| 【Background】Soft tissue defects caused by congenital deformities, trauma and tumorresection has been a big hang-up for the current clinical tissue reconstruction.Adipose tissue has been proposed as a promising filler for the clinicalrestoration of soft tissue defects for the merits of none immunological rejection,abundantresources and easy access. However, the traditional way of autologousfat transplantation has many limitations, such as atrophy, liquefaction, necrosisand the hard manipulation of precise volume need to fill, and in turn wouldbrought great hardship to the majority of patients and physicians.With thecontinuous development of a variety of research techniques, fat grafting hasbeen further development. The result of our pre-research showed that thecombination of platelet-rich fibrin (PRF) and stromal vascular fraction (SVF)has great effect in the improvement of survivorship of transplanted fat tissue.But its mechanism involved in the experimental study is unclear. So in this study,we will try to investigate the mechanism of ASCs and PRF in the process of fat grafting at the basis of angiogenesis.【Objective】To investigate the mechanism of vascularization in fat transplantation withadipose stem cells and platelet-rich plasma，and provide experimental evidencefor the production ofnew composite material in adipose tissue engineering.【Methods】1、ASCs were isolated and cultured from rabbit adiposetissue, the characteristicsof ASCs would be identified by HE staining, MTT assay, adipogenicinduction, osteogenic induction and flow cytometry.2、The release of vascular endothelial growth factor (VEGF) by PRF was testedusing ELISA. The test of PRF and ASCs was divided into four groups:0PRF(blank control),1PRF,3PRF,5PRF. Then the MTT assay, induction ofadipogenic and osteogenic and flow cytometry would be performed.3、The general volume of the graft adipose tissue would be observed by photos.4、The volume of graft fat would be measured by B-mode ultrasound and caliperat different time points after transplantation, the volume of graft was countedby the formula: V=a*b*c*0.5(a: Longitudinal diameter, b:Transversediameter, c: thickness).Survival rate=Vx/V0*100%, and the absorption rateofgraft fat in all of the four groups would be evaluated by the formula: R=(1-Vx/V0)*100%(R: absorption rate, V0: the graft fat volume of thepostoperative day, Vx: the graft fat volume of the harvest day).5、 For histological examination and vessel densities, transplanted fatwasembedded in paraffin, andparaffin sectionsstained with hematoxylin andeosin (H&E) and Immunohistochemicalstaining with antibody for CD31.6、Statistical analysis: For the absorption rate/survival rate of the graft fat, all theevaluations were carried out by the Image-Pro Plus6.0software. Difference among the four groups were examined for statistical significance usingOne-way classification ANOVA with paired t test for statistical comparisonsusing the PASW statistics18software.7、The Integrated optical density (IOD) of capillary and the adipogenic andosteoblast images would be analyzed by the Image-ProPlus6.0. Differenceamong the different groups were examined for statistical significance usingOne-way classification ANOVA with paired t test for statistical comparisonsusing the PASW statistics18.0software.【Results】1、ASCs,which was isolated and cultured from rabbit adipose tissue showedfibroblast-like and spindle shape, strong proliferative ability and activedifferentiation．Flow cytometry showed that ASCs have the expression ofstem cell-specific surface antigen and the stable biological characteristics.2、PRF has the ability to release high quantities of VEGF, and PRF have anobvious and continuous stimulation on proliferation of the ASCs, whichreveals the dose dependent effect. Also, PRF can enhance induceddifferentiation of ASCs to adipogenic andosteoblastic cells.3、The survival rate of Transplanted fatThe survival rate of1,3,6months among the four groups, showed that thePRF+ASCs+AG group has the highest survival rate with (72.50±3.70)%,(70.71±4.08)%(71.78±5.78)%, while the AG group hits the bottom of thefour groups with (50.83±5.97)%(40.45±10.32)%,(34.20±10.61)%.And theresult of the other two groups (ASCs+AG, PRF+AG) are between the previoustwo groups (P<0.05).4、 PRF+ASCs+AG group showed the fastest step in the improvement of tissueconversion and vascularization, and lowest in fibrosis and inflammatory infiltration during the early stage (1W～4W), but an opposite effect tookplace in AG group with highest fibrosis and inflammatory infiltration, andslowest step in the improvement of tissue conversion and vascularization.There was no significant difference between the four groups of thetransplanted fat in histologic featuresat3months after the operation, thehistologic features of transplanted fat was very similar to the normal fatfromthe rabbits.5、The result of IOD analyzed by the Image-Pro Plus6.0showed that thestimulation of differentiation in PRF groupswere significantly higher (p <0.05) than control group, the5PRF group was higher than3PRF (p<0.05),and the same result with3PRF and1PRF (P<0.05). And the IOD of vesseldensities showed that PRF+ASCs+AG group was the highest with(14284.24±4.81*103), and the AG group was loweset with (1921.27±777.72),there was Significant statistical significance among the four groups, p <0.05.【Conclusion】1、PRF and ASCs can greatly reduce the absorption rate of the transplanted fattissue, and maybe have important effect in the vascularization of the fattransplantation.2、PRF has the ability to release high quantities of VEGF, and PRF has anobvious and continuous stimulation on proliferation of ASCs.3、PRF and ASCs have great effect in the improvement of tissue conversion andvascularization, and enhance the survivorship of transplanted fat.4、The PRF+ASCs+AG group showed the best effect among the four groups,and the volume of transplanted fat remained stable in the next3to6monthsafter transplantation. So it would be more accurate to handle the volume of transplanted fat, and better achieve the filling effect of soft tissue defects.Moreover, the combination of autologous PRF and ASCs would holdtremendous promisein inrestoration of soft tissue defects, regenerativemedicine and cosmetic surgery with the advantages of high bio-security, lowcost, easy operation. |
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