The Biological Effect Of The Axial Compression On Tissue-engineered Nucleus Pulposus Tissue Formed On An Interpenetrating Polymer Network Hydrogel Scaffold

Background:Degenerative disc disease(DDD) is a kind of spinal disease, which is centered on nucleus pulposus(NP) degeneration in the intervertebral disc(IVD), leading to NP fragmentation and IVD herniation and consequently a series of symptoms. DDD is of high incidence and seriously affecting the middle aged and aged patients’ quality of life and working ability. With faster population aging, it leads to a large burden to public health and has significant socioeconomic implications. The common treatments of DDD mainly focuses on the pain relief, lacking of etiological treatment strategies of repairing the cataclastic NP and rebuild the biomechanics of IVD. The development of the tissue engineering and regenerative medicine(TERM) strategies has emerged as a promising solution to the repairation and regeneration of the NP.Objective:Biomechanical function is the primary function of the IVD and thus the IVD bears high mechanical load. Specially, the NP supports approximately 70% of compressive axial load exerted on the spine. Therefore, only the tissue-engineered nucleus pulposus(TE-NP) with favorable mechanical properties could maintain a three-dimensional(3D) structure and provide a template for tissue regeneration under the compression in vivo, resulting in restoring the mechanical function of IVD and the biomechanics of the spine. However, the bottleneck of applying hydrogels on load-bearing tissues are the relatively weak mechanical properties. In the study, we aimed to construct a TE-NP with favorable mechanical properties and biocompability. Based on the TN-NP, we aimed to clarify the biologic effect of axial compression on NP cells.Methods:In this study, we developed a novel hierarchically structured biomimetic interpenetrating polymer network(IPN) hydrogel with enhanced mechanical properties, which is suitable for NP regeneration. The IPN hydrogel is composed of 4-arm poly(ethylene glycol)-acrylate(4A-PEG-Acr), oxidative dextran(ODex) and amino gelatin(MGel). We tested the physical and chemical properties of the pore size, porosity, mechanical property, hydration and cytocompability to optimize the best composition of the IPN hydrogel to construct the TE-NP by varying the proportion of the 4A-PEG-Acr in the hydrogel. And then the nucleus pulposus cells(NPCs) is encapsulated in the hydrogel and the proliferation, biochemical content, expression of specific NP genes and extracellular matrix(ECM) related protein of the TE-NP were tested.Thereafter, the hybrid was transferred to our bioreactor system consisting of integrated servomotor and circulating system to apply the different magnitude of dynamic compression on the TE-NP. Though testing proliferation, ECM synthesis, cell phenotype change, anabolic metabolism, catabolic metabolism, inflammatory response, apoptosis and cytoskeleton change, we aimed to clarify the relationship between the magnitude of the axial compression and biological function of TE-NP, and determine the appropriate magnitude of axial compression which could positively regulate the biological function of TE-NP.Results:(1) The hierarchically structured IPN hydrogel that is compose of 4A-PEG-acr, ODex and MGel by two-step crosslink can be a suitable candidate for NP tissue engineering. The IPN hydrogel at the ratio of 2:3:5 exhibited biomimetic 3D structure, macroporous morphology, relative higher stiffness and toughness, NP-like swelling behaviors and good cytocompatibility. After NPCs were seeded in the IPN hydrogel, the NPCs in the TE-NP showed favorable proliferation, natural phenotype and enhanced biosynthesis.(2)There is definite relationship between the magnitude of the axial compression and biological function of TE-NP. The proliferation and anabolism and ECM proteins expression of NPCs in the TE-NP were enhanced while natural circular cellular morphology of the NPCs were maintained by the lower magnitude of the axial compression(1%~10%) especially by the 1% strain amplitude. In contrast, the biological function of TE-NP was negatively affected by the higher magnitude of the axial compression(1%~10%), leading to catabolism, inflammatory response, increasing expression of apoptosis gene and vimentin while cellular morphology of the NPCs were changed to spindle shape.Conclusion:The hierarchically structured IPN hydrogel composed of 4A-PEG-acr、ODex and MGel, which was fabricated by two-step crosslinking, can be a biomimetic candidate for NP tissue engineering. The IPN hydrogel at the ratio of 2:3:5 exhibited enhanced mechanical properties and favorable cytocompability, which would benefi t for bearing the compression.When applying the lower magnitude of the axial compression(1%-10% strain amplitude) especially 1% strain amplitude on the TE-NP formed on the IPN hydrogel, the circular morphology of NPCs was maintained while the proliferation, anabolism and consequently biosynthesis were enhanced. However, when applying the higher magnitude of the axial compression, the bioactivity of the TE-NP was negatively regulated.In this study, we have constructed a novel TE-NP with enhanced mechanical properties and bioactivity, while found the lower magnitude of the axial compression could enhance the biological function of the TE-NP, which would lay the experimental and theoretical foundations for its clinic application.