| Objective: After the dermal tissue and normal saline were mixed,the self-developed Controllable temperature high speed soft tissue homogenizer(hereinafter referred to as homogenizer)was used to prepare injectable human micronized dermal homogenate(hereinafter referred to as dermal homogenate).The preparation conditions were explored by taking 27 G needle as the exclusion standard.The dermal homogenate was observed by naked eye and scanning electron microscope.HE staining section and DAPI staining of dermal homogenate were observed under microscope.The macro structures and micro structures of dermal homogenate were explored.The cytotoxicity of dermal homogenate to fibroblasts was detected by cck-8 kit.Thus,the feasibility of using injectable human micronized dermal homogenate as injectable soft tissue filling material was explored.By observing its degradation in the dermis of Wistar rats,the filling effect of dermis homogenate as injectable soft tissue filling material in animals was studied.In order to explore a good injectable soft tissue filling material.Methods1.In aseptic condition,after removing the extra subcutaneous fat and superficial fascia of skin tissue,the epidermis was removed with 0.5% Dispase ? enzyme.The volume ratio of dermal tissue to normal saline was 1:3,1:5,1:9,the speed of homogenizer was 1000 R / min,3000 R / min,5000 R / min,the time was 7 min,9 min,11 min,and 27 groups of preparation parameters were formed.Under the condition of each group of parameters,the suspension was prepared,and the leather suspension was screened through 80 mesh and 120 mesh,respectively,to prepare injectable human micronized leather homogenate with different particle size range.According to the standard that leather homogenate can pass through 27 G needle without resistance,the appropriate volume ratio,rotation speed and time were obtained,so as to explore the relevant parameters of preparing leather homogenate.In aseptic environment,theinjectable human micronized dermal homogenate was prepared according to the above parameters.The dermal homogenate obtained through 80 mesh and 120 mesh sieve was recorded as liquid a and liquid B respectively.After centrifugation of liquid a and liquid B,part of the supernatant was discarded to make the volume ratio of dermal tissue to normal saline about 1:1.After shaking,it was sealed and stored in a refrigerator at 4 ? In animal experiments.2.The gross appearance of dermal homogenate was observed by naked eyes,he staining and DAPI staining of dermal homogenate were observed under microscope,the particle size and homogeneity of dermal homogenate were observed under scanning electron microscope,and its macro and micro structure were explored;after centrifugation of dermal homogenate,the precipitated dermal tissue was obtained,so as to extract human fibroblasts and expand the culture,and the third generation of human fibroblasts were inoculated into the dermal homogenate In order to detect the cytotoxicity of dermal homogenate to human fibroblasts,we used CCK-8 kit to detect the OD value of cells at 450 nm.3.Take 15 Wistar rats,make 4 circles with diameter of 1cm on the back side of the rats with eyebrow tattoo liquid as the mark,the marked range is the injection area,respectively recorded as group A,B,C and D,use 27 G needle,1ml empty needle,take 0.2ml of liquid a and liquid B respectively and inject them into the skin and dermis deep layer around the experimental range of group A and group B on the back of the rats,multi-point,even and scattered in group C with No.4 half needle,group D does not do any work As a blank control group,5 rats were randomly selected at the 2nd,4th and 6th week after injection.The whole skin layer of each experimental area on the back of the rats was cut and stained with he and Masson.The inflammatory changes,ultrastructure,fibroblasts and collagen hyperplasia of dermis were observed.The thickness of dermis was measured by Image Pro Plus 6.0 software.The results were statistically processed by spss24.0 software.Results: 1.The injectable human micronized dermal homogenate with 27 G needles can be prepared by using a homogenizer.The parameters of the preparation are: the volume ratio of dermal tissue to normal saline is 1:5,the rotating speed is 3000 r / min,and the time is 9min.2.In general,the homogenate of dermis is a milky white homogenous suspension.After centrifugation,it can be divided into two layers: the upper layer is clear clear and colorless supernatant,and the lower layer is white muddy dermis tissue sediment.Inverted phase contrast microscope is used to observe the precipitate HE staining section to see fibroblast nucleus and collagen fibers with loose arrangement.Scanning electron microscope is used to observe the smear of dermis homogenate tosee dermis The size of homogenate particles in the field of vision was measured and the average value was taken.The size of solution a was(982.6 ± 238.5)nm,and that of solution B was(161.7 ± 36.6)nm.3.DAPI staining of dermal homogenate was observed under fluorescence microscope,and the scattered nuclei were observed.Fibroblasts were extracted from dermal homogenate by collagenase digestion,and the activity of fibroblasts was good.The cytotoxicity of dermal homogenate to fibroblasts was detected by CCK-8 kit,and the OD value of 450 nm wave length was measured.The results showed that OD increased with the increase of culture days.The results showed that there was no significant difference between the two groups(P > 0.05).4.After the dermal homogenate was injected into the body of Wistar rats,the experimental area on the back of the rats was observed on the third day or so.It was found that the skin in the injection area of group A and B had slight redness and swelling,no secretion and other inflammatory reactions.The redness and swelling in group C were obvious.After one week,the redness and swelling gradually disappeared,and the skin color at the rear mark gradually returned to normal.5.At the 2nd,4th and 6th week,5.At the 2nd,4th and 6th week,5 rats were randomly selected.No effusion or pyogenesis was found after skin incision.A.The thickness of dermis in group B was thicker than that in group C and Group D.with the change of time,the thickness of dermis in group A and group B was stable at the initial stage,but decreased slightly at the later stage,and remained stable in group C and Group D.Histological observation showed that there were a large number of fibroblasts,neutrophils and lymphocytes in group A,B and C at the early stage.After that,neutrophils gradually decreased and lymphocytes increased.Group D was normal tissue,and no obvious inflammatory cell changes were observed.At 2,4 and 6 weeks after the injection,the dermal thickness of group A was statistically different from that of group C and group D(P < 0.05);the dermal thickness of group B was statistically different from that of group C and group D(P < 0.05);there was no significant difference between group C and group D(P > 0.05).Conclusion1.With the self-developed homogenizer,the injectable human micronized dermal homogenate with 27 G needles can be successfully prepared,and the relevant parameters of preparation are: the volume ratio of dermal tissue to normal saline is 1:5,the rotating speed is 3000 r / min,and the time is 9min.With naked eyes,the homogenate of dermis is a homogenous suspension with milky white color.The particle size of dermis is uniform,reaching nanometer level.Collagen in the homogenate of dermis isloose.The dermal homogenate has good activity of fibroblasts and no cytotoxicity to human fibroblasts,so it is feasible to be used as injectable soft tissue filling material.2.Injection of human micronized dermal homogenate into the deep layer of animal dermis can directly and indirectly promote the thickening of dermis tissue,so as to play the role of anti-aging skin.It is expected to become a new injectable soft tissue filling material with good performance and easy to obtain. |
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