Preparation Of Novel Thermosensitivity Decellularized Nucleus Pulposus Hydrogel And Research On The Treatment Of Intervertebral Disc Degeneration

It is reported that degenerative disc disease(DDD),lead to low back pain,is a main cause of hospitalization and disability.The etiology of disc degeneration is complex,such as genetic,environmental,mechanical,age and other factors.The nucleus pulposus and the surrounding annulus fibrosus together make up the disc.The nucleus pulposus is the inner part of the disc and the region of high water content(rich in proteoglycan and type II collagen),which is generally believed to be the origin of disc degeneration.There are many treatments for back and leg pain caused by disc degeneration.However,no satisfactory solution to regenerate the IVD's natural properties currently.Stem cell-based tissue engineering is a promising method for NP regeneration.Biological scaffolds,act as carrier of stem cells,create a critical microenvironment for the proliferation and differentiation nomatter transplantation after in vitro co-culture or to specific sites in vivo.Adipose tissue-derived stem cells(ADSCs)are widely used in stem cell-based tissue engineering,for they could differentiate into different kinds of cells in specific conduitions include NP-like phenotype.Homogenous or heterogenous sources tissue extracellular matrix hydrogel provide tissue-specific extracellular matrix components that influence host or transplanted cell behavior,such as migration,proliferation,and differentiation.Fryetes et first described that extracellular matrix was digested by enzyme to form soluble polypeptide as thermo-sensitive hydrogel,meanwhile provides a new method of minimally invasive.The technique has been successfully applied to tissues such as cartilage and myocardium.The research team we cooperated have made cardiac tissue acellular hydrogel successfully recently,which played a very protective effect role in myocardial infarction.In the same way,we prepared the matrix decellularized nucleus pulpous hydrogel(DNPH).Compared with synthetic material,extracellular matrix hydrogel has a unique advantage-biochemical complexity as source of organization,the decellularization can remove host cells and antigen in tissues to reduce foreign body reaction.The way of decellularization have important influence to form the physical and chemical properties of the extracellular matrix hydrogel.Therefore,the selection and control of decellularization are very important.Although extracellular matrix hydrogel has excellent biological activity,they also have some typical limitations,such as poor mechanical properties and fast degradation rate.The bio-scaffolds that degrades too fast is difficult to suppourt the long-term cell survival.However,the disc needs to bear stress,so the mechanical properties of the disc tissue engineering reconstruction process are needed.As the "soil" or carrier of cell growth,mechanical signals such as the hardness and elasticity of ECM extracellular matrix will affect cell proliferation,death and differentiation.The mechanical environment of nucleus pulposus requires higher mechanical properties of bio-scaffolds.Mechanical signals as stiff and elasticity of ECM could control cell proliferation,death and differentiation.Multiple studies have shown that YAP/TAZ plays an important regulatory role in this process.Therefore,only stable bio-scaffolds with good mechanical properties can support the survival and maturation of cells effectively.How to overcome its poor mechanical properties? We think of genipin.Genipin,a new type of natural crosslinking agent,has the advantgage of low toxicity,mild crosslinking process,as well as the crosslinking products has a good stability and biological compatibility.Because genipin can improve the stability and mechanical strength of hydrogel.So in our study,we created DNPH;evaluated the effect of decellularization,established three dimensional culture system;crosselinked the DNPH by genipin to maintain its stability and improved elastic modulus to restore the intervertebral height,reduce hydrogel and cell leakage,maintain long-term survival,promote differentiation.The results of our study showed that most of the cells and Deoxyribonucleic Acid(DNA)(93%)were effectively removed after fresh bovine nucleus pulposus was decellularized,and the original structure and components were highy preserved.0.02% genipin crosselinked Decellularized nucleus pulposus hydrogel(GDH)has a good biocompatibility in vivo and in vitro,and the three-dimensional culture of ADSC has a low biotoxicity,and has a certain role in promoting the differentiation of NP cells.Implantation of Genipin crosse-linked decellularized nucleus pulposus hydrogel like-ADSCs(GDHA)into degenerated intervertebral disc can effectively restore intervertebral height and intervertebral disc signal strength,suggesting that it can be used as a potential tissue engineering scaffold to delay the degeneration and regeneration of nucleus pulposus.Part 1: Preparation and verification of a thermosensitive injectable decellularized nucleus pulposus hydrogelObjective: To evaluate the effect of process of decellularization,create the thermosensitive DNPH by enzyme digestion.To constructe ADSCs-DNPH 3D culture system.Methods: Decellularized Nucleus pulposus was prepared from fresh bovine nucleus pulposus by combining freeze-thaw cycle with sodium dodecyl sulfonate(SDS).The composition of decellularized nucleus pulposus was determined by quantitative detection of DNA,glycosaminoglycan and hydroxyproline,and the structure was observed by HE staining,4',6-Diamidino-2-Phenylindole(DAPI)staining and scanning electron microscopy(SEM)to evaluate the overall effect of Decellularized.DNPH was prepared by Fryetes method,hydrogel structure was detected by HE staining and SME,hydrogel properties were detected by gelation