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.     

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.     

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.     

The effects of needle puncture injury on microscale shear strain in the intervertebral disc annulus fibrosus

Background contextNeedle puncture of the intervertebral disc (IVD) is required for delivery of therapeutic agents to the nucleus pulposus and for some diagnostic procedures. Needle puncture has also been implicated as an initiator of disc degeneration. It is hypothesized that needle puncture may initiate IVD degeneration by altering microscale mechanical behavior in the annulus fibrosus (AF).PurposeQuantify the changes in AF microscale strain behavior resulting from puncture with a hypodermic needle.Study designCadaveric IVD tissue explant study.MethodsAnnulus fibrosus explants from bovine caudal IVDs that had been punctured radially with hypodermic needles were loaded in dynamic sinusoidal shear while being imaged with a confocal microscope. Digital image analysis was used to quantify local tissue strain and damage propagation with repeated shearing.ResultsNeedle puncture changed the distribution of microscale shear strains in the AF under load from homogenous (equal to far field) to a distinct pattern of high (4× far field) and low (0.25× far field) strain areas. Repeated loading did not cause further growth of the disruption beyond the second cycle.ConclusionsNeedle puncture results in a drastic alteration of microscale strain behavior in the AF under load. This alteration may directly initiate disc degeneration by being detrimental to tissue-cell mechanotransduction.     

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     

Therapeutic effects of adenovirus-mediated growth and differentiation factor-5 in a mice disc degeneration model induced by annulus needle puncture

Background contextThe therapeutic strategies that have thus far been used for the treatment of intervertebral disc degeneration (IDD) have focused on relieving the symptoms, although reversal of the degeneration remains an important challenge for the effective treatment of IDD. Growth and differentiation factor-5 (GDF5), of which deficiency leads to early disc degeneration changes, has the potential to increase proliferation of disc cells and expression of extracellular matrix proteins.PurposeThe purpose of the study was to develop a lumbar disc degeneration model in mice and determine the effect of adenoviral GDF5 gene therapy.Study designThe study design was to compare the degeneration changes of discs punctured by different-size needles to develop a mice lumbar disc degeneration model and to evaluate the effects of in vivo gene therapy for the mice disc degeneration model by an adenoviral vector carrying GDF5 gene.MethodsA lumbar disc degeneration model was developed by needle punctures to the discs in Balb/c mice. Afterward, a gene therapy treatment to disc degeneration was evaluated. Two of the mice lumbar discs were randomly chosen to be punctured by a 30-gauge needle and then injected with adenovirus that had been engineered to express either the luciferase gene (Ad-Luc) or the GDF5 gene (Ad-GDF5). Animals were analyzed by bioluminescent imaging, radiographic, and magnetic resonance imaging (MRI) scanning, then sacrificed at 1, 2, 4, or 8 weeks after operation, and subjected to histological and biochemical assays.ResultsBy the detection of T2-weighted MRI scanning and histological study, the degeneration was found in all of the discs punctured by different-size needles. But the development of the degeneration in the discs injured by the 30-gauge needle was more reliable and moderate compared with that in other groups. The detection of luciferase activity by bioluminescent imaging revealed that adenovirus survived and the introduced genes were expressed over 6 weeks after injection. There were no T2-weighted MRI signals in the mice injected with either Ad-Luc or Ad-GDF5 up to 4 weeks after operation. At 6 and 8 weeks, T2-weighted signals were detected in the Ad-GDF5 group but none in the Ad-Luc control group. The percent disc height index (%DHI) was significantly decreased (～20%) by 1 week after injury in both groups, indicating the development of disc degeneration. At 2 weeks, the %DHI in the mice injected with Ad-GDF5 increased significantly compared with that of the mice injected with Ad-Luc; the increase was sustained for the rest of the experiment period. The disc histology treated with Ad-GDF5 was improved compared with that in the control group. Glycosaminoglycan (GAG) levels were significantly decreased in the Ad-Luc injection group since 2 weeks after injury, and the DNA content had diminished by 4 weeks after the operation. In contrast, in the discs injected with Ad-GDF5, there was no decrease in the GAG and DNA levels after injury throughout the 8-week treatment period.ConclusionsDisc degeneration animal model can be developed by using needle puncture to the discs in mice. The adenovirus is an effective vehicle for gene delivery with rapid and prolonged expression of target protein and resulting improvement in markers of disc degeneration. Ad-GDF5 gene therapy could restore the functions of injured discs and has the potential to be an effective treatment.     

