Effect of axial vertical vibration on degeneration of lumbar intervertebral discs in modified bipedal rats: An in-vivo study

Objective: To assess the effects of axial vibrations on gene expression and lumbar intervertebral disc degeneration in vivo. Methods: A modified bipedal rat model was established using a brachial plexus rhizotomy approach to imitate human upright posture. The experimental animals were randomly divided into three groups: control, vertical vibration, and whole-body vibration. Gene expression in degeneration of the intervertebral discs was assessed by reverse transcription-quantitative polymerase chain reaction. Results: The expression of aggrecan, Col1α1, Col2α1, and decorin were shown to be up-regulated in 14-week-old rats in the vertical vibration and whole-body vibration groups, whereas biglycan and versican expression was down-regulated in 14-week-old rats of the two experimental groups. Furthermore, biglycan and versican expression levels were shown to be lower in the whole-body vibration group than in the vertical vibration group(P<0.05). Conclusions: This in-vivo study demonstrated that vibrations can influence the expression of anabolic genes. Furthermore, whole-body vibrations seem to have a greater effect in this regard than vertical vibrations. A new method is expected to relieve the low back pain of the patients through our research.

Biocompatibility of KLD-12 Peptide Hydrogel as a Scaffold in Tissue Engineering of Intervertebral Discs in Rabbits

KLD-12 peptide with a sequence of AcN-KLDLKLDLKLDL-CNH2 was synthesized and its biocompatibility was assessed in animals. Rabbit MSCs were cultured in the hydrogel for 2 weeks. Live cells were counted by using Calcein-AM/P1 fluorescence staining. MTT was employed to assess the viability of MSCs cultured in KLD-12 peptide solution of 0.01%, 0.03%, and 0.05%. Hemolysis test, skin irritation test and implantation test were conducted to evaluate its biocompatibility with host tissues. Our results demonstrated that the MSCs in hydrogel grew well and maintained round shape. Cell survival rate was 92.15% （mean： 92.15%±1.17%） at the 7th day and there was no difference in survival rate between day 7 and day 14. Cell proliferation test showed that the A value of the KLD-12 solutions was not significantly different from that of control groups （complete culture media） （P〉0.05） at the 24th and 48th h. The hemolysis rate of KLD-12 solution was 0.112%. Skin irritation test showed that the skin injected with KLD-12 solution remained normal and the score of skin irritation was 0. The histological examination with HE staining exhibited that the skin layers were clear and there was no infiltration with neutrophilic granulocytes and lymphocytes. It is concluded that KLD-12 peptide hydrogel bad a good biocompatibility with host rabbit and MSCs, and KLD-12 pep- tide hydrogel can provide an appropriate microenvironment for MSCs.

The study of migration of bone mesenchymal stem cells transplanted in intervertebral discs of rabbits and expression of exogenous gene

Objective： To explore the survival and migration of bone mesenchymal stem cells transplantated in intervertebral disc of rabbits and expression of the exogenic genes. Methods. Thirty-two rabbits were used, A randomized block design was used and discs in the same rabbit were one block,the lumbar discs from L2-3 to L5-6 were randomly divided into blank group, saline group, cell transplantation group Ⅰand cell transplantation group Ⅱ. The fluorescence microscopy was used to determine the fluorescence of the maker protein GFP and DNA-PCR was used to analyze the copies of DNA of neomycin-resistant gene at 1, 3, 6, months after transplantation. Results： There was fluorescence in cell transplantation group Ⅰ and Ⅱ and none in blank group, saline group at 1, 3, 6 months after transplantation. In cell transplantation groups,the fluorescent distribution was more scatter with time, but no significant difference between cell groups Ⅰ and Ⅱ. The test of neomycin resistant gene expressed in cell transplantation group Ⅰ and Ⅱ and quantitative analysis showed that there was no significant difference between the cell groups Ⅰ and Ⅱ （P〉0.05）. Conclusion： The transplanted bone mesenchymal stem cells can survive, migrate and the transfer genes can express efficiently, it suggests that the BMSC therapy may be effective to prevent and treat intervertebral disc degeneration.

