The Effect And Mechanism Of Tissue Engineering Strategy-based Nucleus Pulposus Regeneration Under The Regulation Of Extracellular Matrix Receptors Intereaction By Low-magnittude Compression

Backgrounds and objects:Nucleus pulposus(NP)degeneration is the core pathological change of intervertebral disc degenerative diseases.Due to the characteristics of upright walking and special anatomic structure of human intervertebral disc,the nucleus pulposus cells(NPCs)lived in a complex physical and chemical in-vivo environment with compressive and hydrogen pressure,limited blood supply and high osmotic pressure,leading to the weak self-repairing capacity of NP tissue.In recent years,rapid development of biomaterials and tissue engineering technology brings new hope for the regeneration and repair of the degenerative NP.However,the poor extracellular matrix(ECM)synthesis ability,weak biomechanical microenvironment adaptability of the seeding cells,and insufficient bionics of scaffold materials etc.are the bottleneck problems of the traditional tissue-engineered nucleus pulposus(TE-NP).As the key structure to buffer the compressive load of spine,the occurrence,development,and degeneration of NP are under regulated by compression stress.Thus,the present study aims to systematically elucidate the regulatory effect of compressive stress on the biological behaviors of NPCs,and constructs a new type of TE-NP with enhanced biological activity and stress microenvironment adaptability under the guidance of the aforementioned regulatory mechanism.This study may provide new ideas and theoretical guidance for realizing the biological treatment of intervertebral disc degenerative diseases.Methods:PART I: Based on our self-developed “Intelligent Bionic Mechanical Compression Loading Tissue Culture System”,the hydrogel-encapsulated rat NPCs were exerted graded axial dynamic compressive loading which could cause different deformation of hydrogel.The senescence and apoptosis of NPCs under graded axial dynamic compressive loading were observed and analyzed.The molecular mechanisms involved in this regulatory process were explored by high-throughput sequencing and functional experiments.PART II: Based on the “Intelligent Bionic hydraulic pressure Loading Tissue Culture System”,graded hydraulic pressure was further performed to the hydrogel-encapsulated rat NPCs and isolated rabbit NP tissues.The NPCs viability and ECM synthesis ability under graded hydraulic pressure were observed.The molecular mechanisms involved in this regulatory process were explored by high-throughput sequencing and functional experiments.PART III: Based on the regulatory mechanism involved in compression induced NP biological behavior changes,we speculated that enhanced cell-cell adhesion could activate ECM-receptors interaction induced ECM anabolic process,which may promote the seeding cell biological viability in TE-NP.Thus,we cultured the NPCs via “suspended autoadhesion system” in vitro to form NPC spheroids to enhance cell-cell contact effect.The NPCs viability and ECM synthesis ability under this culturing mode were observed.The molecular mechanisms involved in this regulatory process were explored by high-throughput sequencing.PART IV: In order to realize the minimally injectable nature and improve the bionic properties,including composition,structure,and mechanical properties of the bioscaffolds used for the TE-NP construction,methylacrylic anhydride(MA)was used to modify the collagen fiber side-chain amino group of the native decellularized NP matrix(DNPM).This MA modified DNPM hydrogel could maintain the original component integrity DNPM,and be crosslinked under ultraviolet radiation.Thus,DNPM-MA hydrogel was endowed with light-responsive cross-linking characteristics and appropriate mechanical strength to encapsulate seeding units for injectable TE-NP construction.It could also imitate the spatial and temporal distribution of ECM anabolism of NPCs.PART V: To verify the regenerative and repairing effect of the NPC spheroids-based TE-NP construction mode,rabbit lumbar and rat tail NP resection models were established.The DNPM-MA hydrogel encapsulated NPCs or NPC spheroids was implanted into the defect position of NP cavity.The regenerative and repairing effect of TE-NP and regulatory mechanism concluded by the first three parts studies were evaluated by the radiological and histological analysis.Results:PART I: Low-magnitude mechanical compression causing 5%hydrogel deformation maintained the normal activity,phenotype and functional ECM homeostasis of NPCs.Whereas high-magnitude mechanical compression causing ≧ 10% shape deformation induced senescence phenotype and apoptosis of NPCs,and further exacerbated functional ECM catabolism.The regulatory mechanism involved in this process was related to oxidative stress-mediated mitochondrial dysfunction in NPCs suffered high-magnitude mechanical compression.PART II: Low-magnitude hydrostatic pressure of 0.5 MPa maintained normal activity,and showed stronger functional ECM anabolic capacity compared with the pressure-free control group.Whereas ≧ 0.8 MPa hydrostatic pressure reduced activity and inhibited functional ECM anabolism.The potential regulatory mechanism of hydrostatic pressure-mediated NPCs biological behavior changes is related to the ECM receptor-related protein N-CDH and ITG β1 interaction.PART III: The N-CDH and ITG β1 interaction mediated cell-to-cell adhesion plays an important regulatory role in compressive stress-induced NP degeneration.Based on this regulatory mechanism,we performed suspending auto-adhesion culturing method to promote intercellular adhesion and form NPC spheroids.By comparing with monolayer-cultured NPCs,we found that NPC spheroids showed stronger functional ECM synthesis ability and low-magnitude compression effective “window”reactivity.PART IV: Photocrosslinkable DNPM hydrogel was successfully synthesized under the modification of MA.Then,the optimal concentration(10%)of DNPM-MA hydrogel used for TE-NP construction was figured out via evaluation of material mechanical properties,cell compatibility,and histocompatibility test.PART V: Transplantation of two types of injectable TE-NP(hydrogel encapsulated NPCs and hydrogel encapsulated NPC spheroids)showed a certain degree of NP regenerative effect.Noticeably,the NPC spheroid-based TE-NP revealed better regenerative effect than the NPC-based TE-NP in both radiological and histological evaluation.Conclusion:Compressive stress has a "window" effect on NPCs biological behaviors.High-magnitude compression exacerbates NPCs senescence,apoptosis,and NP degeneration via inducing oxidative stress damage and mitochondrial dysfunction.Whereas,low-magnitude compression promotes functional ECM anabolism via enhancing N-CDH and ITG β1interaction-mediated cell to cell adhesion effect.Based on the aforementioned regulatory mechanisms,suspending autoadhesion culturing method was performed to promote cell-cell adhesion and form NPC spheroids,which showed better cell viability,compression stress reactivity,and ECM synthesis ability.Then,the native DNPM-based photocrosslinkable hydrogel was synthesized to encapsulate NPC spheroids,and constructed injectable TE-NP.This NPC spheroid-based “bottom-up”TE-NP constructing mode reveals better regenerative effect than the traditional NPC-based TE-NP,and expands the application prospect of microscale tissue engineering-based TE-NP in the biological therapy of disc degenerative diseases.