The Mechanism Of Adipose Tissue Regeneration Induced By Mechanical Forces

Background and ObjectionThe repair of large volume soft tissue defect is the greatest for plastic surgeon. There were 5.76 million cases of reconstructive operation, 76% of which were because of the soft tissue defect after tumor resection.Surgical strategies for tissue loss replacement initially laid on the historical maxim "replace tissue with like-tissue", so autologous adipose tissue is considered as the most ideal soft tissue filling material. However, clinical use of fat transplantationhas been limited by the controversial results in the degree of lasting of corrections, due to fat reabsorption.The traditional autologous skin flap or fat flap for repairing soft tissue defect causes irreversible deformity and scar in donor site.So the emerging field of adipose tissue engineering aims at developing fuctional adipose tissue, which provides a new way for the repair of soft tissue defect of large volume. The key aspects of the adipose tissue engineering include cell source, scaffold biomaterial, and microenvironment to provide the appropriate cues and signals for cell growth and adipose tissue formation.Unlike traditional adipose tissue engineering, Morrison et al. inserted the chamber subcutaneously in the groin enclosing a fat flap based on vessel pedicale, and the fat flap spontanesou grew. This technique is called "tissue engineering chamber", which demonstrated the production of new autologous tissue from minimal starting tissue (5 ml) with no additional cell transplantation or processing. Fat tissue grew over five times its original volume during this period.The technique of tissue engineering chamberproduces large volume and functional adipose tissue with vessel pedicle, which gets closer to clinical application.Adipose tissue engineering chamber takes advantage of the special structure of adipose tissue, which itself includes the three key elements of adipose tissue engineering. The precise mechanism by which tissue growthoccurs in this model remains uncertain, but according to previous studies, the following factors may be involved in the tissue growth:①sufficient blood supply, providing nutrients for tissue growth and recruiting more mesenchymal stem cells to the site,②appropriate adipogenic microenvironment,③loss of cell-to-cell contact,④changes of the mechanical forces.How the changes of the mechanical forces control the adipose tissue growth? Generally, cells in adipose tissues are physiologically exposed to compound mechanical loading:tensile, compressive and shear strains/stresses, which are associated with bodyweight loads andweight-bearing. During sitting for example, the adipose tissues at the buttocks are subjected to peak tensile, compressive and shear strains of ～30%,～45% and ～40%, respectively; a lying posture induces peak strains that are approximately half these magnitudes at the same anatomical location. Recently, the research concerning the molecular and cellular basis of the mechanosensing and subsequent intracellular signaling (hereafter referred to as mechanotransduction) has been preceded by research into the force-receivingand the force-generating cells/tissues such as musculoskeletal, cardiovascular, cartilage and connective tissues.Some studies show that adipocytes and stem cells in adipose tissue have mechanosensibility and mochanoresponsivity. The mechanical forces include tension, shear force, pressure, hydrostatic and osmotic pressure. Mechanotransconduction is by means of cytoskeleton that connects the cellular internal structure.The focal adhesions complexes are specific membrane molecules that anchor the cell to ECMor other cells. The main components of focal adhesions complexes are transmembrane proteins (such as integrins), which internally function as anchors for the cytoskeleton. Other proteins such as protein kinases and stress-activated channels that can act as alternative force receptors also gather at focal adhesions.The cytoskeleton has the capacity to store en- ergy distributed throughout its various compo- nents in an equilibrium of tensile and compressive forces. The energy stored in the cytoskeleton derives from the balance of multiple forces, those applied from the outer environment (ECM and other cells), those produced inside the cell (and cytoskeletal remodeling) and osmotic and gravitational forces, which are thus effectively integrated. Additional external or internal forces alter the established state ofcytoskeletal elements with a shifting of the reciprocal position transmitted globally inside the cell.Thismechanism explainsthe transform of restingtension and its perturbations into biochemical signals.Cells in adipose tissue transfer mechamical stimulus into biolchemical signal and regulate cell proliferation and differentiation by Rho-Rho-kinase pathway and ERK/MAPK signaling, cyclooxygenase pathway, Wnt signal and other signal pathway.The invasive expander has been widely used in plastic surgery. The expander is inserted subcutaneously causes the skin tooverstretch, forcing it to generate new skin to accommodatethe expander. As a direct or indirect resultof this mechanical force, the expanded skin andsubcutaneous fat