| Background and Objection:The reconstruction of large-volume soft tissue defects caused by complex traumas,oncology resections,and congenital abnormalities represents a major problem in plastic surgery.Current therapies include the use of autologous flaps,fat grafting,or allogenic/synthetic fillers.Despite improved surgical techniques,these treatments still result in donor site morbidity and often unsatisfying results.External volume expansion by suction induces adipose tissue regeneration.The commercial external volume expansion device(BRAVA),developed by Khouri et al.,induces subcutaneous adipose tissue regeneration and breast enlargement in clinical settings.However,insufficient volume of adipose tissue expansion and volume setbacks after removal of the device remain as drawbacks that limit the use of this method for reconstructing large-volume soft tissue defects.The combined effects of mechanical tension,edema,and inflammation created by external volume expansion trigger adipogenic differentiation of resident adipose-derived stem cells(ASCs)present in the adipose tissue.Recent fat grafting studies revealed that adipose-derived stem cells are a source of cells for adipose regeneration,and the long-term retained fat is mainly attributable to the repopulation and surviving adipose-derived stem cells left in the dead fat mass.These data suggest that adipose-derived stem cells are closely related to the regenerative process,and adipose derived stem cell pre-expansion in adipose tissue may improve final adipose regeneration.Spatial constraints caused by the presence of neighboring cells control cell proliferation through cell-to-cell contacts,mediated by cell contact inhibition by means of the Hippo/Yes associated protein signal pathway.Thus,we hypothesize that the release of cell-to-cell contacts triggers adipose-derived stem cell proliferation in adipose tissue and expanded adipose-derived stem cells undergo adipogenesis under the adipogenic environment created by external volume expansion.To test this hypothesis,two complementary studies were performed.Various densities of adipose-derived stem cells were prepared in vitro to study the influence of cell-to-cell contacts on proliferation.In vivo,a new method was developed to dissect adipose tissue and to release cell-to-cell contacts.The external volume expansion device was subsequently used to maintain the released state in adipose tissue.Cadherin-mediated cell-to-cell contact is a highly regulated process,which affects Hippo/Yes associated protein 1 cellular localization to modulate cell cycle progression through the Hippo pathway.Hippo/Yes-associated protein 1 localizes to the nucleus and is active in cells undergoing proliferation at low density,but accumulates in the cytoplasm at high cell densities.Cadherin and Hippo/Yes-associated protein 1 expression was evaluated in vitro to study the potential mechanism underlying the results observed in our in vivo model.Methods and materials1?The Various densities of ASCs were prepared to test the influence of cell-to-cell contacts on cell proliferation.Various densities of ASCs(19,36,75,and 93 percent confluence)were seeded in triplicate in culture dishes and Cadherin and YAP were evaluated to test the influence of cell-to-cell contacts on cell proliferation.2?The combination of tissue dissection and external volume expansion induce adipose tissue regeneration.In vivo,dorsal adipose tissue of rabbits was thoroughly dissected and the EVE device was applied to maintain the released state.Pan-Cadherin and YAP expression were evaluated at different time points.3?Statistical analysis.Data were expressed as mean ± SD.results were analyzed with two-way analyses of variance(SPSS,version 21;IBM Corporation,Armonk,NY,USA).Furthermore,an independent Student's t-test of two groups at one time point was performed.A value of P<0.05 was considered statistically signifcant.Results:1?Quantifcation of the adipose-derived stem cell proliferation rate revealed that a higher percentage of proliferative cells was observed at lower cell density,whereas a lower percentage of proliferative cells was observed at high cell density.2?The expression of Cadherin and YAP were evaluated at different time points after the adipose tissue dissection.In vivo Cadherin expression was extremely low in the dissection group at weekl.Opposite dynamic changes of YAP expression in the nucleus were observed between weeks 1 and 12.Ki67-positive cells and small adipocytes were most frequently observed.Similar dynamic changes of cadherin and Hippo/Yes-associated protein expression were observed in an in vitro mode,indicating that tissue dissection can release cell-cell contacts and induce cell proliferation,which is mediated by release of contact inhibition and Hippo/YAP pathway activation.DiscussionsIn our study,adipose tissue dissection released cell-to-cell contacts and triggered adipose-derived stem cell proliferation.Expanded adipose-derived stem cells underwent adipogenesis under the adipogenic environment created by external volume expansion,leading to better adipose tissue regeneration than the control group using external volume expansion alone.The balloon in this study was used to dissect the adipose tissue and induce adipose-derived