Optimized Adipose Tissue Engineering Strategy Based On A Neo-mechanical Processing Method

BackgroundThere is a clear and urgent clinical need for the restoration of soft tissue defects in plastic surgery.The development of adipose tissue engineering techniques using scaffolding materials could provide the means to accommodate this increasing demand.Currently,researchers continue to search for an optimal biomaterial for adipose tissue engineering applications.Decellularized adipose tissue(DAT)represents a promising scaffold for adipose tissue engineering owing to its potential to spontaneously induce in vivo adipose regeneration[1].However,previous studies failed to achieve significant adipogenesis with DAT even with stem cell-assisted DAT transplantation[2].It is generally accepted that conventional hash decellularization methods cause excessive disruption to DAT constituents(i.e.,ultrastructure and composition),and nutrient shortage due to inadequate vascularization further limits the adipoinductive properties of DAT implants in vivo[3-5].Hence,increasing matrix quality and promoting graft vascularization have been emphases for constructing DAT-based engineered adipose tissue in recent years.The removal of substantial quantities of lipid represents a unique challenge for adipose tissue decellularization.Commonly used methods to decellularize adipose tissue require days(120-156 h)and nearly half of the time is spent on the lipid removal process[6].Adipose tissue ECM is composed of collagen,elastin,glycosaminoglycan and ECM-bound soluble adipokines(any substance released by adipose tissue)[7-9].Given that all lipid removal agents inevitably disrupt adipose tissue ECM constituents,minimizing these undesirable effects would theoretically promote engineered adipose tissue construction.We previously utilized a simple mechanical method to selectively destroy most mature adipocytes and eliminate the lipid portion within adipose tissue.The resulting product,an injectable mixture known as extracellular matrix/stromal vascular fraction gel(ECMISVF-gel)is particularly rich in SVF cells and adipose-derived ECM[10].Remarkably,the ECM structure within the SVF-gel was intact after mechanical processing,suggesting that this process may be an ideal choice for rapid lipid removal with no additional damage to the ECM.In this study,we applied this mechanical method to remove lipids from the adipose tissue,and the lipid-devoid adipose tissue was then subjected to a short and mild decellularization procedure to fabricate high-quality DAT(M-DAT).A special adipose liquid extract(ALE)that may contain a high concentration of angiogenic factors was collected during the mechanical processing of adipose tissue.Therefore,the potential of incorporation of ALE into M-DAT to optimize the engineered adipose tissue construction was further tested.Methods and resultsIn this study,we proposed a neo-mechanical protocol for rapidly breaking adipocytes and removing lipid content from adipose tissue.The lipid-depleted adipose tissue was then subjected to a fast and mild decellularization to fabricate high-quality M-DAT and ALE.This developed strategy was evaluated based on decellularization efficiency.Both M-DAT and ordinary DAT samples were fixed and embedded in paraffin.Four-micrometer sections were then cut for hematoxylin and eosin or Masson trichrome staining,respectively.Residual DNA was extracted and then quantified with a microplate reader.Scanning electron microscopy was used to examine M-DAT and ordinary DAT microstructure.Samples were fixed,dehydrated,critical point-dried,coated with gold,and examined under a scanning electron microscope.The retention of several key ECM proteins in M-DAT and ordinary DAT was measured by western blot analysis.Total protein lysates from samples were harvested and protein concentration was estimated.Protein extracts were subjected to SDS-PAGE and then transferred to polyvinylidene difluoride membranes.Membranes were blocked and detected with primary antibodies anti-collagen I,anti-collagen IV,and anti-laminin,respectively.After incubation with secondary antibodies,detection was performed using a WesternBreeze Chemiluminescent Detection Kit.GADPH served as an internal control.Angiogenic factor components(basic fibroblast growth factor,epidermal growth factor,transforming growth factor-β1,and vascular endothelial growth factor)of ALE were evaluated by enzyme-linked immunosorbent assay.The angiogenic potential of ALE was evaluated by tube formation assay using human umbilical vein endothelial cells in vitro.Cells treated with angiogenic growth factors served as a positive control,and those grown in complete medium were used as a negative control.After 24 h,tube formation was observed and photographed.All tube formation assays were performed in quadruplicate.In animal study,ALE derived from this mechanical process was collected and incorporated into M-DAT to further optimize in vivo recellularization.Ordinary DAT was fabricated.M-DAT and rdinary DAT served as controls.Immunofluorescence staining was performed to measure adipogenesis and angiogenesis.Quantitative reverse-transcription polymerase chain reaction was performed to assess relative expression of adipogenic gene.M-DAT achieved the same decellularization efficiency,but exhibited better retention of ECM components and recellularization,compared to those with ordinary DAT.Protein quantification revealed considerable levels of angiogenic factors(basic fibroblast growth factor,epidermal growth factor,transforming growth factor-β1,and vascular endothelial growth factor)in ALE.ALE promoted tube formation in vitro and induced intense angiogenesis in M-DAT in vivo;furthermore,higher expression of the adipogenic factor PPARy and greater numbers of adipocytes were evident following ALE processing,compared to those in the M-DAT group.DiscussionIn this study,we described a simple protocol for decellularizing adipose tissue with a focus on decreasing the lipid removal time using a mechanical processing method.This protocol achieved the goal of successful decellularization and led to better retention of native ECM constituents as compared to commonly used methods.Moreover,ALE,an angiogenic factor-enriched extract,was collected during this mechanical process and was confirmed to optimize the recellularization of M-DAT when incorporated.Adipose-derived ECM proteins are crucial for maintaining the structural integrity of adipocytes and play a pivotal role in adipose tissue development and regeneration strategies[11,12].Most adipose tissue decellularization strategies employ isopropanol as an organic solvent to dissolve lipids[1,13,14].Although highly effective for this purpose,isopropanol tends to result in protein precipitation[1,13,14],leading to disruption of ECM constituents[15-17].Hence,reducing isopropanol processing time by prior lipid removal would likely contribute to the retention of ECM constituents and reduce the presence of residual cytotoxic chemicals in the processed matrix.The measurement of ECM components confirmed better retention of key proteins in the M-DAT scaffold,compared to that in the ordinary DAT scaffold.Moreover,SEM imaging and Masson staining clearly showed the preservation of fine network-type collagens within the M-DAT.These results partly explain the improved adipose regeneration result in the M-DAT group,as compared to that in the ordinary DAT group.Sarkanen et al.[18]previously described the collection of adipose tissue extract(ATE).The ATE was found to contain angiogenic factors and adipogenic factors and was proven to have angiogenic and adipogenic potential in vitro.It is reasonable to suggest that the mechanical processing of adipose tissue proposed in this study led to the rapid rinsing of soluble molecules from adipose tissue ECM.When incorporated in M-DAT,the superior recellularization results suggest that although these ECM-bound soluble molecules are in the minority of total ECM components,they have important biological activity and can participate in constructive tissue remodeling.In this study,the combination of ALE and M-DAT is a novel strategy for optimizing engineered adipose tissue construction,and this strategy could be promising to deliver clinical solutions for soft tissue regeneration.ConclusionsWe present a novel mechanical approach to treat adipose tissue.Simple and efficient mechanical processing of adipose tissue led to the production of high-quality decellularized matrix M-DAT and an angiogenic factors-enriched by-product ALE.The combination of ALE and M-DAT encouraged significant adipose regeneration and angiogenesis in vivo and could serve as a promising strategy for engineered adipose tissue construction.