Transplantation of mesenchymal stem cells in a canine disc degeneration model

Transplantation of mesenchymal stem cells (MSCs) is effective in decelerating disc degeneration in small animals; much remains unknown about this new therapy in larger animals or humans. Fas‐ligand (FasL), which is only found in tissues with isolated immune privilege, is expressed in IVDs, particularly in the nucleus pulposus (NP). Maintaining the FasL level is important for IVD function. This study evaluated whether MSC transplantation has an effect on the suppression of disc degeneration and preservation of immune privilege in a canine model of disc degeneration. Mature beagles were separated into a normal control group (NC), a MSC group, and the disc degeneration (nucleotomy‐only) group. In the MSC group, 4 weeks after nucleotomy, MSCs were transplanted into the degeneration‐induced discs. The animals were followed for 12 weeks after the initial operation. Subsequently, radiological, histological, biochemical, immunohistochemical, and RT‐PCR analyses were performed. MSC transplantation effectively led to the regeneration of degenerated discs. FACS and RT‐PCR analyses of MSCs before transplantation demonstrated that the MSCs expressed FasL at the genetic level, not at the protein level. GFP‐positive MSCs detected in the NP region 8 weeks after transplantation expressed FasL protein. The results of this study suggest that MSC transplantation may contribute to the maintenance of IVD immune privilege by the differentiation of transplanted MSCs into cells expressing FasL. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:589–600, 2008     

大鼠退变腰椎间盘组织的超微结构观察

[目的]观察大鼠腰椎间盘退变动物模型中椎间盘组织的超微结构改变。[方法]32只SD大鼠随机分为实验组和对照组，每组各16只。实验组采用手术方法以L3为中心切除棘突、关节突、棘上、棘间韧带，切断双侧竖棘肌。对照组仅切开皮肤后即缝合。术后8周，应用电镜技术对SD大鼠椎间盘组织进行详细的超微结构观察。[结果]对照组的椎间盘组织超微结构的病理改变不显著，而实验组表现为软骨样细胞减少，出现不同程度地退变、坏死，细胞器数目减少，细胞外周致密颗粒增多；基质中胶原纤维发生不同程度的变性、融合、扭结或钙化，胶原纤维束间裂隙增大；[结论]应用电镜观察腰椎间盘退变动物模型的超微病理改变，可为研究腰椎间盘疾患提供相关的实验依据。     

Effect of cell number on mesenchymal stem cell transplantation in a canine disc degeneration model

Transplantation of mesenchymal stem cells (MSCs) inhibits the progression of disc degeneration in animal models. We know of no study to determine the optimal number of cells to transplant into the degenerated intervertebral disc (IVD). To determine the optimal donor cell number for maximum benefit, we conducted an in vivo study using a canine disc degeneration model. Autologous MSCs were transplanted into degenerative discs at 10⁵, 10⁶, or 10⁷ cells per disc. The MSC‐transplanted discs were evaluated for 12 weeks using plain radiography, magnetic resonance imaging, and gross and microscopic evaluation. Preservation of the disc height, annular structure was seen in MSC‐transplantation groups compared to the operated control group with no MSC transplantation. Result of the number of remaining transplanted MSCs, the survival rate of NP cells, and apoptosis of NP cells in transplanted discs showed both structural microenvironment and abundant extracellular matrix maintained in 10⁶ MSCs transplanted disc, while less viable cells were detected in 10⁵ MSCs transplanted and more apoptotic cells in 10⁷ MSCs transplanted discs. The results of this study demonstrate that the number of cells transplanted affects the regenerative capability of MSC transplants in experimentally induced degenerating canine discs. It is suggested that maintenance of extracellular matrix by its production from transplanted cells and/or resident cells is important for checking the progression of structural disruption that leads to disc degeneration. Published by Wiley Periodicals, Inc. J Orthop Res 28:1267–1275, 2010     

椎间失稳致兔椎间盘退变磁共振影像计量分析

目的 :探讨由于椎间失稳诱发的椎间盘退变在磁共振成像 (magneticresonanceimaging ,MRI)中的表现。方法 :选用新西兰兔 15只 ,随机分为手术组 9只、对照组 6只 ,手术组沿L3～ 6棘突作后正中切口 ,剥离骶棘肌和关节突附丽肌肉 ,切除棘上、棘间韧带和关节突关节外后 1/ 2 ;对照组作相同皮肤切口即缝合。所有动物在标准条件下饲养 ,分别于术后 2、 4、 8个月行腰椎MRI检查及髓核信号值测量。结果 :术后 2～ 8个月 ,对照组腰椎未见异常 ,而手术组L3～ 6椎间盘则相继出现T2 加权像低信号、腰椎后凸畸形、T1加权像低信号、椎间盘后突和硬膜囊受压等改变。对手术组手术节段及其邻近节段椎间盘髓核信号值的定量分析显示 ,T2 加权像信号值减低在术后 2、 4、8个月均具有统计学意义 ,而T1加权像信号值减低在术后 8个月具有统计学意义 ;T2 信号值减低主要发生于术后 2个月 ,T1信号值减低发生于术后 8个月。结论 :脊柱失稳可诱发椎间盘退变。髓核T2 加权像低信号是椎间盘退变的早期和先发征象 ,T1加权像显示形态改变较好 ,但T1信号值在退变早期变化不明显。     