kinetics.ADSCs of SD rat were extracted and identified by tri-directional differentiation culture and flow cytometry.ADSCs was 3D-cultured in DNPH,and cell morphology was observed by laser confocal microscopy.Results: 1.HE staining and DAPI staining showed that only a small amount of nuclei remained after decellularization of the nucleus pulposus.Quantitative DNA testing showed that about 93% of the DNA was removed.The contents of glycosaminoglycan and hydroxyproline showed that collagen and proteoglycan were well preserved.HE staining and SME assay showed that the fibrous structure in the tissue was intact and similar to fresh nucleus pulposus,although it was relatively loose in fresh nucleus pulposus.DNPH precursors were gelatinized at 37° for about 30 min.2.Flow cytometry and tri-directional differentiation culture showed that the stem cells were real ADSCs.3.A three-dimensional culture of Green fluorescent protein(GFP)expressed ADSCs,the laser confocal microscope showed that GFP-ADSCs grow well in the hydrogel.Conclusion: The way of decellularization of bovine nucleus pulposus can effectively remove the cells from the tissue and retain other structures and components.DNPH is a typical grid bio-hydrogel and can be used in the three-dimensional culture of ADSC.Part 2: Construction and characterization of genipin cross-linked decellularized nucleus pulposus hydrogelObjective:(1)to detect the crosslinking degree,microstructure,mechanics and stability of GDH at different concentrations,and to conduct three-dimensional culture of GDHA or proliferation and cytotoxicity detection to determine the optimal concentration of genipin.(2)further biocompatibility test in vivo and in vitro.(3)ADSCs were cultured in three dimensions to preliminarily evaluate the differentiation of ADSCs in GDH.Methods: Different concentrations of GDH were constructed,gel kinetics and ninhydrin test were perform to determine the crosslinking law,and universal material testing machine was used to test the compression properties of each group of hydrogel.Degradation experiments were conducted to determine the stability of the hydrogel.3D culture of GDHA at different concentrations was performed.Cell Counting Kit-8(CCK8)experiment and Live/Dead detection were performed to evaluate biological compatibility.The expression of NP-related genes was detected by reverse transcription-polymerase chain reaction(RT-PCR).The hydrogel was implanted subcutaneously in SD rats,and HE staining was performed to observe the inflammatory reaction and foreign body reaction in vivo.Results: GDH at different concentrations presented different degree of blue after crosslinking.With the increase of genipin concentration,the colour deepened and the crosslinking degree also increased.Electron microscopy confirmed that gnipin was added to DNPH to form new crosslinking,and the mesh area decreased with the increase of crosslinking degree.The results of mechanical testing showed that the elastic modulus of hydrogel increased with the increase of crosslinking degree.When the concentration of gipin was 0.02-0.04%,the elastic modulus was close to that of nucleus pulposus in normal people.The degradation time of GDH was significantly prolonged by collagenase degradation test(P<0.05).The CCK8 experiment showed that(0-0.02%)there was no significant difference in the OD value of GDHA detected on day 1,3 and 7(p >,0.05).However,OD value in 0.04%GDHA showed no significant upward trend(P<0.05).The results showed that :(0-0.02%)at the 5th day of 3d culture in GDHA,ADSCs were mostly green(living cells)and a small amount red(dead cells).However,in 0.04% gel,a large number of red dye cells can be seen.In vivo compatibility tests showed that the inflammatory and foreign body reactions of DNPH and 0.02%GDH were mild.PCR showed that the expression of NP-lated genes(ol-2,Acan,Sox9 m RNA)in 0.02%GDHA was increased,followed by DNPH group,and the expression of cells cultured in adherent culture was the lowest.No significant difference in Col-1 m RNA expression were observed.Conclusion: 0.02%GDH has good biocompatibility in vitro and in vivo,and has elastic modulus similar to that of human nucleus pulposus.The GDH had a fast self-crosslinking process at the first 30 minutes,followed by a slow Genipin crosslinking process.SME showed that GDH had a mesh structure with smaller pores,reflecting a higher degree of crosslinking.The degradation test showed that the GDH was not easy to be degraded,and had better stability,which could last longer in vivo.0.02%GDH has a good effect on promoting the differentiation of ADSCs into NP-like cell.This will lay a foundation for the further study of in vivo disc degeneration.Part 3: Evaluation of the effect of GDHA on the rat model of intervertebral disc degenerationObjective: To construct a rat model of intervertebral disc degeneration by acupuncture.0.02%GDHA was injected into the degenerated intervertebral disc to observe the repair effect in vivo.Methods: The rat model of intervertebral disc degeneration was established,and the negative control group,positive control group,GDH group,ADSCs group and GDHA group were set up.Each intervention was injected into the degenerative disc,and X-ray,MRI and histological staining were performed at 0,4,8,and 12 weeks after the operation,respectively.Intervertebral height,MRI index,and histological morphology were observed.Results: DHI%,MRI index,histological observation and intervertebral disc degeneration index of GDHA all showed significant improvement,and the repair effect was better than other groups except the Sham group.Conclusion: Injection of 0.02%GDHA complex into the degenerative disc can restore the intervertebral height and the signal intensity of the disc to some extent,and also improve the histology.GDHA may be a potential tissue engineering to delay and treat intervertebral disc degeneration.