Regulation Of Neural Stem Cell Fate And Spinal Cord Injury Repair By Nano-hydroxyapatite

The incidence of central nervous system(CNS)diseases increases year by year.Spinal cord injury(SCI)easily leads to varying degrees of limb paralysis,even death.How to effectively repair SCI threatens patients’ lives.The main cause of CNS diseases is the loss or dysfunction of neurons.Thus the therapeutic key is to replenish neurons,and then restore the function of the nervous system.Guiding precursor cells to differentiate into neurons for rebuilding neural connections in the injury is the crucial scheme to achieve nerve repair.With the potential of selfrenewal and multi-differentiation,neural stem cells(NSCs)could differentiate into neurons,astrocytes,oligodendrocytes,etc.But its slow differentiation speed and uncertain differentiation direction limit the therapy of CNS diseases.Therefore,accelerating the differentiation of NSCs into mature neurons has become an important research topic in the field of CNS diseases therapy.It will become the potential way to regulate NSCs fate by applying nanomaterials-mediated chemical and physical microenvironment,taking advantage of the controllable size,adjustable composition,stable performance,and site-specific regulation of nanomaterials.Due to the rich calcium,excellent biocompatibility,and degradability in vivo,nano-hydroxyapatite(HAp)is widely used in bone tissue engineering,especially in the osteogenic differentiation of stem cells.Based on the role of calcium in neural differentiation,the possibility of HAp nanomaterials regulating the fate of NSCs is explored,and the scheme of HAp combined with NSCs for the therapy of CNS diseases is designed,it is expected to provide new ideas for the therapy of neurological diseases.The dissertation focuses on "the regulation of NSCs fate by nano-HAp",exploring the regulation of NSCs neural differentiation by nano-HAp first.The mode and biological mechanism of HAp on NSCs should be clarified,then to accelerate NSCs differentiation into mature neurons to rapidly repair SCI.The research content of dissertation mainly includes the following four parts:(1)HAp nanorods function as safe and effective "inorganic growth factors" regulating neural differentiation and neuron development.The author prepared HAp nanorods with uniform morphology by hydrothermal method.The endocytosis of HAp nanorods by NSCs was found.Real-time quantitative PCR and immunofluorescence staining were used to detect gene and protein expression:the mRNA of stemness marker Nestin decreased than the control group(without HAp nanorods)after NSCs culturing with HAp nanorods for 7 days,the mRNA of neuron markers Tuj1 and MAP2 increased.After culturing with HAp nanorods for 13 days,the proportion of Tuj1 and MAP2 positive cells increased by～13.6%and～35.2%respectively.The results indicated that HAp nanorods can promote NSCs differentiation into mature neurons.The patch-clamp technique was used for electrophysiology:the cells showed the regular electrophysiological behavior of neurons after culturing with HAp nanorods just for 5 days.The gene sequencing was used to analyze the cell signal pathways:the biological mechanism of HAp nanorods promoting NSCs differentiation into neurons is that HAp nanorods release calcium ions after entering lysosomes rich in hydrolytic enzymes,then activate the calcium signaling pathway,MAPK,PI3K-Akt neuron-related signaling pathways,etc.,accelerating NSCs differentiation into neurons.With low-cost,controllable morphology,and rich in calcium,HAp nanorods can function as "inorganic growth factors" to regulate the fate of NSCs.The first application of HAp nanorods in regulating NSCs fate provides a safe and efficient calcium preparation candidate for nerve repair.(2)HAp nanorods functionalized hydrogel acts as "brood pouches" to incubate and programrelease NSCs for SCI repair.The sustained release and rapid differentiation of NSCs are key to neural repair after entry into the organism.But cells are easily dispersed after entry in vivo,and difficult to arrive at the affected area.To improve the compatibility of HAp nanorods and NSCs in vivo,the author designed the HAp-NSCs hydrogel system.HAp and NSCs can be confined in the gel microcavity by sol-gel phase transition.It was found that NSCs grew in the three-dimensional spherical state in the hydrogel and endocytosis surrounding HAp nanorods after 24 h culture.The mRNA of cell stemness marker Oct4/Sox2/Nanog/CD133/Nestin in the hydrogel without HAp was higher than in tissue culture plates and HAp hydrogel groups after 7 days of culture.After that,the cells released from hydrogel were collected and cultured for another 7 days.The mRNA of neuron markers Tuj1 and MAP2 was higher in HAp hydrogel than in hydrogel without HAp,which was consistent with the protein expression.The results of multiple studies demonstrated that hydrogels can maintain cell stemness and vitality.The NSCs endocytosed HAp nanorods were released with the degradation of hydrogels and achieved rapid differentiation into neurons.The subcutaneous metabolism,differentiation simulation,and SCI therapy experiments more directly confirmed that the mixture was converted into a gel state after injection into the affected area.The HAp hydrogel plays a role in maintaining the stemness and vitality of NSCs.Combined with the immunofluorescence staining,basso mouse scale,and electrophysiology,it was found that after 6 weeks of SCI therapy with HAp-NSCs hydrogel,the neural synaptic connections were obvious,and the neural conduction was excellent.The lower limb motor function was repaired.Acting as the "brood pouches" of NSCs,HAp nanorod-functionalized temperature-sensitive hydrogels provide a simple and effective strategy for avoiding the cell mixture dispersion,improving the stem cell survival rate,accelerating neural differentiation,and accelerating SCI repair in vivo.