Optimization of puncture injury to rat caudal disc for mimicking early degeneration of intervertebral disc

The caudal discs of rats have been proposed as a puncture model in which intervertebral disc (IVD) degeneration can be induced and novel therapies can be tested. For biological repair, treatments for ongoing IVD degeneration are ideally administered during the earlier stages. The purpose of this study was to elucidate the optimal puncture needle size for creating a model that mimicked the earlier stages of IVD degeneration. According to the disc height index, histologic score, and MRI grading, a puncture needle sized 21G or larger induced rapid degenerative processes in rat caudal discs during the initial 2–4 weeks. The degenerative changes were severe and continued deteriorating after 4 weeks. Conversely, puncture injury induced by needles sized 25G or smaller also produced degenerative changes in rat caudal discs during initial 2–4 weeks; however, the changes were less severe. Furthermore, the degenerative process became stabilized and showed no further deterioration or spontaneous recovery after 4 weeks. In the discs punctured by 25G needles, the expression of collagen I was increased at 2–4 weeks with a gradually fibrotic transformation thereafter. The expressions of collagen II and SOX9 were enhanced initially but returned to pre‐injury levels at 4–8 weeks. The above‐mentioned findings were more compatible with earlier degeneration in discs punctured by needles sized 25G or smaller than by needles sized 21G or larger, and the appropriate timing for intradiscal administration of proposed therapeutic agents would be 4 weeks or longer after puncture. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:202–211, 2018.     

Needle puncture injury of the rat intervertebral disc affects torsional and compressive biomechanics differently

Needle puncture is a common method of inducing intervertebral disc (IVD) degeneration in small animal models and may have some similarities to IVD injury conditions such as herniation. Yet, the influence of puncture injuries on IVD biomechanics is not well understood. This study quantified the acute effects of anular injury on the biomechanics of rat caudal IVDs in compression and torsion following puncture with 30, 25 and 21 G needles. In compression, puncture injury reduced elastic stiffness by 20% for all needle sizes, but differences between control and punctured discs did not remain after compressive overload. In contrast, torsional parameters associated with anular fiber tension were affected proportionally with needle size. We conclude that IVD injuries that penetrate through the thickness of the annulus affect IVD biomechanics through different mechanisms for compression and torsion. Anular injuries affect torsional properties in a manner directly related to the amount of fiber disruption and compressive properties in a manner that affects pressurization.     

Long‐Term Pathology of Ovine Lumbar Spine Degeneration Following Injury Via Percutaneous Minimally Invasive Partial Nucleotomy

The focus of this work is to assess the long‐term progression of degeneration in the ovine lumbar spine following a minimally invasive model injury comparable to the damage of an intervertebral disc (IVD) herniation. A partial nucleotomy was performed on 18 sheep via the percutaneous dorsolateral approach. The animals were culled at 6 and 12 months to evaluate the damaged and neighboring functional spine units (FSUs) for degenerative characteristics via μ‐CT and histology. Both quantitative μ‐CT and histology investigations demonstrated statistically significant differences between the native and damaged FSUs investigated. Qualitative analysis of μ‐CT revealed numerous pathological markers consistent with intervertebral disc degeneration (IDD), with differences in frequency and severity between the native and damaged FSUs. The annulus fibrosus reforms a pressure seal within 6 weeks, but the extent of the trauma is significant enough to initiate IVD degeneration, which is already clearly visible at 6 months and especially so 12 months post‐op. IDD pathology consistent with signs of a herniation was seen in both the 6‐ and 12‐month groups. This technique provides a useful model injury for the preclinical evaluation of IDD in large animal models, especially in regards to simulating disc herniation as well as for testing the efficacy of associated therapies in the future. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2376–2388, 2019     

Intradiscal injection of simvastatin results in radiologic,histologic, and genetic evidence of disc regeneration in a rat model of degenerative disc disease

