Human intervertebral disc cells are genetically modifiable by adenovirus-mediated gene transfer: implications for the clinical management of intervertebral disc disorders

STUDY DESIGN: Human intervertebral disc cells were cultured in monolayer and treated with adenovirus-containing marker genes to determine the susceptibility of the cells to adenovirus-mediated gene transfer. OBJECTIVES: To test the efficacy of the adenovirus-mediated gene transfer technique for transferring exogenous genes to human intervertebral disc cells in vitro. SUMMARY OF BACKGROUND DATA: Upregulated proteoglycan synthesis after direct in vivo adenovirus-mediated transfer of growth factor genes to the rabbit intervertebral disc has previously been reported. Before contemplating extending this approach to the treatment of human disc disease, it is necessary to demonstrate that human intervertebral disc cells are indeed susceptible to adenovirus-mediated gene transduction. METHODS: Human intervertebral disc cells were isolated from disc tissue obtained from 15 patients during surgical disc procedures. The cells were cultured in monolayer and treated with saline containing five different doses of adenovirus carrying the lacZ gene (Ad/CMV-lacZ), saline containing adenovirus carrying the luciferase gene (Ad/CMV-luciferase), or saline alone. Transgene expression was analyzed by 5-bromo-4-chloro-3-indolyl-beta-galactosidase (X-Gal) staining and luciferase assay. RESULTS: Adenovirus efficiently transferred lacZ and luciferase marker genes to cells from degenerated discs as well as to cells from nondegenerated discs. A minimum dose of 150 MOI Ad/CMV-lacZ was found to be sufficient to achieve transduction of approximately 100% of disc cells-regardless of patient age, sex, surgical indication, disc level, and degeneration grade. No statistically significant difference in the luciferase activities could be detected in disc cell cultures from degenerated and nondegenerated discs treated with Ad/CMV-luciferase. CONCLUSIONS: In vitro transducibility of human intervertebral disc cells by adenovirus is relatively insensitive to disc degeneration grade. Because the rate-limiting step for successful gene therapy is the ability to transfer genes efficiently to the target tissue, the achievement of efficient gene transfer to human intervertebral disc cells(using a direct, adenovirus-mediated approach) is an important and necessary step in the development of gene therapy strategies for the management of human intervertebral disc disorders.     

Expression of Fas receptor and apoptosis in vertebral endplates with degenerative disc diseases categorized as Modic type I or II

Study design

Vertebral endplate tissues were obtained from patients with degenerated lumbar disc classified as Modic type 1 or type 2 and investigated with immunohistochemical staining and the DNA nick end labelling techniques.

Objective

To examine the expression of fas receptor (FasR) and apoptosis in the endplate cells isolated from patients with degenerative disc disease and to see whether they are associated with the pathological change of endplate.

Methods

Fifty-six degenerated lumbar endplate specimens were obtained from the patients with degenerative disc disease categorized as type Modic I or II in magnetic resonance imaging (MRI) and eight nondegenerated specimens as control (vertebra burst fracture patients without degenerative change in MRI) during surgical procedures. Immunohistochemical staining was performed to determine the presence of FasR and apoptosis in sections of those endplate tissues. To investigate whether the FasR expression and apoptosis in endplate were influenced by degeneration and ageing of the discs, the level of FasR expression and apoptosis in the changed and unchanged endplates was analysed.

Results

FasR were expressed in the cytoplasm of the endplate cells. A higher degree of FasR expression and apoptosis in endplate cells in degenerated discs was found than that in nondegenerated discs. In cell culture, the level of FasR expression and apoptosis in cells from the degenerative endplates was higher than those in unchanged endplates. The percentage of FasR-positive endplate and apoptotic endplate cells correlated significantly with the patient's age (r = 0.301, p < 0.05; r = 0.307, p < 0.05. respectively), but not with the degree of disc degeneration in MRI (r = 0.047, p > 0.05; r = 0.066, p > 0.05, respectively).

