【作者】 贺龙龙；

【导师】 刘彦普；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2013， 博士

【摘要】 背景:自体脂肪组织抽吸注射移植是目前软组织修复常用的方法，然而无法预知的吸收率（20%～90%），常常需要多次注射或过度移植，移植效果的个体差异明显。本课题组在研究中发现，在脂肪颗粒中复合PRF和（或）SVF可以有效的降低移植组织吸收率，并且在小动物模型中得到有效的实验论证，现拟通过大动物实验进一步观察这种思路的可行性。目的：建立小型猪脂肪移植的动物模型，制备可注射式AG（脂肪颗粒）复合PRF（富血小板血浆）和（或）SVF（血管基质成分）混合物，在猪耳背部，猪腹部植入皮肤扩张器后的皮下，猪背部软组织缺损皮下进行注射移植，通过观察不同实验组中移植组织的吸收率及组织学检查结果来评价新的移植方式的动物实验效果。方法：1.实验分组：对照组为单纯脂肪颗粒组分；实验组A为脂肪颗粒+PRF组分；实验组B为脂肪颗粒+PRF+SVF组分。2.在贵州小型猪中建立制备AG（脂肪颗粒）复合PRF（富血小板血浆）和（或）SVF（血管基质成分）混合物的手术模型，在猪耳背皮下，猪侧腹部扩张器皮肤下，猪背部软组织缺损皮下注射移植脂肪组织。3.在不同观察点通过卡尺和B超测量观察和统计比较不同实验组的吸收率的变化。4.对组织标本进行组织学检测（HE染色，油红染色，FⅧ因子免疫组化染色，Tunel细胞凋亡检查）比较不同实验组的差别。5.使用SPSS统计学软件对实验数据进行分析。结果：1.在贵州小型猪中建立了AG（脂肪颗粒）复合PRF（富血小板血浆）和（或）SVF（血管基质成分）混合物的手术模型，猪耳背部软组织移植、猪腹部皮肤扩张器皮下软组织移植、猪背部软组织缺损皮下软组织移植的实验模型。2.在猪耳背部皮下移植模型中，按照20ml，10ml和5ml阶梯体积进行注射移植实验，对照组︰实验组A︰实验组B按照1︰1︰1进行，每组6例。单次大剂量注射脂肪组织20m l，移植腔隙为类圆形，直径约5cm，移植物形成高度约2.5cm。对照组移植体积存活率约为6.25%（SD3.59），实验组A存活率约为9.27%（SD3.69），实验组B存活率约为12.96%（SD4.02）。单次大剂量注射脂肪组织10ml，移植腔隙为类圆形，直径约5cm，移植物形成高度约1.3cm。对照组移植体积存活率约为10.28%（SD2.42），实验组A存活率约为21.88%（SD4.34），实验组B存活率约为31.00%（SD4.29）。单次大剂量注射脂肪组织5ml，移植腔隙为类圆形，直径约5cm，移植物形成高度约0.9cm。对照组移植体积存活率约为31.01%（SD10.76），实验组A存活率约为30.80%（SD7.01），实验组B存活率约为46.75%（SD9.04）。对数据进行单因变量（存活率）多因素（阶梯体积和实验组别）方差分析，结果显示实验组B移植存活率要高于实验组A，实验组A移植存活率要高于对照组；5ml移植组存活率高于10ml移植组，10ml移植组存活率高于20ml移植组，其组间差异具有统计学意义。对组织标本检查中发现，对照组组织标本断面出血点较少，标本组织不均一，有坏死变形的脂肪结节，正常的脂肪组织比例较小；实验组A组织标本断面有丰富出血点，血管较粗，大小不均匀，未见明显的坏死变性的脂肪结节，脂肪组织比例较多；实验组B组织标本断面出血丰富，出血点细小均匀，组织均匀一致。在对组织血管数量的单变量多因素分析中，在20ml移植组中，实验组B的血管数量高于实验组A，实验组A高于对照组；在10ml和5ml移植组中对照组的血管数量高于实验组A，高于实验组B，其差异具有统计学意义。Tunel凋亡检测中观察到对照组中所有的凋亡染色均有大量的凋亡细胞，而实验组A和实验组B的切片中凋亡细胞数量明显减少，而5ml，10ml和20ml组间没有明显区别。在油红染色中，对照组移植后的组织中脂滴被染成红色，脂肪泡体积一致性较差，散在分布；在实验组中，脂肪泡体积一致性较好，分布较均匀，脂滴间隔为结缔组织细胞，实验组A和实验组B差异不明显。3.建立9例20ml扩张器模型，移植术后均发生严重吸收，切开皮肤后发现皮肤增厚明显，皮下少量瘢痕结缔组织，脂肪组织存活率低，对照组同实验组A和实验组B无明显差别。4.完成软组织缺损脂肪复合物移植18例，其中6例作为对照组，6例作为实验组A，6例作为实验组B。对照组脂肪移植量为13.60±3.05ml，术前脂肪层厚度为13.85±2.71mm，邻近脂肪层厚度为39.55±2.48mm，移植增加了2.85±0.82mm脂肪层厚度；实验组A脂肪移植量为11.00±2.24ml，术前脂肪层厚度为12.74±1.69mm，邻近脂肪层厚度为39.54±1.97mm，移植增加了11.27±5.51mm脂肪层厚度；实验组B脂肪移植量为11.20±1.30ml，术前脂肪层厚度为13.35±2.00mm，邻近脂肪层厚度为42.34±1.69mm，移植增加了16.73±3.27mm脂肪层厚度。由于移植物区域的面积测量存在困难，仅观测移植区域中心点脂肪层厚度变化来评估移植物的吸收存活情况，对照组中移植物厚度存活率为11.04±2.80%，实验组A移植物厚度存活率为40.94±16.79%，实验组B移植物厚度存活率为56.80±10.19%，一维方差分析组间差异具有统计学意义；LSD两两比较其差异均具有统计学意义。实验组A和实验组B的血管计数少于对照组，实验组B和实验组A同对照组的主要区别是单个棕染的团块细胞数量较少，提示为较小的血管生长丰富。结论：1.使用猪作为实验对象进行脂肪移植是可行的选择之一。2.在脂肪颗粒中复合PRF和（或）SVF在单腔隙注射移植5ml，10ml和20ml的实验观察中，脂肪颗粒中复合PRF和（或）SVF移植的吸收率小于单纯使用脂肪颗粒进行移植的吸收率，脂肪颗粒复合PRF和SVF作为移植材料的吸收率小于脂肪颗粒复合PRF进行移植的吸收率，差异均具有统计学意义。3.在猪腹部制作皮肤扩张器模型，并在腔隙中进行移植实验观察，实验结果显示实验组和对照组移植物均发生严重吸收，提示在此实验构想可行性较低。4.在猪背部制备的软组织缺损模型中进行脂肪组织移植实验观察，实验结果提示在脂肪颗粒中加入PRF和（或）SVF有着较好的移植效果。5.使用自体血液加工制备富含生长因子及纤维蛋白成分的PRF，自体脂肪组织加工制备富含脂肪干细胞及脂肪前体细胞的SVF，将其加入自体脂肪组织作为移植复合物进行移植可以有效的减少移植吸收率，并可能使得存活组织状态更稳定，更成熟。更多还原

【Abstract】 Background: Injectable autologous fat tissue transplantion of liposuction is a commonmethod in soft tissue reconstruction, but it often needs repeat injections or over-filling dueto unpredictable absorption rate (20%～90%). In earlier studies, an improve of survivalpercentage when PRF and (or) SVF was added to adipose granule before transplantion invitro studies and vivo studies in rabbits.Purpose: Establishedment of fat transplantation model in minipig, preparation ofinjective adipose granule, PRF and SVF composites, subcutaneous injection of thecomposites to different sites of the animal