交联透明质酸对颗粒脂肪移植成活影响的实验研究

Experimental study on the effects of cross-linked hyaluronic acid on the survival of microfat grafting

目的探讨交联透明质酸(CLHA)对颗粒脂肪移植成活的影响。方法72只8周龄SPF级雌性BALB/c裸鼠,体重22~24 g分为两部分,其中24只用于确定定制CLHA的代谢情况,48只用于研究CLHA对颗粒脂肪移植后成活的影响。24只裸鼠按随机数字表法分为A、B、C、D组,每组6只;于裸鼠背部皮下注射0.3 ml定制CLHA,术后1、2、4、12周分别处死A、B、C、D组所有裸鼠,测定定制CLHA剩余体积及HE染色情况。48只裸鼠按随机数字表法分为实验组与对照组,每组24只。实验组裸鼠背部皮下移植0.3 ml定制CLHA与0.3 ml人颗粒脂肪混合物,对照组仅移植0.3 ml人颗粒脂肪。各组分别于术后1、2、4、12周处死6只裸鼠,比较两组脂肪组织体积保留率、组织学评分、脂周蛋白-1(Perilipin-1)(+)活脂肪细胞占比、CD31(+)血管数量和血管内皮生长因子(VEGF)光密度值及有效体积保持率。结果随着时间推移,移植后定制CLHA的肉眼观和实测体积均逐渐减小,D组不再可见定制CLHA。A组可见大量成片淡蓝紫色的定制CLHA分布在组织间隙中;B组可见间质出现纤维化和炎症细胞浸润,定制CLHA呈分散分布;C组可见组织间质疏松、增宽,间质纤维化加剧,炎症细胞浸润增加,定制CLHA面积有所减少;D组视野中未见定制CLHA,注射部位皮下结构与正常皮下结构相似。实验组在术后4周时新生血管最多,而术后12周时则较前减少;对照组各时间点移植物整体新生血管分布较均匀。术后1、2、4、12周,实验组体积保留率均低于对照组(P<0.05)。实验组移植物脂肪细胞间可见较多炎症细胞浸润和细胞外基质沉积,新生血管则主要集中于移植物外周;对照组则以结构较完好的脂肪细胞为主,炎症细胞浸润和细胞外基质沉积则较少。术后1、2、4、12周,实验组脂肪细胞完整性评分低于对照组,纤维化区域和炎症细胞浸润评分高于对照组(P<0.05);术后1、4、12周,实验组坏死区域评分高于对照组(P<0.05);术后2、4、12周,实验组油囊/空泡评分低于对照组(P<0.05)。实验组的活脂肪细胞早期体积较小、外形欠规则,但术后2周脂肪细胞体积明显增加、外形趋圆润;对照组的活脂肪细胞形态均一性在各时间点均欠佳,且没有明显的体积增加趋势。在术后1、2周,实验组Perilipin-1(+)活脂肪细胞占比低于对照组(P<0.05);术后4、12周,实验组Perilipin-1(+)活脂肪细胞占比高于对照组(P<0.05)。术后1、2、4、12周,实验组有效体积保持率低于对照组(P<0.05)。术后1、2周,实验组新生血管主要集中于移植物边缘,随时间推移逐渐长入内部;对照组血管分布则始终较为整体、均匀。术后1周,实验组CD31(+)血管数量低于对照组(P<0.05);术后2、4、12周,实验组CD31(+)血管数量高于对照组(P<0.05)。术后1、2、4、12周,实验组VEGF光密度值高于对照组(P<0.05)。结论CLHA的存在不利于移植的颗粒脂肪远期成活,建议临床上待局部CLHA完全代谢或预先使用透明质酸酶溶解后再行颗粒脂肪移植。

