3D Printing Biomaterials Mediated Drug Therapy For Peripheral Nerve Injury

Peripheral nerve injury(PNI)is a common clinical disabling disease.PNI often affects neural signal transduction,leading to the loss of sensory and motor function of patients.The peripheral nervous system has an intrinsic ability for regeneration,but spontaneous nerve repair often reuslts to unsatisfactory functional recovery.The treatment of PNI faces significant clinical needs.As an exogenous scaffold,artificial nerve guide conduit(NGC)which can provide physical support and direction guidance for the regeneration of injured nerves,has become as an important method for PNI therapy.However,the current artificial NGCs has limited therapeutic effects on PNI,especially long-gap PNI,and it is difficult to meet the need of functional recovery.There is an urgent need to construct functional NGCs to provide a good microenvironment for nerve repair,so as to improve the recovery of PNI.Conduit structures,conduit matrix materials and biological cues,including cells,drugs and growth factors are key factors that play the role of NGCs in promoting nerve repair.With the characteristics of rapid construction of personalized complex structures,3D printing technology can integrate biomaterials with cells,drugs and growth factors to construct functionalized scaffolds,providing a platform for the fabrication of functionalized NGCs.The ideal conduit matrix materials usually possess good biocompatibilities,biodegradabilities,mechanical properties and biological functions.However,the properties of most 3D printing materials cannot meet the needs of constructing functional NGCs.There is an urgent need to improve the existing materials or develop new 3D printing materials.In this paper,in order to solve the problems of lack of excellent photocurable biomaterials for peripheral nerve repair,new 3D printing biomaterials for nerve repair were developed,new drug-mediated functional NGCs were constructed by 3D printing technology,and their functions and mechanisms for promoting nerve repair were further studied.The research contents are as follows:(1)Photocurable gelatin-silk fibroin hybrid materialsThe matrix material for PNI repair should possess appropriate mechanical properties that match with the native nerve tissue,so as to better simulate the tissue microenvironment of nerve repair and further promote myelination and axon regeneration.With the good cell adhesion performance,biocompatibility and printability,gelatin methacryloyl(GelMA)has been successfully applied for the construction of 3D printed NGCs.However,the poor mechanical properties of GelMA result in its limited therapeutic effects on PNI.In this study,to enhance the mechanical properties of GelMA,photocurable silk fibroin(SF-MA)was introduced.GelMA and SF-MA were mixed in different ratios,and further photocured to form hydrogels.By soaking the hydrogels in ammonium sulfate,GelMA/SF-MA hybrid materials with different ratios(GelMA,G/F 2:1,G/F1:1,G/F 1:2,SF-MA)were prepared.The mechanical performance test showed that the introduction of SF-MA significantly improved the tensile and compressive properties of GelMA.Furthermore,the ratio of hybrid materials was screened by investigating the effect of materials on Schwann cells.The results indicated that compared with other groups,G/F 1:1 material could promote the proliferation,adhesion,spreading and migration of Schwann cells.The results of RT-q PCR showed that compared with other groups,G/F 1:1 material could up-regulate the gene expression of Schwann cell adhesion,migration and myelination related proteins.The above results demonstrated that G/F 1:1 material could effectively regulate the behavior of Schwann cells.The structure characteristics and degradation performance of G/F 1:1 material were further investigated.The results showed that compared with GelMA,G/F 1:1 material has higher β-sheet structure content,higher crystallinity and denser network structure,indicating that the synergistic enhancement of the mechanical properties of GelMA by SF-MA and ammonium sulfate may be related to the β-sheet structure,crystallinity and the density of network structure.In addition,G/F 1:1 material has good biodegradability and biocompatibility.In summary,this study developed a kind of GelMA/SF-MA hybrid material with enhanced mechanical properties.The GelMA/SF-MA hybrid material can effectively regulate the behavior of Schwann cells.This material exhibits great potential for promoting nerve repair,providing a new choice for the construction of NGCs.