Background contextA large percentage of back pain can be attributed to degeneration of the intervertebral disc (IVD). Bone morphogenetic protein 2 (BMP-2) is known to play an important role in chondrogenesis of the IVD. Simvastatin is known to upregulate expression of BMP-2. Thus, we hypothesized that intradiscal injection of simvastatin in a rat model of degenerative disc disease (DDD) would result in retardation of DDD.PurposeThe purpose of the present study was to develop a novel conservative treatment for DDD and related discogenic back pain.Study design/settingThe setting of this study is the laboratory investigation.MethodsDisc injury was induced in 272 rats via 21-ga needle puncture. After 6 weeks, injured discs were treated with simvastatin in a saline or hydrogel carrier. Rats were sacrificed at predetermined time points. Outcome measures assessed were radiologic, histologic, and genetic. Radiologically, the magnetic resonance imaging (MRI) index (number of pixels multiplied by the corresponding image densities) was determined. Histologically, disc spaces were read by three blinded scorers using a previously described histologic grading scale. Genetically, nuclei pulposi were harvested, and polymerase chain reaction was run to determine relative levels of aggrecan, collagen type II, and BMP-2 gene expression.ResultsRadiologically, discs treated with 5 mg/mL of simvastatin in hydrogel or saline demonstrated MRI indices that were normal through 8 weeks after treatment, although this was more sustained when delivered in hydrogel. Histologically, discs treated with 5 mg/mL of simvastatin in hydrogel demonstrated improved grades compared with discs treated at higher doses. Genetically, discs treated with 5 mg/mL of simvastatin in hydrogel demonstrated higher gene expression of aggrecan and collagen type II than control.ConclusionsDegenerate discs treated with 5 mg/mL of simvastatin in a hydrogel carrier demonstrated radiographic and histologic features resembling normal noninjured IVDs. In addition, the gene expression of aggrecan and collagen type II (important constituents of the IVD extracellular matrix) was upregulated in treated discs. Injection of simvastatin into degenerate IVDs may result in retardation of disc degeneration and represents a promising investigational therapy for conservative treatment of DDD.     

Use of Allogeneic Hypoxic Mesenchymal Stem Cells For Treating Disc Degeneration in Rabbits

Intervertebral discs (IVDs) are important biomechanical components of the spine. Once degenerated, mesenchymal stem cell (MSC)‐based therapies may aid in the repair of these discs. Although hypoxic preconditioning enhances the chondrogenic potential of MSCs, it is unknown whether bone marrow MSCs expanded under hypoxic conditions (1% O₂, here referred to as hypoxic MSCs) are better than bone marrow MSCs expanded under normoxic conditions (air, here referred to as normoxic MSCs) with regards to disc regeneration capacity. The purpose of this study was to compare the therapeutic effects of hypoxic and normoxic MSCs in a rabbit needle puncture degenerated disc model after intra‐disc injection. Six weeks after needle puncture, MSCs were injected into the IVD. A vehicle‐treated group and an un‐punctured sham‐control group were included as controls. The tissues were analyzed by histological and immunohistochemical methods 6 and 12 weeks post‐injection. At 6 and 12 weeks, less disc space narrowing was evident in the hypoxic MSC‐treated group compared to the normoxic MSC‐treated group. Significantly better histological scores were observed in the hypoxic MSC group. Discs treated with hypoxic MSCs also demonstrated significantly better extracellular matrix deposition in type II and XI collagen. Increased CD105 and BMP‐7 expression were also observed upon injection of hypoxic MSCs. In conclusion, hypoxic MSC injection was more effective than normoxic MSC injection for reducing IVD degeneration progression in vivo. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1440–1450, 2019.     

细胞因子IL-1a、IL -6、TNF-a、MMP3与颈椎间盘退变机制的相关性研究

[目的]探讨细胞因子在颈椎间盘退变机制中的作用及其与神经功能的相关性.[方法]实验组椎间盘组织取自46例颈椎病患者,根据术前颈椎MRI及术中椎间盘突出情况分为两组:退变组(24例)和突出组(22例).对照组椎间盘组织取自15例无颈椎病病史的颈椎外伤患者.根据颈椎病患者术前JOA评分分为三组:轻度组(17例),中度组(15例)和重度组(14例).采用酶联免疫吸附法(ELISA法)分别检测不同退变程度颈椎间盘中IL-1a、IL -6、TNF -a和MMP3的表达水平.[结果]对照组、退变组和突出组三组之间比较,IL -1a、IL -6、TNF-a和MMP3的表达有统计学意义(P＜0.05),其表达水平与颈椎间盘退变呈正相关趋势;轻度组、中度组和重度组三组之间比较,MMP3、TNF -a的表达有统计学意义(P＜0.05),其表达水平与JOA评分呈负相关趋势.[结论]IL -1、IL -6、TNF -a和MMP3与颈椎间盘退变密切相关,其表达水平与椎间盘退变呈正相关趋势;TNF -a与神经功能有关,可能在神经损伤中起主导作用;MMP3与椎间盘突出有关,对TNF -a的神经功能损伤可能起促进作用.     

