Improvement Of The Survival Of Rabbit Fat Transplantation By Using Freshly-isolated Adipose Stromal Vascular Fraction Cells

[Background and Objective]Repair of soft tissue defects caused by the congenital deformity,trauma and tumorectomy has been regarded as one of considerable chanllages in plactic and reconstructive surgery.More than 100 years have passed by since Neuber was first to publish findings regarding the use of autologous fat transplantation in 1893.He filled scars with autologous fat.Autologous free-fat transplantation has many potential advantages over other methods of soft-tissue augmentation for small to moderate-sized defects.This tissue is abundant,easily harvested,nonimmunogenic, and has very favourable physical characteristics.However,low survival rate of fat graft and unpredictability of retain volumn limit the clinical application of fat transplantation.Histologically,progressive loss of transplanted adipocytes is noted along with a conversion of the graft to fibrous tissue, often times with cyst formation.It is presumed that the portion of the graft adjacent to the native recipient blood supply nourishes the graft and sustains it.New growth of blood vessels from the surrounding tissue may take 5 days to infiltrate and provide adequate nutrition,which could result in graft central cell death and tissue necrosis.This concept has led to decreasing the size of fat grafts with the hope that a smaller graft will mean more fat is adjacent to viable recipient tissue,resulting in improved availability and diffusion of cellular nutrition until neovascularization occurs.Obviously,the promt and sufficient neovascularization of fat graft plays a key role for improving the survival rate.With development of stem cell research,stem cells are found to promote neovascularization in graft.Despite the ethical quandaries entangling embryonic stem cell usage,there are also key biological limitations with their use as well.Major hurdles such as cell stability,oncogenicity,spontaneous teratoma formation and so on represent profound impediments to clinical use for non-life-threatening indications.As adult stem cells,bone marrow mesenchymal stem cells are able to differentiate toward multiple lineages,such as osteogenic and chondrogenic lineage.However,the use of bone marrow derived stem cells is limited by the quantity of cells that can be collected from the patient and associated donor-site pain and morbidity.Fibroblast-like cells in adipose tissue,adipose-derived stem cells(ASCs),contain a population of cells that can differentiate toward a number of lineages not limited to the osteogenic,myogenic,chondrogenic,adipogenic,and neurogenic lineages. Especially,ASCs seem to have a significant potential for angiogenesis and vasculogenesis,one of the fundamental limitations in the current technique of autologous fat transfer.We confirmed that cell-assisted lipotransfer based on human ASCs can significantly promote the viability of fat graft in rats attributed to its angiopoietic characteristics in our previous report. However,expansion of ASCs in vitro apparently does not meet the need of clinical use.This procedure is time-consuming,complicated and expensive,which limits the cilinic application of cultured ASCs.How to obtain adequate ASCs with biologic activity safely and conveniently is critical to its clinical application.Clinician can achieve the cell-assisted lipotransfer immediately and conveniently once "ASCs bank" could be established.Recent studies showed that ASCs can exhibit a low immunogenic and immunosuppressive profile in both vitro and vivo,which signifies the perspective of allgeneic application.However,clinician prefer autogeneic cells to allgeneic ones due to low security and cross-inflection.To avoid these problems,we choose freshly insolated stromal-vascular fraction cells(SVFs).ASCs can be obtained from the processing of either liposuctioned or excised fat.Due to its ease of harvest and the volume of tissue obtainable from liposuction,actually,freshly isolated SVFs obtains adequate ASCs for clinical application whithout expansion in vitro.The uncomplicated enzyme-based isolation procedures make the cell therapy more attractive for researchers and clinicians.We evaluate the effectiveness of freshly isolated SVFs and allgeneic cultured ASCs in animal models of fat transplantation compared with DMEM medium and autogeneic cultured ASCs by the measure of the wet weight and histological analysis.Here,we provide evidence to support a more convenient and efficacious method of autologous tissue transfer.