Conclusion

This is the first study to compare the expression of FasR and apoptosis on vertebral endplate cells in degenerated disc with in nondegenerated disc. The results show that the endplate in degenerated disc may undergo fas-mediated apoptosis and vice versa, endplate degenerative changes may promotes apoptosis of the endplate cells within degenerated disc.     

终板下椎体缺血诱导兔椎间盘退行性变模型的建立及终板中内皮素1的表达

目的通过终板下注射无水乙醇阻碍椎体-终板营养,建立一种新型兔腰椎椎间盘退行性变模型,并观察终板退行性变过程中内皮素1(ET-1)的表达情况。方法健康4月龄新西兰兔32只,随机分成4组,每组8只,选取L5,6椎体(对应L4/L5及L5/L6椎间盘)注射300μL无水乙醇,选取L4椎体(对应L3/L4椎间盘)注射磷酸盐缓冲液(PBS)作为实验对照,L7椎体(对应L6/L7椎间盘)未注入任何物质作为正常对照。其中1组造模后1个月提取软骨终板细胞,行免疫细胞化学染色检测ET-1表达;余3组分别于造模后1、3和5个月进行椎间盘X线和MRI检查,取椎间盘组织行HE染色观察形态学改变,免疫组织化学染色观察ET-1表达。结果注射无水乙醇后,随着时间进展,X线片显示椎间隙高度显著下降、椎间隙变窄、边缘骨赘增生,MRI T2WI显示椎间盘低信号;苏木精-伊红染色(HE)显示终板的生长板厚度变薄,终板结构破损,同时软骨终板细胞退化、直至消失,髓核中细胞发生转化(由空泡细胞转变为软骨样细胞,进而形成纤维软骨样细胞)造成髓核纤维化,纤维环结构排列紊乱、纤维化程度逐步加重;免疫组织化学染色显示,发生退行性变的终板组织内有ET-1表达,但随着退行性变加剧,ET-1表达强度下降;提取的退行性变软骨终板细胞(造模后1个月)也显示细胞质内ET-1强表达。结论通过注射无水乙醇阻碍椎体-终板营养途径可成功建立兔椎间盘退行性变模型,终板退行性变过程中伴随ET-1的表达。     

退变腰椎间盘组织中碱性成纤维细胞生长因子的表达研究

目的 探讨碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF)在正常和退变椎间盘组织中的表达情况。方法 交30例来源于腰椎间盘突出症患者手术中所取得的椎间盘组织（观察组，男11例，女19例；年龄25－78岁，平均48岁；病程3个月－30年，平均9年11个月）与6例来源于脊柱侧凸患者前路松解术所取得的椎间盘组织（对照组，男女各3例，年龄10－17岁，平均14.2岁）进行对比，首先经病理组织学检查证实为退变椎间盘组织和政党椎间盘组织，然后将两组椎间盘组织分别通过免疫组织化学方法和原位杂交方法，检测各自椎间盘组织中的bFGF及其mRNA的表达。观察组30例均为退变椎间盘组织，免疫组化阳性率为90％（27／30），原位杂交阳性率为20％（6／30）；对照组6例均为正常椎间盘组织，其免疫组及原位杂交均为阴性，两组间免疫组化方法检测阳性率在统计学上差异有非常显著性意义。结论 bFGF在正常和退变椎间盘组织中表达的差异有显著性意义。提示 bFGF可能作为增生刺激因子促进椎间盘组织中的软骨细胞增生和细胞外基质合成，进而加速椎间盘退变。     

Sustained long-term RNA interference in nucleus pulposus cells in vivo mediated by unmodified small interfering RNA