Needle puncture injury causes acute and long‐term mechanical deficiency in a mouse model of intervertebral disc degeneration

Low back pain is a significant socioeconomic burden and intervertebral disc degeneration has been implicated as a cause. A reliable animal model of disc degeneration is necessary to evaluate therapeutics, and functional metrics are essential to quantify their benefit. To this end, needle puncture injuries were created in the caudal intervertebral discs of mice to induce disc degeneration. Compression, torsion, and creep mechanics were assessed both immediately and after eight weeks to distinguish between the effects of injury and the subsequent reparative or degenerative response. Two needle sizes (29 and 26 gauge) were used to determine injury size‐dependence. Compressive stiffness (62%), torsional stiffness (60%), and early damping stiffness (84%) decreased immediately after injury with the large needle (26G). These mechanical properties did not change over time despite structural and compositional changes. At 8 weeks following large needle injury, disc height decreased (37%), nucleus pulposus (NP) glycosaminoglycan content decreased (41%), and NP collagen content increased (45%). The small needle size had no significant effect on mechanics and did not initiate degenerative changes in structure and composition. Thus, the injection of therapeutics into the NP with a minimal needle size may limit damage due to the needle insertion. These findings, along with the wide commercial availability of mouse‐specific biological probes, indicate that the mouse caudal disc model can be a powerful tool for investigating disc degeneration and therapy. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1276–1282, 2013     

NF-κB inhibitor,NEMO-binding domain peptide attenuates intervertebral disc degeneration

BACKGROUND CONTEXTNonphysiological mechanical loading and inflammation are both critically involved in intervertebral disc (IVD) degeneration, which is characterized by an increase in cytokines and matrix metalloproteases (MMPs) in the nucleus pulposus (NP). This process is known to be mediated by the NF-κB pathway.CLINICAL SIGNIFICANCECurrent clinical treatments for IVD degeneration focus on the alleviation of symptoms rather than targeting the underlying mechanism. Injection of an NF-κB inhibitor may attenuate the progression of IVD degeneration.PURPOSETo investigate the ability of the NF-κB inhibitor, NEMO binding domain peptide (NBD), to alter IVD degeneration processes by reducing IL-1β- and mechanically-induced cytokine and MMP levels in human nucleus pulposus cells in vitro, and by attenuating IVD degeneration in an in vivo rat model for disc degeneration.STUDY DESIGNExperimental in vitro and animal model.PATIENT SAMPLEDiscarded specimens of lumbar disc from 21 patients, and 12 Sprague Dawley rats.OUTCOME MEASURESGene and protein expression, cell viability, µMRI and histology.METHODSIL-1β-prestimulated human nucleus pulposus cells embedded into fibrin constructs were loaded in the Flexcell FX-5000 compression system at 5 kPa and 1 Hz for 48 hours in the presence and absence of NBD. Unloaded hNPC/fibrin constructs served as controls. Cell viability in loaded and unloaded constructs was quantified, and gene and protein expression levels determined. For in vivo testing, a rat needle disc puncture model was employed. Experimental groups included injured discs with and without NBD injection and uninjured controls. Levels of disc degeneration were determined via µMRI, qPCR and histology. Funding sources include $48,874 NASS Young Investigator Research Grant and $119,174 NIH 5K01AR071512-02. There were no applicable financial relationships or conflicts of interest.RESULTSMechanical compression of hNPC/fibrin constructs resulted in upregulation of MMP-3 and IL-8. Supplementation of media with 10 μM NBD during loading increased cell viability, and decreased MMP-3 gene and protein levels. IVD injury in rat resulted in an increase in MMP-3, IL-1β and IL-6 gene expression. Injections of 250 µg of NBD during disc injury resulted in decreased IL-6 gene expression. µMRI analysis demonstrated a reduction of disc hydration in response to disc needle injury, which was attenuated in NBD-treated IVDs. Histological evaluation showed NP and AF lesion in injured discs, which was attenuated by NBD injection.CONCLUSIONSThe results of this study show NBD peptide's capacity to reduce IL-1β- and loading-induced MMP-3 levels in hNPC/fibrin constructs while increasing the cells’ viability, and to attenuate IVD degeneration in rat, involving downregulation of IL-6. Therefore, NBD may be a potential therapeutic agent to treat IVD degeneration.     