[Material and Methods]1.Isolation and expansion of rabbit ASCsThe inguinal fat pads are harvested by exairesis from 14 rabbits(The Southern Medical University institutional ethics committee approved all experiments in advance),and then enzyme digestion method are used for primary culture and observe changes in cell morphology and function of cells.Only cultured cells for 4 passages were used in this study in vivo.Then the cells are separated into two half for autogeneic and allogeneic grafting respectively.2.Label of ASCs with Di-IFor tracing ASCs,the three types of cells are labeled by incubated with 5 mg/mL DiI for 1 h before transplantation.Results are observed with fluorescence microscope.3.Rabbit models for cell-assisted lipotransfer(CAL)Freshly isolated SVFs(Group B),autogeneic(Croup A) and allogeneic(Group C) cultured ASCs of passage 4 are respectively mixed with 0.5ml of autogeneic excised fat and then injected under the back skin of rabbit.In the same manner,rabbit fat was injected with complete medium as control(Group D).The four recipient sites on the back are randomly choosed.4.Trace of DiI-labeled ASCs in vivoFat graft of 1 sample is harvested in 1 month post transplantation.Fluorescence trace of DiI-labeled ASCs is concerned with the reveal of vascular endothelial cell by Haematoxylin staining.5.Evaluation of fat graft survivalFat grafts of the rest 13 samples are harvested at 6 months post implantation.Wet weight of fat grafts are mersured In order to characterise the histological differences that could contribute to the different macroscopic aspects in the samples,three anatomic-pathological criteria were considered:ngiopoiesis,viable adipocytes,and fibrous proliferation.For this analysis,the 'point-counting' technique proposed by Gundersen et al.was used,with a reticulum of 100 points and 20 lines.6.Statistical analysisResults were expressed as the mean_standard error.The data were statistically analyzed using a Two-way ANOVA.A LSD test is used for the multiple comparison of the means.A p value less than.05 was considered to represent a statistically significant difference.[Results]1.The wet weight of grafts are(0.2693±0.0673)g in Group A,(0.291±0.0721)g in Group B and(0.2579±0.0715)g in Group C.Al1 the three are significantly larger than conrol group,(0.1778±0.06)g,P＜0.05.The difference of multiple comprare between three experimental groups is not significant,P＞0.05.2.The counts of blood vessel are(12.7±3.1)/HPF in Group A,(13.2±3.7) /HPF in Group B and(11.2±2.5)/HPF in Group C.All the three are significantly larger than conrol group,(6.9±2.8)/HPF,P＜0.05.The difference of multiple comprare between three experimental groups is not significant,P＞0.05.3.The counts of survival adipose cells are(6503±851)/10HPF in Group A,(6820±1021)/10HPF in Group B and(6658±858)/10HPF in Group C.Al1 the three are significantly larger than conrol group,(905±383)/10HPF,P＜0.05.The difference of multiple comprare between three experimental groups is not significant,P＞0.05.4.The counts of fibrous proliferation and steatonecrosis are(6336±848)/10HPF in Group A,(6135±918) / 10HPF in Group B and(6181±829)/10HPF in Group C.All the three are significantly smaller than conrol group,(11694±625)/10HPF, P＜0.05.The difference of multiple comprare between three experimental groups is not significant,P＞0.055.Red fluorescence is observed from parts of vascular endothelial cell in all the three experimental group.As comprared,the same phenomenon is not observed in control group.It suggests that all the three types of ASCs can differentiate to vascular endothelial cells in vivo.[Conclusions]1.CAL based on freshly isolated SVFs,similar to autogeneic expanded ASCs,may improve the survival rate of fat graft.The former is more convenient and easier,which suggests the wide clinical perspective of the CAL method.2.Despite of the immunologic rejection,allgeneic expanded ASCs may efficaciously improve the survival rate of fat graft,which suggests the clinical potential of the cells.3.CAL with the three types of ASCs(i.e.,freshly isolated SVFs,autogeneic and allgeneic cultured ASCs of passage 4) may improve survival rate of fat graft by promote the angiopoiesis and lessen fibrous proliferation and steatonecrosis.The possible mechanism is that ASCs may participate and promote the angiopoietic proceeding of fat graft.