The Study On The Strategy Of Constructing Tissue-engineered Breast Based On Decellularized Adipose Tissue

Purpose:1.In vitro validation of the molecular mechanism of basic fibroblast growth factor(bFGF)for vascularization and lipogenic differentiation;2.Preparation of decellularized adipose tissue(DAT)loaded with bFGF and evaluation of the effectiveness of DAT loaded with bFGF in adipose tissue engineering;3.Validation of the DAT-based and 3D printed tissue engineering chamber for breast tissue engineering strategy.Methods:1.The effects of bFGF on the proliferation,migration,tube formation and expression of related downstream proteins of human umbilical vein endothelial cells(HUVECs)were investigated in vitro by proliferation assay,scratch assay,tube formation assay and western blot(WB);the effects of bFGF on the proliferation,lipogenic differentiation and expression of related downstream proteins in human adipose-derived stem cells(hADSCs)were also investigated by proliferation assay,lipogenic differentiation assay,real-time quantitative PCR(qRT-PCR)and WB;2.Rabbit-derived DAT was prepared using a chemoenzymatic method,and the efficiency of decellularization and extracellular matrix retention was observed by histology(H&E,Masson,Oil Red O and Gomori’s staining),DNA content determination,enzyme linked immunosorbent assay(ELISA)and WB,and scanning electron microscopy(SEM).The heparinized DAT was prepared by chemical cross-linking,loaded with bFGF.In vitro binding and release of bFGF within DAT were determined by ELISA.The biocompatibility and lipogenic induction of DAT were observed by proliferation and lipogenic differentiation assay of hADSCs.3.A biomimetic polycaprolactone(PCL)tissue engineering chamber was prepared by fused deposition modeling and its mechanical properties were measured by an electronic universal testing machine.Finite element analysis was used to simulate the mechanical changes of the chamber after implantation.Rabbit models were used to confirm the feasibility of tissue engineering chamber strategy for the generation of adipose tissue from DAT in vivo.After 12 weeks and 6 months of subcutaneous implantation,the distribution of newly-formed tissue inside the scaffolds was observed using MRI.The distribution of fat,fibrous tissue and blood vessels inside the scaffold was also assessed on histology(H&E,Masson and EVG staining).The expression of lipogenesis-related genes(PPARγ,C/EBPβ,AP-2),inflammation-related gene TNF-β and macrophage marker F4/80 was further investigated using qRT-PCR.In vivo degradation of PCL chambers was analyzed using scanning electron microscopy,differential scanning calorimetry and gel permeation chromatography.Results:1.The live-dead staining and CCK8 assays showed that 10 ng/mL bFGF significantly stimulated the proliferation of HUVECs compared to the blank group and the control group;the scratch assay showed that bFGF significantly promoted the healing of HUVECs(65.5%±5.6%,P<0.001)compared to the blank group(48.3%± 4.8%).The Transwell migration assay showed that bFGF significantly promoted the migration of HUVECs(441 ± 75 cells/field,P<0.001)compared to the blank group(253 ± 36 cells/field).Tube-forming assays revealed that bFGF increased the number and total length of tube-like structures formed by HUVECs within the Matrixgel,with quantitative results showing that more tubelike structures were formed in the bFGF group(101.3 ± 18.2/field,P<0.001)compared to the blank group(41.0 ± 7.4/field).The total length of tubular structures was also longer in the bFGF group(29.5 ± 5.8 μm/field,P<0.001)compared to the blank group(17.7 ± 2.0μm/field).Compared to the untreated blank group,the control group and the 10 ng/mL VEGF group,the live-dead staining and CCK8 assay results showed that 10 ng/mL bFGF treatment significantly promoted the proliferation of hADSCs;bFGF treatment also significantly promoted the lipogenic differentiation performance of hADSCs.On days 12 and 25 of lipogenic differentiation,more hADSCs were observed in the bFGF group,and more and larger lipid droplets were observed.Quantitative analysis of lipid droplet number and area showed that on day 12 after lipogenic induction of differentiation,compared to the blank group(110 ± 38 droplets/field,7270±2640 μm2/field),the bFGF group formed a greater number of lipid droplets(272 ± 79 