(3)Three-dimensional construction of "multi-functional HAp-NSCs" cell spheroids and NSCs neural differentiation for accelerating SCI repair.The thermosensitive and injectable properties of thermosensitive hydrogels help protect cells and prevent them from dispersing freely in vivo,but the hydrogels increase the metabolic pressure of organisms to a certain extent.Biological modules assembled by stem cells and functional materials can effectively solve the above problems and have strong interaction with the surrounding microenvironment.The author assembled ferroferric oxide(Fe3O4)@HAp@polydopamine(PDA)nanobelts and NSCs into "multi-functional HAp-NSCs" cell spheroids.The high-aspect ratio Fe3O4@HAp@PDA nanobelts were stacked with small nanobelts,which showed "haystack" morphology and easily separated under external forces.It contained Fe3O4 for magnetic manipulation of cell spheroids.By immunofluorescence staining and TUNEL cell apoptosis detection,it was found that the expression of hypoxia factor HIF-1α and cell apoptosis was decreased,and the mRNA of neurotrophic factor markers NT-3/NGF/FGF/BDNF/PDGF/EGF was increased in nanobelts-incorporating cell spheroids than that groups without HAp nanobelts.It showed that HAp nanobelts can effectively alleviate the block of nutrient transport,reduce cell apoptosis inside the cell spheroids,and have the function of nerve protection.Based on cell-nanobelt location,the expression of neural differentiation-related markers Tuj1/MAP2/NeuN/GFAP,and the study of cell signaling pathways,it was found that HAp nanobelts entered into the lysosomes of NSCs under the cell action,then degraded and released calcium ions to activate JNK/ERK-p53 signal pathways,thus accelerating the differentiation of NSCs into mature neurons with calcium activity.The SCI model confirmed that the "multi-functional HAp-NSCs"cell spheroids were gathered in the SCI by magnetic manipulation.The NSCs differentiated rapidly into mature neurons,which then established synaptic connections and restored the motor function of the mouse hind limbs.The multi-functional nanobelts have the dual functions of nutrient delivery and nerve differentiation promotion.The three-dimensional construction of"multi-functional HAp-NSCs" cell spheroids provides guidance for SCI repair.(4)"NSCs(HAp)-mesenchymal stem cells" heterogeneous cell microtissue and their multimode regulation of nerve repair.The other cells are crucial for NSCs differentiation and neural development during neural regeneration.Mesenchymal stem cells(MSCs)have powerful paracrine and can secrete various cytokines to regulate nerve regeneration and the immune microenvironment.To achieve better neural repair and microenvironment regulation,the author constructed the "NSCs(HAp)-MSCs"heterogeneous cell microtissues by the three-dimensional assembly of NSCs endocytosed HAp nanorods and MSCs.By detecting the expression of inflammation-related factors IL-6/IL-1β/TNF-α/IL-10,anti-inflammatory cell phenotype(M2-type)markers Arg-1/CD206,effector factor YAP,and astrocyte marker GFAP,it indicated that MSCs in microtissues can avoid neuroinflammatory injury and regulate the neural microenvironment by regulating microglia polarization into M2-type and inhibiting the proliferation of astrocytes.The expression of neurotrophic factors and neural differentiation-related markers in MSCs-containing microtissues was higher than that of groups without MSCs,suggesting MSCs accelerate NSCs differentiation into mature neurons.Single-cell sequencing was used to identify cell subtypes in microtissues,analyze signal pathways,and explore the distribution and proportion of five cell groups of late activated NSCs,neuroblasts,GABAergic neurons,neurogliocytes,and glutaminergic neurons.The GABAergic neurons in MSCs-containing microtissues were increased by～7.82%compared with that without MSCs,and MAPK and cAMP neural signaling pathways were activated during neuronal differentiation.After transplanting "NSCs(HAp)-MSCs" heterogeneous cell microtissues into the SCI site for 4 weeks,the basso mouse scale was higher than other experimental groups,and the electrophysiological behavior was more obvious.These results indicated that the "NSCs(HAp)-MSCs" heterogeneous cell microtissues significantly restored the motor and neural function of mice.It realizes the multi-mode regulation of neuroprotection,regeneration,and the neural microenvironment.The 1cm neuron-like tissues formed after the fusion between the spheroids,tend to be tissue-like and organoid.It brings a new prospect for establishing a drug screening model and the therapy of CNS diseases.In the dissertation,the NSCs neural differentiation regulated by nano-HAp was studied gradually from stem cell types,the functionality of nanomaterials,and spatial dimensions of cell culture.The mechanism of HAp-NSCs and the various cell culture modes were explored.An efficient SCI repair was achieved.It provides a brand-new way for the regulation of functional nanomaterials on the stem cell fate and the therapy of CNS diseases.