| BackgroundAt present there were more than 150 million elderly people over 65 years old in China,accounting for 10.8% of the total population.With the arrival of the aging society in our country,the disease of cervical and low back pain caused by degeneration of intervertebral discs is increasing.IDD seriously troubled the health and quality of life of the elderly life,while for young people in terms of lumbar degenerative disease is also one of the main causes of loss of labor capacity.According to the latest epidemiological survey,80% of people in their lives at least experienced low back pain,which not only take individuals a great physical and psychological pain,but also to the family and society a serious economic burden.Therefore effective treatment for IDD is one of the most important problems in the field of medical needs to be solved.The pathogenesis for intervertebral disc degeneration(IDD)is complex,genetic,obesity,smoking,occupation,age,trauma can induce the disease.IDD is a result of a variety of factors which acting on the nucleus pulposus,annulus and end plate.IDD is a prerequisite and basis for a series of degenerative diseases of the spine.Lumbar disc degeneration pathophysiology mainly manifested as intervertebral disc nucleus pulposus reduction,decreased cell function,intervertebral disc extracellular matrix synthesis decreased,accelerated degradation,loss of metabolism balance.Clinical manifestations of IDD are spinal stenosis,intervertebral disc herniation,waist and low leg pain and other diseases.IDD is a very common disease.At present in clinic there many treatment methods for lumbar disc degeneration disease,including drug and massage,massage-based conservative treatment and intervertebral disc nucleus removal,spinal fusion fixation and artificial intervertebral disc-based surgical treatment.Conservative treatment is symptomatic treatment and the effect is inaccurate which can not delay the process of degeneration of the disc.Surgical treatment such as nucleus removal cause a high degree of disc lossassociated with biomechanical and anatomical changes and may further aggravate disc degeneration and lumbar instability.Spinal fusion and internal fixation only solve the oppression and instability of the lesion,which not only can not maintain the intervertebral disc function,and may even stress concentrated in the adjacent segment and cause adjacent segment disease.Therefore seeking a new treatment is the current urgent task,biological therapy for disc regeneration provide a new methods.The main pathophysiological changes of intervertebral disc degeneration are intervertebral disc cells and cell function reduced,extracellular matrix metabolism imbalance,so the ideal treatment should be able to maintain the number of normal cells in the disc tissue,regulate intervertebral disc cell function,promote extracellular matrix secretion,slow down the degradation of extracellular matrix.Seed cells,growth factors and scaffolds are three major factors in repairing intervertebral disc degeneration.Bone marrow mesenchymal stem cells(BMSCs),derived from the bone marrow stroma,with multi-directional differentiation potential,in different media,BMSCs can be induced to the differentiation of bone cells,chondrocytes,fibroblasts,cardiomyocytes,adipocytes and et al.BMSCs have the advantages of convenient preparation,rapid growth in vitro,abundant sources,easy isolation and culture,strong proliferative ability,and low immunogenicity both in vitro and in vivo or in autologous or allograft.It is one of the most ideal seed cells in tissue engineering.However,which cells the BMSCs can differentiate into in vivo mainly depends on the local environment in which the cells are located.Under the hypoxic microenvironment of the nucleus pulposus and under the induction of transforming growth factor-β1,BMSCs can differentiate into the nucleus pulposus cells.Transforming growth factor-β1(TGF-β1)is the most important member in TGF-βfamily with the highest content and the strongest activity.In the differentiation of BMSCs into chondrocytes,TGF-β1 binds to type Ⅰ and Ⅱ receptors through ligand binding,leading to phosphorylation of type Ⅰ receptors,activation of Smad protein,entry of nucleus,regulation of transcription of target genes.By altering the activity of the protease substrate TGF-β1 stimulates the target cells.TGF-β1 also has the role of promoting chondrocyte proliferation and maintaining chondrocyte phenotype.However,in the early chondrocyte culture,it was found that TGF-β1 had two-way regulation of proliferation and differentiation of chondrocytes,whether it was a inhibition orpromotion by TGF-β1 concentration.TGF-β1 is an exogenous growth factor with short half-life,easy to be hydrolyzed by proteolytic enzymes.TGF-β1 1ocal application has not yet induced the completion of induction and the formation of a certain number of extracellular matrix,biological molecules have been degraded.The key issue in