Induce Intervertebral Disc Degeneration Research Articles

Induction of cervical disc degeneration and discogenic pain by low concentration Propionibacterium acnes infection: an in vivo animal study

BackgroundAlthough cervical intervertebral disc (IVD) degeneration is closely associated with neck pain, its cause remains unclear. In this study, an animal model of cervical disc degeneration and discogenic neck pain induced by a low concentration of Propionibacterium acnes (P. acnes-L) is investigated to explore the possible mechanisms of cervical discogenic pain.MethodsCervical IVD degeneration and discitis was induced in 8-week-old male rats in C3–C6 IVDs through the anterior intervertebral puncture with intradiscal injections of low and high concentrations of P. acnes (P. acnes-L, n = 20 and P. acnes-H, n = 15) or Staphylococcus aureus (S. aureus, n = 15), compared to control (injection with PBS, n = 20). The structural changes in the cervical IVD using micro-CT, histological evaluation, and gene expression assays after MRI scans at 2 and 6 weeks post-modeling. The P. acnes-L induced IVD degeneration model was assessed for cervical spine MRI, histological degeneration, pain-like behaviors (guarding behavior and forepaw von Frey), nerve fiber growth in the IVD endplate region, and DRG TNF-α and CGRP.ResultsIVD injection with P. acnes-L induced IVD degeneration with decreased IVD height and MRI T2 values. IVD injection with P. acnes-H and S. aureus both lead to discitis-like changes on T2-weighted MRI, trabecular bone remodeling on micro-CT, and osseous fusion after damage in the cartilage endplate adjacent to the injected IVD. Eventually, rats in the P. acnes-L group exhibited significant nociceptive hypersensitivity, nerve fiber ingrowth was observed in the IVD endplate region, inflammatory activity in the DRG was significantly increased compared to the control group, and the expression of the pain neurotransmitter CGRP was significantly upregulated.ConclusionP. acnes-L was validated to induce cervical IVD degeneration and discogenic pain phenotype, while P. acnes-H induced was identified to resemble septic discitis comparable to those caused by S. aureus infection.

Regenerative potential of platelet derived growth factor in nicotine induced intervertebral disc degenerative model – In vivo study

Intervertebral disc (IVD) degeneration is an age-related disease affecting the elderly population worldwide. The disease is contributed by degeneration of the cartilage tissue in the vertebral column. The current study explores the possibility of exploiting platelet derived growth factor (PDGF), a growth factor secreted by platelets, as a therapeutic agent against the IVD degeneration. BALB/c mice were exposed to nicotine at higher concentrations to induce IVD degeneration. Three different concentrations of PDGF (1 ng/mL, 2 ng/mL, 3 ng/mL) were used in the study. Oxidative stress parameters were assessed through Lipid peroxidation (LPO), Myeloperoxidase (MPO) and Nitric oxide (NO). Histopathology was done for assessing the extent of degeneration, collagen II, mucin, and calcium deposition by Haematoxylin and Eosin staining, immunohistochemistry, Alcian blue and Alizarin red staining respectively. Gene expression studies were carried out by quantitative real-time Polymerase chain reaction (RT-PCR) for Aggrecan, alkaline phosphatase, RUNX2, Collagen I alpha and osteocalcin. IVD degeneration was prominent and PDGF treatment restored the tissue as evidenced by H&E staining. Oxidative stress was induced by the nicotine treatment and all the biomarkers of oxidative stress were restored back to normal by PDGF in a dose dependent manner. Biomarkers of endochondral bone formation (aggrecan, alkaline phosphatase, RUNX2, Collagen I alpha and osteocalcin) was observed to be normalized by exposing to PDGF in mRNA level though RT-PCR. Mucin, calcium contents were also brought back to normal by PDGF treatment. In conclusion, current work strongly supports that PDGF could be used as a therapeutic agent for the treatment of IVD degeneration.

Effect of Tuina along "bladder meridian" alleviating intervertebral disc degeneration by regulating the transforming growth factor-β1/Smad signaling pathway in a rabbit model.

