A new perspective on intervertebral disc calcification—from bench to bedside

Disc degeneration primarily contributes to chronic low back and neck pain.Consequently,there is an urgent need to understand the spectrum of disc degeneration phenotypes such as fibrosis,ectopic calcification,herniation,or mixed phenotypes.Amongst these phenotypes,disc calcification is the least studied.Ectopic calcification,by definition,is the pathological mineralization of soft tissues,widely studied in the context of conditions that afflict vasculature,skin,and cartilage.Clinically,disc calcification is associated with poor surgical outcomes and back pain refractory to conservative treatment.It is frequently seen as a consequence of disc aging and progressive degeneration but exhibits unique molecular and morphological characteristics:hypertrophic chondrocyte-like cell differentiation;TNAP,ENPP1,and ANK upregulation;cell death;altered Pi and PPi homeostasis;and local inflammation.Recent studies in mouse models have provided a better understanding of the mechanisms underlying this phenotype.It is essential to recognize that the presentation and nature of mineralization differ between AF,NP,and EP compartments.Moreover,the combination of anatomic location,genetics,and environmental stressors,such as aging or trauma,govern the predisposition to calcification.Lastly,the systemic regulation of calcium and Pi metabolism is less important than the local activity of PPi modulated by the ANK-ENPP1 axis,along with disc cell death and differentiation status.While there is limited understanding of this phenotype,understanding the molecular pathways governing local intervertebral disc calcification may lead to developing disease-modifying drugs and better clinical management of degeneration-related pathologies.

Lumbar instability remodels cartilage endplate to induce intervertebral disc degeneration by recruiting osteoclasts via Hippo-CCL3 signaling

Degenerated endplate appears with cheese-like morphology and sensory innervation,contributing to low back pain and subsequently inducing intervertebral disc degeneration in the aged population.1 However,the origin and development mechanism of the cheese-like morphology remain unclear.Here in this study,we report lumbar instability induced cartilage endplate remodeling is responsible for this pathological change.

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.

Expression and significance of VEGF and p53 in degenerate intervertebral disc tissue

Objective:To investigate the mechanism of expression and significance of vascular endothelial growth factor(VEGF) and p53 in degenerate intervertebral disc tissue.Methods:Pathological sections collected from 156 patienls with lumbar disc herniation after surgery were tested by immunohistochemistry method,for evaluation of the expression of VEGF and p53 in degenerate intervertebral disc tissue.Results:98 cases(62.8%) with vascular infiltration phenomenon are found,and positive rates of VEGF and p53 in degenerate intervertebral disc tissue are 73.42% (116/156) and 58.97%(92/156);co-expression rate is 53.2%(83/l56);the expression rates of VEFG and p53 are significantly higher in the tissue with blood vessel infiltration than in the tissue without infiltration;there is a close relationship of VEGF with p53.Conclusions:VEGF and p53 gene synergetic express in degenerate intervertebral disc tissue,working together in neovascularization and infiltration,and accelerating intervertebral disc tissue degeneration.

Expression and significance of VEGF and p53 in rat degenerated intervertebral disc tissues

Objective:To discuss the expression and significance of vascular endothelial growth factor (VEGF) and p53 expression in rat degenerated intervertebral disc(IVD) tissues.Methods:A total of 78 even-aged Sprague-Dawley(SD) rats(observation group) were chosen to establish a rat IVD degeneration model with anterior limbs and tail-removing method.Three months after modeling, rats were executed and degenerated IVD tissues were obtained for pathological biopsy so as to observe VEGF and p53 expression thereof.Meanwhile,IVD tissues from 8 healthy rats were gained for comparison.Results:Capillary infiltration was found in 62.8%of cases in observation group. Positive expression of VEGF and p53 was seen in 74.4%and 59.0%of cases respectively with a co-expression rate of 53.8%.VECF and p53 expression in degenerated IVD tissues with capillary infiltration was higher than that in non-infiltration group(P【0.05).Conclasion:A coordinate expression of VEGF and p53 in rat degenerated IVD tissues is demonstrated,indicating that they are both involved in neovascularization and infiltration,which accelerates IVD degeneration.