RNA interference (RNAi) technology has recently emerged as an important biological strategy for gene silencing. Previously, the efficacies of RNAi in cultured nucleus pulposus cells in vitro have been reported. However, RNAi in the disc in vivo has never been reported. Therefore, the aims of the present study were to establish a method for RNAi in the disc in vivo and to evaluate the applicability of this technique for endogenous genes in the intervertebral discs using Fas Ligand (FasL) as a representative endogenous gene. To evaluate the efficacy of RNAi in vivo, two reporter luciferase plasmids (Firefly and Renilla) were used. These plasmids and unmodified short interference RNA (siRNA) duplex for targeting Firefly luciferase were co-transfected into coccygeal intervertebral disc of Sprague-Dawley rats in vivo using the ultrasound gene transfer technique. To evaluate the RNAi of the endogenous gene in vivo, siRNAs targeting rat FasL were transfected with the same technique. Non-specific siRNA was used as the negative control. The discs receiving no siRNAs were used as the control. The inhibitory effect of Firefly luciferase against Renilla luciferase was obtained using the results of dual-luciferase assay. Down-regulation of endogenous FasL was calculated by the data from real-time PCR. Our results showed that siRNA for Firefly luciferase can dramatically down-regulate the Firefly luciferase gene expression in vivo compared with Renilla luciferase. The inhibitory effects were maintained for at least 24 weeks and at 24 weeks post transfection, the inhibitory rate was 80% compared with the control group. Furthermore, the siRNA co-transfection group inhibited endogenous FasL expression by 53% compared with the control group. The present study demonstrates long-term down-regulation mediated by unmodified siRNA is possible not only for the exogenous reporter gene, but also for endogenous FasL expression in rat discs in vivo. This application of RNAi might be promising as a local therapy for disc degeneration and associated disorders by down-regulating some of the genes that are harmful for the normal physiology of the disc and may cause disc degeneration.     

P物质阳性神经纤维在退变腰椎间盘的分布及意义

目的研究P物质（substance P,SP）阳性神经纤维在腰椎间盘中的出现及分布,以探讨其在椎间盘退变中的潜在作用。方法收集16例患者的21个病变腰椎间盘和来自于新鲜尸体的12个正常对照椎间盘,行组织学检查和SP免疫组织化学染色检查。结果SP阳性神经纤维偶见于正常椎间盘,在病变椎间盘内可见较多的SP免疫反应阳性神经纤维分布,阳性神经纤维数量与腰椎间盘退变程度呈正相关。结论腰椎间盘退变程度和SP阳性神经纤维数量有明显的相关性,SP可能作为神经炎性介质加速了腰椎间盘的退变。     

Lumbar Intervertebral Disc Herniation Following Experimental Intradiscal Pressure Increase

Summary  An experimental biomechanical model of overload and rupture of the annulus fibrosus (AF) and lumbar disc herniation was achieved by increasing intradiscal pressure while keeping disc height constant in 69 motion segments at the L4–L5 level excised from cadaveric spines. The experiments were made on 53 specimens in neutral posture and on 16 specimens in flexion posture.  The values found for the rupture intradiscal pressure (RIP) ranged from 750 to 1300 kPa for neutral posture and the maximum RIP in anterior flexion was 1177 kPa.  The degree of disc degeneration was assessed by vertebral transcorporeal discography (previous to experiment) and by sectioning the intervertebral disc after the experiment.  The herniated lumbar intervertebral disc model by intradiscal pressure increase makes possible these assertions: –*The correlation between the degree of AF degeneration and the RIP is significant: the maximum RIP corresponds to a nondegenerated AF and the less RIP can tear only a degenerated AF; so disc herniation only occurs to discs with torn AF. –*AF breaking is more often paramedian, left or right. The place of AF breaking was paramedian in 70.3% cases, median in 9.45% cases and posterolateral in 20.25% cases.     

Localization of cathepsins D, K, and L in degenerated human intervertebral discs.