The aim of this study was to investigate the protective effects of Tuina (a traditional Chinese massage therapy) on intervertebral disc (IVD) degeneration and the regulatory mechanisms of the transforming growth factor-β1 (TGF-β1)/small mothers against decapentaplegic (Smad) signaling pathway. Thirty New Zealand white rabbits were randomized into five groups: the control group, model group, model + Tuina group (Tuina group), model + TGF-β1 group (TGF-β1 group), and model + TGF-β1 inhibitor SB431542 group (SB431542 group). The model was established by posterolateral annulus fibrosus puncturing (AFP). Recombinant TGF-β1 and inhibitor SB431542 was injected into the TGF-β1 group and SB431542 group with a microsyringe, respectively. The rabbits in the Tuina group received Tuina treatment along the bladder meridian for 4 weeks. Magnetic resonance imaging (MRI) was performed on rabbits before AFP and after 4 weeks of intervention. Lumbar IVDs (L2-L3 to L4-L5) were harvested after intervention. Histopathological changes in the IVDs were measured by hematoxylin and eosin (HE) staining. Type I collagen was analyzed by immunohistochemistry detection. The expression level of matrix metalloproteinase-3 (MMP3) was determined by enzyme-linked immunosorbent assay. Cell apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated nick end labeling and Western blotting. Real-time polymerase chain reaction and Western blotting were used to analyze the expression of TGF-β1 and Smad2/3/4 and a disintegrin and metalloproteinase with thrombospondin motifs 5. Posterolateral AFP induced IVD degeneration in rabbits with histopathological damage and noticeable changes in MRI images. Tuina alleviated histo-pathological changes and reversed the expression of extracellular matrix degeneration-related molecules and apoptosis-related proteins. Furthermore, AFP induced the activation of TGF-β1 and Smad2/3/4, whereas Tuina therapy markedly reduced the protein expression of Smad2/3 and the gene expression of TGF-β1 and Smad2/3/4. Additionally, the TGF-β1/Smad signaling pathway was activated in the TGF-β1 group, while the TGF-β1/Smad signaling pathway was inhibited in the SB431542 group. Posterolateral AFP induced disc degeneration as determined by MRI assessment and histological analysis. Tuina alleviated disc degeneration, possibly by inhibiting the fibrotic response mediated by the TGF-β1/Smad pathway, thus alleviating extracellular matrix degeneration and reducing cell apoptosis.

Advanced glycation end products promote intervertebral disc degeneration by transactivation of matrix metallopeptidase genes

Examine the mechanism by which advanced glycation end products (AGEs) induce intervertebral disc degeneration (IDD) in C57BL/6J mice. Matrix metallopeptidase (MMP) gene mRNA levels were assessed using RT-qPCR. Immunoprecipitation and co-immunoprecipitation were performed to identify the transcriptional complex regulating MMP expression due to AGEs. The preventive effects of inhibitors targeting this complex were tested in mice on high AGE diets. IDD and AGE accumulation were evident in mice on high-AGE diets (HAGEs), persisting across dietary shifts but absent in mice exclusively on low-AGE diets. Molecularly, HAGEs activated p21-activated kinase 1 (PAK1), prompting peroxisome proliferator-activated receptor gamma coactivator-related protein 1 (PPRC1) phosphorylation. Ubiquitin-specific protease 12 (USP12) interacted with the phosphorylated PPRC1 (pPPRC1), safeguarding it from proteasomal degradation. This pPPRC1, in collaboration with two histone acetyltransferases p300/CREB-binding protein (CBP) and a transcription factor activator protein 1(AP1), enhanced the expression of 12 MMP genes (MMP1a/1b/3/7/9/10/12/13/16/19/23/28). In vitro AGE exposure on nucleus pulposus and annulus fibrosus cells replicated this gene activation pattern, driven by the PAK1/pPPRC1-p300/CBP-AP1 pathway. The application of PAK1, p300, and AP1 inhibitors reduced pPPRC1-p300/CBP-AP1 binding to MMP promoters, diminishing their expression. These inhibitors effectively thwarted IDD in HAGE mice. Our results revealed that HAGEs instigate IDD via the PAK1/pPPRC1-p300/CBP-AP1 signaling pathway. This insight can guide therapeutic strategies to slow IDD progression in prediabetic/diabetic patients.