droplets/field,P<0.05)with larger total area of lipid droplets(18770 ± 6540 μm2/field,P<0.05).After 25 days of lipogenic differentiation,the effect of bFGF in promoting lipogenic differentiation of hADSCs was more significant.Compared with the blank group(1369 ± 112 lipid droplets/field,100890 ± 12350 μm2/field),the bFGF group formed more lipid droplets(2347±178 lipid droplets/field,P<0.0001)and larger total area of lipid droplets(157690 ± 23100 μm2/field,P<0.0001).WB analysis showed that the binding of bFGF to FGFR-2 activated the phosphorylation of AKT and ERK and upregulated the expression of FAK and p38 MAPK,thereby promoting the expression of HUVECs.The binding of bFGF to FGFR-2 activated the phosphorylation of AKT and ERK and up-regulated the expression of JNK,thus promoting the proliferation and lipogenic differentiation of hADSCs.2.4,6-diamidino-2-phenylindole(DAPI)staining revealed no obvious nuclei within rabbit-derived DAT.Similarly,H&E or Oil Red O-stained DAT samples confirmed effective elimination of oil and cellular debris,and Masson staining and modified Gomori’s staining showed that DAT consisted mainly of a densely packed collagen network.DNA quantification showed a significant reduction in DNA content(56.97 ± 19.95 ng/mg,dry weight)compared to native rabbit adipose tissue(296.6 ± 85.1 ng/mg,dry weight).ELISA assays showed a moderate decrease in elastin and laminin content,but no impairment in collagen(COL)and a slight increase in glycosaminoglycans(GAGs)during decellularization.Western blot assay showed good retention of angiogenic factors such as vascular endothelial growth factor A(VEGFA)and transforming growth factor-β1(TGFβ1),while fibroblast growth factor 2(FGF-2)and tumor necrosis factor alpha(TNF-α)were not detected after decellularization.Live/dead staining showed that hADSCs cultured on DAT scaffolds had a more spindle-shaped cell morphology and showed a larger cell spreading area compared to those cultured on COL scaffolds.CCK-8 assays showed that hADSCs seeded on DAT scaffolds had significantly higher cell densities(from 1 to 7 days),in addition,oil red O-stained sections showed that hADSCs cultured on both coatings were successfully induced into adipocytes following lipogenesis induction,while the DAT coatings exhibited greater accumulation of lipid droplet and larger intracellular lipid droplets compared to the COL coating.Toluidine blue O(TBO)staining results showed significantly darker staining and higher levels of cross-linked heparin(77.07 ± 5.17 μg/100 mg,wet weight)for heparinized DAT(Hep-DAT)relative to the blank control.The 14-day in vitro release assays showed that compared to non-heparinized(control,50.2 ± 4.2 ng bFGF per 100 mg wet weight),Hep-DAT could load with more bFGF after washing(125.6± 17.3 ng bFGF per 100 mg).Specific lipid droplet fluorescence staining results showed that after 3 days of proliferation culture and subsequent 14 days of lipogenic induction,compared to DAT alone,more differentiated hADSCs and greater number of lipid droplets within individual cells were observed on bFGF-loaded DAT coatings.3.The fabricated PCL chambers had a porosity of 84.3%±1.6%and a stiffness of 4.6±0.3 MPa.12 weeks after subcutaneous implantation in the New Zealand rabbits,MRI results showed a homogeneous distribution of adipose tissue observed in the PCL/DAT group and especially in the PCL/DAT+group where a large amount of de novo adipose tissue production could be seen on MRI images.H&E staining confirmed that both the interior and exterior of the chamber of the PCL/DAT+group had been partially occupied by newly-formed adipose tissue.Masson and EVG staining showed that the PCL/DAT+group had many round or polygonal morphology of adipocytes accompanied by infiltrating blood vessels.A significantly higher overall adipogenesis rate in the PCL/DAT+group(34.2%±1.6%)were observed relative to the PCL/DAT group(21.5%± 1.1%)and the PCL group(12.0%±5.6%).No significant differences were found in the overall fibrous tissue and vasculatures among all groups,although inside cavity regions(19.15%±17.70%)were significantly fewer in the PCL/DAT+group than PCL/DAT(64.64%± 11.41%)and the PCL(70.39%±12.10%)groups.qRT-PCR-based analysis of expression of adipogenic-and inflammatory-related genes,at 12-weeks,revealed significant upregulation of CCAATenhancer-binding proteins