layer become thinne. Khouri et al. increased breast size by negative pressure suction and achieve breast enlargement without surgery. The clinical studies proved that the volume of breast incrased at least one cup by treatment of this device for 10 hours everyday.Tissue edema and elastic deformation caused by suction or adipose tissue regeneration lead to the increase of breast volume.If it can be proven that the tissue growth is adipose tissue regeneration induced and regulated by mechanical forces, then how the mechanical forces regulate the process of adipose tissue regeneration?This study aimed at the mechanism of adipose tissue regeneration induced by mechanical forces and investgated the effects of mechanical forces on proliferation, differentiation and section of pro-inflammatory cytokines of ASCs. By negative pressure suction induced adipose tissue regeneration of SD rat model, mechanism of of adipose tissue regeneration induced by mechanical forces is explained. This is a very important study for achieveing non-invasive construction of vascularized adipose tissue and provides a new ideal and experimental basis for repair of large volume soft tissue defect.Methods and Materials1. The effects of mechanical force on the proliferation, adipogenic differentiation, and section of pro-inflammatory cytokines of ASCs(1) Mechanical forces were loaded on ASCs. The experimental groups were as follows:①control group:no stretch; ②stretch group:12%,10cycles/min stretch; ③stretch+cytochalasin D group:12%,10cycles/min stretch and culture with 2μM cytochalasin D. The cell morphology, cytoskeleton (stained with pholloidin) and proliferation were observed and tested after 48h stretch.(2) ASCs of the 3 groups were cultured in adipogenic induction medium. PPARy gene expression was tested by Real-Time PCR after 5 days adipogenic induction, and LPL gene expression was tested by Real-Time PCR and Oil Red O stain after 12 days adipogenic induction.(3) The intercelluar expression of MIF was analysed by Western blotting, and the concentration of MIF and IL-6 were analysed by ELISA.(4) The effect of pro-inflammatory cytokines released by ASCs subjectd to mechanical forces on migration of macrophages (induced from THP-1) was tested by Transwell chamber.(5) Statistical analyses:numerical data in each group are presented as mean ± standard deviation (SD). Comparisons between multiple groups were performed with one-way analysis of variance (ANOVA), followed by the Student-Newman-Keuls (SNK) multiple comparison procedure. All statistical analyses were performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Differences were considered to be significant at P< 0.05.2. The expanded prefabricated adipose tissue structured by mechanical forces(1) The inguinal fat flaps were transferred to abdomen, and then negatice pressure suction devices were placed on fat flaps.(2) The fat were harvested at 0 day,1 week,8 weeks, and 12 weeks after suction in both control group and suction group. In suction group, the devices were removaled at 12 weeks and the fat were harvested after 4weeks.(3) The adipose tissue samples were analysed as following:①volume of adipose tissue, ②H&E staining, ③immunofluorescenct staining of CD31 to analyse vascular density, ④immunofluorescenct staining of CD34 and perilipin to analyse adipose tissue regereneration, ⑤expression of adipogenic gene tested by Real-Time PCR, ⑥section of IL-1β, IL-6, TNF-α, and MIF tested by ELISA.(4) Statistical analyses:numerical data in each group are presented as mean ± standard deviation (SD). Comparisons between multiple groups were performed with one-way analysis of variance (ANOVA), followed by the Student-Newman-Keuls (SNK) multiple comparison procedure. All statistical analyses were performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Differences were considered to be significant at P<0.05.Results1. Mechanical forces promoted proliferation, inhibited adipogenic differentiation, and up-regulated pro-inflammatory cytokines of ASCs① The changes of cytoskeleton:in the control group, the ASCs were randomly aligned and actin stress fibers were distributed randomly. In the stretched group, ASCs became arranged nearly perpendicular to the direction of applied stretching, while parallel arrays of actin stress fibers within the individual ASCs also became aligned in this direction. In the cytochalasin D treated group, the poorly organized and mostly subplasmalemmal actin cytoskeleton is entirely dispersed into clusters by cytochalasin D treatment.② Cell proliferation tested by MTS:the cyclic group showed a higher proliferation profile than the control and cytochalasin D groups both in 48h and 72h analysis (P<0.05=.③ Oil Red O staining:ASCs in stretch group began to show adipogenic differentiation after adipogenic induction 12 days, later than control group and cytochalasin D group. The cytochalasin D group showed the most lipid drops stained by Oil Red O.④ Real-Time PCR:the expression of PPARyand LPL was highest in cytochalasin D group compared with other two groups (P<0.05=.⑤ The MIF expression of ASCs at intercellular and extracellular level:the stretch group showed the highest MIF expression level in the three groups at intercellular and extracellular level (P<0.05=. However, the cytochalasin D treated group still showed higherMIF expression level than control group.