A Minimally Invasive Annulus Fibrosus Needle Puncture Model of Intervertebral Disc Degeneration in Rats

The needle puncture model in rats has been accepted as an ordinary model to induce intervertebral disc degeneration (IVDD). However, the model primarily penetrated the whole intervertebral disc, resulting in injury to the nucleus pulposus (NP) and annulus fibrosus (AF). The intention of this research was to explore a minimally invasive approach through needle puncture of the AF percutaneously in rats. Twenty SD rats underwent puncture at Co8/9 via a 20G percutaneous needle. The needle was slowly advanced perpendicular to the tail skin to penetrate the whole AF without damaging the NP limited by a hand-made sheath. The X-rays and magnetic resonance imaging T2 relaxation was evaluated at 1, 2, and 3weeks to assess the disc height index and signal changes. Histological and immunohistochemical staining of the IVD were obtained under a light microscope. X-rays showed that the disc height had progressively narrowed to 49% of baseline 3weeks after injury. magnetic resonance imaging evaluation demonstrated that the mean T2-weighted signal intensity at 3weeks was 43% of that in the uninjured control group at the Co8/9 level. Histological staining demonstrated disorganized lamellae in the AF and decreased proteoglycan content and cellularity within the NP in the injured discs. The present research demonstrates a reliable and convenient approach to induce an AF tear in rats through percutaneous needle puncture. This model reduces harm to the experimental animals significantly while imitating the progressive degeneration process. More importantly, the model confirmed that AF damage alone could lead to IVDD and provided a research method for AF degeneration in IVDD.

Lentivirus-mediated transfer of gene encoding fibroblast growth factor-18 inhibits intervertebral disc degeneration.

Fibroblast growth factor 18 (FGF-18) is a well-characterized anabolic growth factor involved in cartilage homeostasis. However, the effect of FGF-18 on intervertebral disc (IVD) degeneration has not been investigated. The present study aimed to investigate the role of FGF-18 in the process of rabbit IVD degeneration. In vitro, primary nucleus pulposus cells (NPs) were cultured and transfected with a lentivirus. Tert-butyl hydroperoxide (TBHP) was used to induce apoptosis in NPs on the second passage, while overexpression of FGF-18 in NPs attenuated TBHP-induced apoptosis. A rabbit annular puncture model was generated to induce IVD degeneration in vivo. The discs were injected with an FGF-18-overexpression lentivirus or a negative control lentivirus. In the sham group, the discs were exposed and not punctured. Disc degeneration was evaluated using H&E staining and a histological grading system. Reverse transcription-quantitative PCR was used to detect the expression of the extracellular matrix-degrading enzymes matrix metalloproteinase-3 (MMP-3) and A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5). Nucleus pulposus apoptosis was detected via western blotting, immunohistochemical methods and TUNEL staining. Histologic examination showed that disc degeneration was attenuated after FGF-18 overexpression treatment. At 8 weeks after surgery, the expression of MMP-3 and ADAMTS-5 in the annular puncture groups was higher compared with in the sham group. FGF-18 treatment inhibited the expression of MMP-3 and ADAMTS-5 at the mRNA level. Western blot assays indicated that the expression level of Bax was significantly reduced in the FGF-18 groups, and that the expression level of Bcl-2 was significantly increased compared with those in the control group. Moreover, immunohistochemical analysis indicated that the FGF-18 group exhibited a lower percentage of cleaved caspase 3-positive NPs. Quantification of the TUNEL staining demonstrated that the FGF-18 group had fewer apoptotic NPs than the control group. These findings indicated that FGF-18 could delay IVD degeneration by inhibiting the apoptosis of NPs and the expression of matrix-degrading enzymes.