β(C/EBP-β,P<0.0001)in all experimental groups,relative to endogenous adipose tissues.Notably,we found no significant differences in expression of peroxisome proliferator-activated receptor γ(PPAR-γ)and F4/80 between newly formed and endogenous adipose tissues in the PCL/DAT+group.However,significant upregulation and downregulation of adipocyte protein-2(AP-2,1.81 ± 0.20-fold,P<0.0001)and tumor necrosis factor α(TNF-α,0.50 ± 0.07-fold,P<0.05)were observed,respectively.4.Long-term adipogenic efficacy was further observed up to 6 months.MRI confirmed that adipose tissues with high signal could be found in all the observed sections and an increase in adipogenesis in all groups were observed relative to 12 weeks.Importantly,all representative sections of T1-weighted images showed that the PCL/DAT+group was dominated by high signals,suggesting that PCL chambers containing bFGF-binding DAT were occupied with newly-formed adipose tissue.In contrast,only a small amount of fat was observed in the PCL group.Although a substantial amount of adipose tissue was observed in the PCL/DAT group,cavities appeared at the edge or the very core of the chambers.Further validation of this phenomenon,via post-explantation images and histology,revealed abundance of adipose tissue both inside and outside chambers in the PCL/DAT+group,relative to the other groups,with no visible cavity observed after 6 months’ incubation.Considerable adipogenesis was also observed in the PCL/DAT group,but partially implanted DAT remained unchanged at the core of the chambers.As expected,very little adipose tissue was found at the edge of chambers in the PCL group,due to infiltration of surrounding endogenous adipose tissue,while the inside of the chambers was dominated by fibrous tissue.Quantitatively,significantly more adipose tissue both inside and outside was found in the PCL/DAT+group(82.54%±10.48%and 54.81%±4.31%for inside and outside,respectively)than the PCL(20.99%±11.93%and 23.03%±10.16%for inside and outside,respectively)and PCL/DAT(45.38%± 14.38%and 21.99%± 10.79%for inside and outside,respectively)groups.Furthermore,the PCL/DAT+group exhibited fewer overall fibrous tissue and cavity regions,as well as significantly more vasculatures than the other groups.All groups exhibited significant upregulation of adipogenic genes at 6 months,compared to endogenous adipose tissues.Specifically,we found significant upregulation of PPAR-γ(3.08±0.77-fold,P<0.0001),C/EBP-β(2.50±0.07-fold,P<0.0001)and AP-2(3.51 ± 0.55-fold,P<0.0001)in the PCL/DAT+group compared to endogenous adipose tissues.With respect to inflammation-related genes,F4/80 was significantly downregulated(0.73 ± 0.07-fold,P<0.001),while there was no significant difference in TNF-α expression.5.Degradation studies showed that the overall gross morphology of the implanted PCL chamber had no apparent differences but microcracks,and there was virtually no molecular weight change of chambers at each time point.Conclusion:1.The binding of bFGF to FGFR-2 activated the phosphorylation of AKT and ERK and up-regulated the expression of FAK and p38 MAPK,thus promoting the proliferation,migration and tube formation of HUVECs.Furthermore,bFGF could enhance the proliferation and lipogenic differentiation of hADSCs by activating AKT,ERK and JNK;2.The rabbit-derived DAT was prepared and characterized for the first time,based on previous studies based on human-derived DAT preparation,and its cellular components were completely removed while the extracellular matrix components were well preserved.In vitro studies have also demonstrated the good biocompatibility and lipogenic induction properties rabbit-derived DAT,which are further enhanced by loading with bFGF;3.An improved TEC strategy for soft tissue regeneration was successfully developed by combining biodegradable chambers and bFGF-binding DAT.When compared to other reported methods,this approach saves specialized harvesting of autologous pedicles and/or adipose tissue or reoperation for removal of the chamber.This concept’s feasibility was validated using long-term in vivo assays in rabbit models.Taken together,this new approach can generate multifold volume increased highly vascularized adipose tissue de novo from DAT.The concept described here could be assist in the endeavor to translate breast tissue engineering into clinical practice.