Biomimetic Construction Of Neural Extracellular Matrix Microenvironment Based On Hyaluronan Hydrogels

Nerve injuries as global problems bring great burdens to society and patients due to the limited endogenous recovery.On the one hand,the elevated inflammatory responses expand the injured area,which impedes functions of normal nerve tissue and suppresses the growth of endogenous neural cells.On the other hand,the lack of healthy extracellular matrix(ECM)in diseased microenvironment leads to absence of sufficient inductive cues.Thus,for the repair of nerve injuries,it is crucial to build permissive repairing microenvironment for endogenous neural cells to relieve the growth limits,recapitulate features of biomimetic and healthy cellular niche as well as provide inductive biochemical and biomechanical signals for axonal growth.Via endowing specific physical and chemical features to biomaterials,tissue engineering method is capable of mimicking native neural cellular microenvironment,which not only provides biochemical-mechanical cues for neural cells,but also is availiable for regulating the immune microenvironment.Therefore,tissue engineering is an effective strategy integrating nerve protection and inducing regeneration to enhance the repair of nerve injury.Native nerve tissue is hierarchically oriented,therein,neurons,as basic units,are surrounded by the viscoelastic and soft extracellular matrix(ECM)composited by vast of hyaluronan and bits of adhesive fibrillar proteins,additionally are constantly communicated with macrophages and myelin sheath which wraps axons.These extracellular cues play vital roles in the morphological maturation and functional expression of neurons.Thus,designing hydrogels mimetic to neural ECM for repair of nerve injury should take comprehensive considerations into composition,mechanical properties,topographic structure,myelination and immune-regulation.Hyaluronan,as dominant component of neural ECM,is crucial in neuronal development and functional modulation,moreover holds high chemical reactivity due to the abundance of hydroxyl,carboxyl,acetylamino groups,and reductive terminals,which is easy to functionalize and generate various derivatives.Thus,hyaluronan hydrogels are suitable options for biomimetic construction of neural ECM microenvironment.However,challenges are still faced in building neural ECM-mimicked materials:1)difficulties in independent control of certain features for matrix materials,leading to troubles in decouple and reveal of the effects of these features on neural behaviors;2)dilemmas in fabricating hydrogels with multiple inductive mechanical/biochemical traits for recapitulating native ECM properties comprehensively,resulting in unsatisfied repairing of nerve injuries.Collectively,on the basis of principles for tissue engineering,firstly,this work takes the avails of biocompatible and reactive hyaluronan for functionalization,hybrid and biofabrication,aims to design hydrogel matrices appropriate for nerve tissue engineering.Then,utilizing hydrogels recapitulating native neural ECM features such as composition,mechanics,topography and cellular communication(myelination),the influence of neural ECM characteristics on neural morphology and function is explored.Hydrogel matrices able to induce nerve regeneration together with specific physical/chemical properties are also screened to establish biomimetic and inductive ECM microenvironment.In addition,based on resulting optimized materials supplemented with immune-regulatory capability,the co-effects of protection and inducing regeneration are expected to explore.Main results are shown as follows.(1)Inspired by the native hierarchically oriented nerve tissue,we utilizing microfluidic technology build neural ECM-mimetic hydrogel with ability to guide alignment of neural cells,and investigate the effects of extracellular topography on neural cells with morphology and function included.Firstly,via photo-triggered polymerization and thrombin crosslinking processes,hyaluronan/fibrin(HA-MA/Fb)hybrid hydrogels are prepared,the composition,stiffness and degradation kinetics of which meet the demands of nerve tissue engineering.Then,we propose an alginateassistant solidication strategy,which gives microfluidic manufacturability to slowlygelatinized HA-MA/Fb hydrogels.Also,modified microfluidic method is developed to prepare nano-aligned hydrogel microfibers,which is profiting to elucidate the relevance between the degree of nano-orientation and composition,microfluidic parameters,rotary speed.Finally,with the avails of neural cells in situ encapsulated into resulting hydrogels,we reveal the contact guidance of matrix topography on neural cells,and find the correlation between matrix nano-orientation and axonal elongation even the expression of neurogenic genes/proteins.