Construction And Experimental Study Of Tissue-Engineered Breast Based On Microsphere Printing Technology

BackgroundBreast absence after radical mastectomy is a common clinical problem,while current clinical breast reconstruction methods have limited effects.Breast tissue engineering can solve the limitations of autologous tissue and implant reconstruction,and is an ideal breast reconstruction method.3D printing technology can realize the personalized customization of breast scaffolds,however 3D printed breast scaffolds with soft tissue mechanical properties need to be developed.Granular hydrogels have printability,soft-tissue-like mechanical strength,and suitable pores for cell growth,which have great potential in breast tissue engineering applications.PurposeTo explore the feasibility of constructing tissue-engineered breast using microsphere printing technology and to verify the effectiveness of the constructed tissue-engineered breast in vivo.Method1.Preparation of Breast Scaffold by Microsphere Printing Technology:Prepare GelMA hydrogel microspheres by coaxial flow microfluidic emulsification technology.Explore the influence of needle inner diameter,water and oil phase flow rate ratio,swelling,penetration and extrusion on the size of GelMA microspheres.Verify the shear thinning and self-healing properties of GelMA granular hydrogel ink through rheological tests.Prepare breast scaffold by GelMA granular hydrogel ink using extrusion 3D printing technology.Explore the effects of nozzle temperature,slice layer height and extrusion speed on printing accuracy.Evaluate the mechanical strength of the scaffold by compression detection.Observe the surface topography of the printed scaffold by stereo microscopes,SEM and laser microscopes.Use a laser confocal microscope to observe the internal pores of the printed scaffold.2.Construction of Tissue-Engineered Breast in Vitro:Extract human adipose derived stem cells(hADSC)from adipose tissue obtained by liposuction,amplify and seed hADSC on the surface of the printed scaffold,and induce hADSC to adipogenitic differentiation to construct tissue-engineered breasts.Observe the effect of cell seeding density and microspheres size on cell distribution and infiltration by cytoskeletal fluorescence staining.Explore the cell growth and proliferation on scaffolds by cell counting and CCK-8 cell viability proliferation assays.Explore the influence of scaffold on cell differentiation behavior by lipid droplet detection.3.Application of Tissue-Engineered Breasts in vivo:Implant the tissue-engineered breast subcutaneously on the back of 8-week-old female nude mice,and use the scaffold without cells as the control group.Take HE staining,Masson staining,immunohistochemical staining and immunofluorescence staining after 4 weeks to observe the adipogenesis,neovascular growth and immune response of the two groups.Results1.Size-tunable GelMA microspheres with sizes at 145.9～416.4μm can be successfully prepared by coaxial flow microfluidic emulsification technology.The granular hydrogel ink prepared by GelMA microspheres is shear-thinning and self-healing,which making it possible for extrusion 3D printing.GelMA granular hydrogel ink can be extruded to prepare porous hydrogel breast scaffold,and the best printing accuracy can be reached by setting the nozzle temperature at 24℃,the slice height at 0.62 mm(that is,8 layers),and the extrusion speed at 1.50 mm~3/s.The compressive modulus of the printed scaffold is close to that of human adipose tissue,which is 9.53±4.65 k Pa,and the scaffold has pores conducive to gas and substance exchange.2.The hADSC can be seeded on the surface of the porous hydrogel breast scaffold to successfully construct a tissue-engineered breast.The cell density is positively correlated with cell distribution width and the infiltration depth.On the 7th day after seeding with 10M/m L cell suspension density,cells can infiltrate into the interior of 2～3 mm.After adipogenic induction,intracellular lipid droplets can be observed in hADSC and can increase with induction period going on.hADSC could grow,adhere,proliferate and undergo adipogenesis differentiation on the scaffold.3.After 4 weeks of implantation of the tissue-engineered breast into nude mice,adipose tissue formation and neovascular growth are visible in the scaffold pores,while only fibrous connective tissue is visible in the pores of the control group,which indicates that the tissue-engineered breast has certain application effect in vivo.ConclusionThe novel GelMA granular hydrogel ink can successfully be used with extrusion 3D printing technology to construct breast scaffolds,which can simulate the mechanical strength of human adipose tissue,provide pores suitable for cell growth.Seeding hADSC and inducing adipogenic differentiation in vitro can construct tissue-engineered breast,which has a certain in vivo regenerative effect and has a good application prospect in breast tissue engineering.The construction strategy of microsphere printing technology has a good application prospect in breast tissue engineering.