Joint analysis of IVD herniation and degeneration by rat caudal needle puncture model

Intervertebral disc (IVD) degeneration is responsible for various spine pathologies and present clinical treatments are insufficient. Concurrently, the mechanisms behind IVD degeneration are still not completely understood, so as to allow development of efficient tissue engineering approaches. A model of rat IVD degeneration directly coupled to herniation is here proposed in a pilot study. Disc injury is induced by needle puncture, using two different needles gauges: a low caliber 25‐G needle and a high caliber 21‐G needle. Histological, biochemical, and radiographic degeneration was evaluated at 2 and 6 weeks post‐injury. We show that the larger caliber needle results in a more extended histological and radiographic degeneration within the IVD, compared to the smaller one. TUNEL quantification indicates also increased cell death in the 21‐G group. Analyses of collagen type I (Picrosirius red staining), collagen type II (immunofluorescence), and GAG content (Blyscan assay) indicate that degeneration features spontaneously recover from 2 to 6 weeks, for both needle types. Moreover, we show the occurrence of hernia proportional to the needle gauge. The number of CD68+ macrophages present, as well as cell apoptosis within the herniated tissue are both proportional to hernia volume. Moreover, hernias formed after lesion tend to spontaneously diminish in volume after 6 weeks. Finally, MMP3 is increased in the hernia in the 21‐G group at 2 weeks. This model, by uniquely combining IVD degeneration and IVD herniation in the same animal, may help to understand mechanisms behind IVD pathophysiology, such as hernia formation and spontaneous regression. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:258–268, 2017.

     

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.     

经皮穿刺纤维环建立兔腰椎间盘退变模型

目的探讨使用自制穿刺针经皮穿刺纤维环制备兔腰椎间盘退变模型的可行性。方法将18只新西兰大白兔分为实验组、假手术组及空白对照组。实验组及假手术组使用自制穿刺针穿刺L_(3~4)、L_(4~5)和L_(5~6)椎间盘位置,实验组穿刺椎间盘深度为5 mm,假手术组钝性穿刺但不损伤椎间盘,空白对照组不作处理。术后3、6、9周每组取2只兔麻醉后行腰椎MRI检查,处死行大体观察并取椎间盘行HE染色及髓核蛋白多糖含量测定。结果术后3周开始实验组MRI信号强度、髓核蛋白多糖含量与其他两组相比明显下降(P0.05),其后呈逐渐下降趋势,大体观察及HE染色显示实验组髓核及纤维环呈逐渐退变趋势。结论自制穿刺针经皮穿刺纤维环法能成功建立兔腰椎间盘退变模型,具有操作简单、损伤小、动物存活率高等优点。     

Heme oxygenase-1 modulates degeneration of the intervertebral disc after puncture in Bach 1 deficient mice

Purpose

Intervertebral disc degeneration is considered to be a major feature of low back pain. Furthermore, oxidative stress has been shown to be an important factor in degenerative diseases such as osteoarthritis and is considered a cause of intervertebral disc degeneration. The purpose of this study was to clarify the correlation between oxidative stress and intervertebral disc degeneration using Broad complex-Tramtrack-Bric-a-brac and cap‘n’collar homology 1 deficient (Bach 1?/?) mice which highly express heme oxygenase-1 (HO-1). HO-1 protects cells from oxidative stress.

Methods

Caudal discs of 12-week-old and 1-year-old mice were evaluated as age-related models. Each group and period, 5 mice (a total of 20 mice, a total of 20 discs) were evaluated as age-related model. C9–C10 caudal discs in 12-week-old Bach 1?/? and wild-type mice were punctured using a 29-gauge needle as annulus puncture model. Each group and period, 5 mice (a total of 60 mice, a total of 60 discs) were evaluated. The progress of disc degeneration was evaluated at pre-puncture, 1, 2, 4, 8 and 12?weeks post-puncture. Radiographic, histologic and immunohistologic analysis were performed to compare between Bach 1?/? and wild-type mice.

