Injection of human umbilical tissue–derived cells into the nucleus pulposus alters the course of intervertebral disc degeneration in vivo

Background contextPatients often present to spine clinic with evidence of intervertebral disc degeneration (IDD). If conservative management fails, a safe and effective injection directly into the disc might be preferable to the risks and morbidity of surgery.PurposeTo determine whether injecting human umbilical tissue–derived cells (hUTC) into the nucleus pulposus (NP) might improve the course of IDD.DesignProspective, randomized, blinded placebo–controlled in vivo study.Patient sampleSkeletally mature New Zealand white rabbits.Outcome measuresDegree of IDD based on magnetic resonance imaging (MRI), biomechanics, and histology.MethodsThirty skeletally mature New Zealand white rabbits were used in a previously validated rabbit annulotomy model for IDD. Discs L2–L3, L3–L4, and L4–L5 were surgically exposed and punctured to induce degeneration and then 3 weeks later the same discs were injected with hUTC with or without a hydrogel carrier. Serial MRIs obtained at 0, 3, 6, and 12 weeks were analyzed for evidence of degeneration qualitatively and quantitatively via NP area and MRI Index. The rabbits were sacrificed at 12 weeks and discs L4–L5 were analyzed histologically. The L3–L4 discs were fixed to a robotic arm and subjected to uniaxial compression, and viscoelastic displacement curves were generated.ResultsQualitatively, the MRIs demonstrated no evidence of degeneration in the control group over the course of 12 weeks. The punctured group yielded MRIs with the evidence of disc height loss and darkening, suggestive of degeneration. The three treatment groups (cells alone, carrier alone, or cells+carrier) generated MRIs with less qualitative evidence of degeneration than the punctured group. MRI Index and area for the cell and the cell+carrier groups were significantly distinct from the punctured group at 12 weeks. The carrier group generated MRI data that fell between control and punctured values but failed to reach a statistically significant difference from the punctured values. There were no statistically significant MRI differences among the three treatment groups. The treated groups also demonstrated viscoelastic properties that were distinct from the control and punctured values, with the cell curve more similar to the punctured curve and the carrier curve and carrier+cells curve more similar to the control curve (although no creep differences achieved statistical significance). There was some histological evidence of improved cellularity and disc architecture in the treated discs compared with the punctured discs.ConclusionsTreatment of degenerating rabbit intervertebral discs with hUTC in a hydrogel carrier solution might help restore the MRI, histological, and biomechanical properties toward those of nondegenerated controls. Treatment with cells in saline or a hydrogel carrier devoid of cells also might help restore some imaging, architectural, and physical properties to the degenerating disc. These data support the potential use of therapeutic cells in the treatment of disc degeneration.     

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.     

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.     

NSAID use in intervertebral disc degeneration: what are the effects on matrix homeostasis in vivo?

Background Context

Non-steroidal anti-inflammatory drugs (NSAIDs) are a widely used treatment for low back pain (LBP). Literature on NSAID use in articular cartilage has shown detrimental effects; however, minimal data exist to detail the effects of NSAIDs in intervertebral disc degeneration (IDD). As IDD is a major cause of LBP, we explored the effects of indomethacin, a commonly used NSAID, on disc matrix homeostasis in an animal model of IDD.

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.     

腺相关病毒介导的骨形态发生蛋白-2体内转染兔退变椎间盘的实验研究

目的 观察腺相关病毒介导的骨形态发生蛋白-2(AAV-BMP-2)基因对兔退变腰椎间盘的治疗作用.方法 将36只新西兰大白兔L2-3、L3-4、L4-5、L5-6椎间盘针刺制造退变模型后随机分为3组,其中12只注射AAV-BMP-2作为实验组,12只注射AAV作为实验对照组,12只注射生理盐水作为空白对照组.注射后的2、4、8周各组随机抽取4只兔行腰椎MRI扫描,扫描后将其处死,取L2-3、L3-4、L4-5、L5-6椎问盘髓核组织,用间苯三酚分光光度法检测髓核组织中的蛋白多糖含量.结果 在MRI影像学Thompson分级评估中,实验组各时间点椎间盘MRI信号比较,差异无统计学意义.实验对照组和空白对照组各时间点MRI信号比较,差异有统计学意义.实验组和实验对照组、实验组和空白对照组各时间点MRI髓核信号比较,差异有统计学意义;实验对照组和空白对照组比较,差异无统计学意义.在蛋白多糖含量测定中,实验组蛋白多糖含量在各时间点均高于实验对照组和空白对照组,实验对照组和空白对照组蛋白多糖均随时间的变化逐渐减少,各时间点两组比较无明显区别.结论 AAV-BMP-2对兔退变的腰椎间盘有治疗作用.     

