Retention Mechanism Of Large-volume Fat Grafting Based On A Parabiotic Animal Model

Background and Objection:The most representative mass fat transplantation in clinic is fat transplantation for breast augmentation.Its first report was in 1987,and the early diagnosis of breast cancer may be affected by widespread resistance.With the increasing evidence from subsequent laboratories,the safety and efficacy of the breast cancer have been gradually accepted by the public.Finally,the American Association of plastic surgeons released the proposal of autologous fat transplantation for breast augmentation in 2009.In the following ten years,the breast augmentation technology of autologous fat transplantation developed rapidly,including Brava method and beauli method ? A variety of fat transplantation techniques,including method,have achieved good clinical results.Although the mass fat transplantation represented by breast augmentation has achieved good clinical results,the retention mechanism after transplantation has not been clear.There seems to be a general consensus in the industry that bulk fat grafting works by the same mechanism as granular fat grafting.At present,the more recognized mechanism of granular fat graft retention is the three-zone regeneration theory proposed by Yoshimura's team in 2012.The outermost layer is the fat survival zone,with a thickness of 0.1-0.3mm,and the middle layer is the regeneration zone,with a thickness of 0.6-1.2mm,the innermost layer is the necrotic area;in other words,if the fat granules are simplified as spheres,according to the three-zone regeneration theory,the maximum radius for maintaining fat vitality is 1.5mm,and necrosis will occur in the middle of the fat globules beyond 1.5mm.The definition of large volume of fat has not yet been unified.Some authors believe in their article that fat transplantation with a unilateral injection of more than 250 ml is called megavolume or large volume;in the clinical practice of our experimental team,the unilateral injection volume is often more than 300 ml.Another finding of our team's daily clinical practice is that after large-volume fat transplantation,our team found that the fat survival rate behind the pectoralis major muscle is often high,and the central area after injection did not appear as predicted by the three-zone regeneration theory.A condition in which large areas of fat cells and tissue are necrotic.This fact is counter-intuitive:it stands to reason that the central area of fat behind the pectoralis major cannot fully survive the single-point subcutaneous injection of less than 0.6 ml in the three-zone regeneration theory,but in fact the amount of fat retained still sufficient.In view of the above clinical findings,this study believes that there may be a new retention mechanism in large-volume fat transplantation,which is different from the three-zone regeneration theory,and obtained clinical specimens for testing experiment.Methods1.Theoretical modeling of three-zone regeneration by computer simulationIn this study,the modeling software 3ds max 2020 model was used to simulate,and the fat was completely modeled according to the three-zone regeneration theory to verify what kind of granular fat model was in line with clinical practice.2.Clinical observation of adipose tissue preservation after autologous fat transplantation for breast augmentationIn view of the fact that the team found that the large amount of fat in the single cavity after injection into the pectoralis major muscle survived better in clinical practice,the fat in the central region did not have a large amount of necrosis as expected by the three-region regeneration theory.Therefore,the team recruited volunteers in the clinic,and obtained the central and marginal transplanted tissues of the pectoralis major muscle by biopsy and puncture under the guidance of ultrasound at different times after autologous breast fat transplantation.The tissue was further observed by gross,HE staining and immunofluorescence staining:HE initially observed tissue morphology;Perilipin staining was used to observe fat regeneration;CD31 staining was used to observe angiogenesis;Mac2 and CD206 staining were used to observe the infiltration and polarization of macrophages.3.Construction,experimentation,and detection of animal modelsAnimal experiments were designed according to the above experimental results.In this experiment,a new animal model was constructed through a chamber with a diameter of 0.8 microns and a diameter of 2 cm to simulate the condition of no blood supply but tissue fluid infiltration in the early stage after transplantation.First,the function of the chamber was verified,and animal experiments were carried out after verifying that it met the needs.1ml of Coleman's fat prepared according to the standard procedure was injected into the chamber and the chamber was implanted into the back of nude mice by surgery.In order to simulate the process of blood vessels gradually growing into the graft in clinical practice,the chamber was opened again after 7 days,and the fat in the chamber was implanted again.Into the back of nude mice to provide a material basis for blood vessels to infiltrate the graft,this group was used as the experimental group.At the same time,1 ml of Coleman fat was directly injected into the back of nude mice as a control group.Then,the samples were taken at 7,30,60,and 90 days respectively,the volume was measured by the drainage method and the retention rate was calculated,and the fat regeneration status was observed by the gross,HE and immunofluorescence results.Results1.According to the 3D modeling results,it is found that the three-zone regeneration theory cannot be fully satisfied in the clinical practice of large-volume fat transplantation,and there must be a centimeter-level transplanted fat mass.According to the three-zone regeneration theory,this will lead to a large amount of necrosis.However,in clinical practice did not appear in.This study speculates that in large-volume fat transplantation,there may be a retention mechanism that is different from the three-zone regeneration theory.2.There was no significant difference between the central area and the marginal area in the whole period after transplantation,and both showed normal fat