bone tissue engineering research is how to make growth factors continue to play a role.Gelatin is the preferred material for the production of microspheres with good biological characteristics and is currently the focus of drug release system.The method of making gelatin microspheres has been relatively mature and has been widely used in the field of drug release carrier.Gelatin microspheres with the diameter of about 10μm and completely degraded about 2 months or so can be used as intervertebral disc tissue engineering scaffold material.TGF-β1-loaded gelatin microspheres were used to induce the differentiation of BMSCs in vivo for a long time,which effectively inhibited the degeneration of intervertebral disc and had wide application prospect in intervertebral disc tissue engineering.In this study,we constructed the intervertebral disc tissue engineering material in vitro and transplanted into the rabbit degeneration discs.TGF-β1 with gelatin microspheres induced BMSCs to differentiate into nucleus pulposus cells for a long time and promote the proliferation of nucleus pulposus cells and synthetic protein polysaccharide and type II collagen.The combination of the two material can effectively delay the disc degeneration.Part 1: in vitro culture and identification of rabbit bone marrow mesenchymal stem cells(BMSCs)Objective:To culture and identify BMSCs from bone marrow of fetal rabbits by density gradient centrifugation,and to provide experimental basis for the study of the combination of gelatin microspheres combined with TGF-β1 for repairing intervertebral disc degeneration.Methods: BMSCs were cultured in vitro.The morphology of P3 and P5 cells was observed under inverted microscope.The growth curves of P3 generation BMSCs were observed by density gradient centrifugation.The CD105,CD90,CD31 and CD14 of BMSCs were identified by flow cytometry.BMSCs were induced to differentiate into osteoblasts,adipocytes,alizarin red stained calcium nodules,and oil red O stained cells drop.Results: BMSCs were successfully extracted by density gradient centrifugation.Under the microscope of inverted phase contrast microscope,some cells of BMSCs began to adhere to the cells after 24 h.Primary BMSCs were passaged to the third generation by selective digestion.The cell purity was about 90 % Of BMSCs and the fifth generation BMSCs were observed under inverted microscope.The proliferation activity of CCK-8 cells showed that the cell proliferation activity of P3 generation BMSCs was good,and the cells entered the logarithmic growth phase on the second day The expression of CD90 and CD105 were 93.67% and 92.03%,respectively.The negative markers were CD14 and CD31,which were 0.11% and 1.08%,respectively.P3 generation BMSCs induced by osteoblasts and adipogenic inducers,which could differentiate into osteogenic and adipogenic cells.Alizarin red staining found that calcium nodules and oil red O staining showed intracellular lipid droplets.Conclusion: BMSCs can be successfully extracted by density gradient centrifugation in fetal rabbit bone marrow,and then purified by selective digestion to obtain high proliferation activity and high purity BMSCs.Through the identification of cell surface molecules and multi-directional differentiation potential,it was confirmed that the extracted cells were BMSCs.And to provide experimental basis for the subsequent treatment of intervertebral disc degeneration with BMSCs combined with gelatin microspheres with TGF-β1.Part 2: Preparation of TGF-β1 gelatin microspheres,in vitro release of TGF-β1and proliferation of BMSCsObjective: To investigate the production process and in vitro release of TGF-β1gelatin microspheres and its effect on the proliferation of BMSCs.Methods: The gelatin microspheres were prepared by emulsion cross-linking method.Morphological characteristics and particle size of gelatin microspheres were observed by electron microscopy.TGF-β1 was loaded into blank microspheres by shaking-centrifugation method.The effect of CCK-8 on the proliferation of BMSCs was studied by in vitro release.Results: Gelatin microspheres were prepared by emulsion cross-linking.Under the microscope,the microspheres were observed under the microscope.After the freeze-dried microspheres,the particle size was uniform and the globularity was good and no adhesion.Under the scanning microscope,The average particle size was 10.55± 1.25μm.The encapsulation efficiency of 60 ng TGF-β1 was more than 98%,the drugloading was 58.76±0.026 ng,and the drug loading was 5.9%.TGF-β1 was released in vitro for 14 days,and the cumulative release of TGF-β1 was more than 96%.TGF-β1was slowly released from TGF-β1 gelatin microspheres and to promote the proliferation of BMSCs.Conclusion: The gelatin microspheres were successfully prepared by emulsion method.The microspheres were rounded,the surface was smooth and the particle size was uniform.TGF-β1 sustained release for 14 days and co-cultured with BMSCs.Can significantly