Construction Of Vascularized Adipose Tissue Based On Microsphere Embedded Bioprinting Technology And Application In Vivo

Background:The soft tissue defect caused by tumor,congenital malformation and trauma is a common and intractable problem for plastic surgeons.The construction of engineered adipose tissue by tissue engineering technique has a good prospect for clinical application.However,there are still two problems that lead to poor regeneration effect of adipose tissue in existing studies:(1)the lack of embedded vascular networks in large-scale tissue constructs;(2)the physiological activities of cells are restricted since they are encapsulated in traditional bulk hydrogels.Compared to the bulk hydrogels,the packed hydrogel microspheres have significant porosity,which can be turned to support cells migration and nutrients exchange.Besides,the embedded bioprinting technique can be used for the construction of vascular networks within tissue constructs.Above all,it could be hypothesized that a combination of hydrogel microspheres and embedded bioprinting technique would be more beneficial to vascularization of adipose tissue and promote effect of adipose tissue regeneration.Purpose:1.To explore the feasibility of constructing complex vascular channels by embedded bioprinting using matrix bath based on hydrogel microspheres.2.To explore the feasibility of the prevascular channel scaffold for adipose tissue engineering.Methods:1.Fabrication and characterization of hydrogel microspheres: Gel MA hydrogel microspheres were generated by microfluidic emulsions and crosslinked by cooling.The swelling property and monodispersity of hydrogel microspheres were evaluated by particle size analysis.The porosity of the packed microspheres was assessed by incubating with 70 k Da fluorescein labeled dextran solution.The Rheological behaviors of microspheres ink were tested by rheological experiment.The mechanical properties of bulk hydrogels and hydrogel microspheres were tested by compression experiments.The biocompatibility of the microspheres was assessed by culturing cells on the surface of the microspheres.2.Construction and Characterization of porous scaffolds with complex branched vascular channels based on microspheres matrix bath: Optimal concentration and printing temperature of gelatin sacrificial ink were determined by rheological experiment and extrusion experiment.Printing accuracy was explored by changing the size of the microspheres matrix bath,diameter of the needle and printing rate.Then Gel MA hydrogel microspheres and gelatin were used as matrix bath and sacrificial ink for embedded printing.The patency of the vascular channel was tested by infusing Microfil injection compounds.The permeability of the porous scaffold was tested by perfusion of the fluorescein labeled dextran solution.The morphology of cells in the scaffold was observed by scanning electron microscope.A vascularized porous scaffold with multistage vascular network was formed by seeding HUVECs into the channel and dynamic culture with a customized bioreactor.3.Construction of vascularized adipose tissue in vitro and evaluation of the efficacy in adipose tissue regeneration in vivo: Dome-shaped porous scaffold within vascular channel was printed based on microspheres matrix bath.Adipose stem cells were extracted from adipose tissue from patients undergoing liposuction.Vascularized engineering adipose tissue was constructed by co-culturing adipogenic inducted ADSCs and HUVECs.Cell viability,adhesion and lipid droplet formation were evaluated by Live/Dead viability,phalloidin and BODIPY staining.PCL tissue engineering chamber was printed by fused deposition modeling.To analyze the functionality of vascularized adipose tissue in vivo,composite scaffolds including PCL tissue engineering chamber and microsphere construct was implanted into subcutaneous of nude mouse.The regeneration efficiency and angiogenesis ability of adipose tissue in vivo were evaluated by HE,Masson staining,immunofluorescence staining,q RT-PCR and Western Blot.Results:1.Gel MA hydrogel microspheres were successfully generated by microfluidic emulsions with a controllable diameter range of 200-500μm and stable crosslinked at a low temperature(4℃).The hydrogel microspheres had good mono dispersity.The porosity and pore size of packed Gel MA microspheres were correlated with microsphere size.Gel MA microsphere inks had rheological behaviors such as shear thinning and selfhealing.Compared with the bulk hydrogels,Gel MA microspheres had enhanced mechanical properties.Gel MA microspheres had good biocompatibility and supported cell adhesion and proliferation.2.Porous scaffolds with different vascular channels were successfully constructed by microspheres matrix bath,and the internal channels were completed and perfusable.The width of the printing channels was related to the size of the microsphere matrix bath,inner diameter of the printing needle and the printing rate.The microsphere scaffold had higher permeability than the bulk hydrogels.,and cells could migrate to microsphere pores through the lumen.3.Dome-shaped porous scaffold with vascular channel was successfully printed based on microsphere matrix bath.After co-culture of ADSCs and HUVECs,immunofluorescence staining showed that adipocytes and vascular endothelial cells coexisted in the void space of scaffold,indicated that the vascularized adipose tissue was successfully constructed in vitro.At scheduled times(2 and 6 weeks),the implanted samples were harvested.A number of functional blood vessels could be observed at the border region of the scaffolds and only a small amount of adipocytes existed at 2 weeks.While a large number of adipocytes and blood vessels were observed within central region and border region of the scaffolds at 6 weeks,the relative expression of adipogenic-related protein(PPARγ)was 6 times as the blank scaffold group,which proved that the engineered adipose tissue constructed using our strategy had a good effect of promoting angiogenesis and adipose tissue regeneration in vivo.Conclusion:1.Gel MA hydrogel microspheres were successfully generated by microfluidic emulsions with controllable size,which could be two-step crosslinked and had rheological properties of self-healing and shear thinning.2.Porous scaffolds containing different branched vascular channels were successfully constructed by using microsphere matrix bath,which had great advantages in nutrients exchange and cell migration.3.Experiments in vitro and in vivo showed that the scaffold could promote adipose tissue regeneration and angiogenesis,which might provide a new approach for the vascularization of adipose tissue engineering.