The effect of needle size inducing degeneration in the rat caudal disc: evaluation using radiograph,magnetic resonance imaging,histology, and immunohistochemistry

Background context

The rat caudal disc has been increasingly used in studying of disc degeneration because of its simplicity, low cost, and efficiency. However, the reproducibility and standardization are essential to facilitate the investigations of biologic therapeutics at different stages of degeneration.

Purpose

To identify the effect of different needle gauges to the degenerative response in rat caudal discs and to examine its pathogenesis by looking at the cellular and matrix changes.

Study design

In vivo study of injury-induced rat caudal disc degeneration using needle puncture.

Patient sample

Thirty-six Lewis rats aged 12–14 weeks.

Outcome measures

The induced degenerative discs were analyzed by plain radiograph, magnetic resonance imaging (MRI) and histological examination. Proteoglycan content was assessed by alcian blue stain. Immunohistochemistry using aggrecan, collagen II, and Sox-9 was also evaluated to investigate cell differentiation and matrix changes.

Methods

All rats were divided into three groups according to different needle gauges (18G, 20G, and 22G). Caudal discs were punctured percutaneously under image guidance. Radiographs and MRI were obtained at 2 weeks interval until 8 weeks. At each time point, three rats from each group were sacrificed for histological analysis and immunohistochemistry.

Results

Larger needle gauges, especially 18G, produced more deterioration of the disc when compared with smaller sizes, particularly with time. Significant differences were identified in almost all parameters compared between 18G and 22G at the 8-week time point. For the effect of time in the same needle size, the differences occurred between 2- or 4-week and 8-week time point in the 18G and 20G groups. The proteoglycan and aggrecan stain gradually decreased over time. Chondrogenic differentiation was identified within the degenerative disc by detecting Sox-9 positive cells and collagen II accumulation increased as degeneration progressed.

Conclusions

The puncture-induced degenerative changes in rat caudal discs can imitate the human degenerative cascade as observed in plain radiograph, MRI, histology, and immunohistochemistry. We suggest that needle size affects the occurrence of progression of degeneration; thus, the large needle size was required to accelerate the deterioration. The size of needle and time point after injury should be considered when investigating the effect of therapeutic materials to retard degeneration or regenerate the intervertebral disc.     

Lactate down‐regulates matrix systhesis and promotes apoptosis and autophagy in rat nucleus pulposus cells

The intervertebral disk (IVD) is avascular and anaerobic glycolysis has been recognized as the main source of energy. Due to anaerobic glycolysis, there are high levels of lactate production in disk. Previous study shows lactate concentration is elevated in the degenerated IVD. However, it is not clear how lactate causes degeneration of disks. In this study, we found that 2 mM lactate promote proliferation of NP cells, while 6 mM lactate slightly inhibit their proliferation. By detection under transmission electron microscopy, and western bolt for autophagy related protein beclin‐1, LC3 and p62, we demonstrated that 6 mM lactate leads to autophagy induction of NP cells. TUNEL results showed that the apoptosis incidence was increased. High lactate concentration induced the degradation in protein expression and mRNA level of GAG content, type II collagen and slight increase of type I collagen. Based on these observations, we conclude that high lactate concentration is a pathogenic factor for IVD degeneration, and lactate metabolism may be a new therapeutic target for IVD degeneration. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:253–261, 2014.     

Long-term histological analysis of innervation and macrophage infiltration in a mouse model of intervertebral disc injury–induced low back pain