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。     

中药抑制椎间盘退变的分子机制研究

椎间盘退变(intervertebral disc degeneration,IVDD)是一系列脊柱退行性疾病的病理基础,发病率高,严重影响患者的生活。中药治疗椎间盘退变临床应用广泛,疗效较好,对其作用机制研究也越来越深入。本文就单味药、复方以及中成药治疗IVDD的实验药理机制研究进行了归纳和总结,为临床提供参考。     

Calcification in human intervertebral disc degeneration and scoliosis

Calcification is a pathological process that may lead to impairment of nutrient supply and disc metabolism in degenerative and scoliotic intervertebral discs (IVDs). The purpose of this study was to assess the calcification potential of IVDs in degenerative disc disease (DDD) and adolescent idiopathic scoliosis (AIS). For this purpose, 34 IVDs from 16 adult patients with DDD and 25 IVDs from 9 adolescent patients with AIS were obtained at surgery. The concave and convex parts of the scoliotic discs were analyzed separately. Von Kossa staining was performed to visualize calcium deposits, while type X collagen (COL X) expression associated with endochondral ossification was measured by immunohistochemistry. Alkaline phosphatase activity and calcium and inorganic phosphate concentrations were used as indicators of calcification potential. Results showed the presence of calcium deposits and COL X in degenerative and scoliotic IVDs, but not in control discs, and the level of the indicators of calcification potential was consistently higher in degenerative and scoliotic discs than in control discs. The results suggest that disc degeneration in adults is associated with ongoing mineral deposition and that mineralization in AIS discs might reflect a premature degenerative process. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1888–1895, 2011     

组织蛋白酶L与人腰椎间盘退变关系的研究

[目的]探讨Cathepsin L与人腰椎间盘退变的关系.[方法]实验组标本来源于腰间盘突出症的患者,在外科手术过程中取得的退变间盘组织,并根据术中所见将腰间盘突出分为非包含型组和包含型组;对照组间盘来源于胸腰段骨折行前路手术患者手术中切除的间盘组织,以上标本采用.HE染色组织学观察椎间盘的退变特征,免疫组织化学S-P法和逆转录-聚合酶链式反应(RT-PCR)法检测人腰椎间盘细胞Cathepsin L蛋白表达.[结果]HE染色组织学观察在退变组间盘中,各标本均表现出一定的退变特征纤维环板层结构紊乱,软骨细胞巢状增生,纤维环粘液瘤样变,髓核软骨细胞减少,纤维细胞增生,玻璃样变及纤维化,髓核毛细血管的侵入,炎性细胞的浸润等.在未包含型组与包含型组比较显示了更多的髓核毛细血管侵入和炎性细胞浸润,髓核及纤维环退变更严重,纤维细胞增生及瘢痕形成.免疫组织化学染色显示组织蛋白酶L阳性率在退变组(90.68±7.61)%明显高于对照组(10.00±8.69)%,并且在非包含型组(95.45±4.63)%明显高于包含型组(86.93±7.49)%.RT-PCR结果显示组织蛋白酶L的mRNA表达水平在退变组(1.445±0.206)%较对照组(0.526±0.378)%明显上调,并且在非包含型组(1.608±0.044)%明显高于包含型组(1.282±0.166)%.以上结果均经统计分析证实组间差异有统计学意义.[结论]Cathepsin L与人腰椎间盘退变有关.     

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.     

Inflammatory cytokine and catabolic enzyme expression in a goat model of intervertebral disc degeneration

Intervertebral disc degeneration is implicated as a leading cause of low back pain. Persistent, local inflammation within the disc nucleus pulposus (NP) and annulus fibrosus (AF) is an important mediator of disc degeneration and negatively impacts the performance of therapeutic stem cells. There is a lack of validated large animal models of disc degeneration that recapitulate clinically relevant local inflammation. We recently described a goat model of disc degeneration in which increasing doses of chondroitinase ABC (ChABC) were used to reproducibly induce a spectrum of degenerative changes. The objective of this study was to extend the clinical relevance of this model by establishing whether these degenerative changes are associated with the local expression of inflammatory cytokines and catabolic enzymes. Degeneration was induced in goat lumbar discs using ChABC at different doses. After 12 weeks, degeneration severity was determined histologically and using quantitative magnetic resonance imaging (MRI). Expression levels of inflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin-1β [IL-1β], and IL-6) and catabolic enzymes (matrix metalloproteinases-1 [MMPs-1] and 13, and a disintegrin and metalloproteinase with thrombospondin type-1 motifs-4 [ADAMTS-4]) were assessed as the percentage of immunopositive cells in the NP and AF. With the exception of MMP-1, cytokine, and enzyme expression levels were significantly elevated in ChABC-treated discs in the NP and AF. Expression levels of TNF-α, IL1-β, and ADAMTS-4 were positively correlated with histological grade, while all cytokines and ADAMTS-4 were negatively correlated with MRI T2 and T1ρ scores. These results demonstrate that degenerate goat discs exhibit elevated expression of clinically relevant inflammatory mediators, and further validate this animal model as a platform for evaluating new therapeutic approaches for disc degeneration.     

