| Objective:Human acellular dermal matrix（ADM）is prepared and homogenized by a developed high-speed soft tissue homogenizer with controllable temperature,and to explore suitable preparation parameters of the injectable human ADM filling materials.The injectable human ADM filling material is evaluated through macroscopic observation,scanning electron microscope（SEM）observation,the measurement of particle size and particle size distribution.At the same time,L929 cells are used to detect the cytotoxicity of the injectable filling material and its effect on cell proliferation.The intradermal injection experiment of the prepared injectable human ADM filling material is performed on Wistar rats to judge the filling effect and the impact on the skin at the injection site of the injectable filling material prepared by the developed high-speed soft tissue homogenizer with controllable temperature by observing the rat skin thickness changes over time,skin histologic analysis at the injection site after intradermal injection.Explore a new approach to preparing injectable soft tissue filling materials and provide a reference of the preparation and application of clinical autologous soft tissue filling materials.Method of the experiment:This experiment includes two parts1.Preparation and evaluation of the injectable human ADM filling materialMethods:prepare human ADM with the Dispase-Triton method by using clinical abandoned full-thickness skin.ADM is homogenized by the developed high-speed soft tissue homogenizer with controllable temperature under the condition of different parameters to prepare injectable human ADM filling material whose exclusion criteria is to pass 27g needles after passing the 80 mesh and 120 mesh sieve,named ADM homogenate tissue I and ADM homogenate tissue II.And determine the preparation conditions of injectable human ADM filling material through the above step.The prepared materials are observed by eye and SEM,the particle size and distribution are determined,and the prepared human ADM is stained by HE and DAPI to realize the basic properties of the prepared filling materials.2.Cytotoxicity and in vivo experiment of the injectable human ADM filling materialMethods:the injectable human ADM filling materials prepared by the above methods,named ADM homogenate tissue I and ADM homogenate tissue II,are sterilized by 60Co 1kGy irradiation.When L929 cells are cultured to generation 10-13,the morphology of fibroblasts is basically stable,and logarithmic growth cells are selected.ADM homogenate tissue I and ADM homogenate tissue II suspensions are prepared.L929 cells are co-cultured with different concentrations of ADM homogenate tissue I suspensions and ADM homogenate tissue II suspensions on a 24-well culture plate,and blank control group,100%ADM homogenate tissue group,75%ADM homogenate tissue group,50%ADM homogenate tissue group,25%ADM homogenate tissue group and negative control group are set.On day 1,day 4,and day 7 after co-culture,the OD value of the cells at the wavelength of 450nm is detected by CCK-8.The OD value of each experimental group and the negative control group minus the OD value of the blank control group represents the corresponding actual OD value of the group,which is the cytotoxicity and biocompatibility of the injectable human ADM filling material.In addition,ADM homogenate tissue I and ADM homogenate tissue II are sterilized by 60Co 1kGy irradiation for intradermal injection experiments,Eight circular areas with a diameter of about 1.5cm are selected from both sides of the spine of fifteen adult male Wistar rats of clean grade and marked with markers,and eight injection sites are selected from both sides of the spine of adult clean male Wistar rats for intradermal injection,four on each side,with bilateral symmetry.From head to tail,followed by A group（experimental group 1）:intradermal injection of ADM homogenate tissue I（injection quantity:0.2 ml/site）,group B（experimental group 2）:intradermal injection of ADM homogenate tissue II（injection quantity:0.2 ml/site）,group C（control group）:No.4 and a half needles are evenly inserted into the dermis within the marked range without any injection（20 times/site）,group D（blank control group）:no treatment.Rats are numbered and feed separately.Five rats are randomly selected in 2w,4w and 6w after injection to get samples（n=10）and are generally observed at the injection site skin,whether there is inflammation reaction of surrounding tissue.HE staining and Masson staining are done to evaluate the degree of inflammation and fibrosis.HE staining and Image-Pro Plus 6.0 software are used to measure the thickness of dermis,compare the changes of dermal thickness over time to observe the effect of different