appearance.In the early(7 and 30 days)HE staining,the adipocytes in the central area were more regular and tidy,and there were more cell debris and inflammatory reactions in the marginal area.The tissue showed more CD31 positive vascular structures and more Mac2 and CD206 expression.In the late post-transplantation period(90 days),immunofluorescence showed that there were fewer Perilipin positive areas in the central area,and CD31 positive and Mac2 positive were similar to those in the marginal area.It shows that the fat in the central area is in a special environment with poor blood supply and less macrophage infiltration in the early post-transplantation period,and the fat is in an parabiotic environment where the active part is reduced.With the passage of time,in the late stage of transplantation,the blood vessels gradually grow in,the macrophages gradually infiltrate,and the fat in the central area is converted into the fat in the marginal area.For further experiments in animals,we made a new animal model based on the above characteristics.3.In the experiment to verify the function of the chamber,the appearance of the complete chamber graft has no obvious change within 30 days after transplantation,and there is no vascular infiltration on the surface;clear and intact adipocytes can be seen under HE,with rare blood vessels,broken cells,fibrous tissue,etc.;immune Perilipin positivity was generally weak under fluorescence but did not change with time.CD31 positivity was rare,and CD206 and Mac2 positivity was rare.In this experiment,it is believed that the cell can keep the internal fat structure basically intact for a long time after transplantation(within 30 days),without blood supply and without macrophage infiltration,and the fat activity is acceptable,and there is no large amount of necrosis.requirements,followed by animal experiments.At 90 days post-transplant,the volume retention rate of the intergenic fat group(Coleman fat+chamber)(42%±2.3%)was lower than that of the normal fat group(Coleman fat)(55%± 1.6%)(p<0.05),in general,there was no significant difference between the ecological fat group and the normal fat group,with less oil sacs and inflammatory reactions,showing a healthy fat appearance.HE showed that the ecological fat group had a more regular and regular adipocyte structure,less oil sac and inflammatory cell infiltration in the early post-transplantation period(7 days and 30 days)than the normal fat group,and the ecological fat group in the late posttransplantation period(90 days)Compared with the normal fat group,there were more oil pockets;immunofluorescence showed that the ecological fat group had fewer CD31 positive areas in the early post-transplantation period(7 days and 30 days)than the normal fat group,and the ecological fat group in the late posttransplantation period(90 days)Partial Perilipin positive area could still be observed in the fat group,but its intensity was weaker than that of the normal fat group;the Mac2 positivity of the parabiotic fat group was weaker than that of the normal fat group in the early post-transplantation(7 days),and the late post-transplantation(90 days)Compared with the early stage of transplantation,more Mac2-positive areas and some CD206-positive areas were observed in the parabiotic fat group.It suggested that the adipose of the parabiotic adipose group had slower macrophage infiltration after 7 days of closed chamber treatment.ConclusionThis study is based on the fact that my clinical team found in the clinical practice of large-volume fat transplantation that a large amount of single-chamber fat injected into the pectoralis major survived well,and the central area did not have a large number of necrosis as described in the three-zone regeneration theory.Start the design.First,it was verified by computer modeling that large-volume fat transplantation cannot be injected in accordance with the three-zone regeneration theory in clinical practice,and will inevitably produce centimeter-scale transplanted fat masses.However,the expected large amount of necrosis was not observed in practice.It is speculated that there may be a preservation mode different from the three-zone regeneration theory in this process.Subsequently,it was confirmed by the recruitment of clinical volunteers that the fat activity in the posterior central area of the pectoralis major muscle was good,and at the same time,it was verified that there was an early parabiotic fat retention characterized by no blood supply,no macrophages but with tissue fluid infiltration in large-volume fat transplantation.mechanism.Finally,a new parabiotic animal model was designed to simulate this process and verified by animal experiments.Although the parabiotic fat group had a worse late retention rate than the normal fat group(p<0.05),the parabiotic fat group had a worse late-stage retention rate after transplantation.After revascularization,it returned to a near-normal fat group.This study believes that the mechanism behind this phenomenon is that after 7 days of pretreatment with no blood supply,no inflammatory infiltration,and only tissue fluid infiltration in the closed chamber,the parabiotic fat group showed slower blood vessels than the normal fat group.Infiltration and inflammatory infiltration,and in the early post-transplantation period,due to the late arrival of blood vessels and inflammation,the necrotic part of the interfacial fat could not be removed in time,while maintaining a relatively intact structure.With the slow growth of blood vessels in the later stage,the parabiotic fat gradually turned into a near-normal adipose tissue.SignificanceUnder the full understanding and recognition of the three-zone theory,which is widely recognized by the academic community,this study was inspired by clinical observations to design an ecological animal model between large-volume fat transplantation,which filled the gap of animal models in this field.Through animal experiments and clinical specimens,the three-zone regeneration theory is organically supplemented in the field of large-volume fat transplantation,which lays a good foundation for further exploration of the retention mechanism of largevolume fat transplantation and possible in vitro pre-culture of fat and related clinical applications.