Correlation between Osteoporosis and Degeneration of Intervertebral Discs in Aging Rats

This study examined the correlation between osteoporosis and the degeneration of intervertebral discs. Sprague-Dawley rats were maintained up to 22 or 28 months. The femoral bone, tibial bone and lumbar vertebra were histologically studied and the expression of collagen type Ⅱ and Ⅹ in intervertebral discs was immunohistochemiscally determined. Several indices for the degeneration of intervertebral discs and osteoporosis and the correlation among them were then analyzed. Close correlations were found among the indices for the degeneration of intervertebral discs, including the relative area of the vascular bud, the ratio of the uncalcified and the calcified layers, the expression of collagen type Ⅱ and Ⅹ. The correlation with collagen type Ⅹ was negative. There existed positive correlations among the indices for osteoporosis, including the thickness ratio of cortical bone, the relative area of bone trabecula, the density of femoral and vertebral body bones, and the maximum stress and strain on bone. Analysis on the relationship of osteoporosis and the disease on disc showed that the indices of osteoporosis were negatively correlated with the indices of the degeneration of intervertebral discs but the expression of collagen type Ⅹ was positively correlated, with the density of vertebral body bones having the strongest dependence on collagen type Ⅹ. The maximum stress and strain bore no correlation with the degeneration of intervertebral discs. These results suggest that osteoporosis was negatively correlated with the degeneration of intervertebral discs.

Ad/CMV-hTGF-β1 Treats Rabbit Intervertebral Discs Degeneration in Vivo

Summary: To investigate therapeutic efficiency of Ad/CMV-hTGF-β1 gene for rabbit intervertebral disc degeneration model. 60 Japanese white rabbits were selected to form the L5-L6 Anterior-Lateral-Anulus-Fibrosus-Incision-Induced model in order to simulate human intervertebral disc degeneration. 36 rabbits, whose corresponding intervertebral discs were injected with 20 μl (10×106 pfu)of Ad/CMV-hTGF-β1 gene, constituted the therapy group, 12 were injected with 20 μl (10×106 pfu)of Ad/CMV-LacZ gene as comparison group, while 12 were only injected with equivalent capacity of saline for empty comparison group. 3 weeks after injection, examples were taken for investigation of HE staining, MRI, Western Blotting and immunohistochemical research TGF-β1. Wide distribution of TGF-β1 was detected by immunohistochemical research in the degenerated annulus fibrosus after injection. Western Blotting research showed significant increase of TGF-β1 content in intervertebral discs treated with TGF-β1 gene than comparison groups. MRI signal transformed from low to comparatively high and that intervertebral disc pathological degree improved. Ad/CMV-hTGF-β1 gene transfection is a potential method to increase TGF-β1 content and reverse intervertebral disc degeneration.

Experimental Study on Stress Relaxation and Creep Properties of Human Thoracolumbar Vertebral Bodies and Intervertebral Discs

Objective:Underwater shock can produce extremely high accelerations, resulting in severe human injuries on shipboard, and human thoraco lumbar spines are prone to suffer from injuries by ship shock motion. To observe the viscoelasticity of thoracolumbar of young fresh cadavers, and to provide biomechanical parameters for both research and clinical practice. Materials and Methods:5 fresh young male cadavers (aged 22 to 31 years) were provided, and 15 thoracolumbar spinal anatomies of 5 samples were harvested within 1 hour of death. WE-10A universal testing machine was used for creep and relaxation tests.Results:Stress relaxation and creep deformation equations are derived from the biomechanics model and the measured and simulated curves are compared. The creep in vertebral bodies and intervertebral discs exhibited significantly changes in the first 5 min and 10 min, respectively. The stress rapidly decreased in the first 2 min, and then gradually went balance during the relaxation process. Conclusion:The change in creep rate is significant at early stage,and gradually slows down.This indicates that the differences between internal pressure and local pressure are decreased until balance. The simulated curve derived from equation coincides with the experimental data to a large degree, which states that the equation is rational and reliable.