enhance the proliferation of BMSCs.Part3: Construction of intervertebral disc degeneration animal modelObjective: To explore the feasibility of establishing the animal model of lumbar intervertebral disc degeneration by nucleus pulposus extraction and the identification of the model by imaging and molecular biology.Methods:The intervertebral disc degeneration model was established with 18 G needle puncture L3 / 4,L4 / 5,L5 / 6 intervertebral disc,depth of about 5mm suction time of about 20 seconds.L3/4,L4/5,L5/6 intervertebral disc magnetic resonance index were measured by magnetic examination in the 1w,2w,3w,4w after surgery.The expression of proteoglycans in the nucleus pulposus was detected by immunohistochemistry.HE staining oberve cell arrangement and morphological changes.Results: The nucleus pulposus of L3/4,L4/5 and L5/6 intervertebral discs were drawn and the average quality of nucleus pulposus was 10 mg,and no accident death.After 1 week to 4 weeks after operation the magnetic resonance index continued to decrease,the difference was statistically significant.At the beginning of the second week,the proteoglycan expression in the intervertebral disc degeneration model began to appear,and the difference was more obvious with the prolongation of time,Nucleus pulposus group,with the extension of time,nucleus pulposus and fibula branch blunt,nucleus pulposus cells decreased,uneven distribution,abnormal nucleus pulposus structure disorder.Conclusion: The model of intervertebral disc degeneration was successfully established by nucleus pulposus method.This method has the advantages of simple operation,short operation time,good reproducibility and convenient development.The infection rate of the experimental animals was low and the success rate was high in thelumbar disc degeneration model constructed by this method.Through the imaging,histomorphology,molecular biology of the established model to assess,are consistent with the signs of early disc degeneration,can be used as an intervertebral disc degeneration study of animal models.Part 4: Experimental study of BSMCs combined with TGF-β1 gelatin microspheres in the treatment of intervertebral disc degenerationObjective: To investigate the feasibility of BSMCs combined with TGF-β1gelatin microspheres in the treatment of intervertebral disc degeneration,and to provide some reference for the study of intervertebral disc degeneration.Methods:The tissue engineering materials were constructed by bone marrow mesenchymal stem cells(BMSCs)combined with TGF-β1 gelatin microspheres in vitro.This material was transplanted into rabbit models 4 weeks after making the model.Sixty New Zealand white rabbits were randomly divided into 5 groups and 12 animals in each group:Group A blank control group;group B saline group;group C BMSCs group;group D BMSCs + non-adsorbed TGF-β1 gelatin microspheres group;group E BMSCs + gelatin / TGF-β1.The degree of repair of intervertebral disc was evaluated by MRI,RT-qPCR,HE staining and immunohistochemistry(IHC)at3w,6w and 12 w after transplantation.Results: MRI examination showed that,except for group A,the nucleus pulposus signals were reduced in different degrees,B group decreased the most obvious,E group of signals reduce the slowest.The MRI indexes were compared at 3 weeks,6weeks and 12 weeks after transplantation.The result showed that at 3w only A group and B group have significant difference compared with each other;at 6w in addition to group A MRI index were reduced in varying degrees,compared with the B,C,D groups,the difference was statistical.But there was no significant difference between group A and group E.Compared with group A,the MRI index continued to decrease at12 weeks.The total RNA was extracted from the nucleus pulposus of the intervertebral discs at 3w,6w and 12 w,and the proteoglycan and type II collagen mRNA were detected by RT-qPCR.Statistical analysis showed that the expression level of proteoglycan and type II collagen in group E was the highest,and the difference was statistically significant.The expression of proteoglycans and type II collagen in each group showed no significant difference between group A and group E.At 12 weeks,there was no difference between group A and group E,and B、C、D group degeneration was obvious.Conclusion: The intervertebral disc tissue engineering material was transplanted into the degenerated intervertebral disc,and the disc degeneration was significantly slowed.It is observed by MRI that transplanted tissue engineering materials can significantly delay the loss of water content in the intervertebral disc.RT-qPCR and immunohistochemical detection of extracellular matrix(proteoglycan,type II collagen)were significantly higher than those in other intervention groups.Through the pathological examination,tissue engineering materials can maintain the structure of the disc,delayed further degeneration of the disc. |
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