⑥ The IL-6 expression in ASCs culture supernates:stretch promoted IL-6 expression level increasing by 2.2-fold to control group (P<0.05=. Meanwhile, IL-6 expression of ASCs loaded stretch and treated with cytochalasin D is still higher than control group.⑦ The role of pro-inflammatory cytokines released by ASCs subjectd to mechanical forces on migration of macrophages:the number of macrophages in stretchgroup that migrate through the membrane was the most in the three groups (P <0.05). However, the cytochalasin D treated group still showed higher number of migrated macrophages than control group.2. Adipose tissue regeneration induced by mechanical forces① The volume of fat flap:statistical analysis revealed that the volume of fat flap in control group didn’t increase with time, while the volume of fat flap of suction group increased from 4 weeks and maintained at 8 weeks and 12 weeks (P<0.05=. The volume of fat flap lightly decreased when suction device was removed for 4 weeks.② H&E staining:there were no significant differences of tissue structure between two groups and normal adipose tissue at every time point.③ Vascular density:compaired with control group, the number of vessels of suction group maintained at a higher level from 4 week to 12 weeks (P<0.05=, which returned to normal level after removal of device for 4 weeks.④ Cell proliferation:the number of Ki67 positive cells in suction group was more than control group (P<0.05=. Some of Ki67 positive cells were CD34 positive at the same time.⑤ The expression of adipogenic genes:compaired with control group, the expression of PPARγ and CEBPβ in suction group were higher from 1 week to 12 weeks (P<0.05=. The expression return to normal level and is no significantly different with control group after removal of device for 4 weeks.⑥ The expression of inflammatory cytokines:the expression trends of IL-1β, IL-6, TNF-α, and MIF in adipose tissue of suction group are similar. The expression level peaked at 1 week and lightly decreased but maintained at a higher level until 12 weeks, but returned to control group level when the suction device was removed for 4 weeks.DiscussionsAll organisms living on the earth are subjected tovarious mechanical forces, such as gravity, tension, and compression. This, in turn, affects the morphologyand function of our bodies. How do the cells sense and response to the mechanical forces? Donald Ingber explains the complexmechanotransduction pathways by the concept of tensegrity. Accroding to this theory, the cell is considered as tensegrity structure of multifunctional moduls. Microtubules are stiff scaffolds, and microfilaments and intermediate filaments are elastic stuctures. The balance between the tension and the pressure of these structures maintains the three-dimensional structure of the cell structure and shape. The focal adhesions complexes are specific membrane molecules that anchor the cell to the extracellular matrix (ECM) or other cells. The main components of focal adhesions complexes are transmembrane proteins (such as integrins), which internally function as anchors for the cytoskeleton. Other proteins such as protein kinases and stress-activated channels that can act as alternative force receptors also gather at focal adhesions. The cytoskeleton is built by filaments and tubules that vary in size and stiffness, some acting more like uncompressible struts, others as elastic strings. The cytoskeleton has the capacity to store energy distributed throughout its various components in an equilibrium of tensile and compressive forces. The energy stored in the cytoskeleton derives from the balance of multiple forces, those applied from the outer environment (ECM and other cells), those produced inside the cell (cytoskeletal remodeling) and osmotic and gravitational forces, which are thus effectively integrated. Additional external or internal forces alter the established state ofcytoskeletal elements with a shifting of the reciprocal position transmitted globally inside the cell. The shape and relative position of all molecules and organelles connected to the cytoskeleton are affected, changing the likeliness of reactions to occur and providing a mechanism to transform restingtension and its perturbations into biochemical signals. Further, the isolated nucleus itself behaves as a structure built according to tensegrity principles, and its inner framework is in close interaction with the cytoskeleton via anchorage to the nuclear membrane. Mechanical stimuli can be directly transmitted to the DNA influencing gene transcription.There is close relationship of cell shape and cell function. Cell shape was a critical factor in determining whether cells would proliferate or stay quiescent. In vitro study showed that cells that cannot elongate tend toward apoptosis; increasing stretching stimulates differentiation and induces cells to proliferate. We got the similar results in this study. ASCs subjected to cyclic uniaxial stretch became arranged nearly perpendicular to the direction of applied stretching, while parallel arrays of actin