Biomechanical analysis of bone mesoscopic structure and mechanical properties of vertebral endplates of degenerated intervertebral discs in rabbits

In previous studies of disc degeneration,the structural and mechanical properties of the endplate were often neglected.In this paper,the station legislation was used to construct an animal model of minor trauma disc degeneration,and the mechanism of disc degeneration was further investigated by observing the changes of mesoscopic structure and developing the mechanical properties of endplate bone.Twenty-eight 6-month-old Japanese white rabbits were divided into two groups:control group and experimental group.An animal model of intervertebral disc degeneration was established by upright experiment in the experimental group.The bone mesoscopic structures in different areas of each endplate were observed by histological and imaging methods,and the mechanical properties of the endplates were measured by indentation method.The two groups of data were compared by one-way ANOVA.After the experimental animals stood for 17 weeks,The experimental group showed the characteristics of early disc degeneration.The microstructure of the degenerative group showed that the end plate mineralization degree was higher,the bone mass was larger,and the number and thickness of bone trabeculae were larger.The results of indentation test showed that the mechanical properties of the degeneration group were enhanced,and the lower endplate was obviously enhanced.We successfully established a model of human disc degeneration with non invasive trauma and more consistent with the process of human disc degeneration through the standing experiment.In the experimental group,the internal structure of the endplate was dense and pore distance was reduced.The change of bone mesoscopic structure further affects the endplate,resulting in the enhancement of the mechanical properties of the endplate after intervertebral disc degeneration.The reduction of the pore distance and the narrowing of the internal channel structure of the endplate also hinder the nutrition supply of the intervertebral disc,which may be the key reason affecting the degeneration of the intervertebral disc.A biomechanical method for investigating the mechanism of intervertebral disc degeneration can be provided in this paper.

Injectable bioorthogonal hydrogel (BIOGEL) accelerates tissue regeneration in degenerated intervertebral discs

Intervertebral disc(IVD)degeneration is a leading cause of back pain and precursor to more severe conditions,including disc herniation and spinal stenosis.While traditional growth factor therapies(e.g.,TGFβ)are effective at transiently reversing degenerated disc by stimulation of matrix synthesis,it is increasingly accepted that bioscaffolds are required for sustained,complete IVD regeneration.Current scaffolds(e.g.,metal/polymer composites,non-mammalian biopolymers)can be improved in one or more IVD regeneration demands:biodegradability,noninvasive injection,recapitulated healthy IVD biomechanics,predictable crosslinking,and matrix repair induction.To meet these demands,tetrazine-norbornene bioorthogonal ligation was combined with gelatin to create an injectable bioorthogonal hydrogel(BIOGEL).The liquid hydrogel precursors remain free-flowing across a wide range of temperatures and crosslink into a robust hydrogel after 5-10 min,allowing a human operator to easily inject the therapeutic constructs into degenerated IVD.Moreover,BIOGEL encapsulation of TGFβpotentiated histological repair(e.g.,tissue architecture and matrix synthesis)and functional recovery(e.g.,high water retention by promoting the matrix synthesis and reduced pain)in an in vivo rat IVD degeneration/nucleotomy model.This BIOGEL procedure readily integrates into existing nucleotomy procedures,indicating that clinical adoption should proceed with minimal difficulty.Since bioorthogonal crosslinking is essentially non-reactive towards biomolecules,our developed material platform can be extended to other payloads and degenerative injuries.

Apparent diffusion coefficient in normal and abnormal pattern of intervertebral lumbar discs: initial experience

The aim of the present study was to compare the relationship of morphologically defined non-bulging/herni-ated, bulging and herniated intervertebral lumbar discs with quantitative apparent diffusion coefficient （ADC）. Thirty-two healthy volunteers and 28 patients with back pain or sciatica were examined by MRI. All intervertebral lumbar discs from L1 to S1 were classified according to morphological abnormality and degenerated grades. The ADC values of nucleus pulposus （NP） were measured and recorded. The significant differences about mean ADC values of NP were found between non-bulging/herniated discs and bulging discs as well as herniated discs （P 0.05）, whereas there were no significant differences in ADC values between bulging and herniated discs （P 0.05）. Moreover, statistically significant relationship was found in the mean ADC values of NP between ＂non-bulging/herniated and non-degenerated discs＂ and ＂non-bulging/herniated degenerated discs＂ as well as herniated discs （P 0.05）. Linear regression analysis between ADC value and disc level revealed an inverse correlation （r = -0.18）. The ADC map of the NP is a potentially useful tool for the quantitative assessment of componential and molecular alterations accompanied with lumbar disc abnormalities.