(2)3D printed drug-releasing NGCsArtificial NGCs can only provide physical support and direction guidance for the regeneration of defective nerves.Lacking the guidance of biochemical cues for nerve regeneration,artificial NGCs exhibit limited therapeutic effects on PNI,especially long-gap PNI.Biological cues such as cells,drugs and growth factors are benificial to build a microenvironment that could promote nerve regeneration and improve the efficiency of nerve repair.7,8-Dihydroxyflavone(7,8-DHF)is an agonist of tropomyosin receptor kinase B(Trk B),which can promote neuronal axon elongation.In this study,in order to improve the water solubility of 7,8-DHF,the prodrug selfassembly strategy was used to prepare 7,8-DHF-OA nanoparticles.Furthermore,using G/F 1:1 material as the matrix material,combined with 7,8-DHF-OA nanoparticles,a functional NGC was fabricated by DLP 3D printing technology,and its performance was investigated.The results demonstrated that the functional NGC can release 7,8-DHF slowly,thus promote the axon elongation of rat adrenal pheochromocytoma(PC12)cells,and up-regulate the gene expression of GDNF and NGF in Schwann cells,which are benificial to build a good microenvironment for peripheral nerve repair.Finally,a long-gap(12 mm)rat sciatic nerve defect model was constructed to evaluate the therapeutic effect of the functional NGC in vivo.The results indicated that,compared with the blank NGC group,the functional NGC can promote the repair and reconnection of defect nerves,and realize the recovery of electrophysiological function.The functional NGC can promote axon elongation and myelination and relieve gastrocnemius atrophy,of which the effect is comparable to that of the autologous nerve graft group.The results of tissue immunofluorescence showed that compared with the blank NGC group,the sciatic nerve of the functional NGC group expressed more GAP-43 proteins,indicating that the functional NGC can promote axon regeneration.Histopathological studies on the main organs of rats indicated that the functional NGC has good biological safety.In summary,a new type of functional NGC was constructed by combining biomaterials and prodrug nanoassemblies with3 D printing technology,which can promote the regeneration and functional recovery of PNI,providing a new strategy for the treatment of long-gap PNI.(3)Photocurable plasma materialsMatrix materials for nerve repair should possess appropriate biological functions to promote tissue repair and regeneration.However,till now,most photopolymerized biomaterials lack effective biological functions,and there is an urgent need to develop new functional biomaterials.Plasma is an important part of blood.It contains a variety of proteins and endogenous growth factors.The endogenous growth factors in plasma are rich in components,and their types,quantities,composition ratios can replicate those of physiological conditions.Plasma exhibits multiple biological functions,providing potential new options for the development of 3D printing biomaterials.In this study,photocurable plasma materials with different degrees of substitution(DS)were prepared via the direct reaction between plasma and methacrylic anhydride.The results showed that the physical properties of plasma hydrogels could be regulated by adjusting the DS of plasma materials.Investigation of the effect on human umbilical vein endothelial cells(HUVEC)showed that plasma materials with different DS have good cytocompatibilities,and can promote HUVEC cells to form tubes,indicating that plasma materials with different DS have good biological effects.Follow-up studies was performed on plasma materials with a moderate degree of substitution(49.45±5.66%).It was found that plasma hydrogels can continuously release vascular endothelial growth factor(VEGF).Plasma hydrogels have good cell compatibilities with Schwann cells and PC12 cells,and can promote the axon elongation of PC12 cells,exhibiting the potential to promote nerve repair.Furthermore,the results of subcutaneous implantation studies in vivo indicatied that the plasma material is biodegradable and almost completely degrades after three weeks of implantation.Meanwhile,the plasma material can induce cell infiltration and promote the formation of new blood vessels.Finally,investigation of the 3D printing performance showed that the plasma material has good printing performance and can be used for the construction of personalized complex structures.In summary,this study developed a new photocurable plasma material that can release endogenous growth factors,which has good biological effects such as inducing angiogenesis and promoting axonal elongation of PC12 cells,exhibiting great potential in nerve repair and other tissue repair fields.In summary,in order to solve the problems in peripheral nerve repair,this paper applied digital light processing based 3D printing technology to the research of peripheral nerve repair materials,designed and constructed new types of photocurable biomaterials,and 3D printed a new type of drug released NGCs,which can promote the repair of PNI.Firstly,GelMA/SF-MA hybrid materials with enhanced mechanical properties were designed and prepared,which could regulate the behavior of Schwann cells to promote myelination,exhibiting great potential to promote peripheral nerve repair.Furthermore,functionalized NGCs were constructed based on GelMA/SF-MA materials and prodrug nanoassemblies.The functional NGCs can slowly release drugs,and promote axon elongation and remyelination,thus improving the functional recovery of PNI.Finally,a new type of functional photocurable plasma material was developed.Plasma materials can release endogenous growth factors and have a variety of biological effects,providing new ideas for the construction of new 3D printing bioinks and exhibiting application potential in nerve repair and other tissue repair fields.The above research provides new methods for the construction of functional NGCs and new ideas for the treatment of PNI.