Preparation And Characterization Of Decellularized Adipose-derived Matrix Using An Enzyme-free Procedure

Background:Autologous tissue transplantation is an important part of plastic surgery.However,the lack of graft tissue has been a problem for a long time.Recently,extensive research has been performed in tissue regeneration for tissue reconstruction.The decellularized scaffold prepared by decellularization technology has a physical structure and active ingredients that are similar to natural materials,which provides a microenvironment for cell growth and become a new option for repair tissue defects.Adipose tissue provides enough materials for making decellularized adipose-derived matrix(DAM),which was easily obtained through liposuction.Decellularization technologies have advantages and disadvantages in terms of decellularization effect,physical properties,cytotoxicity,and biocompatibility,but without exception,multiple enzymes are required in all the decellularization procedures.Since the enzymes are extracted from animals,which raises the risk of xenoantigens and restricts the clinical application.To this end,this study proposes a low-cost enzyme-free procedure mainly based on physical treatment.The present study demonstrates its feasibility as a tissue engineering scaffold through in vitro and in vivo experiments.Objective:1.Establish an enzyme-free procedure and verify its decellularized effect and the biological characteristics of DAM.2.To verify the cell compatibility of DAM prepared by enzyme-free procedure.3.To investigate the potential to induce adipose regeneration of the DAM prepared by the enzyme-free procedure.Methods:1.Introduce the technique of enzyme-free procedure and compare the DAM prepared by the enzyme-free procedure with the classic procedure(Flynn method).The enzyme-free procedure mainly based on physical treatment is used to decellularize adipose tissue without using any enzymes.DAM prepared by the classic method(Flynn method)was the control group.Compare the DAM prepared by two procedures in the following field.Ultrastructure of DAM which identity as the proportion of pore diameter ranging from 60 to 200 ?m.The physical property which is water uptake ratio.DNA residual,and glycosaminoglycan(GAG)content which were quantified by PicoGreen analysis and glycosaminoglycans quantitative experiments.2.In vitro experimentsHuman-derived adipose stem cells(hADSCs)were co-cultured with two kinds of DAM,and each DAM was prepared into blocks and powders.Live/dead staining was performed on days 1,6,13,and 19 to determine the cytotoxicity of the DAM.PrestoBlueTM quantitative analysis of the cell adhesion rate and cell proliferation within 7 days.Enzyme-linked immunosorbent assay to quantitatively analyze the content of vascular endothelial growth factor(VEGF)and basic fibroblast growth factor(bFGF)in the supernatant within 7 days.Adipogenic differentiation was performed after 7 days of co-culture,and oil red staining was used to quantitatively analyze the adipogenic differentiation of hADSC s.3.In vivo experimentsThe two kinds of DAM powders were injected in the back of nude mice.Histologic analysis with perilipin immunohistochemistry was performed at 1,2,and 4 weeks.And weighing the DAM samples at the same time points.Results:1.DAM prepared by the enzyme-free procedure is composed of collagen type ?-?,elastin,fibronectin,and laminin.Histological staining proved there are no intact adipocytes,nuclei,and oil residues.The proportion of pore diameter ranging from 60 to 200?m within DAM prepared by the enzyme-free procedure is higher than DAM prepared by the classic procedure(41±7.3)%(P<0.01).There is no significant differences in the water uptake ratio in either kind of DAM(P>0.05).DNA residue of DAM prepared by the enzyme-free procedure is less than that prepared by the classic procedure(58±0.4)ng/mg(P<0.001).And the GAG content is lower than that of the classic procedure(P<0.001).2.In vitro experiments implied that when the DAM are in bulk form,DAM prepared by the enzyme-free procedure have a higher cell adhesion rate,which is(30.6±8.7)%(P<0.01).When an equal number of hADSCs adhered to the DAM,the one prepared by the enzyme-free procedure promotes cell proliferation.When the DAM are in powder form,there are no significant differences in hADSCs proliferation.There are no significant differences in adipogenic differentiation in the two kinds of DAM(P>0.05).DAM prepared by the enzyme-free procedure can promote hADSCs to secrete VEGF and that prepared by the classic procedure can promote hADSCs to secrete bFGF.3.In vivo,DAM prepared by the enzyme-free procedure maintained a higher weight than DAM which prepared by the classic procedure at 1,2,and 4weeks.The perilipin positive cells could be seen in the DAM at 2 weeks which were adipocytes.Until 4 weeks,adipocytes were overgrown in the DAM prepared by the enzyme-free procedure(P<0.001).Conclusions:1.The enzyme-free procedure can effectively remove the cellular components and genetic material of adipose tissue,while retaining the ultrastructure,various types of collagen components and GAG.2.The DAM prepared by the enzyme-free procedure has good cell compatibility which is similar to the one prepared by the classic procedure,and provides a microenvironment for biological behaviors such as cell adhesion,proliferation,and differentiation.3.The DAM prepared by the enzyme-free procedure has good biocompatibility and the weight of the DAM remains stable.It shows a stronger capacity to induce regeneration of adipocytes than the one prepared by the classic procedure.