Reabsorption of intervertebral disc prolapse after conservative treatment with traditional Chinese medicine:A case report

BACKGROUND Conservative treatments have been reported to diminish or resolve clinical symptoms of lumbar intervertebral disc herniation(LIDH)within a few weeks.CASE SUMMARY Computed tomography and magnetic resonance imaging(MRI)of the lumbar region of a 25-yearold male diagnosed with LIDH showed prolapse of the L5/S2 disc.The disc extended 1.0 cm beyond the vertebral edge and hung along the posterior vertebral edge.The patient elected a conservative treatment regimen that included traditional Chinese medicine(TCM),acupuncture,and massage.During a follow-up period of more than 12 mo,good improvement in pain was reported without complications.MRI of the lumbar region after 12 mo showed obvious reabsorption of the herniation.CONCLUSION A conservative treatment regimen of TCM,acupuncture,and massage promoted reabsorption of a prolapsed disc.

How to enhance the ability of mesenchymal stem cells to alleviate intervertebral disc degeneration

Intervertebral disc(ID)degeneration(IDD)is one of the main causes of chronic low back pain,and degenerative lesions are usually caused by an imbalance between catabolic and anabolic processes in the ID.The environment in which the ID is located is harsh,with almost no vascular distribution within the disc,and the nutrient supply relies mainly on the diffusion of oxygen and nutrients from the blood vessels located under the endplate.The stability of its internal environment also plays an important role in preventing IDD.The main feature of disc degeneration is a decrease in the number of cells.Mesenchymal stem cells have been used in the treatment of disc lesions due to their ability to differentiate into nucleus pulposus cells in a nonspecific anti-inflammatory manner.The main purpose is to promote their regeneration.The current aim of stem cell therapy is to replace the aged and metamorphosed cells in the ID and to increase the content of the extracellular matrix.The treatment of disc degeneration with stem cells has achieved good efficacy,and the current challenge is how to improve this efficacy.Here,we reviewed current treatments for disc degeneration and summarize studies on stem cell vesicles,enhancement of therapeutic effects when stem cells are mixed with related substances,and improvements in the efficacy of stem cell therapy by adjuvants under adverse conditions.We reviewed the new approaches and ideas for stem cell treatment of disc degeneration in order to contribute to the development of new therapeutic approaches to meet current challenges.

EFFECT OF ACUPUNCTURE ON POST-OPERATIVE FUNCTIONAL RECOV-ERY IN PATIENTS WITH LUMBAR INTERVERTEBRAL DISC PROTRUSION

Objective To observe the influence of acupuncture on the post-operative rehabilitation in patients undergoing vertebral pulp extraction due to lumbar intervertebral disc protrusion （LIDP） so as to evaluate its efficacy in the treatment of lumbago induced by LIDP. Methods A total of 69 patients undergoing vertebral pulp extraction were randomized into acupuncture group （ n = 35） and control group （ n = 34）. Patients of acupuncture group were treated with routine rehabilitation method and electroacupuncture （EA） stimulation on Zhìbiān （秩边 BL54） and Wěizhōng （委中 BL40）, acupuncture on Shènshū （ 肾俞 BL23）, Dàchángshū （大肠俞 BL25）, BL54, BL40, Jiájǐ （夹脊 EX-B 2）, Shàngliáo （上髎 BL31）, etc., and cupping on BL40, with 15 days being a therapeutic course and for average 7. 36 courses. Patients of control group were treated with simple rehabilitation method. Functional recovery state was judged before and after surgery by using the standards of Japanese Orthopedic Association （JOA）. Results The average recovery rates of 3 months, 6 months and one year after surgery were 49.93% ,90.31% and 95.08% separately in acupuncture group, and 26.24% ,63.42% and 71.36% successively in control group. The recovery rates of acupuncture group were significantly higher than those of control group at the same time-points （P〈0. 05）. Conclusion Acupuncture has a definite effect on promoting post-operative functional recovery in LIDP patients undergoing vertebral pulp extraction and spinal fusion.

