Microfluidic Technology-Based Injectable Alginate/Gelatin Microrods For Tissue Engineering Vascularization

Despite the significant advancements in fabricating various scaffolding systems over the past decade,the generation of functional vascularized tissues remains challenging for the currently available biofabrication approaches.On the other hand,the applicability of traditional surgical transplantation of vascularized tissue constructs is limited due to the sophisticated surgical procedures,which are invasive,leading to increased risks of scar formation and infection.Hydrogels are polymer networks with a highly hydrated microenvironment that can easily transport nutrients,oxygen and metabolic waste,and are chemically similar to extracellular matrix?ECM?for tissue engineering.Due to their small size,minimally invasive microrods can be injected directly into the body,avoiding the surgical process.Therefore,in this thesis,we used sodium alginate?SA?and gelatin?Gel?.Then,SA/Gel composite hydrogel microrods were prepared using microfluidic technology,and biological studies were carried out by external loading and inclusion of human umbiliical vein endothelial cells?HUVECs?.Further,the efficacy of these injectable microfibers was explored in vivo in mice to observe vascularization,which could find a convenient solution to the problem of tissue engineering vascularization.Initially,we demonstrated the preparation and characterization of SA/Gel composite microrods.Herein,poly?methyl methacrylate??PMMA?was used as a template material to design a micro-channel with a double-T-shaped structure with a spindle channel diameter of 400?m and an inlet of 200?m.Further,the whole set-up of template material was established on a polydimethylsiloxane?PDMS?chip.The hydrophobicity of the chip was improved by octadecyltrichlorosilane?OTS?.SA/Gel composite microrods were prepared by using a PDMS chip and droplet fusion principle.The preparation was optimized by Minitab full factorial design.The experimental results showed that 5%calcium chloride?CaCl₂?could be optimum as cross-linking agent.Moreover the optimum concentration of the formulation parameters include the SA of 1.75%,and Gel of 4%,at a ratio?SA:Gel?of 3:1.When the phase flow ratio of was 3.5:1,the obtained microrods resulted in uniform size,and a good morphology at a length of about 750?m,a width of about 250?m,and an ratio of about 3,.Secondly,we demonstrated the fabrication of externally loaded HUVECs microrods and their biological performance were explored.The study of the growth curve of HUVECs confirmed that the population doubling time of the cells was 71 h,and the optimal inoculation density of the cells was 5×10⁴ cells/mL.Fourier transform infrared spectroscopy?FT-IR?results indicate successful preparation of oxidized sodium alginate?OSA?and cross-linking of OSA with Gel.The cell adhesion experiment showed that the microrods after cross-linking Gel with OSA resulted in excellent adhesion ability,which increased from 7.5 to 25.6%,and the proliferation of cells outside the microrods was also significantly augmented.At the same time,the cells secreted the significant amounts of protein on the fiber scaffold after fixation of Gel.Moreover,Scanning electron microscopy results showed that cells could achieve significant growth on microrods.The degradation efficiency in vitro of the scaffold reached 80%in 2 months,indicating that the scaffold has good degradation ability.Thirdly,we demonstrated the fabrication of microrods wrapped with HUVECs and their biological performance were explored.The prepared scaffolds were fixed at different times for 0,10and 30 min,respectively,to achieve the differences in the internal and external structure of microrods.Gel release experiments and degradation experiments in vitro have demonstrated that different cross-linking levels could be achieved by controlling the cross-linking time of genipin.FT-IR results showed that gelatin was successfully cross-linked in the 10-min group and 30-min group.Further,Cell Counting Kit-8?CCK-8?and Nitric Oxide?NO?chemistry showed that cells in the10-min and 30-min groups showed higher activity in microrods.The growth of the cells in the microrods was observed by confocal laser scanning microscopy?CLSM?.The results showed that the microrods in the 10-min group migrated from the inside to the outside after 15 d of culture to form a vascular structure.Hematoxylin-eosin?HE?staining and immunohistochemical staining results indicated that the cells migrated from the inside to the outside in the microrods.On the other hand,Immunofluorescence experiments showed that cells in the 10-min group expressed Platelet endothelial cell adhesion molecule-1?CD31?in a large amount in microrods.The expression levels of angiogenic genes were also increased in the microrods as the culture time increased.Finally,we investigated the biocompatibility and vascularization of injectable microrods.The results of cell experiments,acute toxicity tests and hemolysis experiments of injectable microrods indicated that the injectable microrods have good biocompatibility.Injectable microrods containing HUVECs were injected into mice to observe the vascularization.The experimental results showed that there were a large number of inflammatory signs around the injected microrods,and capillary angiogenesis at that sites revealing that the tendency of neovascularization increasing the coordination with the surrounding tissues.In summary,the microfluidic chip technology could be used to successfully fabricate short SA/Gel microrods;for the externally loaded HUVECs microfibers with good cell adhesion,and HUVECs-laden microrods for vascularization;Injectable microrods have excellent biocompatibility and could result in neo-vascularization and establish coordination with the tissue surrounding at the injection site,which provides a way of thinking to the problems associated with vascularization in tissue repair.