Objective To investigate the effects of cross-linked hyaluronic acid(CLHA)on the survival of microfat grafting.Methods Seventy-two eight-week-old SPF female BALB/c nude mice weighting 22-24 g were divided into two parts,of these,24 were used only to determine the metabolism of customized CLHA,and 48 were used only to study the effect of CLHA on survival after microfat grafting.Twenty-four nude mice were divided into groups A,B,C,and D according to the random number table method,with 6 mice in each group;0.3 ml customized CLHA was injected subcutaneously into the back of nude mice,all nude mice in groups A,B,C,and D were necropsied at 1,2,4,and 12 weeks postoperatively,and the remaining volume of customized CLHA was determined and HE staining were determined.Forty-eight nude mice were divided into the experimental group and the control group according to the random number table method,with 24 mice in each group.The experimental group received a mixture of 0.3 ml customized CLHA and 0.3 ml human microfat subcutaneously in the back,while the control group received only 0.3 ml of human microfat.Six nude mice were necropsied at 1,2,4,and 12 weeks postoperatively in each group,and fat tissue volume retention,histologic scores,Perilipin-1(+)live adipocytes percentage,CD31(+)vessels number,and vascular endothelial growth factor(VEGF)optical density values,and effective volume retention were compared between the two groups.Results Over time,the visual and measured volume of the transplanted customized CLHA gradually decreased,and customized CLHA was no longer visible in group D.In group A,a large number of pale blue-purple customized CLHA were distributed in the tissue interstitium;in group B,fibrosis and inflammatory cell infiltration were seen in the interstitium,and customized CLHA were scattered;in group C,the interstitium of the tissues were loosened and widened,the interstitial fibrosis was intensified,and the infiltration of inflammatory cells was increased,and the area of customized CLHA was reduced;in group D,no customized CLHA were seen in the field of view,and the subcutaneous structure of the injection site was similar to that of the normal subcutaneous structure.The experimental group had the most neovascularization at 4 weeks postoperatively,whereas it decreased at 12 weeks postoperatively compared with the previous period;and the overall neovascularization of the grafts in the control group was more evenly distributed at all time points.At 1,2,4,and 12 weeks postoperatively,the volume retention rate in the experimental group was lower than that in the control group(P<0.05).In the experimental group,more inflammatory cell infiltration and extracellular matrix deposition were seen among the adipocytes of the grafts,and neovascularization was mainly concentrated in the periphery of the grafts;in the control group,the adipocytes were predominantly structurally intact,and inflammatory cell infiltration and extracellular matrix deposition were less.At 1,2,4,and 12 weeks postoperatively,the adipocyte integrity scores of the experimental group were lower than those of the control group,and the fibrosis regions and inflammation cell infiltration scores were higher than those of the control group(P<0.05);at 1,4,and 12 weeks postoperatively,the necrosis regions scores of the experimental group were higher than those of the control group(P<0.05);at 2,4,and 12 weeks postoperatively,the oil sac/void scores of the experimental group were lower than those of the control group(P<0.05).The live adipocytes in the experimental group were smaller and less regular in shape in the early stage,but the adipocytes increased significantly in volume and tended to be more rounded in shape at 2 weeks postoperatively;in the control group,the morphologic homogeneity of the live adipocytes was poorer at all time points and there was no obvious tendency for the volume to increase.At 1 and 2 weeks postoperatively,Perilipin-1(+)live adipocytes percentage in the experimental group was lower than that in the control group(P<0.05);at 4 and 12 weeks postoperatively,Perilipin-1(+)live adipocytes percentage in the experimental group was higher than that in the control group(P<0.05).At 1,2,4 and 12 weeks postoperatively,the effective volume retention rate of the experimental group was lower than that of the control group(P<0.05).At 1 and 2 weeks postoperatively,neovascularization in the experimental group was mainly concentrated at the edges of the grafts and gradually grew into the interior over time;in the control group,the distribution of vasculature was consistently more holistic and homogeneous.At 1 week postoperatively,CD31(+)vasculature number in the experimental group was lower than that in the control group(P<0.05);at 2,4,and 12 weeks postoperatively,CD31(+)vasculature number in the experimental group was higher than that in the control group(P<0.05).At 1,2,4,and 12 weeks postoperatively,the VEGF optical density value of the experimental group was higher than that of the control group(P<0.05).Conclusion The presence of CLHA is detrimental to the long-term survival of microfat grafting,and it is recommended to wait until the local CLHA has been completely metabolized or pre-solubilized with hyaluronidase before granular fat grafting is performed in the clinic.