3D Printed RGO Composite Hydrogel Scaffold For Multicellular Delivery And Bone Repair

Background and objective:Bone tissue is a complex part of the human body structure,and the nerves and blood vessels in the bone are widely distributed in the Haversian canal and Volkmann canal of the cortical bone.On the one hand,sensory and sympathetic nerves participate in bone growth and repair by secreting neurotransmitters,neuropeptides and other substances.On the other hand,the nerves in the bone sense changes in the bone by conducting mechanical or electrical signals,so as to maintain its physiological activities within the normal range.Sensory denervation may result in loss of trabecular bone,leading to loss of bone strength and impairment of new bone formation.In addition,due to the lack of precise and timely conduction to external stimuli,bone that lacks bone perception may produce pathological responses.Therefore,simultaneous repair of intraosseous nerves is critical for functional bone regeneration.Cell therapy is considered as a strategy to replace,repair or enhance the biological function of damaged tissues or systems by autologous or allogeneic cells.Among them,bone marrow mesenchymal stem cells(BMSC)are stem cells extracted from bone marrow with self-proliferation ability and multidirectional differentiation potential.They are often supported by bone tissue regeneration scaffold materials for osteogenesis research.Schwann cells(SC)are myelinating cells of the peripheral nervous system that digest myelin debris from distal nerve segments during functional restoration of damaged peripheral nerves as part of Wallerian degeneration.Following disassembly and phagocytosis of the distal segment,SC in the distal nerve dedifferentiate into non-myelinating Schwann cells and proliferate to form Büngner’s ribbons,which guide regenerated axons from the proximal segment to denervated targets.Bone marrow mesenchymal cells and Schwann cells can promote differentiation mutually.Studies have shown that SC co-cultured with BMSC on osteogenic scaffolds exhibit better proliferation ability and expression of NGF,BDNF,Trk A,and S100.Moreover,Trk A,Trk B and Trk C have also been reported in osteoblasts,which are receptors for NGF,BDNF and NT-3,respectively.The presence of Schwann cells also significantly induced the expression of osteogenic marker genes,including alkaline phosphatase and COL-1,and enhanced the proliferation of primary osteoblasts through the NGF pathway.Here,we combined 3D printing technology to prepare a rGO composite hydrogel scaffold to simultaneously load SC and BMSC to achieve the purpose of bone and nerve synergistic regeneration.Methods:1.Preparation of Hydrogel Bioink.2.Material characterization experiments,including SEM detection,mechanical properties,rheological properties,degradation rate and expansion rate.3.Use CCK-8,live-death staining,and cytoskeleton fluorescent staining to verify the in vitro cytocompatibility of the material scaffold.4.Alkaline phosphatase activity assay,alizarin red staining and q RT-PCR were used to detect the gene expression levels of stem cells in the direction of osteogenesis and stem cells in the direction of neurogenesis in vitro.5.To verify the biocompatibility and osteogenic differentiation potential of the biomaterial in the in vivo experiment-rat subcutaneous transplantation model.Results:1.rGO can be well dissolved in the GelMA precursor solution by circulating ultrasound and stirring,so that it can flow out of the nozzle of the printer smoothly.2.The results showed that with the addition of rGO,the surface porosity of the hydrogel scaffold was improved,and the mechanical properties and rheological properties were also improved.3.Both CCK-8 and live-death staining showed the good cytocompatibility and proliferation ability of the material scaffold.At the same time,the cytoskeleton fluorescent staining also showed that the cells adhered and stretched better on the surface of the material.4.Both alkaline phosphatase and alizarin red staining indicated its osteogenic potential.q RT-PCR further confirmed the induction of differentiation at the gene level.5.No obvious verification response was found in the rat subcutaneous transplantation model.At the same time,the induction ability of osteogenesis/neurogenesis was confirmed by immunofluorescence and immunohistochemistry.Conclusions:The rGO/GelMA scaffold was fabricated by applying 3D printing technology.Scaffolds with suitable pore sizes showed excellent biocompatibility and proliferation ability.The 0.05% rGO/GelMA scaffold has good mechanical properties,can promote the adhesion of BMSC and SC,and maintain high stemness and continuous proliferation within 7 days.In vitro experiments,Alizarin red and alkaline phosphatase staining demonstrated its excellent osteoinductive ability.In rats,0.05% rGO/GelMA scaffolds loaded with both BMSC and SC achieved osteogenic and neural differentiation 2 months after transplantation.This is attributed to the high adhesion ability and osteogenic/neural differentiation potential of the scaffold material.Due to their flexibility as bioinks in 3D printing,GelMA hydrogels mixed with rGO are expected to have broad applications in the fields of multicellular delivery and functional tissue regeneration.