Low back pain (LBP) is a leading cause of global disability. Multiple anatomical, cellular, and molecular factors are implicated in LBP, including the degeneration of lumbar intervertebral discs (IVDs). We previously described a mouse model that displays behavioral symptoms of chronic LBP. Here, we investigated the development of pathological innervation and macrophage infiltration into injured IVDs following a puncture injury in mice over 12 months. 2-month old CD1 female mice underwent a single puncture of the ventral L4/5 IVD using a 30G needle, and were sacrificed 4 days and 0.5-, 3-, 6- and 12-months post-injury. Severity of disc degeneration was assessed using colorimetric staining. IVD innervation was measured by PGP9.5-immunoreactivity (-ir) and calcitonin gene-related peptide-ir (CGRP-ir). Macrophage accumulation into IVDs was detected by F4/80-ir. Mechanical IVD injury resulted in severe degeneration and increased PGP9.5-ir nerve fiber density starting at 4 days that persisted for up to 12 months and dorsal herniations began to occur at 3 months. CGRP-ir was also upregulated in injured IVDs, with the largest increase at 12 months after injury. Infiltration of F4/80-ir macrophages was observed in injured IVDs by day 4 both dorsally and ventrally, with the latter diminishing in the later stage. Persistent LBP is a complex disease with multiple underlying pathologies. By highlighting pathological changes in IVD innervation and inflammation, our study suggests that strategies targeting these mechanisms might be useful therapeutically.     

Nucleus pulposus cells expressing hBMP7 can prevent the degeneration of allogenic IVD in a canine transplantation model

We have previously explored the possibilities of allogenic intervertebral disc (IVD) curing disc degeneration disease in clinical practice. The results showed that the motion and stability of the spinal unit was preserved after transplantation of allogenic IVD in human beings at 5‐year follow‐up. However, mild degeneration was observed in the allogenic transplanted IVD cases. In this study, we construct the biological tissue engineering IVD by injecting the nucleus pulposus cells (NPCs) expressing human bone morphogenetic protein 7 (hBMP7) into cryopreserved IVD, and transplant the biological tissue engineering IVD into a beagle dog to investigate whether NPCs expressing hBMP7 could prevent the degeneration of the transplanted allogenic IVDs. At 24 weeks after transplantation, MRI scan showed that IVD allografts injected NPCs expressing hBMP7 have a slighter signs of degeneration than IVD allografts with NPCs or without NPCs. The range of motion of left–right rotation in the group without NPCs was bigger than that of two cells injection group. PKH‐26‐labeled cells were identified at IVD allograft. The study demonstrated that NPCs expressing hBMP7 could survive at least 24 weeks and prevent the degeneration of the transplanted IVD. This solution might have a potential role in preventing the IVD allograft degeneration in long time follow‐up. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1366–1373, 2013     

A comparison of intravenous and intradiscal delivery of multipotential stem cells on the healing of injured intervertebral disk

A major hurdle of cellular therapy for biological treatment of intervertebral disk (IVD) degeneration is the delivery method where current delivery methods are limited to intradiscal injection which can potentially cause further degeneration. Recent studies indicated that multipotential stem cells (MPSCs) from human umbilical cord blood home to injured sites and induce local therapeutic changes, thereby potentially addressing the drawbacks of direct delivery. We tested the effects of these cells on injured IVD using a mouse model of puncture‐induced degeneration via two delivery methods. Caudal IVD underwent needle puncture, and MPSCs were injected indirectly (intravenously), or directly (intradiscally) into the nucleus pulposus. IVD were harvested for histological, gene and protein analysis after 14 weeks. Our finding showed limited homing ability of the MPSCs. However, regardless of delivery method, no engraftment or expansion of MPSCs was observed at the injured site. Contrasting to direct injection, intravenous injection neither improved the degeneration status, nor preserve disk height, however, both delivery methods increased glycosaminoglycan (GAG) protein and Acan gene expression relative to controls, suggesting possible paracrine effects. Identifying the mechanisms by which MPSCs act on endogenous IVD cells would provide insights into the potential of these cells to treat IVD injuries and degeneration. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:819–825, 2014.     

Influence of age‐related degeneration on regenerative potential of human nucleus pulposus cells

Nucleus pulposus (NP) cells, sourced from herniation surgeries, may be used as a cell‐based therapy for intervertebral disc (IVD) degeneration. But, both the regenerative potential of these degenerative adult NP cells and how to stimulate optimum matrix synthesis is not yet clear. The purpose of the current study was to understand the different phenotypic behaviors between degenerative adult NP cells and normal adolescent NP cells. Degenerative adult NP cells produced a significantly higher amount of proteoglycans and collagens than adolescent cells. Insulin‐like growth factor‐1 was the only anabolic cytokine with increased endogenous expression in degenerative adult NP cells. TGF‐β1 treatment of degenerative NP cells promoted matrix synthesis but stimulated too much type I collagen and suppressed type II collagen and aggrecan. Adult degenerative NP cells possess upregulated regenerative potential, but stimulation in addition to TGF‐β1 is needed to enhance matrix productivity and optimize the collagen expression profile. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:379–383, 2010     

