms of quality of a material soft and rich flexibility,good gloss,implant surface by capillary bag around,no obvious fat liquefaction,necrosis,cyst,induration,calcified form;At the second week,the gross appearance and texture of the grafts in each group were similar to those in the first week.A little fat liquefaction was observed in the grafts in all three groups,and the formation of oil sacs was visible to the naked eye,but no cyst,sclerosis or calcification was found in the grafts.There were more capillaries around the surface of the grafts in the three groups than that in the first week.At the 4th week,the gross appearance and texture of the grafts were similar to those of the 2nd week.There were more fat liquefaction in the grafts of the three groups without cyst,sclerosis or calcification.At 6-8 weeks,there was calcification of homogenate fat and granular fat in 1 case,and the texture became hard.No obvious calcification or sclerosis was observed in the other grafts,and a small amount of blood vessel wrapping was observed on the graft surface.(2)Graft mass,volume and volume retention: The variation trend of graft mass,volume and volume retention was basically the same.At the first week,the mass,volume and volume retention of the three groups were higher than the initial level of transplantation.At the second week,the mass,volume and volume retention of the three groups were close to the initial level of the graft.At 4-8 weeks,the mass,volume and volume retention of the grafts in the three groups decreased gradually.(3)HE staining of the graft: At the first week,adipocytes in the center of the graft were necrosis.Peripheral adipocytes were different in size and irregular in arrangement,and the interstitial of adipocytes was widened.Inflammatory cell infiltration and neovascularization were observed around the graft,and hyperplasia of fibrous connective tissue was observed.At the second week,the adipocytes were different in size and irregular in arrangement,and some of the vacuoles fused into large oil sacs.There were more inflammatory cell infiltration,more fibrous connective tissue and new blood vessels in the peripheral and center of the graft.At week 4-8,the adipocytes were irregular in size,and some of the vacuoles fused into larger oil sacs with less inflammatory infiltration and less fibrous connective tissue than before.(4)Masson staining of grafts: Over time,a large amount of fibrous connective tissue hyperplasia was observed in all three groups,reaching a peak at the second week,followed by a gradual decrease in collagen fiber content in all three groups.(5)Immunohistochemical CD31 staining of the graft showed that vascular endothelial cells were stained brown.Some endothelial cells formed microvessels,some showed single or clustered endothelial cells,and some showed cord shape without obvious lumen.During the 1-2 weeks,the number of graft blood vessels in the three groups gradually increased,the homogenate fat group and the granulated fat group reached the peak in the 2nd week,and the nanofat 2.0 group reached the peak in the 4th week,and then the number of graft blood vessels in the three groups gradually decreased.(6)Immunohistochemical CD68 staining of the graft showed the brownish-yellow staining of macrophages.As time went on,the number of graft macrophages in the three groups gradually increased and reached a peak in the second week,then the number of graft macrophages in the three groups gradually decreased.Conclusions:1.Human adipose tissue homogenate that could pass through a 27 G needle was successfully prepared by using a self-developed high-speed soft tissue homogenizer with controllable temperature.The optimal parameters of preparation were as follows: the optimal volume ratio of fat to normal saline was 1:1,the optimal rotation speed of the homogenizer was 4000 rpm,and the optimal homogenizing time was 120 seconds.2.The prepared homogenous human adipose tissue homogenous emulsified,yellow and white appearance,the extracellular matrix structure was complete,there were some alive adipose cells,with some activity.3.The homogenate of human adipose tissue was injected subcutaneously into the back of wistar rats,which could play a certain filling effect and was expected to be a new injectable soft tissue filling material. |
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