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.     

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。     

阻断双侧终板营养途径构建山羊椎间盘退变模型

目的通过建立山羊腰椎双侧终板营养途径阻断的动物模型,观察椎间盘退变(IDD)的情况,研究椎间盘营养途径与IDD的相关性。方法选取8只24月龄雌性关中山羊,每只山羊L2~3、L3~4作为实验椎间盘,麻醉后在平行于终板2 mm的椎体骨质处造成骨缺损,并使用骨水泥填塞,阻断椎体和终板之间的营养通路,L1~2、L4~5作为对照椎间盘。分别于术后4、12、24、48周行X线、MRI检查,各时间点随机处死2只山羊,采集椎间盘标本,计算骨水泥有效阻断面积、椎间高度指数(DHI)和Pfirrmann分级,并行HE、Masson三色、蛋白多糖、番红O染色组织学检查。结果术后骨水泥有效阻断面积达49.6%~69.6%(60.7%±5.3%)。术后48周时实验椎间盘DHI百分比为60.5%~81.7%(72.7%±5.6%),椎间高度丢失较对照差异有统计学意义(P<0.01);术后48周时实验椎间盘Pfirrmann分级为3~5(4.0±0.7)分,较对照差异有统计学意义(P<0.01)。组织学检查证实,实验椎间盘术后12周即发生退变,并随时间(24、48周)逐步加重。结论骨水泥填塞阻断双侧终板营养途径可以构建山羊IDD的动物模型,阻断终板营养途径可以导致IDD发生。     

Development of a rat model with lumbar vertebral endplate lesion

Purpose

Vertebral endplate lesion (EPL) caused by severe disc degeneration is associated with low back pain. However, there is no suitable animal model to elucidate the pathophysiology of EPL. This study aimed to develop a rat model of EPL and evaluate rat behavior and imaging and histological findings.

Methods

The L4-5 intervertebral discs of Sprague–Dawley rats were transperitoneally removed, except for the outer annulus fibrosus and cartilage endplate, in the EPL group. The L4-5 discs were not removed and simply exposed in the sham group. Changes around the vertebral endplate on magnetic resonance imaging (MRI) and computed tomography (CT) were evaluated. Additionally, pain-related behavioral and histological assessments were performed.

Results

In the EPL group, a low-signal area around the vertebral endplate was observed on T1-weighted and T2-weighted fat-saturated MRI at 8 weeks or later after surgery. In the same group, CT showed osteosclerosis around the vertebral endplate at 12 weeks after surgery. The sham group did not show abnormal imaging features on the MRI and CT. Behavioral evaluation showed that the EPL group had a significantly longer grooming time than the sham group. Conversely, the 12-week postoperative locomotion time and the 1- and 12-week postoperative standing times were significantly shorter in the EPL group than in the sham group. Histological evaluation showed a high degree of vertebral endplate degeneration and an increased number of osteoclasts and proportion of nerve fibers expressing calcitonin gene-related peptide in the EPL group compared to those in the sham group.

Conclusion

Our rat EPL model showed pain-related behavioral patterns and an increased expression of pain-related neuropeptide. This model could contribute to the study of the pathophysiology of EPL and will help in the treatment of low back pain in the future.

     

自体富血小板血浆干预兔早期椎间盘退变的初步研究

目的富血小板血浆(platelet-rich plasma,PRP)具有刺激椎间盘细胞增殖、促进细胞外基质合成代谢及抑制纤维环细胞凋亡等作用。通过观察自体PRP干预兔早期椎间盘退变,明确其治疗效果,为临床应用提供理论依据。方法取健康成年新西兰大白兔45只,体重2.5～3.0 kg,雌雄不限;随机分为实验组、对照组、假手术组(n=15)。取实验组兔耳中央动脉血,采用Landesberg等方法制备PRP,同时对全血及PRP行血小板计数。实验组及对照组采用纤维环针刺法建立L4、5及L5、6椎间盘退变模型,造模2周后于L4、5及L5、6椎间隙分别注入100μL自体PRP及100μL PBS液;假手术组仅分离暴露椎间盘,不作处理。观察实验动物造模后一般情况;造模2周及干预1、2周时各组取5只实验动物行腰椎MRI、HE染色及Ⅱ型胶原免疫组织化学染色观察,腰椎MRI退变程度分级及Ⅱ型胶原阳性积分吸光度(IA)值检测。结果兔PRP中血小板计数约为外周血的4.92倍。实验动物均存活至实验完成。造模2周时,与假手术组相比,实验组和对照组椎间盘信号降低,髓核细胞减少,基质退变,Ⅱ型胶原表达降低。腰椎MRI退变程度分级及Ⅱ型胶原阳性IA值结果显示,各时间点实验组、对照组与假手术组相比差异均有统计学意义(P<0.05),干预1、2周时,实验组MRI退变程度分级显著低于对照组(P<0.05),但仍与假手术组有差异(P<0.05);干预1、2周时,实验组髓核细胞及软骨样基质较对照组增多,基质纤维化程度轻,Ⅱ型胶原表达明显强于对照组(P<0.05)。结论椎间盘内注射自体PRP可终止甚至一定程度逆转兔早期椎间盘退变,可能与PRP含有多种生长因子调控细胞功能、改善组织微环境、促进组织再生修复有关。     