Platelet-Derived Biomaterials Inhibit Nicotine-Induced Intervertebral Disc Degeneration Through Regulating IGF-1/AKT/IRS-1 Signaling Axis

Apart from aging process, adult intervertebral disc (IVD) undergoes various degenerative processes. However, the nicotine has not been well identified as a contributing etiology. According to a few studies, nicotine ingestion through smoking, air or clothing may significantly accumulate in active as well as passive smokers. Since nicotine has been demonstrated to adversely impact various physiological processes, such as sympathetic nervous system, leading to impaired vasculature and cellular apoptosis, we aimed to investigate whether nicotine could induce IVD degeneration. In particular, we evaluated dose-dependent impact of nicotine in vitro to simulate its chronic accumulation, which was later treated by platelet-derived biomaterials (PDB). Further, during in vivo studies, mice were subcutaneously administered with nicotine to examine IVD-associated pathologic changes. The results revealed that nicotine could significantly reduce chondrocytes and chondrogenic indicators (Sox, Col II and aggrecan). Mice with nicotine treatment also exhibited malformed IVD structure with decreased Col II as well as proteoglycans, which was significantly increased after PDB administration for 4 weeks. Mechanistically, PDB significantly restored the levels of IGF-1 signaling proteins, particularly pIGF-1 R, pAKT, and IRS-1, modulating ECM synthesis by chondrocytes. Conclusively, the PDB impart reparative and tissue regenerative processes by inhibiting nicotine-initiated IVD degeneration, through regulating IGF-1/AKT/IRS-1 signaling axis.

Danshen Attenuates Intervertebral Disc Degeneration via Antioxidation in SD Rats.

Objective To investigate the effects of Danshen on the imaging and histological parameters, expression levels of ECM-associated proteins and inflammatory factors, and antioxidative activity in the degenerated intervertebral disc (IVD) of SD rats. Methods Sixty male rats were randomly divided into three groups (control, IDD, and Danshen IDD). Percutaneous needle puncture in Co8-9 intervertebral disc was conducted in all rats of the IDD and Danshen IDD groups to induce intervertebral disc degeneration (IDD). After operation, animals of the Danshen IDD group were administrated with Danshen granules (3 g/kg body weight ) by gavage once a day. Four weeks later, the coccygeal vertebrae were harvested and used for imaging (disc height and MR signal), histological, immunohistochemical, and biochemical [water content, glycosaminoglycans (GAG), superoxide dismutase (SOD2), glutathione (GSH), and malondialdehyde (MDA)] analyses. Results The puncture induced significant decreased IVD space and MR T2 signal at both 2 and 4 weeks, which were attenuated by Danshen treatment. The disc degeneration in the IDD group (HE and Safranin O-Fast Green histological staining was markedly more serious compared with that in the control group. Four weeks of Danshen treatment significantly alleviated this degeneration compared with the IDD group. Needle puncture resulted in the upregulation of IL-1β and TNF-α, MMP-3, and downregulation of COL2 and aggrecan in the IDD group. However, this change was significantly weakened by Danshen treatment. Significantly lower water and GAG content, as well as the SOD2 and GSH levels, in the IDD group were found compared with those in the control group. However, the above parameters of the Danshen IDD group were significantly higher than those of the IDD group. Danshen treatment significantly decreased the content of MDA which was increased by needle puncture in the IDD group. Conclusion Danshen can attenuate intervertebral disc degeneration in SD rats by suppressing the oxidation reaction.

Advanced Glycation End Product Inhibitor Pyridoxamine Attenuates IVD Degeneration in Type 2 Diabetic Rats.