应用微创技术建立恒河猴腰椎间盘早期退变模型

目的 应用CT定位,经皮穿刺纤维环诱导恒河猴腰椎间盘退变,建立灵长类动物腰椎间盘早期退变模型.方法 恒河猴13只,随机分为三组:(1)造模组:在CT定位下,用20G穿刺针从左侧后方入路经皮穿刺L1,2:(n=12),L2,3、L3,4、L4,5、L5,6(n=13)椎间盘的纤维环全层至椎间盘髓核正中,共64个椎间盘.(2)穿刺对照组:15G穿刺针穿刺1只猴的L1,2椎间盘.(3)正常对照组:L6,7,L7-S1,共26个椎间盘.造模前及造模后4、8、12周对各组椎间盘行MRI检查,并行HE、Masson、番红O、免疫组织化学染色组织学观察.结果 (1)MRI:20G穿刺针穿刺的造模组椎间盘造模前及造模后4、8、12周,椎间盘信号强度按Pfirmann分级均为Ⅰ级.15G穿刺针穿刺椎间盘4周时信号降低(Pfirrmann Ⅲ级),8周时为黑色椎间盘(Pfirmann Ⅳ级).正常对照组椎间盘为Pfirmann Ⅰ级.(2)组织学:造模组椎间盘造模后4周未见改变,8周时HE染色示髓核内细胞数减少,12周时较为明显.Masson染色4周未见改变,8周时各层纤维间出现裂隙,12周时裂隙增宽.番红O染色见8、12周髓核内蛋白聚糖进行性减少.免疫组织化学结果显示4周和8周时同正常椎间盘比较差异无统计学意义(P＞0.05),12周时,Ⅱ型胶原合成减少(P＜0.05).15G穿刺对照组在8周时HE染色见髓核内细胞减少明显,Masson染色见纤维环各层间裂隙明显,呈波浪状.番红O染色示髓核内蛋白聚糖数量明显减少.免疫组织化学染色示Ⅱ型胶原合成减少.正常对照组在各时间点未见到形态学改变.结论 20G穿刺针可以诱发椎间盘缓慢进展的轻度退变.MRI平均信号强度观察椎间盘轻度退变时,不是敏感的指标,需要依靠组织学证实.     

Organ culture stability of the intervertebral disc: Rat versus rabbit

There is a need to develop mechanically active culture systems to better understand the role of mechanical stresses in intervertebral disc (IVD) degeneration. Motion segment cultures that preserve the native IVD structure and adjacent vertebral bodies are preferred as model systems, but rapid ex vivo tissue degeneration limits their usefulness. The stability of rat and rabbit IVDs is of particular interest, as their small size makes them otherwise suitable for motion segment culture. The goal of this study was to determine if there are substantial differences in the susceptibility of rat and rabbit IVDs to culture‐induced degeneration. Lumbar IVD motion segments were harvested from young adult male Sprague–Dawley rats and New Zealand White rabbits and cultured under standard conditions for 14 days. Biochemical assays and safranin‐O histology showed that while glycosaminoglycan (GAG) loss was minimal in rabbit IVDs, it was progressive and severe in rat IVDs. In the rat IVD, GAG loss was concomitant with the loss of notochordal cells and the migration of endplate (EP) cells into the nucleus pulposus (NP). None of these changes were evident in the rabbit IVDs. Compared to rabbit IVDs, rat IVDs also showed increased matrix metalloproteinase‐3 (MMP‐3) and sharply decreased collagen type I and II collagen expression. Together these data indicated that the rabbit IVD was dramatically more stable than the rat IVD, which showed culture‐related degenerative changes. Based on these findings we conclude that the rabbit motion segments are a superior model for mechanobiologic studies. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 838–846, 2013     

The Effects of Platelet‐Rich Plasma on Halting the Progression in Porcine Intervertebral Disc Degeneration

