The Mechanism Of Adipose Tissue Regeneration Induced By External Volume Expansion System

Background and AimsSoft tissue defects resulting from tumor resection,trauma,and congenital deformities represent an ongoing challenge in reconstructive surgery.Such defects can be repaired by surgical reconstruction with autologous soft tissue flaps;however,the limited availability of qualitative autologous flaps as well as donor site morbidity significantly limit this approach.Tissue engineering of bioactive tissue constructs that can regenerate adipose tissue in both structure and function has offered solutions to soft tissue defects.In 2000,Khouri introduced an external negative tissue expansion(EVE)system for nonsurgical breast enlargement.EVE devices mechanically stretch and stimulate tissues by suction in a non-invasive manner.In recent years,the clinical application of EVE has been further expanded,and it has been used to correct thoracic deformity,augment breast after prosthesis removal,and reconstruct breast Recent researches found that EVE led to the recruitment of mesenchymal stem cells and the vascular remodelling with increased blood vessel density.However,the specific mechanism underlying adipose tissue regeneration by EVE remains unclear.We previously used an EVE device to construct expanded prefabricated adipose tissue in rats,resulting in significant growth of adipose tissue,and we found that adipose-derived stem cells(ASCs)tend to proliferate in the early stage of adipose tissue regeneration(at week 1)and undergo adipogenic differentiation at a later stage(at week 8).However,the cause of the cell fate change at a certain time point was not clear.Indeed,adipose tissue is considered to be a highly expandable connective tissue,and both cells and their pericellular matrices were deformed when adipose tissue was being stretched.Recently,it has been demonstrated that deformation of cells or tissues induced by an external force can control cell proliferation and differentiation.Moreover,ASCs in adipose tissue are mechano-sensitive and-responsive,and they can express different kinds of mechanosensitive proteins depending on whether they are exposed to mechanical stimulus or not.Integrins,which act as mechanosensors alone or in concert with cytoskeletal proteins,are one of the major components involved in mechanotransduction of stem cells.It is apparent that ASCs are able to sense and respond to mechanical forces through the regulation of specific proteins and the induction of cellular program.We hypothesized that the application of mechanical force to fat flap using an EVE device induces tissue deformation at the level of individual cells,leading to the stretch of ASCs,thereby inducing cell proliferation and adipogenesis and hence promoting fat regeneration.Angiogenesis is also closely related to adult neo-adipogenesis in fat regeneration.We previously observed that EVE induced the formation of special cell clusters in the well-vascularized connective tissue,and special cell clusters could secret some kinds of chemokines.However,the origin and the function of these special cell clusters remains unknown.We hypothesized those special cell clusters might promote vascular remodelling with increased blood vessel density by secreting corresponding chemokines.To test this hypothesis,we used an EVE device to construct EPAT in rats.The deformations of the adipose tissue and the proliferation and adipogenic differentiation of cells,as well as the expression of integrin β1were detected over time.The proliferation and adipogenic differentiation of ASCs under different mechanical strains were also investigated in vitro.We also investigated the properties of these cells by detecting specific cell markers.In addition,through examining the paracrine functions of these cells,we explored their effects on regulating the angiogenesis during adipose tissue regeneration.Materials and Methods1.The effect of tissue deformation on the proliferation and differentiation of cells in fat tissueWe used an EVE device to construct expanded prefabricated adipose tissue(EPAT)in rats.We demonstrated the deformations of the adipose tissue by real-time shear wave elastography(RT-SWE),and then detected the proliferation and adipogenic differentiation of cells,as well as the expression of integrin β1 over time.2.The effect of cell deformation on the proliferation and differentiation of ASCs in vitroThe proliferation and adipogenic differentiation of adipose-derived stem cells(ASCs)under different deformation levels were investigated in vitro using a custom-built Flexcell Device.Integrin β1 was knocked down to investigate its role in determining the cell fate of ASCs.3.Detection the properties of special cell clusters and their roles in fat regeneration The structure and origin of the special cell clusters were determined by hematoxylin and eosin staining,and immunohistochemistry;their role in adipose tissue regeneration was investigated by immunohistochemistry and western blot analyses.Results1.In the EPAT model,the deformation levels of fat flaps increased at week 1 and then decreased over time under EVE device.Cells were inclined to proliferate under a large deformation level at week 1,but shifted to adipogenic differentiation under a small defoamation level after week 4.2.In vitro,ASC proliferation rate under 12%deformation was higher than that under 6%and 0%,and inversely,adipogenesis was compromised.Integrin β1 expression positively correlated with proliferation,but negatively with adipogenic differentiation.Furthermore,knockdown of integrin β1 affected mechanical strain-mediated ASC proliferation and adipogenic differentiation.3.Special cell clusters began to increase at week 1 with a peak at week 4,and then receded from weeks 8 to 12.Clusters were identified as glandular epithelial cells as determined by their gland-like structure and expression of specific markers.The cell clusters induced significant infiltration of macrophage antigen-2(Mac-2)positive macrophages by secreting monocyte chemoattractant protein-1(MCP-1)at the early stage of suction.Subsequently,these infiltrated macrophages expressed massive vascular endothelial growth factor(VEGF)to promoted angiogenesis.ConclusionsOur findings reveal a potential mechanism of tissue deformation in EVE action.The deformation levels of fat flaps increased at week 1 and then decreased over time under EVE device.A large deformation level promotes ASC proliferation,whereas a small deformation level accelerates adipogenesis,and this cell-fate switch was mediated by the integrin β1 expression level.EVE generated glandular epithelial cell clusters.witch recruited macrophages to promote angiogenesis and subsequent adipose tissue regeneration.These findings shed light on the mechanisms underlying the effects of EVE devices on adipose tissue regeneration.