Type 2 diabetes mellitus (T2DM) is associated with advanced glycation end product (AGE) enrichment and considered a risk factor for intervertebral disc (IVD) degeneration. We hypothesized that systemic AGE inhibition, achieved using pyridoxamine (PM), attenuates IVD degeneration in T2DM rats. To induce IVD degeneration, lumbar disc injury or sham surgery was performed on Zucker Diabetic Sprague Dawley (ZDSD) or control Sprague Dawley (SD) rats. Post-surgery, IVD-injured ZDSD rats received daily PM dissolved in drinking water or water only. The resulting groups were SD uninjured, SD injured, ZDSD uninjured, ZDSD injured, and ZDSD injured + PM. Levels of blood glycation and disc degeneration were investigated. At week 8 post-surgery, glycated serum protein (GSP) levels were increased in ZDSDs compared to SDs. PM treatment attenuated this increase. Micro-MRI analysis demonstrated IVD dehydration in injured versus uninjured SDs and ZDSDs. In the ZDSD injured + PM group, IVD dehydration was diminished compared to ZDSD injured. AGE levels were decreased and aggrecan levels increased in ZDSD injured + PM versus ZDSD injured rats. Histological and immunohistochemical analyses further supported the beneficial effect of PM. In summary, PM attenuated GSP levels and IVD degeneration processes in ZDSD rats, demonstrating its potential to attenuate IVD degeneration in addition to managing glycemia in T2DM.

Selection of the Optimal Puncture Needle for Induction of a Rat Intervertebral Disc Degeneration Model

Background: The incidence of intervertebral disc (IVD) degeneration has increased in recent years. A simple, reliable, and reproducible animal model is critical for understanding the underlying mechanisms of IVD degeneration. The caudal discs of rats have been proposed as a common puncture model in which to induce IVD degeneration. However, there is still no consensus on the size of needle to be used. Objectives: The present study aimed to identify the appropriate needle size to establish an IVD degeneration model. Study Design: A randomized, experimental trial. Setting: Department of Orthopaedic Surgery, The First Affiliated Hospital of Soochow University, China. Methods: Validity was verified by magnetic resonance imaging (MRI) and histology. Results: From T2-weighted MRI imaging and histological examination, the IVD punctured by the 16-gauge needle degenerated acutely one week after the operation, whereas the 26-gauge needle puncture did no harm to the IVD. An 18-gauge needle showed a progressive degeneration in IVD. Limitations: The observation period was not very long (4 weeks). Conclusions: An 18-gauge needle can be used to induce IVD degeneration in rats. Therefore, an 18-gauge needle is the optimal selection to establish the degenerative IVD model on rats, whereas the 26-gauge needle failed to cause IVD degeneration. Thus, to study the prevention and treatment of IVD degeneration, a 26-gauge needle can be used for IVD injection of growth factors, plasmids, and drugs. A 16-gauge needle may be used to induce acute disc injury, but not IVD degeneration. Key words: Low back pain, degenerative intervertebral disc, animal model, puncture needle, rat model, optimal choice

Longitudinal characterization of intervertebral disc remodeling following acute annular injury in a rat model of degenerative disc disease

ABSTRACT Objective: Characterize 3D remodeling of the rat intervertebral disc (IVD) following acute annular injury via in vivo micro-computed tomography (µCT), ex vivo contrast-enhanced (CE)-µCT, and histology. Design: Female Lewis rats (N = 4/group) underwent either sham surgery or anterior annular puncture to L3-L4 and L5-L6 (n = 8 IVDs/group) to induce IVD degeneration. Rats were allowed ad libidum cage activity before and after surgery and underwent in vivo µCT scanning at baseline and every 2 weeks post-op for 12 weeks to characterize longitudinal changes in IVD height. At 12 weeks, lumbar spines were dissected and underwent CE-µCT scanning to characterize endpoint glycosaminoglycan distribution and nucleus pulposus (NP) volume ratio. Spines were processed for safranin-O-stained sagittal histology, and IVD degeneration was graded via the Rutges scale. Results: Puncture IVDs exhibited loss of IVD height at all time points from 4 weeks onward compared to Sham—the most severe height loss occurred posteriorly, with significant changes also occurring in the NP and laterally. Puncture IVDs exhibited higher CE-µCT attenuation, indicative of lower glycosaminoglycan content, and reduced NP volume ratio compared to Sham. Histologically, Puncture IVDs had higher Rutges damage scores and exhibited reduced NP cellularity and hydration, disorganized annulus fibrosus (AF) lamellae with evidence of the stab tract, and indistinct AF-NP border compared to Sham. Conclusions: Characterization of the complex, 3D alterations involved in the onset and early progression of IVD degeneration can foster greater understanding of the pathoetiology of IVD degeneration and may inform future studies assessing more sensitive diagnostic techniques or novel therapies.