两种骨水泥渗漏对椎间盘影响的实验研究

目的 探讨椎体成形术时骨水泥渗漏是否会引起椎间盘退变，以及椎间盘退变程度与骨水泥类型是否相关。方法 选用8只成年家犬，以每只犬L2-3、L3-4、L4-5椎间盘为实验对象，随机分为对照组、聚甲基丙烯酸甲酯（polymethylmethacrylate，PMMA）与磷酸钙骨水泥（calcium phosphate cement，CPC）3组。对照组仅行椎间盘穿刺，不注入任何物质，PMMA组及CPC组均各向椎间盘注入0．1ml骨水泥。术前及术后24周摄正、侧位X线片，计算椎间盘高度指数百分数（disc height index percentage，DHIP）。术后24周行MR检查，计算MRI指数。组织学检查参照Masuda标准对椎间盘退变程度评分并分析。结果 术后24周X线片显示对照组椎间隙无狭窄，病理学检查未见椎间盘退变。PMMA、CPC组椎间盘MRI显示：椎间隙有狭窄，R加权像髓核信号不同程度降低且不均一，其相对高信号区面积减小，髓核形态不规则，纤维环与髓核界限不清。组织学检查显示髓核细胞数量不同程度减少，空泡变小。髓核的细胞外基质不同程度压缩，纤维环断裂或扭转。3组DHIP、MRI指数、组织学评分的差异均有统计学意义（P〈0.01）。结论 PMMA、CPC注入椎间盘会导致椎间盘退变，PMMA所致椎间盘退变较CPC更为严重.     

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.     

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.     

髓核内注射过氧化还原酶Ⅱ对兔椎间盘退变过程的影响

目的探讨髓核内注射人重组过氧化还原酶（Peroxiredoxin,Prx）对兔椎间盘退变过程的影响。方法通过纤维环穿刺法建立新西兰大白兔椎间盘退变模型（L3～4～L5～6）,24只兔随机分为高浓度组、低浓度组和对照组,每组各8只。4周后MRI显示造模成功,然后行L3～4～L5～6椎间盘内注射,高浓度组和低浓度组分别注射1 mg/mL、10μg/mL Prx蛋白,对照组注射0.1%磷酸盐缓冲液,注射量均为25μL。在注射后2、4、12、16周每组分别随机取2只兔行脊柱MRI检查,采用硫酸咔唑法检测髓核蛋白多糖的含量,免疫组化检测型胶原含量,并做病理切片HE染色观察组织及细胞的改变情况。结果蛋白注射后三组椎间盘均发生不同程度退变,注射后16周,高浓度组髓核内水分的含量明显低于低浓度组和对照组（P〈0.05）;髓核内蛋白多糖含量高浓度组显著低于低浓度组和对照组（P〈0.05）;细胞外基质型胶原灰度值高浓度组显著低于低浓度组和对照组（P〈0.05）,而低浓度组和对照组比较差异没有统计学意义（P〉0.05）。结论高浓度（1 mg/mL）Prx在椎间盘退变过程中起促进退变作用。     

Novel stepwise model of intervertebral disc degeneration with intact annulus fibrosus to test regeneration strategies