褪黑素在椎间盘退变中的研究进展

椎间盘退变是下腰痛的主要因素之一,随着老年社会的到来,其发病率逐年升高,由于椎间盘退变(intervertebral disc degeneration,IDD)致病因素众多,其发病机制尚不清楚,目前仍无有效治疗药物.褪黑素(melatonin,MT)作为松果体分泌的一种神经内分泌激素,因其卓越的抗氧化应激、抗炎及抗细...     

Human intervertebral disc internal strain in compression: The effect of disc region,loading position,and degeneration

The primary function of the disc is mechanical; therefore, degenerative changes in disc mechanics and the interactions between the annulus fibrosus (AF) and nucleus pulposus (NP) in nondegenerate and degenerate discs are important to functional evaluation. The disc experiences complex loading conditions, including mechanical interactions between the pressurized NP and the surrounding fiber‐reinforced AF. Our objective was to noninvasively evaluate the internal deformations of nondegenerate and degenerate human discs under axial compression with flexion, neutral, and extension positions using magnetic resonance imaging and image correlation. The side of applied bending (e.g., anterior AF in flexion) had higher tensile radial and compressive axial strains, and the opposite side of bending exhibited tensile axial strains even though the disc was loaded under axial compression. Degenerated discs exhibited higher compressive axial and tensile radial strains, which suggest that load distribution through the disc subcomponents are altered with degeneration, likely due to the depressurized NP placing more of the applied load directly on the AF. The posterior AF exhibited higher compressive axial and higher tensile radial strains than the other AF regions, and the strains were not correlated with degeneration, suggesting this region undergoes high strains throughout life, which may predispose it to failure and tears. In addition to understanding internal disc mechanics, this study provides important new data into the changes in internal strain with degeneration, data for validation of finite element models, and provides a technique and baseline data for evaluating surgical treatments. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29: 547–555, 2011     

脂肪细胞因子在椎间盘退变中的作用及其机制的研究进展

目的对近年来脂肪细胞因子在椎间盘退变(intervertebral disc degeneration,IVDD)过程中的作用及其机制的研究进展进行综述。方法广泛查阅近年国内外与脂肪细胞因子在IVDD过程中作用研究有关的文献,对脂肪细胞因子种类及功能、在IVDD过程中的作用及机制,以及在椎间盘生物治疗中的应用前景进行总结。结果脂肪细胞因子是一种主要由脂肪组织分泌的生物活性物质,在骨关节疾病、代谢性疾病、乳腺癌等疾病中具有重要作用。在IVDD过程中,多数脂肪细胞因子通过与自身受体结合来激活多种信号传导途径,引起椎间盘内细胞增殖及凋亡失调、促炎及抗炎因子分泌紊乱,进而导致椎间盘内代谢失衡和初始炎症环境的建立。结论脂肪细胞因子作为一种具有代谢和免疫调节功能的生物活性物质,在IVDD的发生、发展及生物治疗等方面发挥着重要作用。     

Histological and reference system for the analysis of mouse intervertebral disc

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

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     

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。     

MRI evaluation of spontaneous intervertebral disc degeneration in the alpaca cervical spine

Animal models have historically provided an appropriate benchmark for understanding human pathology, treatment, and healing, but few animals are known to naturally develop intervertebral disc degeneration. The study of degenerative disc disease and its treatment would greatly benefit from a more comprehensive, and comparable animal model. Alpacas have recently been presented as a potential large animal model of intervertebral disc degeneration due to similarities in spinal posture, disc size, biomechanical flexibility, and natural disc pathology. This research further investigated alpacas by determining the prevalence of intervertebral disc degeneration among an aging alpaca population. Twenty healthy female alpacas comprised two age subgroups (5 young: 2–6 years; and 15 older: 10+ years) and were rated according to the Pfirrmann‐grade for degeneration of the cervical intervertebral discs. Incidence rates of degeneration showed strong correlations with age and spinal level: younger alpacas were nearly immune to developing disc degeneration, and in older animals, disc degeneration had an increased incidence rate and severity at lower cervical levels. Advanced disc degeneration was present in at least one of the cervical intervertebral discs of 47% of the older alpacas, and it was most common at the two lowest cervical intervertebral discs. The prevalence of intervertebral disc degeneration encourages further investigation and application of the lower cervical spine of alpacas and similar camelids as a large animal model of intervertebral disc degeneration. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1776–1783, 2015.     

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.

     

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。