PMMA Bone Cement Acts on the Hippo/YAP Pathway To Regulate CTGF and Induce Intervertebral Disc Degeneration.

Kyphoplasty is a novel approach treating osteoporotic vertebral compression fractures and poly(methyl methacrylate) (PMMA) is the most common bone cement used for kyphoplasty. Cement leaked into an intervertebral disc (IVD) usually causes problems. Herein, we explored the effects of PMMA particles on the biological behavior of nucleus pulposus cells and proved the inhibiting effect of PMMA particles in a dose-dependent manner. Besides, PMMA particles led to cell cycle arrest and the abnormal expression of several apoptosis associated gene. Futhermore, we found PMMA particles upregulated the expression of p-Mst1/2, p-Lats1, and p-YAP, whereas they downregulated the expression of YAP and CTGF, suggesting the activing effect on the Hippo/YAP/CTGF signaling pathway. By overexpression of YAP, PMMA-induced nucleus pulposus cell degeneration was reversed. In vivo study also showed that recombinant CTGF protein could alleviate PMMA-induced IVD degeneration in rats. To sum up, our study revealed the role of PMMA particles in inducing IVD degeneration by downregulating CTGF through Hippo/YAP pathway.

Human iPSCs can be differentiated into notochordal cells that reduce intervertebral disc degeneration in a porcine model.

Introduction: As many as 80% of the adult population experience back pain at some point in their lifetimes. Previous studies have indicated a link between back pain and intervertebral disc (IVD) degeneration. Despite decades of research, there is an urgent need for robust stem cell therapy targeting underlying causes rather than symptoms. It has been proposed that notochordal cells (NCs) appear to be the ideal cell type to regenerate the IVD: these cells disappear in humans as they mature, are replaced by nucleus pulposus (NP) cells, and their disappearance correlates with the initiation of degeneration of the disc. Human NCs are in short supply, thus here aimed for generation of notochordal-like cells from induced pluripotent cells (iPSCs).Methods: Human iPSCs were generated from normal dermal fibroblasts by transfecting plasmids encoding for six factors: OCT4, SOX2, KLF4, L-MYC, LIN28, and p53 shRNA. Then the iPSCs were treated with GSK3i to induce differentiation towards Primitive Streak Mesoderm (PSM). The differentiation was confirmed by qRT-PCR and immunofluorescence. PSM cells were transfected with Brachyury (Br)-encoding plasmid and the cells were encapsulated in Tetronic-tetraacrylate-fibrinogen (TF) hydrogel that mimics the NP environment (G'=1kPa), cultured in hypoxic conditions (2% O2) and with specifically defined growth media. The cells were also tested in vivo in a large animal model. IVD degeneration was induced after an annular puncture in pigs, 4 weeks later the cells were injected and IVDs were analyzed at 12 weeks after the injury using MRI, gene expression analysis and histology.Results: After short-term exposure of iPSCs to GSK3i there was a significant change in cell morphology, Primitive Streak Mesoderm (PSM) markers (Brachyury, MIXL1, FOXF1) were upregulated and markers of pluripotency (Nanog, Oct4, Sox2) were downregulated, both compared to the control group. PSM cells nucleofected with Br (PSM-Br) cultured in TF hydrogels retained the NC phenotype consistently for up to 8 weeks, as seen in the gene expression analysis. PSM-Br cells were co-cultured with bone marrow (BM)-derived mesenchymal stem cells (MSCs) which, with time, expressed the NC markers in higher levels, however the levels of expression in BM-MSCs alone did not change. Higher expression of NC and NP marker genes in human BM-MSCs was found to be induced by iNC-condition media (iNC-CM) than porcine NC-CM. The annular puncture induced IVD degeneration as early as 2 weeks after the procedure. The injected iNCs were detected in the degenerated discs after 8 weeks in vivo. The iNC-treated discs were found protected from degeneration. This was evident in histological analysis and changes in the pH levels, indicative of degeneration state of the discs, observed using qCEST MRI. Immunofluorescence stains show that their phenotype was consistent with the in vitro study, namely they still expressed the notochordal markers Keratin 18, Keratin 19, Noto and Brachyury.Conclusion: In the present study, we report a stepwise differentiation method to generate notochordal cells from human iPSCs. These cells not only demonstrate a sustainable notochordal cell phenotype in vitro and in vivo, but also show the functionality of notochordal cells and have protective effect in case of induced disc degeneration and prevent the change in the pH level of the injected IVDs. The mechanism of this effect could be suggested via the paracrine effect on resident cells, as it was shown in the in vitro studies with MSCs.

