Feasibility of fat particles cryopreserved for different time for use as subcutaneous fillers

doi:10.3969/j.issn.2095-4344.1095

Abstract

Abstract:

BACKGROUND: In the case of not adding protective agent, whether excessive adipose tissue cryopreserved for long time can be used for the second transplantation and achieve good filling effect is worthy to be investigated.
OBJECTIVE: To investigate the transplantation effect of fat particles cryopreserved for different time, and the feasibility of clinical application.
METHODS: Without addition of protectant, fat particles were cryopreserved at a final temperature of -80℃. There were four groups: fresh fat, cryopreservation for 1, 3, and 6 months groups. Twelve 8-10-week-old SPF nude mice weighting 20 g provided by the Laboratory Animal Center of Xuzhou Medical University were included in this study. After cryopreserved for different time, fat particles were separately implanted into the back of nude mice. Tissue samples were removed at 4 and 8 weeks after transplantation. Appearance, weight, and volume of graft were compared among groups. The survival of fat tissue was determined by MTT assay, hematoxylin-eosin staining and immunohistochemical staining.
RESULTS AND CONCLUSION: After transplantation of cryopreserved fat particles, there were surviving adipocytes, and new blood vessels were observed between cells. The transplantation effect of fat particles cryopreserved for 6 months was superior to that of fat particles cryopreserved for 1 and 3 months. Therefore, fat particles cryopreserved for different time without addition of protectant can be transplanted without safety concern, which can obtain satisfactory filling outcomes.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Adipose Tissue, Cryopreservation, Transplantation, Tissue Engineering

CLC Number:

R446
R318

Zheng Wanling1, Zhang Aijun2, Wang Hao1, Wang Pingping1, Li Jianhua1, Wen Minmin1, Jin Peisheng2. Feasibility of fat particles cryopreserved for different time for use as subcutaneous fillers [J]. Chinese Journal of Tissue Engineering Research, 2019, 23(11): 1706-1710.

Figures/Tables 1

2.1 脂肪组织移植前观察 2.1.1 细胞学观察新鲜脂肪组织中提取的细胞原代培养12 h内即可见细胞贴壁生长，较多红细胞悬浮，培养2周后可见大量细胞贴壁生长，见图1A。未添加保护剂直接冻存3个月的脂肪颗粒在细胞提取72 h后可见极少量细胞贴壁，细胞呈长梭形,，成纤维细胞样特征，呈放射状排列，培养2周后细胞增殖能力良好，见图1B。 MTT实验可见冻存后的脂肪组织中提取的细胞生长曲线良好，呈“S”形，接种后前2 d为缓慢生长期，第3天开始对数生长，第8天达顶点，第9天略有减少后进入平台期，同新鲜脂肪干细胞的生长曲线相仿[5]，见图2。 2.1.2 组织学观察苏木精-伊红染色显微镜下可见来自新鲜脂肪组织的移植物细胞排列紧密，胞质饱满，细胞核分布均匀；来自低温保存1，3个月的移植物脂肪细胞形态尚可，而未添加保护剂冻存6个月后的脂肪组织框架结构维持良好，可见成形脂肪细胞，排列稀疏，胞核分布不均，边缘欠光滑，然未见明显变性和坏死，见图3。 2.2 移植物观察 2.2.1 大体观察术后2个月可见裸鼠背部皮肤光滑，无浅表组织破溃，移植物无钙化硬结，填充效果良好，见图4A；移植物表面均有完整包膜，与周围组织分界清晰，易于剥离，表面有新鲜血管长入，见图4B。4组标本均质地柔软，新鲜脂肪组移植物颜色鲜黄，色泽明亮；冻存1个月组和冻存3个月组虽体积维持良好，但液化均较明显，且2组间似无差异；冻存6个月组除脂肪团表面略暗，缺少光泽，并无其他明显差别，体积亦维持良好。 2.2.2 体积分析脂肪移植后1个月，4组体积维持率依次为：新鲜脂肪组(41.75±3.60)%，冻存1个月组(25.03± 8.54)%，冻存3个月组(27.9±9.67)%，冻存6个月组(35.2± 1.13)%。其中，冻存1个月组、冻存3个月组体积维持率均显著低于新鲜脂肪组，差异有统计学意义(P < 0.05)，冻存1个月组、冻存3个月组间差异不显著；冻存6个月组体积维持率略低于新鲜脂肪组，但差异无显著性意义(P > 0.05)。脂肪移植后移植物体积随时间延长逐渐缩小，术后2个月新鲜脂肪组的体积维持率均高于冻存脂肪组，4组体积差异无显著性意义(P > 0.05)，见图5。 2.2.3 移植物苏木精-伊红染色苏木精-伊红染色可见新鲜脂肪移植物细胞多结构完整，胞质饱满，大小分布较为均匀。冻存脂肪纤维结缔组织增生及坏死凋亡细胞相对较多，冻存6个月组可见移植物内存在囊腔和空泡，然移植物内亦可见较多结构完整的脂肪细胞成团状或散在分布，冻存1个月，冻存3个月组间未见明显差异，见图6。 2.2.4 移植物成活细胞百分数通过计数苏木精-伊红染色切片中存活的脂肪细胞及结构不完整的脂肪空泡，测出4组脂肪细胞的成活率依次为：新鲜脂肪组(93.93±5.25)%，冻存1个月组(81.48±5.04)%，冻存3个月组(80.53± 2.37)%，冻存6个月组(90.66±2.30)%。冻存1个月组、冻存3个月组2组比较差异无显著性意义(P > 0.05)，但均显著低于新鲜脂肪组(P < 0.05)；冻存6个月组成活细胞百分率仅次于新鲜脂肪组，且两组间差异无显著性意义(P > 0.05)，见图7。 2.2.5 CD31免疫组织化学显示新鲜脂肪组织移植物和冻存脂肪移植物内均有成形血管，见图8。"

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