treatments on dermal thickness.Result:1.ADM prepared by Dispase-Triton method is porcelain white,transparent,with visible collagen fibers separated from the edges.It is soft,elastic and has good toughness.HE staining and DAPI staining show no obvious cell residues.The injectable human ADM filling materials（ADM homogenate tissue I and ADM homogenate tissue II）prepared by high-speed soft tissue homogenizer with controllable temperature are milky and in state of suspension.2.Scanning electron microscopy（SEM）observation shows that ADM homogenate tissue I and ADM homogenate tissue II have heterogeneous particles,and a network of collagen fibers is observed to interlace with each other.After the particle size is determined by Image J software,Graph Pad Prism 8 software analyzes the data,and the particle size of ADM homogenate tissue I is（0.5470±0.2156）μm,while that of ADM homogenate tissue II is（0.4415±0.2128）μm.3.The co-culture results of ADM homogenate tissue I and II suspensions with L929 cells at different concentrations show that the cells present a trend of proliferation with the increasing days of culture.25%and 50%homogenate tissue I suspension have a certain effect on cell proliferation,while 75%and 100%homogenate tissue I suspension have a certain effect on cell proliferation in the early stage and a certain promoting effect in the later stage.Statistical analysis is carried out between them and the negative control group,and the difference is not statistically significant（P>0.05）.The 75%and 100%homogenate tissue II suspension have a certain effect on cell proliferation,and the 25%and 50%homogenate tissue II suspension hadve a certain promoting effect,which are statistically analyzed with the negative control group,and the difference is not statistically significant（P>0.05）.Moreover,statistical analysis is conducted between the ADM homogenate tissue group with different concentrations,and the differences are not statistically significant（P>0.05）,suggesting that the suspension of ADM homogenate tissues I and II with different concentrations have certain effects on cell proliferation,but there is no significant difference.4.The dermal thickness of group A and group B is thicker than that of group C and group D after that ADM homogenate tissue I and ADM homogenate tissue II are intradermally injected into Wistar rats,and the dermal thickness of group A is thicker than that of group B.With the change of time,the dermal thickness of group A and group C gradually decreases at 2w,4w and 6w postoperatively,while that of group B decreases at 2w to 4w postoperatively,but increases at 4w to 6w postoperatively.In addition,the difference of dermal thickness between group A and group C and group D is statistically significant at 2w,4w and 6w postoperatively（P<0.05）.The difference in dermal thickness between group B and group C and group D is statistically significant at2w and 6w postoperatively（P<0.05）,but not at 4w postoperatively（P>0.05）.The difference in dermal thickness between group A and group B is statistically significant at4w postoperatively（P<0.05）,but not at 2w and 6w postoperatively（P>0.05）.The difference in dermal thickness between group C and group D is not statistically significant at 2w,4w,and 6w postoperatively（P>0.05）.Histological observation shows that various inflammatory cells cluster in group A and group B,with more neutrophils in the early stage,lymphocytes in the later stage,and eosinophils and foreign body giant cells,while there are a small number of inflammatory cells in group C.In both group A and group B,A large number of fibroblasts are generated and fibrous tissue is generated in the early stage of the skin tissue.In the later stage,fibroblasts are reduced,and the fibrous tissue proliferates and become collagenous gradually,and the structure remodels,while a small number of fibroblasts are seen in the early stage.Conclusions:1.The injectable human ADM filling material with good performance can be prepared by the high-speed soft tissue homogenizer with controllable temperature developed by ourselves.2.The injectable human ADM filling material has no obvious cytotoxicity,does not affect cell proliferation,and has good biocompatibility.3.After the intradermal injection of the prepared injectable human ADM filling materials,the dermal thickness can be increased.And the dermal thickness increases significantly after intradermal injection of ADM homogenate tissue I,showing a better filling effect,suggesting that the size of ADM particles can affect the increase of dermal thickness.4.Collagen proliferation and tissue remodeling may occur after intradermal injection of the injectable human ADM filling material. |
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