Stem cells sources for intervertebral disc regeneration

Intervertebral disc regeneration field is rapidly growing since disc disorders represent a major health problem in industrialized countries with very few possible treatments.Indeed, current available therapies are symptomatic, and surgical procedures consist in disc removal and spinal fusion, which is not immune to regardable concerns about possible comorbidities, cost-effectiveness, secondary risks and long-lasting outcomes. This review paper aims to share recent advances in stem cell therapy for the treatment of intervertebral disc degeneration. In literature the potential use of different adult stem cells for intervertebral disc regeneration has already been reported. Bone marrow mesenchymal stromal/stem cells, adipose tissue derived stem cells, synovial stem cells, muscle-derived stem cells, olfactory neural stem cells, induced pluripotent stem cells, hematopoietic stem cells, disc stem cells, and embryonic stem cells have been studied for this purpose either in vitro or in vivo. Moreover, several engineered carriers(e.g., hydrogels), characterized by full biocompatibility and prompt biodegradation, have been designed and combined with different stem cell types in order to optimize the local and controlled delivery of cellular substrates in situ. The paper overviews the literature discussing the current status of our knowledge of the different stem cells types used as a cell-based therapy for disc regeneration.

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.

Effect of osteoporosis and intervertebral disc degeneration on endplate cartilage injury in rats

Objective:To investigate the effect of osteoporosis and intervertebral disc degeneration on the endplate cartilage injury in rats.Methods:A total of 48 female Sprague Dawley rats(3 months)were randomly divided into Groups A,B,C and D with 12 rats in each group.Osteoporosis and intervertebral disc degeneration composite model,simple degeneration model and simple osteoporosis model were prepared in Groups A,B and C respectively.After modeling,four rats of each group at 12th.18th and 24th week were sacrificed,Intervertebral height of cervical vertebra C6/C7 was measured.Micro-CT was used to image the endplate of cephalic and caudal cartilage at C6/C7 intervertebral disc.Abraded area rate of C6 caudal and C7 cephalic cartilage endplate was calculated,and then C6/C7 intervertebral disc was routinely embedded and sectioned.stained with safranin O to observe histological changes microscopically.Results:At 12,18 and24 weeks,intervertebral disc height of C6/C7 were(0.58±0.09)mm,(0.53±0.04)mm and(0.04±0.06)mm in Group A rats,(0.55±0.05)mm,(0.52±0.07)mm and(0.07±0.05)mm in Group B rats.At 24th week.intervertebral disc height of Group A rats was significantly lower than that of Group B rats(P<0.05);intervertebral disc height of Groups A and B rats at each time point were significantly lower than that of Groups C and D(P<0.05).There was no significantly statistical difference of intervertebral disc height between Groups C and D(P>0.05).At 12 and 18 weeks,the abraded rate of C6 caudal and C7 cephalic cartilage endplate in Group A rats were significantly higher than that in Groups B.C and D rats(P<0.05);the abraded rate in Group B was significantly higher than that in Groups C and D(P>0.05).Microscopic observation of CT showed that ventral defects in C6caudal or C7 cephalic cartilage endplate in Groups A and B appeared after 12 weeks of modeling;obvious cracks were found in front of the C6 and C7 vertebral body,and cartilage defect shown the trend of"repairing"at 18 and 24 weeks after modeling.Conclusions:Intervertebral disc degeneration and osteoporosis can cause damage to the cartilage endplate.Co-existence of these two factors can induce more serious damage to the endplate.which has possitive correlation with intervertebral disc degeneration.Osteoporosis plays a certain role in intervertebral disc degeneration process,and accelerates the degeneration of intervertebral disc in a specific time window.

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.

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.

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.

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.

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.

Mesenchymal stem cell tracking in the intervertebral disc

Low back pain is a common clinical problem, which leads to significant social, economic and public health costs. Intervertebral disc(IVD) degeneration is accepted as a common cause of low back pain. Initially, this is characterized by a loss of proteoglycans from the nucleus pulposus resulting in loss of tissue hydration and hydrostatic pressure. Conservative management,including analgesia and physiotherapy often fails and surgical treatment, such as spinal fusion, is required. Stem cells offer an exciting possible regenerative approach to IVD disease. Preclinical research has demonstrated promising biochemical, histological and radiological results in restoring degenerate IVDs. Cell tracking provides an opportunity to develop an in-depth understanding of stem cell survival, differentiation and migration, enabling optimization of stem cell treatment. Magnetic Resonance Imaging(MRI) is a non-invasive, non-ionizing imaging modality with high spatial resolution, ideally suited for stem cell tracking. Furthermore, novel MRI sequences have the potential to quantitatively assess IVD disease, providing an improved method to review response to biological treatment. Superparamagnetic iron oxide nanoparticles have been extensively researched for the purpose of cell tracking. These particles are biocompatible, non-toxic and act as excellent MRI contrast agents. This review will explore recent advances and issues in stem cell tracking and molecular imaging in relation to the IVD.

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.