stress fibers within the individual ASCs also became aligned in this direction. However, the poorly organized and mostly subplasmalemmal actin cytoskeleton is entirely dispersed into clusters by cytochalasin D treatment.The data of Oil Red O staining and Real-Time PCR showed that cyclic uniaxial stretc inhibited adipogenic differentiation of ASCs, which is relative with the changes of cytoskeleton.The promotion of proliferation and inhibition of adipogenic differatiation induced by cyclic uniaxial stretch were blocked by the disruption of cytoskeleton.The mechanical forces significantly effect on adipogeic differentiation depending upon the type, magnitude, frequency, and duration of applied stimulation. Generally, cyclic stretch promotes osteogenic differentiation and inhibits adipogeic differentiation. Shoham et al. reported thataccelerated lipid production of differentiating 3T3-L1 cells by the static, which concerted with our results that static stetch promoted adipogenic differentiation of ASCs. Mechanical forces not only change the shape and aligment of cells, but also regulate cell proliferation, differentiation and section of cytokines. Previous research showed that hASCs secreted an array of cytokines when exposed to 12% multitiaxial stretch. In this study, we also prove that mechanical stretch directly induced secretion of pro-inflammatory cytokines, IL-6 and MIF by hASCs. The secretion of cytokines reduced when hASCs were treated with cytochalasin D even subjected to stretch, which means that mechanical stretch induces secretion via rearrangement of cytoskeleton. However, the expression of IL-6 and MIF was partially blocked, which suggests that mechanical force regulates the expression of IL-6 and MIF independently through cytoskeleton.Obesity is not only the increase of volume of adipose tissue, but also relative with the chronic inflamamation of adipose tissue. The inflammatory environment plays a key role in regeneration of soft tissue in tissue engineering chamber model. The feature of inflammation includes pro-inflammatory cytokines expression and inflammatory cells recruitment. MIF investigated in this study is an important pro-inflammatory cytokine and has following function:①promoting the adipogenesis of preadipocytes and increases the size and number of mature adipocytes, ②contributing to chronic adipose tissue inflammation,③acting as a chemoattractant, ④directing ATMs into an M1 polarization status, ⑤up-regulating pro-inflammation cytokines. Stretch (high expression of IL-6 and MIF in stretch group) promoted macrophage migration has been proven in this study. In adipose tissue, the migrated macrophages become another main source of pro-inflammation cytokines, such as IL-6, which recruit additional leukocytes, so maintaining and expanding the inflammatory process.In the SD rat model of adipose tissue regeneration induced by negative pressure suction, the volume of adipose tissue increased and lighly decreased after device removal, but still larger than the initial volume, which means that the increase of adipose tissse volume is the real tissue growth, not the elastic deformation and edema. The number of Ki67+ cells is more in suction group than control group. We suggest that this is relative with the mechanical forces, which is proven in the in vitro study. The proliferated cells may include stem cells and vascular endothelial cells, because the number of CD31+ and CD34+ increased in suction group. Moreover, Real-Time PCR showed that the expression of adipogenic gene increased. The mechanical forced induced by suction included various different forces, so which promoted cell proliferation, and promoted adipogenic differentiation at the same. This is not contradictiory and provides two necessary elements for adipose tissue regeneration: adipogenesis and angiogenesis.Mechanical forces directly or indirectly cause inflammatory of adipose tissue, which is associated with adipose tissue regeneration. Mechanical forces lead to hypoxia and edema, which result in inflammatory of adipose tissue. However, from another point of view in this study, direct effects of mechanical forces are investigated. We suggest that mechanical forces direct up-regulated the section of pro-inflammatory cytokines (MIF and IL-6), which recruited more inflammatory cells and up-regulated other inflammatory cytokines. MIF not noly is a pro-inflammatory cytokines, but also promot adipogenesis.Conclusion1. Mechanical forces promoted proliferation and inhibited adipogenic differentiation of ASCs, which was close relative with the change of cell shape and the rearrangement of cytoskeleton, as well as blocked by disruption of cytoskeleton (ASCs treated with cytochalasin D).2. Mechanical forces up-regulated pro-inflammatory cytokines (MIF and IL-6) of ASCs, which promoted macrophage migration.3. Mechanical forces induced by suction promoted cell proliferation (ASCs and vascular endothelial cells) through rearrangement of cytoskeleton at early stage, which provides sufficient stem cells and blood supply for adipose tissue regeneration. MIF released by adipose tissue promoted inflammatory response and induced adipogenic differentiation when the proliferation was at peak level.