Disc degeneration and the subsequent herniation and/or rupture of the intervertebral disc (IVD) are due to a failure of the extracellular matrix of the annulus to contain the contents of the nucleus. This results from inadequate maintenance of the matrix components as well as the proteolytic activity of matrix metalloproteinases (MMPs) that degrade matrix molecules. Arresting progression of disc degeneration in the annulus holds greater clinical potential at this point than prevention of its onset in the nucleus. Therefore, in this study, we have therapeutic aims that would decrease levels of the cytokines and growth factors that indirectly lead to disc degeneration via stimulating MMP and increase levels of several beneficial growth factors, such as transforming growth factor‐β, with the addition of platelet‐rich plasma (PRP) that would stimulate cell growth and matrix synthesis. For this study, we attempted to address these imbalances of metabolism by using tumor necrosis factor‐α treated annulus fibrosus cells isolated from porcine IVD tissue and incubating the cells in a growth factor rich environment with PRP. These results indicate that the PRP in vitro increased the production of the major matrix components (type II collagen and aggrecan) and decreased the inhibitory collagenase MMP‐1. This application will address a therapeutic approach for intervening early in the degenerative process.     

Interleukin‐6 and interleukin‐6 receptor expression,localization, and involvement in pain‐sensing neuron activation in a mouse intervertebral disc injury model

The pathological mechanism of intractable low back pain is unclear. However, intervertebral disc (IVD) degeneration is a primary cause of low back pain, and pain‐related mediators, such as interleukin‐6 (IL‐6), have been correlated with discogenic pain. The objective of this study is to elucidate the mechanism of local IL‐6 and IL‐6 receptor (IL‐6R) expression after IVD injury as well as determine the involvement of IL‐6/IL‐6 signaling in discogenic pain. To do this, quantitative and immunohistological analyses in a mouse model of IVD injury were performed. Firstly, we measured the local expression levels of IL‐6 and IL‐6R in IVDs by enzyme‐linked immunosorbent assay (ELISA). Secondly, we immunohistochemically confirmed their localization in injured IVDs. Lastly, we evaluated the effects of intradiscal injection of an IL‐6 inhibitor by evaluating pain‐related protein, calcitonin gene‐related peptide (CGRP), expression in dorsal root ganglia (DRG) neurons that innervate IVDs. Injured IVDs showed increased production of IL‐6 and IL‐6R. IL‐6 and IL‐6R expression in the injured IVD were predominantly localized in the annulus fibrosus and endplate, and intradiscal injection of the IL‐6 inhibitor suppressed CGRP expression in the DRG neurons. These results show that IL‐6 and IL‐6R expression levels are responsive to IVD injury and that inhibition of IL‐6/IL‐6R signaling may be a promising analgesic treatment for degenerative disc diseases. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1508–1514, 2015.     

Apoptosis of human intervertebral discs after trauma compares to degenerated discs involving both receptor‐mediated and mitochondrial‐dependent pathways

Post‐traumatic disc degeneration with consecutive loss of reduction and kyphosis remains a debatable issue within both the operative and nonoperative treatment regimen of thoracolumbar spine fractures. Intervertebral disc (IVD) cell apoptosis has been suggested to play a vital role in promoting the degeneration process. To evaluate and compare apoptosis‐regulating signaling mechanisms, IVDs were obtained from patients with thoracolumbar spine fractures (n = 21), patients suffering from symptomatic IVD degeneration (n = 6), and from patients undergoing surgical resection of a primary vertebral tumor (n = 3 used as control samples). All tissues were prospectively analyzed in regards to caspase‐3/7, ‐8, and ‐9 activity, apoptosis‐receptor expression levels, and gene expression of the mitochondria‐bound apoptosis‐regulating proteins Bax and Bcl‐2. Morphologic changes characteristic for apoptotic cell death were confirmed by H&E staining. Statistical significance was designated at p < 0.05 using the Student's t‐test. Both traumatic and degenerative IVD demonstrated a significant increase of caspase‐3/7 activity with evident apoptosis. Although caspase‐3/7 activation was significantly greater in degenerated discs, both showed equally significant activation of the initiator caspases 8 and 9. Traumatic IVD alone demonstrated a significant increase of the Fas receptor (FasR), whereas the TNF receptor I (TNFR I) was equally up‐regulated in both morbid IVD groups. Only traumatic IVD showed distinct changes in up‐regulated TNF expression, in addition to significantly down‐regulated antiapoptotic Bcl‐2 protein. Our results suggest that post‐traumatic disc changes may be promoted and amplified by both the intrinsic mitochondria‐mediated and extrinsic receptor‐mediated apoptosis signaling pathways, which could be, in part, one possible explanation for developing subsequent disc degeneration. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:999–1006, 2008