3D assessment of intervertebral disc degeneration in zebrafish identifies changes in bone density that prime disc disease

Back pain is a common condition with a high social impact and represents a global health burden.Intervertebral disc disease(IVDD)is one of the major causes of back pain;no therapeutics are currently available to reverse this disease.The impact of bone mineral density(BMD)on IVDD has been controversial,with some studies suggesting osteoporosis as causative for IVDD and others suggesting it as protective for IVDD.Functional studies to evaluate the influence of genetic components of BMD in IVDD could highlight opportunities for drug development and repurposing.By taking a holistic 3D approach,we established an aging zebrafish model for spontaneous IVDD.Increased BMD in aging,detected by automated computational analysis,is caused by bone deformities at the endplates.However,aged zebrafish spines showed changes in bone morphology,microstructure,mineral heterogeneity,and increased fragility that resembled osteoporosis.Elements of the discs recapitulated IVDD symptoms found in humans:the intervertebral ligament(equivalent to the annulus fibrosus)showed disorganized collagen fibers and herniation,while the disc center(nucleus pulposus equivalent)showed dehydration and cellular abnormalities.We manipulated BMD in young zebrafish by mutating sp7 and cathepsin K,leading to low and high BMD,respectively.Remarkably,we detected IVDD in both groups,demonstrating that low BMD does not protect against IVDD,and we found a strong correlation between high BMD and IVDD.Deep learning was applied to high-resolution synchrotron\iCJ image data to analyze osteocyte 3D lacunar distribution and morphology,revealing a role of sp7 in controlling the osteocyte lacunar 3D profile.Our findings suggest potential avenues through which bone quality can be targeted to identify beneficial therapeutics for IVDD.

Application of Finite Element Analysis for Investigation of Intervertebral Disc Degeneration:from Laboratory to Clinic

Due to the ethical concern and inability to detect inner stress distributions of intervertebral disc(IVD),traditional methods for investigation of intervertebral disc degeneration(IVDD)have significant limitations.Many researchers have demonstrated that finite element analysis(FEA)is an effective tool for the research of IVDD.However,the specific application of FEA for investigation of IVDD has not been systematically elucidated before.In the present review,we summarize the current finite element models(FEM)used for the investigation of IVDD,including the poroelastic non linear FEM,difTusive-reactive theory model and cell-activity coupled mechano-electrochemical theory model.We further elaborate the use of FEA for the research of IVDD pathogenesis especially for nutrition and biomechanics associated etiology,and the biological,biomechanical and clinical influences of IVDD.In addition,the application of FEA for evaluation and exploration of various treatments for IVDD is also elucidated.We conclude that FEA is an excellent technique for research of IVDD,which could be used to explore the etiology,biology and biomechanics of IVDD.In the future,FEA may help us to achieve the goal of individualized precision therapy.

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.

Autologous fibroblasts induce fibrosis of the nucleus pulposus to maintain the stability of degenerative intervertebral discs

Lumbar degenerative disc diseases cause low back pain(LBP). The maintenance of the height and stability of the intervertebral disc(IVD) space is an effective treatment for LBP. The following study evaluated the effects of fibroblast injection on intervertebral disc degeneration(IDD) in a preclinical setting. Compared with the IDD group, the fibroblast treatment group demonstrated effective maintenance of IVD height, reduced endplate degeneration, and improved nuclear magnetic resonance signals and overall histological structure. In doing so, fibrotic IVDs maintained the stability and biomechanics of the vertebra. This finding is in agreement with clinical findings that human nucleus pulposus(NP) fibrosis is essential for the maintenance of IVD height and mechanical properties in patients following percutaneous endoscopic lumbar discectomy(PELD). Mechanistically, we demonstrated that injected fibroblasts not only proliferated but also induced NP cells to adopt a fibrotic phenotype via the secretion of TGF-β.Finally, to better mimic human conditions, the efficacy of autologous fibroblast injection in the treatment of IDD was further examined in a nonhuman primate cynomolgus monkey model due to their capacity for upright posture. We showed that the injection of fibroblasts could maintain the IVD height and rescue IVD signals in cynomolgus monkeys. Taken together, the results of our study reveal that autologous fibroblast injection can enhance the natural process of fibrosis during acute and subacute stages of stress-induced IDD. Fibrotic IVDs can maintain the stability, biological activity, and mechanical properties of the intervertebral space, thus providing a new direction for the treatment of intervertebral space-derived lumbar degenerative diseases.

Treatment of Protrusion of the Lumbar Intervertebral Disc by TCM Massage

Protrusion of the lumbar intervertebral disc is a common disorder frequently encountered in clinic. With TCM massage and the maneuver of manual reduction, we have treated 82 cases of protrusion of the lumbar intervertebral disc, with satisfactory therapeutic results reported in the following.……