Results

In the age-related model, there were no significant differences between Bach 1?/? and wild-type mice radiologically and histologically. However, in the annulus puncture model, histological scoring revealed significant difference at 8 and 12?weeks post-puncture. The number of HO-1 positive cells was significantly greater in Bach 1?/? mice at every period. The apoptosis rate was significantly lower at 1 and 2?weeks post-puncture in Bach 1?/? mice.

Conclusions

Oxidative stress prevention may avoid the degenerative process of the intervertebral disc after puncture, reducing the number of apoptosis cells. High HO-1 expression may also inhibit oxidative stress and delay the process of intervertebral disc degeneration.     

大鼠腰椎间盘针刺退变模型的建立

[目的]建立大鼠腰椎间盘针刺退变模型并进行MRI和组织学观察。[方法]Sprague-Dawley大鼠32只,20只进行腰椎间盘纤维环厚度测量,确定针刺深度;12只经腹膜手术,L3、4、L4、5、L5、6椎间盘使用21G针分别行纤维环部分针刺或全层针刺,L6S1椎间盘作为对照,术前及术后4、8周行MRI检查,然后处死,椎间盘行HE染色。[结果]测量大鼠腰椎间盘纤维环厚度后,确定适用于L3、4、L4、5、L5、6、L6S1椎间盘纤维环部分针刺深度为1.5mm,纤维环全层针刺深度为2.3mm。MRI检查在纤维环全层针刺4周,部分针刺8周后的椎间盘信号强度显著降低。组织学检查发现纤维环全层针刺和部分针刺椎间盘4周时都发生退变,而纤维环全层针刺退变较严重。[结果]纤维环部分针刺或全层针刺方法可以成功建立大鼠腰椎间盘退变模型,方法可靠、有效、重复性好。     

Disc degeneration reduces the delamination strength of the annulus fibrosus in the rabbit annular disc puncture model

Background contextDegenerative disc disease is a common pathologic disorder accompanied by both structural and biochemical changes. Changes in stress distribution across the disc can lead to annulus fibrosus (AF) damage that can affect the strength and integrity of the disc. Given that some present degeneration therapies incorporate biological regrowth of the nucleus pulposus (NP), it is crucial that the AF remains capable of containing this newly grown material.PurposeTo examine the resistance of AF to delamination using an adhesive peel test in experimentally degenerated rabbit discs.Study designExperimentally induced disc degeneration; excised AF tissue study.MethodsDisc degeneration was induced in eight New Zealand white rabbits by annular puncture; four additional rabbits served as controls. In experimental rabbits, an 18-gauge needle was inserted into the anterolateral AF region of levels L2–L3 and L4–L5, and disc height was monitored by X-ray. Animals were sacrificed at 4 and 12 weeks postsurgery and magnetic resonance images and X-rays were taken. Four discs were excised from the experimental animals; two punctured (L2–L3 and L4–L5) and two controls (L3–L4 and L6–L7). The same four discs were also excised from the age-matched control animals and served as nonpunctured control discs. To determine resistance to delamination, AF samples were dissected from each disc and subjected to a mechanical peel test at 0.5 mm/s.ResultsMagnetic resonance imaging and X-ray images confirmed dehydration of the NP and reduced disc height, similar to that found in clinical degeneration. Resistance to delamination was significantly lower in punctured/degenerated discs compared with both the nonpunctured discs from the same animal (27% lower) and the nonpunctured control discs (30% lower) (p=.024).ConclusionsThe findings of this study suggest that degeneration increases the potential for delamination between AF layers. Given this substantial change to the integrity of the AF after degeneration, clinical treatments should not only target rehydration or regrowth of the NP, but should also target repair and strengthening of the AF to confine the NP.     