(2)Based on HA-MA/Fb hybrid hydrogels and microfluidic technology,we construct myelin-sheath-like structure possessing native partitioning distribution of heterotype cells,also explore the influence of tissue engineered myelin-sheath-like structure on neural cellular behaviors.Firstly,utilizing resulting hydrogels in last chapter,we demonstrate the three-dimensional aligned nano-topography is capable of inducing myelinating maturation of Schwann cells,with establishment of polarity,upregulated genes/proteins related to myelin structure and neurotrophic factors contained.Then,using microfluidic devices supplemented with triple-channeled co-axial microfluidic chip and rotary collection,we prepared HA-MA/Fb microfibers with core-shell laminar structure and high nano-alignment.Afterwards,with in situ encapsulation of neural cells and Schwann cells,we fabricate myelin-sheath like constructs which have wraps of tubular distributed Schwann cells on aligned neural cells.Therein,we also find expression of myelination-related proteins induced by nano-alignment of matrix,which shows bio-mimics of native myelin-sheath from structural level to functional level.Moreover,we elucidate that the resulting myelinsheath-like constructs are propitious to nerites extension and neurogenic genes/protein expression,with also enhanced myelination of Schwann cells.(3)Viscoelasticity is a distinct trait of native nerve tissue,however the roles played by viscoelasticity in neuronal behaviors still remain obscure.Thus,in this chapter,we engineer neural ECM mimetic hyaluronan/collagen hydrogels with independently controlled viscoelasticity also explore the effects of extracellular viscoelasticity on neuronal behaviors and the underlying mechanisms.Firstly,series of difunctional hyaluronan/collagen(DHA/Col)composite hydrogels were prepared by a proposed static-dynamic strategy.The hydrogels show aldehyde concentrationrelied viscoelasticity and constant initial elastic modulus,fibrillar morphology,swelling and degradation kinetics.Then,utilizing resulting materials,we find that higher viscoelasticity of matrix makes no differences in proliferation of neural cells but does enhance neurites elongation and expression of neurogenic proteins.Additionally,we modify the motor-clutch model with a tension dissipation component and demonstrate the molecular mechanism of viscoelasticity-dependent neural responses.Moreover,with the avails of spinal cord injured rats,cell-free viscoelastic grafts show abilities to enhance repair of nerve injury including endogenous neuronspecific differentiation,axonal regeneration and re-establishment of functional axons.(4)On the basis of pathologic microenvironmental features including the absence of inductive signals and the presence of growth limit,we construct regenerative microenvironment with the combination of viscoelasticity,nano-alignment and immune-regulation,then explore the synergistic effects of multiple biomimetic extracellular cues on neural cell/tissue,elucidate the co-effects of strategy integrating protection and induction on the repair of nerve injury.Firstly,DHA/Col hydrogels are prepared by the combination of microfluidic and static-dynamic strategy,and the hydrogels show high viscoelasticity and nano-orientation.In vitro results proved the synergistic effects of viscoelasticity and nano-alignment of matrix on neurites extension and neurogenesis.Then,by incorporation of ZIF-8/IL-4 nanocarriers into the viscoelastic and nano-aligned hydrogels,hydrogels combined immune-regulation and inductive cues are fabricated.Utilizing spinal cord injured rats,we demonstrate the co-effects of viscoelasticity and nano-alignment on endogenous neuronal differentiation and nerve regeneration.Moreover,the protective and inductive hydrogels show extra capabilities to reduce inflammation and protect neural cells,eventually lead to promoted structural and functional recovery of spinal cord injury.Hence,hyaluronan-based hydrogels recapitulating multiple native ECM features and immune-regulatory cues are effective tools for construction and regulation of neural regenerative micro-environment,which not only hold promising prospective in nerve repair,but also rich the principles of neuroscience and tissue engineering.