Effect of Thrombin-Induced MCP-1 and MMP-3 Production Via PAR1 Expression in Murine Intervertebral Discs

Structural changes in nucleus pulposus cells induce intervertebral disc (IVD) degeneration as a consequence of cytokine generation, biochemical products, and changes in the local environment. We have previously shown that inflammatory cytokines induce murine IVD (mIVD) angiogenesis and macrophage migration. Although the physiological roles of thrombin, a known proinflammatory factor, are documented, its relationship to IVD degeneration remains largely unexplored. Thrombin mediates cellular responses via the activation of protease-activated receptors such as PAR1 which has been studied in numerous cell types, but not extensively in IVD cells. This study was designed to investigate the endogenous expression of thrombin, tissue factor, and PAR1 in cultured coccygeal mIVDs. Thrombin exclusively induced MCP-1 via the MAPK-ERK and PI3K-AKT pathways. MCP-1 produced by mIVDs induced macrophage migration and thrombin treatment increased MMP-3 production to induce mIVD degeneration. These effects of thrombin on mIVDs were abrogated by a PAR1 inhibitor and suggest that thrombin may be a novel factor capable of stimulating cytokine activity implicated in the regulation several aspects of mIVDs. Mechanisms governing mIVDs, which are regulated by thrombin/PAR1 signaling, require elucidation if our understanding of IVD degenerative mechanisms is to advance.

Histological and reference system for the analysis of mouse intervertebral disc.

A new scoring system based on histo-morphology of mouse intervertebral disc (IVD) was established to assess changes in different mouse models of IVD degeneration and repair. IVDs from mouse strains of different ages, transgenic mice, or models of artificially induced IVD degeneration were assessed. Morphological features consistently observed in normal, and early/later stages of degeneration were categorized into a scoring system focused on nucleus pulposus (NP) and annulus fibrosus (AF) changes. "Normal NP" exhibited a highly cellularized cell mass that decreased with natural ageing and in disc degeneration. "Normal AF" consisted of distinct concentric lamellar structures, which was disrupted in severe degeneration. NP/AF clefts indicated more severe changes. Consistent scores were obtained between experienced and new users. Altogether, our scoring system effectively differentiated IVD changes in various strains of wild-type and genetically modified mice and in induced models of IVD degeneration, and is applicable from the post-natal stage to the aged mouse. This scoring tool and reference resource addresses a pressing need in the field for studying IVD changes and cross-study comparisons in mice, and facilitates a means to normalize mouse IVD assessment between different laboratories. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:233-243, 2018.

Intradiscal application of rhBMP-7 does not induce regeneration in a canine model of spontaneous intervertebral disc degeneration.