model. The absorption rates of the experimentalgroup and the control group were calculated and compared to evaluate this news ways ofadipose tissue transplantion.Methods:1. Establishedment of fat transplantation model in minipig: a. the preparation ofinjective adipose granule, PRF and SVF composites; b. subcutaneous injection of thecomposites in the ears of minipigs; c. injection of the composites in the cavity in theabdominal prepared by a soft tissue expander; d. subcutaneous injection of the compositesin the soft tissue defect in the back of the minipigs.2. Caliper and B-mode ultrasound were used to measure the volume changes of thegraft materials.3. The tissue specimens were prepared for histological examination: hematoxylin andeosin (H&E) staining; Oil-red O staining; Factor VIII immunohistochemical staining forVessel count; Cell apoptosis using Tunel technique. Differences between the experimentalgroup and the control group were analyzed.Results:1. The fat transplantation models in minipig were established: a. the preparation of injective fat particles, PRF and SVF composites; b. subcutaneous injection of thecomposites in the ears of minipigs; c. injection of the composites in the cavity in theabdominal prepared by a soft tissue expander; d. subcutaneous injection of the compositesin the soft tissue defect in the back of the minipigs.2. Subcutaneous injection of the composites in the ear of minipigs: The ratio of controlgroup, experimental group A and group B was1︰1︰1, each group included6cases.When20ml of the adipose composites was injected, the diameter of the donor site wasabout5cm, the height of the donor site was about2.5cm, the survival percentage of thecontrol group, experiment group A and experiment group B was6.25%(SD3.59),9.27%(SD3.69) and12.96%(SD4.02), respectively; When10ml of the adipose composites wasinjected, the diameter of the donor site was about5cm, the height of the donor site wasabout1.3cm, the survival percentage of the control group, experiment group A andexperiment group B was10.28%(SD2.42),21.88%(SD4.34) and31.00%(SD4.29),respectively; When5ml of the adipose composites was injected, the diameter of the donorsite was about5cm, the height of the donor site was about0.9cm, the survival percentageof the control group, experiment group A and experiment group B was31.01%(SD10.76),30.80%(SD7.01) and46.75%(SD9.04), respectively.Single dependent variable multi-factor ANOVA analyses show that the surviralpercentage of experiment group B was higher than experiment group A, the surviralpercentage of experiment group A was higher than control group; the surviral percentageof5ml group was higher than10ml group, the surviral percentage of10ml group washigher than20ml. All the differences between groups were statistically significant.The vessel counts of the specimen after Factor VIII immunohistochemical stainingwere anaylized by Single dependent variable multi-factor ANOVA, the results show that in20ml graft groups, the experiment group B had higher vessel count than the experimentgroup A, the experiment group A had higher vessel count than the control group, while in10ml and5ml graft groups, the control group count highest, then the experiment group A,the experiment group B count lowest. The differences between groups were statisticallysignificant.Cell apoptosis using Tunel technique showed that there were a lot more