Human bone morphogenetic protein 7 transfected nucleus pulposus cells delay the degeneration of intervertebral disc in dogs

The main reason for intervertebral disc (IVD) degeneration is the decrease in the quantity and activity of IVD cells with subsequent reduction of the extracellular matrix (ECM). In this study, we investigated a cell‐based repair strategy by injecting nucleus pulposus cells (NPCs) transduced with human bone morphogenetic protein (hBMP7) by adeno‐associated virus‐2 into the canine degenerative IVD to determine whether NPCs expressing hBMP7 could delay the degeneration of the IVD. Fourteen canines received annular punctures to induce disc degeneration. Eight weeks later, saline (group A), allogeneic NPCs (group B), or allogeneic NPCs transduced with hBMP7 (group C) were injected into the degenerative discs. Twelve weeks after the injection, MRI scan showed that the degeneration process of groups C was slower and less severe compared with that of groups B and C. The IVD stability in group C was superior to that in groups A and B in left‐right bending and rotation. HE, safranin‐O staining, and ELISA indicated that the degenerative degree of the IVD in group C was significantly milder than that in groups A and B. The study demonstrated that the implantation of NPCs‐hBMP7 could effectively maintained the structural integrity, ECM, and biomechanical properties of the canine degenerated discs. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1311–1322, 2017.

     

Lovastatin prevents discography-associated degeneration and maintains the functional morphology of intervertebral discs

Background context

Discography is an important diagnostic approach to identify the painful discs. However, the benefit of discography, a procedure involving needle puncture and injection of the diagnostic agent into the intervertebral disc, is controversial and has been reported to be associated with accelerated degeneration.

Purpose

To investigate the effect of lovastatin on the prevention of degeneration caused by a discography simulation procedure in rat caudal discs.

Study design

In vivo study using rat caudal discs.

Methods

A single flexible 27-gauge needle puncture into rat caudal discs was performed under fluoroscopic monitoring. Different concentrations (0.1, 1, 5, and 10 μM) of lovastatin were prepared and injected into randomly chosen caudal discs. RNA expression of selected genes, histologic, and immunohistochemical staining were performed to evaluate the phenotypic effects of lovastatin on rat caudal discs.

Results

Simulation of the discography procedure by puncturing the rat caudal discs with a 27-gauge needle and injection of saline solution induced degenerative changes in the nucleus pulposus with minimal damage to the annulus fibrosus. Aggrecan, Type II collagen, and SOX9 expressions were upregulated, whereas Type I collagen expression was significantly suppressed in discs treated with 5 and 10 μM lovastatin. Discs treated with 5 and 10 μM lovastatin were subjected to alcian blue staining and immunohistochemistry that revealed higher levels of glycosaminoglycans and an increase in the number of cells producing S-100 proteins, Type II collagen, and bone morphogenetic protein-2 (BMP-2), respectively. The most effective phenotypic repair was observed in discs treated with 10 μM lovastatin.

Conclusions

Intradiscal administration of lovastatin solution upregulated the expressions of BMP-2 and SOX9 and promoted chondrogenesis of rat caudal discs after needle puncture and substance injection. Therefore, we suggest that lovastatin promotes disc repair and can be used as a potential therapeutic agent for biological repair of disc degeneration after the diagnostic discography procedure.     

Injection of AAV2-BMP2 and AAV2-TIMP1 into the nucleus pulposus slows the course of intervertebral disc degeneration in an in vivo rabbit model