IntroductionStrategies for biological repair and regeneration of the intervertebral disc (IVD) by cell and tissue engineering are promising, but few have made it into a clinical setting. Recombinant human bone morphogenetic protein 7 (rhBMP-7) has been shown to stimulate matrix production by IVD cells in vitro and in vivo in animal models of induced IVD degeneration. The aim of this study was to determine the most effective dose of an intradiscal injection of rhBMP-7 in a spontaneous canine IVD degeneration model for translation into clinical application for patients with low back pain.MethodsCanine nucleus pulposus cells (NPCs) were cultured with rhBMP-7 to assess the anabolic effect of rhBMP-7 in vitro, and samples were evaluated for glycosaminoglycan (GAG) and DNA content, histology, and matrix-related gene expression. Three different dosages of rhBMP-7 (2.5 μg, 25 μg, and 250 μg) were injected in vivo into early degenerated IVDs of canines, which were followed up for six months by magnetic resonance imaging (T2-weighted images, T1rho and T2 maps). Post-mortem, the effects of rhBMP-7 were determined by radiography, computed tomography, and macroscopy, and by histological, biochemical (GAG, DNA, and collagen), and biomolecular analyses of IVD tissue.ResultsIn vitro, rhBMP-7 stimulated matrix production of canine NPCs as GAG deposition was enhanced, DNA content was maintained, and gene expression levels of ACAN and COL2A1 were significantly upregulated. Despite the wide dose range of rhBMP-7 (2.5 to 250 μg) administered in vivo, no regenerative effects were observed at the IVD level. Instead, extensive extradiscal bone formation was noticed after intradiscal injection of 25 μg and 250 μg of rhBMP-7.ConclusionsAn intradiscal bolus injection of 2.5 μg, 25 μg, and 250 μg rhBMP-7 showed no regenerative effects in a spontaneous canine IVD degeneration model. In contrast, intradiscal injection of 250 μg rhBMP-7, and to a lesser extent 25 μg rhBMP-7, resulted in extensive extradiscal bone formation, indicating that a bolus injection of rhBMP-7 alone cannot be used for treatment of IVD degeneration in human or canine patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0625-2) contains supplementary material, which is available to authorized users.

Stem cell therapy for intervertebral disc regeneration: obstacles and solutions.

Intervertebral disc (IVD) degeneration is frequently associated with low back and neck pain, which accounts for disability worldwide. Despite the known outcomes of the IVD degeneration cascade, the treatment of IVD degeneration is limited in that available conservative and surgical treatments do not reverse the pathology or restore the IVD tissue. Regenerative medicine for IVD degeneration, by injection of IVD cells, chondrocytes or stem cells, has been extensively studied in the past decade in various animal models of induced IVD degeneration, and has progressed to clinical trials in the treatment of various spinal conditions. Despite preliminary results showing positive effects of cell-injection strategies for IVD regeneration, detailed basic research on IVD cells and their niche indicates that transplanted cells are unable to survive and adapt in the avascular niche of the IVD. For this therapeutic strategy to succeed, the indications for its use and the patients who would benefit need to be better defined. To surmount these obstacles, the solution will be identified only by focused research, both in the laboratory and in the clinic.

Dynamic and static overloading induce early degenerative processes in caprine lumbar intervertebral discs.

Mechanical overloading of the spine is associated with low back pain and intervertebral disc (IVD) degeneration. How excessive loading elicits degenerative changes in the IVD is poorly understood. Comprehensive knowledge of the interaction between mechanical loading, cell responses and changes in the extracellular matrix of the disc is needed in order to successfully intervene in this process. The purpose of the current study was to investigate whether dynamic and static overloading affect caprine lumbar discs differently and what mechanisms lead to mechanically induced IVD degeneration. Lumbar caprine IVDs (n = 175) were cultured 7, 14 and 21 days under simulated-physiological loading (control), high dynamic or high static loading. Axial deformation and stiffness were continuously measured. Cell viability, cell density, and gene expression were assessed in the nucleus, inner- and outer annulus. The extracellular matrix (ECM) was analyzed for water, glycosaminoglycan and collagen content. IVD height loss and changes in axial deformation were gradual with dynamic and acute with static overloading. Dynamic overloading caused cell death in all IVD regions, whereas static overloading mostly affected the outer annulus. IVDs expression of catabolic and inflammation-related genes was up-regulated directly, whereas loss of water and glycosaminoglycan were significant only after 21 days. Static and dynamic overloading both induced pathological changes to caprine lumbar IVDs within 21 days. The mechanism by which they inflict biomechanical, cellular, and extracellular changes to the nucleus and annulus differed. The described cascades provide leads for the development of new pharmacological and rehabilitative therapies to halt the progression of DDD.