apoptotic cellsin control groups than experimental groups, but no obvious difference between5ml,10mland20ml groups. Oil-red O staining of the specimen showed that the fat lipid droplets were dyed red,the fat bubbles consistency was poor in control groups, the fat bubbles consistency wasbetter in experiment groups, but no siginificant difference between the experiment group Aand group B.3. Injection of the composites in the cavity in the abdominal prepared by soft tissueexpander:9models were set up,3for control group,3for experiment group A, and3forexperiment group B, but all the cases were found severe absorbed, no obvious adiposetissue were found during ultrasound exam and sample surgery, no statistics differencebetween experiment group A and group B.4. Subcutaneous injection of the composites in the soft tissue defect in the back of theminipigs:18cases of the models were set up,6for control group,6for experiment groupA, and6for experiment group B. In the control group, the volume of tissue graft was13.60ml (SD3.05), the thickness of the adipose tissue before the transplantation was13.85mm (SD2.71), and the augmented soft tissue was2.85mm (SD0.82); In theexperiment group A, the volume of tissue graft was11.00ml (SD2.24), the thickness ofthe adipose tissue before the transplantation was12.74mm (SD1.69), and the augmentedsoft tissue was11.27mm (SD5.51); In the experiment group B, the volume of tissue graftwas11.20ml (SD1.30), the thickness of the adipose tissue before the transplantation was13.35mm (SD2.00), and the augmented soft tissue was16.73mm (SD3.27).There was no statistic difference between the groups of volume of tissue graft, andthere was no statistic difference between the groups of the thickness of the adipose tissuebefore the transplantation, but there was statistic difference between the groups of theaugmented soft tissue.The survival percentage of the augmented thickness of adipose tissue of the controlgroup, the experiment group A and B was11.04%(SD2.80),40.94%(SD16.79) and56.80%(10.19) respectively. ANOVA analyse showed there was statistic differencebetween groups.The vessel count showed that the experiment group A and B had less vessel numberthan control group. The difference between the experiment group A and B was that the cellnumber of each vessel count less in the experiment group B than group A, which maightindicate the adipose tissue in experiment group B was more mature and stable.Conclusions:1. Using minipig as one of the subjects of adipose tissue transplant is a viable option. 2. Subcutaneous injection of5ml,10ml and20ml of the composites in the ears ofminipigs shows injection of adipose granule+PRF and adipose granule+PRF+SVF havebetter result than injection of adipose granule only, and injection of adiposegranule+PRF+SVF shows the best results.3. The idea of injection of the composites in the cavity in the abdominal prepared bya soft tissue expander may not be a viable option.4. Experiments of subcutaneous injection of the composites in the soft tissue defect inthe back of the minipigs show that adipose granule+PRF+SVF composites have betterresult in adipose transplantation.5. When PRF and (or) SVF is added in adipose granule during transplantation, thesurvival percentage of the graft material is greatly increased, and the augmented tissuelooks more autologous tissue alike, maybe more mature and stable.更多还原