Preparation And Research Of Bioinspired Integrated Hydrogel Based On Additive Manufacturing

Objective To fabricate a personalized integrated hydrogel,which is composed of a bone filling scaffold and a barrier membrane.To explore its feasibility of novel guided bone regeneration（GBR）materials by characterizing microstructure,testing mechanical property,experiment of rat bone marrow mesenchymal stem cells and in vivo experiment of rabbit cranial bone defect models.Methods The synthetic laponite（LPN）solution and the chitosan（CS）solution were mixed in a certain ratio through ultrasonication,and the nacre-mimetic hydrogel（NMH）membrane was prepared via a well-developed evaporation-induced self-assembly technique.Then,by utilizing the thermoresponsive nature of Gel MA,we selected a mixture of Gel MA and HA as the printed bio-ink and added a photoinitiator（lithium phenyl-2,4,6-trimethylbenzoylphosphinate,LAP）to the ink to successfully prepare the porous hydrogel scaffold on the NMH membrane through computer-aided-design（CAD）and additive manufacturing（AM）technology.The fabricated integrated hydrogel and its components were analyzed by scanning electron microscope（SEM）,transmission electron microscope（TEM）and rheometer.The mechanical tester was used to test mechanical performances of hydrogels and simulated body fluid（SBF）was selected for in vitro degradation experiments.Cell counting kit-8（CCK-8）and Live/dead cell staining were used to evaluate cytocompatibility of integrated hydrogels.The in vitro osteogenic property of hydrogels was explored by alkaline phosphatase（ALP）assay and alizarin red staining（ARS）.The osteogenic effect and degradability of the integrated hydrogel were further studied in vivo experiments of rabbit cranial bone defect models.Results The customized scaffold of the bioinspired integrated hydrogel was firmly bonded to the barrier membrane without separation.Owing to the thermosensitivity of Gel MA and the photosensitivity of LAP,the hydrogel scaffold was stable,and the crosssection image exhibited a full interconnected grid structure;the side view of film indicated a uniform and orderly arrangement of LPN nanosheets in the CS polymer matrix,which made the membrane translucent as well as dense.Besides,the tensile strength of membrane reached 97.2 MPa,which is 15 times higher than that of Bio-Gide membrane.Both CCK-8 experiments and live/dead cell staining experiments showed that the bioinspired integrated hydrogel processed good cytocompatibility.The ALP staining experiment revealed that the positive ALP stained area of the integrated hydrogel group was increased,and the ALP activity level was significantly enhanced（P < 0.05）,compared with the control group and the hydrogel group without HA.The ARS experiments showed that the integrated hydrogel group had osteogenic properties.Micro-CT and histological staining exhibited that,compared with the blank group,the experimental group had a large amount of new bone tissue filling the defect space,which indicated that integrated hydrogel can promote new bone formation.Conclusions For a bone defect in a patient,the customized hydrogel with an all-in-one structure can be prepared with the guidance of CAD processing,and it can be constructed with an appropriate shape and size and precisely match the defect area.When the mass ratio of CS to LPN is 5: 5,and the mass fraction of HA is 1%,the bioinspired integrated hydrogel exhibits the best mechanical strength and has an ability to maintain space for new bone regeneration.In vitro and in vivo experiments show that the integrated hydrogel possesses good biocompatibility,osteogenic properties and degradability,which lays a foundation for further research and the development of novel GBR materials.