Novel preclinical models that do not damage the annulus fibrosus (AF) of the intervertebral disc are required to study the efficacy of new regenerative strategies for the nucleus pulposus (NP). The aim of the study was to characterize a preclinical ovine model of intervertebral disc degeneration (IDD) induced by endplate (EP) damage and repair via the transpedicular approach, with or without partial nucleotomy, while keeping the AF intact. Twelve adult sheep were used. By the transpedicular approach, a 2 mm tunnel was drilled to the NP through the EP. A partial‐nucleotomy was performed. The tunnel was sealed using a polyurethane scaffold. Lumbar discs were assigned to different groups: L1‐2: nucleotomy; L2‐3: EP tunnel; L3‐4: nucleotomy + EP repair; L4‐5: EP tunnel + repair; L5‐6: control. X‐Ray and MRI were performed at 0, 1, 3, and 6 months after surgery. Disc height and MRI indexes were calculated. Macro‐ and micro‐morphology were analyzed. Pfirrmann and Thompson grades were assigned. The treated discs exhibited a progressive decrease in NP signal intensity and MRI index, displaying specific grades of degeneration based on the surgical treatment. According to Pfirrmann and Thompson grades different procedures were staged as: EP tunnel + repair: grade‐II; EP tunnel: grade‐III, nucleotomy + EP repair: grade‐IV; nucleotomy: grade‐V. A new stepwise model of IDD to study and test safety and efficacy of novel strategies for NP regeneration has been characterized. The different degrees of IDD have been observed similar to Pfirrmann and Thompson grading system. The intact AF allows for loading studies and eliminating the need for AF closure. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2460–2468, 2018.

     

AAV2-hVEGF165与AAV2-hTGFβ1对退变兔椎间盘纤维环细胞的生物学效应

目的采用基因治疗方法，把AAV2-hVEGF165和AAV2-hTGFβ1共转染兔纤维环细胞，观察其生物学活性，并进一步评价人血管内皮生长因子165（hVEGF165）和人转化生长因子β1（hTGFβ1）逆转椎间盘退变的可行性。方法分离并培养兔椎间盘纤维环细胞，以腺相关病毒（AAV2）为载体，分别携带hVEGF165和hTGFβ1eDNA，转染兔纤维环细胞。用Westernblotting检测hVEGF165和hTGFβ1的表达，并用同样的方法检测纤维环细胞中Ⅰ型胶原的表达。结果AAV2能够感染兔椎间盘细胞。用Westernblotting可以检测到AAV2-hVEGF165和AAV2-hTGFβ1在退变椎间盘细胞中的表达，并且，两种因子联合转染纤维环细胞时Ⅰ型胶原的表达要比分别转染是显著增高。结论hVEGF165和hTGFβ1共同转染培养的兔退变纤维环细胞，Ⅰ型胶原的表达显著增高。     

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.     

Part 1: dual-tuned proton/sodium magnetic resonance imaging of the lumbar spine in a rabbit model

STUDY DESIGN.: Development of a dual-tuned proton/sodium radiofrequency (RF) coil for magnetic resonance imaging (MRI) of the rabbit spine and quantification of sodium concentration in intervertebral discs. OBJECTIVE.: To develop the dual-tuned proton/sodium MRI of rabbit lumbar spine to investigate proteoglycan matrix content and intervertebral disc degeneration (IDD). SUMMARY OF BACKGROUND DATA.: IDD is a common chronic condition that may lead to back pain, limited activity, and disability. Early-stage IDD involves the loss of proteoglycan matrix and water content in the disc. Sodium MRI is a promising noninvasive technique for quantitative measurement of proteoglycan changes associated with IDD. The combined structural (proton) and biochemical (sodium) MRI facilitates the investigation of morphological and molecular changes associated with degeneration of discs. METHODS.: Multichannel dual-tuned proton/sodium transceiver RF coil of the rabbit spine was developed and optimized at 3T human scanner-8 channels allocated for the sodium coil and 4 channels for the proton coil. High-resolution anatomy proton images of the discs were acquired using turbo spin echo and dual echo steady state sequence. Sodium concentration of the discs was quantified from sodium magnetic resonance (MR) images that were calibrated for signal attenuation because of RF field inhomogeneity, sodium MR relaxation times, and disc thickness. Twelve rabbits (～1-yr old, female, 5.2 ± 0.4 kg) were used for measuring disc sodium concentration. RESULTS.: High-resolution in vivo proton and sodium MR images of rabbit discs (≤2-mm thickness) were successfully obtained using an in-house dual-tuned proton/sodium RF coil at 3T. The total acquisition time for each set of images was approximately 40 minutes. Sodium concentration of normal rabbit lumbar discs was measured at 269.7 ± 6.3 mM, and this measurement was highly reproducible, with 5.3% of coefficient of variation. CONCLUSION.: Sodium concentrations of rabbit lumbar discs were reliably measured using our newly developed dual-tuned multichannel proton/sodium RF coil at 3T.