The Research On The Vascularization And Scaffold Functionalization For Breast Tissue Engineering

Purpose: 1.Explore the pro-angiogenic function of Hypoxia Inducible Factor stabilizer FG-4592 and verify the feasibility of local immobilized FG-4592 for the promotion of vascularization in breast tissue engineering;2.Explore the feasibility of crystal lattice-like structural design for the additive manufacturing of mechanical bionic tissue engineered breast scaffold and verify its superiority for the in vivo fabrication of tissue engineered breast;3.Explore whether the surface functionalization by Graphene would endow the breast scaffold photothermal anti-tumor effect and verify its safety and efficiency for the prevention of breast cancer recurrence.Methods: 1.The effects of FG-4592 with different concentration(0,5,20,50 u M)on the migration,tube formation,HIF-1α and VEGF expression were preliminarily observed in vitro by human umbilical vein endothelial cells(HUVEC)scratch test,tube formation test,q RTPCR and Western Blot.The effect of FG-4592 on the expression of inflammatory cytokines were observed in RAW264.7 cell line by q RT-PCR.Thereafter,an in vivo breast tissue engineering model was established by loading FG-4592 with different concentration(0,24,48 m M)in Fibrin gel in a breast tissue engineering chamber and subcutaneously implanting the chamber at the dorsal of SD rat.Tissue samples were collected 3,7,and 14 days later and HE staining as well as immunohistochemical staining(CD31,HIF-1α,VEGF)were used to observe the effect of FG-4592 on the oxygen sensing pathway and tissue engineering vascularization in vivo.2.Breast scaffold models were designed using UG NX10.0 computer aided design software to mimic the crystal lattice structure of isometric crystal system.Then breast scaffolds were additive manufactured using fused deposition modeling 3D printer and polyurethane printing material.The structural and mechanical property of the scaffolds were characterized using Micro-Computed Tomography(Micro-CT)and All-Electric Dynamic Test Instrument,respectively.The different structured scaffolds were screened and the one with the compressive modulus closest to native breast tissue was selected as experimental group and the one with the highest compressive modulus as control group.To fabricate tissue engineered breasts,the scaffolds were implanted beneath the dorsal skin of nude rats and human adipose tissue were injected into the scaffolds two weeks later.Three months after fat injection,the animals were subject to magnetic resonance imaging(MRI)、Micro-CT angiography and histological evaluation(HE、Perilipin,CD31,MAC2,CD206)to evaluate the fabricating effect of tissue engineered breast.Finite element analysis(FEA)were performed using Ansys16.0 software to simulate the mechanical condition of the in vivo implanted scaffolds.3.Different concentration(0,100,200,500ppm)of graphene nanoparticle(GN)suspensions were used to functionalize the surface of 3D printed polyurethane scaffolds(Gf S)by concussion and heat drying.Human adipose derived stem cells(ADSC)were seed on the scaffolds and live/dead staining as well as cytoskeletal staining were performed 7 days later.Scaffolds were subcutaneously implanted beneath the dorsal skin of rats for 1 weeks and 12 weeks and subsequently subject to hematoxylin and eosin(HE)staining.808 nm laser was used to illuminate the scaffolds and temperatures were recorded to evaluated the photothermal effect.In vitro photothermal breast cancer cell ablating effect of the scaffolds were assessed using live/dead staining and CCK8 assay.Lastly,in Balb/C breast cancer model divided in four groups((1)PU scaffold without laser,PU-NL or(2)with laser,PU-L;(3)Graphene functionalized PU scaffold without laser,Gf PU-NL or(4)with laser,Gf PU-L),in vivo breast cancer recurrence preventive effect of the scaffolds was evaluated by tumor recurrence curve,tumor in vivo imaging and tumor anatomical study.Results1.FG-4592 can significantly promote the migration(0u M:45.84±5.92%;50u M:66.61±6.09%)and tube formation(0u M:28.67± 9.02 count/field;20u M:52.90 ±1.41 count/field)of HUVEC,up-regulate the protein expression levels of HIF-1α and VEGF as well as the m RNA expression level of VEGF in vitro;FG-4592 treatment did not significantly alter the Tnfα m RNA level in RAW264.7 macrophage cell lines and the Il1β m RNA level was slightly up-regulated.Fourteen days after implantation of the breast tissue engineering chamber model,we