Background contextIntervertebral disc degeneration (IDD) is a common cause of back pain. Patients who fail conservative management may face the morbidity of surgery. Alternative treatment modalities could have a significant impact on disease progression and patients’ quality of life.PurposeTo determine if the injection of a virus vector carrying a therapeutic gene directly into the nucleus pulposus improves the course of IDD.Study designProspective randomized controlled animal study.MethodsThirty-four skeletally mature New Zealand white rabbits were used. In the treatment group, L2–L3, L3–L4, and L4–L5 discs were punctured in accordance with a previously validated rabbit annulotomy model for IDD and then subsequently treated with adeno-associated virus serotype 2 (AAV2) vector carrying genes for either bone morphogenetic protein 2 (BMP2) or tissue inhibitor of metalloproteinase 1 (TIMP1). A nonoperative control group, nonpunctured sham surgical group, and punctured control group were also evaluated. Serial magnetic resonance imaging (MRI) studies at 0, 6, and 12 weeks were obtained, and a validated MRI analysis program was used to quantify degeneration. The rabbits were sacrificed at 12 weeks, and L4–L5 discs were analyzed histologically. Viscoelastic properties of the L3–L4 discs were analyzed using uniaxial load-normalized displacement testing. Creep curves were mathematically modeled according to a previously validated two-phase exponential model. Serum samples obtained at 0, 6, and 12 weeks were assayed for biochemical evidence of degeneration.ResultsThe punctured group demonstrated MRI and histologic evidence of degeneration as expected. The treatment groups demonstrated less MRI and histologic evidence of degeneration than the punctured group. The serum biochemical marker C-telopeptide of collagen type II increased rapidly in the punctured group, but the treated groups returned to control values by 12 weeks. The treatment groups demonstrated several viscoelastic properties that were distinct from control and punctured values.ConclusionsTreatment of punctured rabbit intervertebral discs with AAV2-BMP2 or AAV2-TIMP1 helps delay degenerative changes, as seen on MRI, histologic sampling, serum biochemical analysis, and biomechanical testing. Although data from animal models should be extrapolated to the human condition with caution, this study supports the potential use of gene therapy for the treatment of IDD.     

The intervertebral disc from embryonic development to disc degeneration: insights into spatial cellular organization

Background contextLow back pain is commonly attributed to intervertebral disc (IVD) degeneration. IVD resembles articular cartilage in its biochemical and cellular composition in many ways. For articular cartilage, degeneration stage-specific characteristic spatial chondrocyte patterns have recently been described.PurposeThis study addresses how spatial chondrocyte organization in the IVD changes from early embryonic development to end stage degeneration.Study designEx vivo immunohistochemical analysis.MethodsWe immunohistochemically investigated bovine IVD-tissue (n=72) from early embryonic development to early disc degeneration and human adult IVD-tissue (n=25) operated for trauma or degeneration for cellular density and chondrocyte spatial organization. IVD samples were sectioned along the main collagen fiber orientation. Nuclei were stained with DAPI and their number and spatial patterns were analyzed in an area of 250,000 µm² for each tissue category.ResultsThe initially very high cellular density in the early embryonic bovine disc (11,431 cells/mm²) steadily decreases during gestation, growth and maturation to about 71 cell/mm² in the fully grown cattle. Interestingly, in human degenerative discs, a new increase in this figure could be noted (184 cells/mm). The IVD chondrocytes appear to be predominantly present as single cells. Especially in the time after birth, string-formations represent up to 32% of all cells in the anulus fibrosus, although single cells are the predominant spatial pattern (>50%) over the entire time. With increasing degeneration, the relative proportion of single cells in human IVDs continuously decreases (12%). At the same time, the share of cells organized in clusters increases (70%).ConclusionSimilar to articular cartilage, spatial chondrocyte organization appears to be a strong indicator for local tissue degeneration in the IVD.Clinical SignificanceIn the future these findings may be important for the detection and therapy of IVD degeneration in early stages.     

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.     

The molecular basis of intervertebral disc degeneration

BackgroundIntervertebral disc (IVD) degeneration remains a clinically important condition for which treatment is costly and relatively ineffective. The molecular basis of degenerative disc disease has been an intense focus of research recently, which has greatly increased our understanding of the biology underlying this process.PurposeTo review the current understanding of the molecular basis of disc degeneration.Study designReview article.MethodsA literature review was performed to identify recent investigations and current knowledge regarding the molecular basis of IVD degeneration.ResultsThe unique structural requirements and biochemical properties of the disc contribute to its propensity toward degeneration. Mounting evidence suggests that genetic factors account for up to 75% of individual susceptibility to IVD degeneration, far more than the environmental factors such as occupational exposure or smoking that were previously suspected to figure prominently in this process. Decreased extracellular matrix production, increased production of degradative enzymes, and increased expression of inflammatory cytokines contribute to the loss of structural integrity and accelerate IVD degeneration. Neurovascular ingrowth occurs, in part, because of the changing degenerative phenotype.ConclusionsA detailed understanding of the biology of IVD degeneration is essential to the design of therapeutic solutions to treat degenerative discs. Although significant advances have been made in explaining the biologic mediators of disc degeneration, the inhospitable biochemical environment of the IVD remains a challenging environment for biological therapies.     