Electroacupuncture improves microcirculation and neuronal morphology in the spinal cord of a rat model of intervertebral disc extrusion

Most studies on spinal cord neuronal injury have focused on spinal cord tissue histology and the expression of nerve cell damage and repair-related genes. The importance of the microcirculation is often ignored in spinal cord injury and repair research. Therefore, in this study, we established a rat model of intervertebral disc extrusion by inserting a silica gel pad into the left ventral sur-face of T13. Electroacupuncture was used to stimulate the bilateralZusanlipoint （ST36） and Neiting point （ST44） for 14 days. Compared with control animals, blood lfow in the ifrst lumbar vertebra （L1） was noticeably increased in rats given electroacupuncture. Microvessel density in the T13 segment of the spinal cord was increased significantly as well. The number of normal neurons was higher in the ventral horn of the spinal cord. In addition, vacuolation in the white matter was lessened. No obvious glial cell proliferation was visible. Furthermore, hindlimb motor function was improved signiifcantly. Collectively, our results suggest that electroacupuncture can improve neuronal morphology and microcirculation, and promote the recovery of neurological functions in a rat model of intervertebral disc extrusion.

Mechanosignaling activation of TGFβmaintains intervertebral disc homeostasis

Intervertebral disc （IVD） degeneration is the leading cause of disability with no disease-modifying treatment. IVD degeneration is associated with instable mechanical loading in the spine, but little is known about how mechanical stress regulates nucleus notochordal （NC） cells to maintain IVD homeostasis. Here we report that mechanical stress can result in excessive integrin αvβ6-mediated activation of transforming growth factor beta （TGFβ）, decreased NC cell vacuoles, and increased matrix proteoglycan production, and results in degenerative disc disease （DDD）. Knockout of TGFβ type II receptor （TβRII） or integrin αv in the NC cells inhibited functional activity of postnatal NC cells and also resulted in DDD under mechanical loading. Administration of RGD peptide, TGFβ, and αvβ6-neutralizing antibodies attenuated IVD degeneration. Thus, integrin-mediated activation of TGFβ plays a critical role in mechanical signaling transduction to regulate IVD cell function and homeostasis. Manipulation of this signaling pathway may be a potential therapeutic target to modify DDD.

Exosomes derived from stem cells as an emerging therapeutic strategy for intervertebral disc degeneration

Intervertebral disc(IVD)degenerative diseases are a common problem in the world,and they cause substantial social and economic burdens for people.The current methods for treating IVD degenerative diseases mainly include surgery and conservative treatment,which cannot fundamentally restore the normal structure of the disc.With continuous research on the mechanism of degeneration and the development of regenerative medicine,rapid progress has been made in the field of regenerative medicine regarding the use of stem cell-derived exosomes,which are active biological substances used in intercellular communication,because they show a strong effect in promoting tissue regeneration.The study of exosomes in the field of IVD degeneration has just begun,and many surprising achievements have been made.This paper mainly reviews the biological characteristics of exosomes and highlights the current status of exosomes in the field of IVD degeneration,as well as future developments regarding exosomes.

Karacoline,identified by network pharmacology, reduces degradation of the extracellular matrix in intervertebral disc degeneration via the NF-κB signaling pathway

Karacoline is a compound found in the plant Aconitum kusnezoffii Reichb.Although Aconitum kusnezoffii Reichb is widely used for the treatment of pain,very few studies have been carried out on the use of karacoline due to its potential toxicity.In this study,we selected key matrix metalloproteinases(MMPs),collagen II,and aggrecan as targets due to their association with intervertebral disc degeneration(IDD).Using these targets,we then used network pharmacology to predict a series of molecules that might exert therapeutic effects on IDD.Of these molecules,karacoline was predicted to have the best effect.Tumor necrosis factor(TNF)-a is known to promote the degeneration of the extracellular matrix in IDD.We therefore applied different concentrations of karacoline(0,1.25,or 12.88 mM)along with 100 ng/mL TNF-a to rat nucleus pulposus cells and found that karacoline reduced the expression of MMP-14 in IDD by inhibiting the nuclear factor(NF)-κB pathway,while collagen II and aggrecan expression was increased.This suggested that extracellular matrix degradation was inhibited by karacoline(P<0.05).Our data therefore reveal a new clinical application of karacoline and provide support for the use of network pharmacology in predicting novel drugs.