observed that comparing to the negative control group,Fibrin gel loaded with FG-4592 promoted the generation of fibrovascular tissue(0m M:488.24±40.11um;24m M:571.76±51.18um;48m M:734.12±104.35um).CD31 immuno-histochemical staining showed that 48 m M FG-4592 treatment decreased the vessel number(0m M: 84.50±12.15 count/field;48m M: 46.75±7.63 count/field)but increased the vascular lumen size(0m M: 213.12±23.02 um2;48m M:326.75±44.90 um2)in the fibrovascular tissue.HIF-1α and VEGF staining showed that experimental group with different concentrations of FG-4592 had greater number of HIF-1α(+)cells and VEGF(+)cells comparing to negative control.2.Based on the crystal lattice of face centered cubic and body centered cubic,both of which belong to isometric crystal system,we designed four kinds of breast scaffolds.According to the numbers of ordered arranged nodes(N)and interconnected struts(S)in their unit cells,the scaffolds were termed as N5S4,N9S8,N7S6 and N4S6,respectively.Mechanical tests revealed that the compressive moduli of the scaffolds were diverse(N5S4:31.74±3.38KPa;N7S6:79.57±4.42KPa;N9S8:81.44±1.96KPa;N4S6:113.10±2.49KPa),in which the compressive modulus of N5S4 scaffold was within the range of native breast.N5S4 scaffold were used in experimental group and N4S6 scaffold in control group for animal study.MRI revealed that the volume retention rate of injected adipose tissue in N5S4 group was significantly higher than that in N4S6 group(73.1±25.5% vs 45.6±14.0%).HE staining revealed that in comparison to N4S6 group,N5S4 group possessed higher proportion of adipose tissue(64.6 ± 5.8% vs 34.7 ± 15.0%)and lower proportion of fibrotic tissue(8.5 ± 2.3% vs 12.6 ± 1.8%).In addition,more Perilipin(+)adipocytes(62.1±12.5count/field vs 16.6±6.3count/field)and fewer proportion of MAC2(+)CD206(+)M2 macrophages(7.3±4.0% vs 25.3±4.7%)could be observed in N5S4 groups than in N4S6 group.FEA results indicated that after in vivo implantation,N4S6 scaffold would generate more high deformation region(50.0% vs 32.9%)and high-stress region(13.51% vs 9.92%)than N5S4 scaffold.3.On the basis of the additive manufactured polyurethane breast scaffolds,we modified the surface of the scaffolds using different concentration of Graphene nanoparticle suspensions(0、100、200、500ppm)to fabricate Graphene functionalized scaffolds(Gf S).Live/Dead cell and cytoskeletal staining revealed that the graphene concentration as high as 500 ppm would inhibit the survival rate of ADSC on the surface of the scaffold(0ppm:99.17±0.72%;100ppm:96.32±2.48%;200ppm:84.01±3.32%;500ppm:20.98±6.71%).HE staining of the subcutaneous implanted scaffolds revealed that Gf S with Graphene concentration no higher than 200 ppm could integrated well with adjacent tissue without significant foreign body reaction.Under 808 nm laser exposure,Gf S could be heated and ablate the adjacent MDA-MB-231 breast cancer cells.The breast cancer cell survival rate was negatively correlated with graphene concentration(0ppm:100.00±20.46%;100ppm:66.93±27.19%;200ppm:59.95±18.99%;500ppm:28.81±24.67%)and light intensity(0m W/cm2:100.00±6.29%;650m W/cm2:96.51±10.77%;1210m W/cm2: 41.11±16.70%;1490m W/cm2: 19.68±16.05%).Lastly,the tumor recurrence curve,tumor volume curve,tumor in vivo imaging and histological study revealed that Gf S could effectively inhibit the tumor recurrence of 4T1 breast cancer in Balb/C mice model(recurrence rate: PU-NL:100%;PU-L:87.5%;Gf PU-NL:87.5%;Gf PU-L:25%).Conclusion 1.By stabilizing the HIF-1α protein and upregulating the expression of pro-angiogenic growth factor VEGF,the small molecule drug FG-4592 immobilized in Fibrin scaffold could promote the vascularization in a breast tissue engineering setting.2.The mechanical bionic scaffold(N5S4 scaffold)could be designed by mimicking the crystal lattice structure and be fabricated using fused deposition modeling technique.The mechanical bionic scaffold is advantageous for the in vivo fabrication of tissue engineered breast.3.The polyurethane scaffolds could be effectively functionalized using 200 ppm Graphene nanoparticle suspension without compromising its biocompatibility.Take the advantages of photothermal effect,the scaffolds could effectively ablate tumor cells and prevent the recurrence of breast cancer.4.With the help of the pro-vascularization strategy and mechanical bionics and tumor preventive scaffolds involve in this study,the effectiveness of tissue engineering based post-mastectomy breast reconstruction could be improved.