STUDY DESIGN: Localization of cathepsins D, K, and L in degenerated intervertebral discs was examined by immunohistochemistry. OBJECTIVES: To determine the involvement of cathepsins in the pathomechanism of intervertebral disc degeneration by monitoring the immunolocalization of cathepsins in degenerated intervertebral disc tissue. SUMMARY OF BACKGROUND DATA: Cathepsins D, K, and L are enzymes that contribute to the matrix destruction seen in the articular cartilage affected by osteoarthritis and rheumatoid arthritis. However, little is known about the contribution of these cathepsins to intervertebral disc degeneration. METHODS: Paraffin-embedded sections of degenerated intervertebral disc tissue collected at the time of surgery (13 discs from 12 patients) were immunohistochemically stained with antibodies for cathepsins D, K, and L. For further characterization of the stained cells, immunohistochemical detection of CD68 and TRAP staining were performed. RESULTS: Hematoxylin and eosin staining revealed obvious signs of degeneration in all sections. Cathepsins D and L were immunolocalized in disc fibrochondrocytes at various sites exhibiting degeneration. Cathepsins K were found in tartrate-resistant acid phosphatase-positive multinucleated cells, in particular near the cleft within the cartilaginous endplate. However, few cells were positive for these cathepsins in anulus fibrosus that maintained the lamellar structure of collagen fibers. CONCLUSIONS: Marked expression of cathepsins D and L was observed at the site of degeneration. Cathepsins D and K localized in tartrate-resistant acid phosphatase-positive multinucleated cells existed at the cleft between the cartilaginous endplate and vertebral body. The site-specific localization of these cathepsins suggests the association of these proteinases with endplate separation and disorganization of the anulus fibrosus in degenerative spinal disorders.     

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。     

一氧化氮在突出腰椎间盘中的表达及其意义

目的 :研究一氧化氮 (NO)在突出腰椎间盘组织中的含量及组织学定位 ,并对其意义进行探讨。方法 :对 32例腰椎间盘突出患者的突出间盘组织采取两种方法进行研究 :(1) 12例做体外培养 ,用分光光度法测定培养液上清中NO含量 ;(2 ) 2 0例用免疫组化方法对产生NO的细胞类型及组织学定位进行研究。同时对取自 4具新鲜尸体的 12个正常椎间盘采用相同方法做为对照。结果 :突出腰椎间盘组织产生NO的量为 2 0 0 70± 6 5 5 5nmol/g ,正常对照组的NO量为 76 31± 19 49nmol/ g ,两者统计学有显著性差异 (P <0 0 0 1)。免疫组化结果发现 ,2 0例患者椎间盘组织中一氧化氮合成酶表达阳性 16例 ,12个正常椎间盘组织中无表达阳性细胞。结论 :诱导型一氧化氮合成酶主要由突出椎间盘周围的肉芽组织产生 ,阳性细胞主要以成纤维细胞、软骨细胞及淋巴细胞为主。腰椎间盘可自身合成NO ,NO可能在椎间盘退变中起重要作用 ,突出腰椎间盘中的NO主要由突出腰椎间盘周围的肉芽组织产生。     

