| Oxygen（O2）is vital for the survival of all organisms,and a steady supply of O2 in organism assists the body in converting nutrients into energy,efficiently.However,in pathological situations,inflammation or blood flow disturbances will lead to a decrease in the partial pressure of O2 in the organism.Persistent or severe hypoxia will activate anaerobic metabolism and inhibit mitochondrial respiration,causing oxidative cellular damage and even inducing cellular necrosis and apoptosis.In addition,according to reports,the diffusion distance of O2 in vivo is about 100～200μm,which is much smaller than the size of conventional three-dimensional（3D）repair scaffolds and tissue-like organs.Therefore,how to meet the supply of O2 in clinical disease treatment and in vitro tissue-like scaffold construction is one of the current challenges to be solved in the biomedical field.Current research mainly uses O2-releasing biological materials,such as perfluorocarbon oxygen carriers or biological materials with peroxides,to achieve local oxygen supply.Although these methods are easy to operate and have excellent oxygen release capacity,there are still problems such as difficulty in controlling the oxygen release time and oxygen release rate.Currently,new strategies such as oxygenated microbubbles and photosynthetic algae are gradually attracting the attention of researchers.Hence,the main objective of this study is to develop hydrogels with controlled oxygen release and excellent cytocompatibility based on the performance of oxygen production by microalgae and to explore their applications in cell co-culture,mitigation of hypoxic damage,and 3D bioprinting.In this thesis,Chlorella Vulgaris was first selected as model algae and modified by the layer-by-layer self-assembly（LbL）technique.The morphology,activity,and surface zeta potential of microalgae were characterized;the principles of microalgae modification and the effects of modification on the proliferation activity and photosynthetic activity of microalgae were investigated.Hydrogels loaded with Chlorella were developed using sodium alginate hydrogels as carrier materials,and the O2-producing properties and cytocompatibility of the microalgae hydrogels（Algae-gels）were characterized;cellular hypoxia/reoxygenation（H/R）damage models and cellular scratch models were constructed,and the potential of Algae-gels for hypoxic damage mitigation and wound repair was evaluated.The potential of Algae-gels for hypoxic injury mitigation and wound repair was evaluated;microalgae bioinks were developed and their 3D printing capability was preliminarily investigated.The principal results and conclusions of this paper are as stated below:（1）Chlorella in suspension culture was in the environmental adaptation period at 1～4 d,logarithmic growth period at 4～15 d,and then entered the senescence period after a short plateau period of 1～2 d.The FT-IR and zeta potential results showed that gelatin and sodium alginate were adsorbed on the surface of Chlorella layer by layer through electrostatic interaction.The biomass characterization of Chlorella showed that surface modification was effective in slowing down the proliferation of Chlorella,but did not affect its photosynthetic activity.（2）Microalgae hydrogels containing Chlorella were prepared by Ca2+cross-linking.The results of CLSM and SEM showed that the microalgae hydrogels were loose and porous with a uniform dispersion of Chlorella.The results of biomass and dissolved oxygen（DO）measurements showed that the microalgal hydrogel had a good oxygen production capacity,the DO content in the medium was increased by 4.9 mg/L,and the average oxygen production rate was 0.61mg·L-1·h-1.Results of cell experiments showed that under two-dimensional（2D）and three-dimensional（3D）co-culture conditions,HUVECs in the co-culture group had good proliferation activity compared with the control group,and the optimal cell density ratios（Chlorella:HUVECs）were 5:1 and 20:1 for 2D and 3D co-cultures,respectively,which indicated that the oxygen demand of cells was different in 2D and 3D spaces,and gas exchange was difficult in 3D hydrogels.Thus,microalgal hydrogels allow for in situ oxygenations of cells.（3）A hypoxia/reoxygenation（H/R）injury model of HUVECs cells was successfully constructed by the annealing package method.the proliferative activity and density of H/R model cells were decreased,the levels of LDH and ROS were increased and the cells were damaged.After co-culture of microalgal hydrogels with H/R model cells,the proliferative activity of HUVECs cells was significantly increased and the levels of LDH and ROS were remarkably decreased.The results of various characterizations of the microalgae bioink show that the pre-crosslinked bioink has good injectability and shear thinning behavior,as well as good light transmission.and good light transmission.In addition,the molding properties of the bioinks can be significantly improved by adding gellan gum（GG）.Therefore,microalgae hydrogels can also be used as bioinks for 3D bioprinting.In summary,this study initially achieved the control of Chlorella proliferation rate by surface modification of Chlorella through the layer-by-layer self-assembly technique.Further,photosynthetic O2-producing Algae-gels were developed using Chlorella vulgaris and sodium alginate.After that,preliminary exploration of the application of Algae-gels was carried out,and the results showed that Algae-gels have good biological activity and are expected to promote the repair of damaged cells,providing new ideas for the research of novel O2-producing biomaterials and 3D bioprinting inks. |
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