Analysis of Gene Expression Pattern of Lumbar Intervertebral Disc Degeneration in Human

ObjectiveTo investigate the gene expression changes in normal and degeneration lumbar intervertebral disc in humans, providing information for clinical. MethodsThe PCR products of 4096 human genes were spotted onto a kind of chemical-material-coated-glass slides. The total RNAs were isolated from the tissues. Both the mRNAs from the degeneration and normal lumbar intervertebral disc in humans were reversely transcribed to the cDNAs, which used as the hybridization probes with the incorporations of fluorescent dUTP. The mixed probes were then hybridized to the cDNA microarray. After high-stringent washing, the cDNA microarray was scanned for the fluorescent signals and analyzed with computer image analysis. ResultsAmong the 4096 targets, there were 706 genes whose expression levels differed between the degeneration and normal lumbar intervertebral disc in all cases, comprising 298 up-regulated and 358 down-regulated ones. ConclusionDNA microarray technology is an effective technique in screening for differently expressed genes between the degeneration and normal lumbar intervertebral disc. Cell apoptosis plays an important role in the process of lumbar intervertebral disc degeneration.

Regulating the fate of stem cells for regenerating the intervertebral disc degeneration

Lower back pain is a leading cause of disability and is one of the reasons for the substantial socioeconomic burden.The etiology of intervertebral disc(IVD)degeneration is complicated,and its mechanism is still not completely understood.Factors such as aging,systemic inflammation,biochemical mediators,toxic environmental factors,physical injuries,and genetic factors are involved in the progression of its pathophysiology.Currently,no therapy for restoring degenerated IVD is available except pain management,reduced physical activities,and surgical intervention.Therefore,it is imperative to establish regenerative medicine-based approaches to heal and repair the injured disc,repopulate the cell types to retain water content,synthesize extracellular matrix,and strengthen the disc to restore normal spine flexion.Cellular therapy has gained attention for IVD management as an alternative therapeutic option.In this review,we present an overview of the anatomical and molecular structure and the surrounding pathophysiology of the IVD.Modern therapeutic approaches,including proteins and growth factors,cellular and gene therapy,and cell fate regulators are reviewed.Similarly,small molecules that modulate the fate of stem cells for their differentiation into chondrocytes and notochordal cell types are highlighted.

Transcription regulators differentiate mesenchymal stem cells into chondroprogenitors,and their in vivo implantation regenerated the intervertebral disc degeneration

BACKGROUND Intervertebral disc degeneration(IVDD)is the leading cause of lower back pain.Disc degeneration is characterized by reduced cellularity and decreased production of extracellular matrix(ECM).Mesenchymal stem cells(MSCs)have been envisioned as a promising treatment for degenerative illnesses.Cell-based therapy using ECM-producing chondrogenic derivatives of MSCs has the potential to restore the functionality of the intervertebral disc(IVD).AIM To investigate the potential of chondrogenic transcription factors to promote differentiation of human umbilical cord MSCs into chondrocytes,and to assess their therapeutic potential in IVD regeneration.METHODS MSCs were isolated and characterized morphologically and immunologically by the expression of specific markers.MSCs were then transfected with Sox-9 and Six-1 transcription factors to direct differentiation and were assessed for chondrogenic lineage based on the expression of specific markers.These differentiated MSCs were implanted in the rat model of IVDD.The regenerative potential of transplanted cells was investigated using histochemical and molecular analyses of IVDs.RESULTS Isolated cells showed fibroblast-like morphology and expressed CD105,CD90,CD73,CD29,and Vimentin but not CD45 antigens.Overexpression of Sox-9 and Six-1 greatly enhanced the gene expression of transforming growth factor beta-1 gene,BMP,Sox-9,Six-1,and Aggrecan,and protein expression of Sox-9 and Six-1.The implanted cells integrated,survived,and homed in the degenerated intervertebral disc.Histological grading showed that the transfected MSCs regenerated the IVD and restored normal architecture.CONCLUSION Genetically modified MSCs accelerate cartilage regeneration,providing a unique opportunity and impetus for stem cell-based therapeutic approach for degenerative disc diseases.