人退变腰椎间盘中细胞凋亡与Bax及半胱胺酸蛋白酶蛋白3基因的表达

目的 检测人腰椎间盘组织中的细胞密度、细胞凋亡率以及Bax和半胱氨酸蛋白酶蛋白3(Caspase-3)的表达,为深入了解人腰椎间盘退变的机制提供实验依据,并为将来通过生物学方法 延缓腰椎间盘退变提供新的思路.方法 实验组30个椎间盘标本(L2～S1)来自27例行后路腰椎间盘切除椎间融合手术自愿捐赠的患者,男18例,女9例;年龄30～72岁,平均51.09岁.所有病变节段均经MRI证实,患者术前均未接受椎间盘造影、胶原酶髓核溶解或椎间盘激光汽化术.对照组20个标本(L2～S1)来自5例青年男性意外死亡者新鲜尸检腰椎间盘;年龄24～37岁,平均30.83岁.采用HE染色观察凋亡软骨细胞、凋亡小体和细胞密度,TUNEL染色法检测人腰椎间盘中的凋亡细胞率,用SP免疫组织化学法检测Bax及Caspase-3表达,图像分析Bax及Caspase-3的平均灰度值.结果 HE染色示对照组、实验组软骨终板和髓核内的平均细胞密度分别为(17.16±1.22)、(12.41±0.95)个/HP,二者差异有统计学意义(P＜0.01).TUNEL染色观察示对照组的软骨终板与髓核内的平均凋亡细胞率为6.97%±0.92%,低于实验组的12.59±0.95%(P＜0.01).SP免疫组织化学染色示对照组髓核内Bax、Caspase-3染色阳性细胞率分别为11.02%4±1.18%和9.01%±1.00%,均低于实验组的19.29%±1.18%和15.07%±0.97%,差异均有统计学意义(P＜0.01).对照组腰椎间盘髓核内Bax、Caspase-3的平均灰度值分别为187.33±7.88和185.68±3.26,实验组分别为124.98±6.69和160.13±4.37,两组间比较差异有统计学意义(P＜0.01).将全部腰椎间盘标本进行Pearson相关分析,对照组和实验组腰椎间盘凋亡细胞率与细胞密度间成负相关,相关系数r分别为-0.88和-0.93(P＜0.01)两组髓核内Bax、Caspase-3阳性细胞率与凋亡细胞率之间成正相关,相关系数r分别为0.83和0.91(P＜0.01).结论 细胞密度下降参与了人腰椎间盘的退变过程,Bax和Caspase-3的表达上调在人腰椎间盘髓核细胞的凋亡过程中有一定作用.     

Gene expression profile of degenerated cervical intervertebral disc tissues in rats

egenerationofintervertebraldisciscausedbymanyfactorsandisacomplicatedwebsystemofdifferentialexpressionofmanygenes .Throughstudyingthegeneexpressionprofile ,theanalysisresultsofaseriesofphysiologicalstatesofintervertebraldisccellsortissuescanbeobtainedsensitivelyandcompletely .1,2 Thesedataaremuchvaluabletotheearlydiagnosis ,thecharacteristicpreventionandtherealizationandpopularizationoftherapeuticmethodsforthisdisease ,aswellasprovideprerequisiteandimportantbasisforstudyingthetherapeuticdrugsan…     

退变颈椎间盘组织中骨形态发生蛋白的基因表达

目的：了解骨形态发生蛋白（BMP）在椎间盘退变、突出过程中是否有表达。方法：采用颈椎间盘突出症前路钻孔减压术中所得椎间盘标本,进行组织学检查、BMP免疫组化和分子原位杂交研究。结果：突出的椎间盘与后纵韧带可融合发生部分骨化,形成的骨赘中BMP免疫组化染色及分子原位杂交阳性。上下软骨板和与之相邻的椎间盘组织BMP表达阳性。但椎间盘中心BMP表达阴性。自椎体骨和软骨板发出一些散在的呈“指状”的骨突嵌入椎间盘;其表面骨组织和周围的椎间盘组织BMP免疫组化染色及分子原位杂交阳性。结论：在突出颈椎间盘、软骨板、后纵韧带、骨膜中发现了BMP的阳性表达,但其来源、激活机制与意义尚需进一步研究。     

瘦素缺乏小鼠椎间盘退变的组织学观察

目的探讨瘦素在椎间盘退变中的可能作用。方法采用HE染色观察6月龄雄性ob/ob小鼠（瘦素缺乏小鼠）和野生型小鼠（C57BL小鼠）椎间盘的形态学;免疫组织化学检测Ⅱ型胶原、蛋白聚糖的表达;Real-time PCR检测Ⅱ型胶原、Ⅹ型胶原及蛋白聚糖的基因表达。结果与野生型小鼠相比,ob/ob小鼠椎间盘HE染色表现为椎间盘组织的胶原结构紊乱、髓核碎裂、椎间盘高度降低,免疫组化检测显示Ⅱ型胶原、蛋白聚糖表达减少,Real-time PCR检测显示Ⅱ型胶原、蛋白聚糖基因表达下调而Ⅹ型胶原基因表达上调,差异有统计学意义（P〈0.05）。结论活体内瘦素缺乏可能加速小鼠椎间盘退变。     

