| Autologous fat transplantation is popular for the repair of soft-tissuedefects in clinical practices. It can be widely applied in soft-tissueaugmentation, such as hemifacial atrophy, breast augmentation, facialrejuvenation, rhinoplasty, the labiomandibular fold, lips, chin, and the jawaugmentation. Autologous fat transplantation can be an excellent techniquewith the theoretical advantages of being available in abundance, easy toharvest, relatively cheap and autogenic, without the possible side effects andpotential risks of implants and allogenic fillers. However, the rate of fat graftsurvival is low and there are some postoperative complications which perplexthe choice of plastic surgeons, such as fat calcification, necrosis, scleroticnodules, and oil cysts. The absorption rates of fat graft still range from20%to90%which were reported inconsistently in different articles. How to decreasethe absorption rate and complications is the key point of autologous fattransplantation. There are still disputations about the survival andneovascularization of fat graft. And until now, we have not found direct proofs in literature confirming fat graft survival and origin of the blood supply of thegraft. About fat transplantation there have been3views: host replacementtheory, adipose cells survival theory, and ASCs alternative theory, which needto be further discussed. Stem cells therapy, especially includingadipose-derived stem cells and stromal vascular fraction, are proved effectivein fat transplantation by several teams. Then what about other stem cells suchas bone marrow-derived mesenchymal stem cells? Is there difference of thepromotion on fat survival between fresh stem cell components (SVF or BM)and stem cell expanded ex vivo? Our objective is to confirm the survival ofthe fat graft and origin of the blood supply; to compare in vitro culturecharacteristics between human and rabbit ASCs under the same conditions;and to compare the effect of ASCs, SVF, BMSCs and BM on fat graftsurvival.Experiment1Observations on the survival and neovascularizationof fat grafts interchanged between C57BL/6-gfp and C57BL/6miceObjective:To confirm whether the fat grafts have survived or have been replaced inthe recipient sites and how the vessels grow.Methods:After the C57BL/6-gfp mice (GFP mice for short) and C57BL/6mice(B6mice for short) were paired randomly, the inguinal fat was excised and cutinto pieces with scissors, and the adipose granules about0.2ml (0.195g) weretransplanted subcutaneously with syringes to the dorsa of the paired mice. Samples were obtained at different time intervals:3days,7days,2weeks,4weeks,2months,3months, and4months after the transplantation. Each fatsample was weighed to evaluate the graft volume. Histology, origin anddensities of neovascularization were examined by immunohistochemistrystaining.Results:At the time point of4months, there was no significant difference ineither graft survival or histology evaluation. Histological evaluationmanifested the normal physiological process of inflammation,neovascularization, remodeling and maturity at different time intervals. At theend point, the results of CD34immunohistochemistry staining showed that thecapillary density of fat graft was31.3±3.9capillaries/mm2on the dorsa ofGFP mice,29.6±3.2capillaries/mm2on the dorsa of B6mice, there existingno statistical significance. The α-SMA staining indicated that there wereneovascularized vessels in both GFP and B6fat grafts.Conclusion:Fat grafts can survive and neovascularized vessels can grow from therecipient sites. Fat transplantation is feasible and will be more widely appliedif fat graft survival is improved.Experiment2Comparison of ex vivo culture characteristicsbetween human and rabbit adipose-derived stem cells Objective:To compare in vitro culture characteristics between human and rabbitadipose-derived stem cells under the same conditions.Methods:Human ASCs (hASCs) were isolated by collagenase digestion fromintact fat of abdomen harvesting of skin grafts. Rabbit ASCs (rASCs) werederived from subcutaneous fat tissue of dorsa. After digestion, the two kindsof ASCs were cultured at37℃in a5%CO2humidified incubator. Observe thegrowth morphology and passage them when reaching90%confluence. Applythe third passage cells to compare the ability of growth and proliferation, stemcell surface marker flow cytometry, and induction to fat and bone.Results:Fibroblast-like adherent cells can be isolated from both human and rabbitfat. hASCs need grow about6-8days to reach90%confluence but rabbitabout4-5days. MTT testing results manifested that the growth climax ofhASCs was the fourth day, whereas rASCs was the sixth day. Flow cytometryboth showed CD29＋CD31－.Under the same inducing condition, hASCs canbe induced to fat and bone, but rASCs were induced faster to fat and poorly tobone.Conclusion:Both hASCs and rASCs are characterized by adherent growth,multipotent differentiation and stem cell phenotype; in comparison withhASCs, rASCs have more powerful ability of proliferation and induction to fat, whereas the ability of induction to bone is poor. Rabbit is a good choice oflaboratory animal for fat transplantation research.Experiment3Comparison between the effect of ASCs andBMSCs on fat graft survivalObjective:To compare the effect of ASCs, SVF, BMSCs and BM on fat graftsurvival.Methods:rASCs were derived from subcutaneous fat tissue of dorsa and isolated,cultured ex vivo. Bone marrow was extracted by operation and BMSCs werescreened by adherent culture. The third passage of rASCs and rBMSCs wereconducted on the induction to fat and bone, and flow cytometry of CD29andCD31. There were5groups in nude mice experiment of fat transplantation:ASCs, SVF, BMSCs, BM and DMEM/F12as a blank control. SVF and bonemarrow was prepared for flow cytometry of CD29and CD31. There were16injection points on the paravertebral dorsa of4nude mice in each group. Thetranplanted mixture consisted of0.3ml AG and0.2ml cell components.4months after the transplantation, the mice were euthanized and the fat graftswere dissected out, weighed, and analyzed by HE staining andimmunohistochemistry staining of CD31.Results:rASCs and rBMSCs were successfully isolated and cultured in vitro, and the third passage of them were both fibroblast-like adherent cells. Under thesame inducing condition, rASCs and rBMSCs can be induced to fat and bone.Flow cytometry indicated that: ASCs group: CD29:96.2%, CD31:3.8%; SVFgroup: CD29:17.0%, CD31:1.3%; BMSCs group: CD29:45.0%, CD31:1.6%; BM group: CD29:24.6%, CD31:1.8%. The survival of the fat graft ineach group of ASCs, SVF, BMSCs, BM and blank control group were0.1798±0.0033g,0.2096±0.0024g,0.1761±0.0014g,0.2052±0.0015g,0.1350±0.0020g, there existing statistical significance between multiplecomparison. HE staining showed that the fat graft in group of SVF and BMexhibited good survival, integrity, uniform lipid droplet and rich blood vessels;fat graft in group of ASCs and BMSCs exhibited survival, less integrity, lessuniform lipid droplet and connective tissue septa; the blank control groupexhibited some large bubbles, all sizes lipid droplet, and significant fibroussepta. In each group the vascular density was29.3±1.6capillaries/mm2,32.6±2.1capillaries/mm2,27.2±1.3capillaries/mm2,30.4±1.5capillaries/mm2,23.3±1.9capillaries/mm2, there existing statistical significancebetween multiple comparison.Conclusion:(1) Both ASCs and BMSCs are characterized by adherent growth,multipotent differentiation and stem cell phenotype;(2) SVF can improve thesurvival of the fat graft;(3) and BM show the improvement too;(4)mesenchymal stem cells including ASCs and BMSCs do good to fattransplantation;(5) fresh stem cell components are more helpful than stemcells expanded ex vivo for fat transplantation. |
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