stem cell proliferation,contributing to the stem cell pool.Quite a few studies have used stem cells seeded onto scaffolds or extracellular matrices,which are then implanted in vivo for engineered adipose tissue construction.However,a main limitation for using these cells is the issue of safety of the cell preparation.Enzymatic isolation changes the biological properties of the isolated cells,which then do not pass minimal manipulation standards.Moreover,separate injection of these cells often results in unexpected migratory outcomes,creating adverse complications(i.e.,ectopic fibrosis and lymphadenopathy).Thus,it would be a valuable alternative for adipose regeneration if tissue-resident adipose-derived stem cells of adipose tissue are preexpanded.The balloon dilatation technique used in this study is a new method aimed to mechanically dissect adipose tissue,and it proved to be effective in inducing tissue-resident adipose-derived stem cell proliferation.The tissue engineering chamber technique proposed by Morrison's group is another technique that is able to induce adipose-derived stem cell proliferation in the adipose tissue.However,implantation of exogenous chambers would lead to severe foreign body reactions and limit the volume growth at the late stage.Some studies have proposed that physical factors such as spatial constraints/cell-to-cell contacts or local tissue mechanics regulate cell division.Cells in tissues are mechanically coupled through their cell-to-cell contacts,mediated by cadherins.Cell contact inhibition,caused by cell to cell contacts,is thought to be continuously active,and its loss usually triggers the switch from cell cycle arrest to proliferation.Cell proliferation because of the release of cell contact inhibition is characterized by the nuclear expression of Hippo/Yes-associated protein 1 and the Hippo/Yes-associated protein pathway activation.Our in vitro results suggested that adipose-derived stem cells at low cell density may have activated the Hippo/Yes associated protein pathway,but that high adipose derived stem cell density may have impeded it.Similar dynamic changes of cadherin expression,Hippo/Yes-associated protein 1 expression,and proliferation results were observed in the in vivo samples,indicating that adipose tissue dissection in our model may release cell-to-cell contacts and induce adipose-derived stem cell proliferation,and this process is possibly controlled by the release of cell contact inhibition and the Hippo/Yes-associated protein pathway activation.The mechanism of effect of external volume expansion has been discussed for years.Researchers have reported that external volume expansion creates mechanical stimulation and induces subcutaneous edema,ischemia,and inflammation,and the suggestion has been proposed that these maintain an adipogenic environment conducive to adipogenesis.Inflammation and adipogenesis are closely interrelated.Inflammation up-regulates peroxisome proliferator-activated receptor-yand promotes the mobilization of adipose precursor cells to form new adipose tissue.A similar external volume expansion model described by Lancerotto et al.indicated that external volume expansion-treated tissues displayed a signifcantly increased inflammatory infltrate by the end of the 2-hour stimulation and remained elevated for 2 days after treatment.Thus,we suggested that inflammation could be one of the adipogenic factors created by external volume expansion in our model.Edema is a well-known effect of external volume expansion both clinically and in experimental models.Similarly,edema could be found within the sample in our model at the early stage.Edema is often accompanied by adipose tissue formation in the affected body part in patients.Levi et al.demonstrated that adipose-derived stem cells from lymphedema-associated adipose tissue expressed much more adipogenic genes and had an enhanced ability to undergo adipogenic differentiation.Harvey et al.demonstrated that functional inactivation of the homeobox gene Proxl led to adult-onset obesity in mice,because of the ruptured lymphatic system and lymphedema.Our model is in agreement with these observation,and adipogenesis could be confirmed by expression of peroxisome proliferator-activated receptor-y and accumulation of small adipocytes among mature adipocytes.Moreover,mechanical stimulation imposed by external volume expansion may also influence the adipogenesis process.In vitro studies reported that sustained static mechanical stretching accelerates lipid production in adipocytes and induced adipogenic differentiation in preadipocytes,indicating an adipogenic effect of static stretching.External volume expansion device applied in our model mechanically stretch and stimulate tissues by suction,creating a sustained static tension circulation,which may promote adipogenesis to some extent.Conclusion1.Adipose tissue dissection releases cell-cell contacts and induces ASCs proliferation,and this process is possibly controlled by the release of cell contact inhibition and the Hippo/YAP pathway activation.2.Expanded ASCs in adipose tissue undergo adipose regeneration under the adipogenic environment created by external volume expansion. |
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