纤维环穿刺诱导椎间盘退变动物模型的实验研究

目的：探讨纤维环穿刺诱导椎间盘退变建立动物模型的可行性。方法：新西兰大白兔24只，用持针器夹持18G皮肤穿刺针从左前外侧刺人L3/4、L4/5、L5/6椎间盘的纤维环，深度控制在5mm。术前及术后3、6、10周对造模后的椎间盘及对照的椎间盘（L2／3）行MRI检查，并行免疫组化及组织学观察。结果：术后第3周到第10周，造模后的椎间盘MRI T2WI信号呈现持续减弱趋势，免疫组化及组织学观察发现髓核细胞的数量及Ⅱ型胶原含量较对照间盘进行性减少（P〈0．01）。结论：纤维环穿刺法可以诱导兔椎间盘的缓慢退变，为研究椎间盘的退行性变提供有效的动物模型。     

Bone morphogenic protein-2 signaling in human disc degeneration and correlation to the Pfirrmann MRI grading system

BACKGROUND CONTEXTBack and neck pain secondary to disc degeneration is a major public health burden. There is a need for therapeutic treatments to restore intervertebral disc (IVD) composition and function.PURPOSETo quantify ALK3, BMP-2, pSMAD1/5/8 and MMP-13 expression in IVD specimens collected from patients undergoing surgery for disc degeneration, to correlate ALK3, BMP-2, pSMAD1/5/8 and MMP-13 expression in IVD specimens to the 5-level Pfirrmann MRI grading system, and to compare ALK3, BMP-2, pSMAD1/5/8 and MMP-13 expression between cervical and lumbar degenerative disc specimens.STUDY DESIGNAn immunohistochemical study assessing ALK3, BMP-2, pSMAD1/5/8, and MMP-13 expression levels in human control and degenerative IVD specimens.METHODSHuman IVD specimens were collected from surgical patients who underwent discectomy and interbody fusion at our institution between 1/2015 and 8/2017. Each patient underwent MRI prior to surgery. The degree of disc degeneration was measured according to the 5-level Pfirrmann MRI grading system. Patients were categorized into either the 1) control group (Pfirrmann grades I-II) or 2) degenerative group (Pfirrmann grades III-V). Histology slides of the collected IVD specimens were prepared and immunohistochemical staining was performed to assess ALK3, BMP-2, pSMAD1/5/8, and MMP-13 expression levels in the control and degenerative specimens. Expression levels were also correlated to the Pfirrmann criteria. Lastly, the degenerative specimens were stratified according to their vertebral level and expression levels between the degenerative lumbar and cervical discs were compared.RESULTSFifty-two patients were enrolled; however, 2 control and 2 degenerative patients were excluded due to incomplete data sets. Of the remaining 48 patients, there were 12 control and 36 degenerative specimens. Degenerative specimens had increased expression levels of BMP-2 (p=.0006) and pSMAD1/5/8 (p<.0001). Pfirrmann grade 3 (p=.0365) and grade 4 (p=.0008) discs had significantly higher BMP-2 expression as compared to grade 2 discs. Pfirrmann grade 4 discs had higher pSMAD1/5/8 expression as compared to grade 2 discs (p<.0001). There were no differences in ALK3 or MMP-13 expression between the control and degenerative discs (p>.05). Stratifying the degenerative specimens according to their vertebral level showed no significant differences in expression levels between the lumbar and cervical discs (p>.05).CONCLUSIONSBMP-2 and pSMAD1/5/8 signaling activity was significantly upregulated in the human degenerative specimens, while ALK3 and MMP-13 expression were not significantly changed. The expression levels of BMP-2 and pSMAD1/5/8 correlate positively with the degree of disc degeneration measured according to the Pfirrmann MRI grading system.CLINICAL SIGNIFICANCEBMP-SMAD signaling represents a promising therapeutic target to restore IVD composition and function in the setting of disc degeneration.