Negative effects of ADAMTS‐7 and ADAMTS‐12 on endplate cartilage differentiation

The roles of ADAMTS‐7 and ADAMTS‐12 in disc degeneration have not been previously examined. The purpose of this study was to examine the expression of ADAMTS‐7 and ADAMTS‐12 in the endplate cells isolated from patients with degenerative disc disease and to see whether they are associated with the pathological change of endplate. Sixty‐four degenerated lumbar endplate specimens were obtained from the patients with degenerative disc disease categorized as type Modic I or II in magnetic resonance imaging (MRI) and 12 nondegenerative specimens as control (vertebra burst fracture patients without degenerative change in MRI) during surgical procedures. The expression of ADAMTS‐7 and ADAMTS‐12 was examined by real‐time PCR and Western blotting. A statistically significant increase in mRNA expression of ADAMTS‐7 and ADAMTS‐12 was observed in the endplate cells in degenerative discs compared with nondegenerative discs. The corresponding protein levels of ADAMTS‐7 and ADAMTS‐12 had the same expression patterns. Moreover, ADAMTS‐7 and ADAMTS‐12 down‐regulated the expression of Col II, Sox9, and Col X the marker genes for chondrogenesis. Our results indicate that ADAMTS‐7 and ADAMTS‐12 appear to be potent negative regulators of endplate cartilage development. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1238–1243, 2012     

移植骨髓间充质干细胞对兔退变椎间盘髓核细胞凋亡的影响

目的 观察骨髓间充质干细胞(MSCs)移植对兔退变椎间盘髓核细胞凋亡的影响.方法 以各兔L2/3、L3/4、L4/5、L5/6节段分为正常组、退变组、成纤维细胞(SFs)移植对照组、MSCs移植治疗组.MSCs和SFs分别经绿色荧光蛋白(GFP)转染后,注射植入退变椎间盘的髓核.通过透射电镜观察退变椎间盘凋亡髓核细胞形态;用实时定量聚合酶链反应(PCR)检测退变组织中髓核细胞凋亡相关基因bcl-2和box mRNA的表达;免疫荧光法标记髓核细胞凋亡相关蛋白Caspase-3,并通过TUNEL法标记凋亡髓核细胞,激光共聚焦显微镜检测髓核细胞凋亡蛋白表达率和细胞凋亡比率.结果 透射电镜下,退变椎间盘中凋亡髓核细胞呈现出核染色质边集,空泡形成,核膜断裂,凋亡小体形成等变化.MSCs移植治疗组bcl-2 mRNA的表达量高于退变组和SFs移植对照组(P<0.05),bax mRNA的表达量与退变组差异无统计学意义(P>0.05).MSCs移植治疗组细胞凋亡率和Caspase-3表达率均高于正常组[细胞凋亡率分别为(16.75±2.14)%和(6.86±1.08)%;Caspase-3表达率分别为[(20.34±1.03)%和(6.09±0.77)%](P<0.05),低于退变组和SFs移植对照组[细胞凋亡率分别为(31.87±4.16)%和(29.02±2.16)%;Caspase-3表达率分别为(31.50±3.78)%和(30.20±4.93)%](P<0.05).结论 髓核细胞凋亡在椎间盘退变过程中起重要作用.MSCs移植能有效抑制椎间盘髓核细胞凋亡,延缓椎间盘退变过程.     

Lumbar disc and back muscle degeneration on MRI: correlation to age and body mass.

Lumbar intervertebral discs and paraspinal muscles in 74 healthy volunteers ranging in age from 19 to 74 years were evaluated with MRI, and the occurrence of degeneration was correlated to age and body mass. Muscle size and the amount of fat in the muscles was studied from MRI cross sections. When the back muscles were degenerated, they were small and contained fat deposits. By contrast, the psoas muscles never showed gross fat deposits. Degeneration of both the lumbar discs and muscles increased with age. No correlation was found between muscle degeneration and overweight. Muscle degeneration is as common as disc degeneration in the lumbar area. MRI is an excellent method to assess both muscle and disc degeneration.     

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.