A small-molecule inhibitor of the Wnt pathway,lorecivivint (SM04690), as a potential disease-modifying agent for the treatment of degenerative disc disease

BACKGROUND CONTEXTAbnormal Wnt signaling in intervertebral discs (IVDs) progresses degenerative disc disease (DDD) pathogenesis by impairing nucleus pulposus cell function, decreasing matrix deposition, and accelerating fibrosis.PURPOSEThis study was conducted to evaluate the effects of lorecivivint (LOR; SM04690), a small-molecule Wnt pathway inhibitor, on IVD cells and in an animal model of DDD.STUDY DESIGNWe used in vitro assays and a rat model of DDD to test the effects of LOR on nucleus pulposus cell senescence and viability, annulus fibrosus (AF) cell fibrosis, and cartilage regeneration and protection.METHODSWnt pathway gene expression was measured in human NP and AF cell cultures treated with LOR or DMSO (vehicle). Chondrocyte-like differentiation of rat and human NP cells, NP cell senescence and protection, and AF cell fibrosis were assessed using gene expression and immunocytochemistry. Disc and plasma pharmacokinetics were analyzed following intradiscal LOR injection in rats. In vivo effects of LOR and vehicle on AF integrity, AF/NP junction, NP cellularity and matrix, and disc height were compared using histopathology and radiography in a rat coccygeal IVD needle-puncture model of DDD.RESULTSIn NP and AF cell cultures, LOR-inhibited Wnt pathway gene expression compared with vehicle. In NP cells, LOR inhibited senescence, decreased catabolism, and induced differentiation into chondrocyte-like cells; in AF cells, LOR decreased catabolism and inhibited fibrosis. A single intradiscal LOR injection in rats resulted in therapeutic disc concentrations (~30 nM) for >180 days and minimal systemic exposure. DDD-model rats receiving LOR qualitatively demonstrated increased cartilage matrix and reduced AF lamellar disorganization and fragmentation with significantly (p<.05) improved histology scores and increased disc height compared with vehicle.CONCLUSIONSLOR showed beneficial effects on IVD cells in vitro and reduced disease progression in a rat model of DDD compared with vehicle, suggesting that LOR may have disease-modifying therapeutic potential.CLINICAL SIGNIFICANCEThe current therapeutic options for DDD are pain management and surgical intervention; there are no approved therapies that alter the progression of DDD. Our data support advancing LOR into clinical development as an injectable, small-molecule, potential disease-modifying treatment for DDD in humans.     

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.     

Therapeutic effects of cell therapy with neonatal human dermal fibroblasts and rabbit dermal fibroblasts on disc degeneration and inflammation

BACKGROUND CONTEXT

Increasing evidence suggests transplanting viable cells into the degenerating intervertebral disc (IVD) may be effective in treating disc degeneration and back pain. Clinical studies utilizing autologous or allogeneic mesenchymal stem cells to treat patients with back pain have reported some encouraging results. Animal studies have shown that cells injected into the disc can survive for months and have regenerative effects. Studies to determine the advantages and disadvantages of cell types and sources for therapy are needed.

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     

Polarization of infiltrating macrophages in the outer annulus fibrosus layer associated with the process of intervertebral disc degeneration and neural ingrowth in the human cervical spine

BACKGROUND CONTEXTAs no infiltrating macrophages exist in healthy discs, understanding the role of infiltrating macrophages including their polarity (M1 and M2 phenotypes) in intervertebral discs (IVDs) is important in the assessment of the pathomechanisms of disc degeneration.PURPOSETo determine the relationship between infiltrating macrophage polarization and the progression of human cervical IVD degeneration.STUDY DESIGNHistopathological study using harvested human cervical IVDs.METHODSIVDs collected during anterior cervical decompression from 60 patients were subjected to immunostaining and immunoblotting. The samples were classified as type 0–3 according to the percentage of CD16- and CD206-positive cells to CD68-positive cells in the outer annulus fibrosus layer. The number of vessels and nerve fibers and the severity of chronic inflammation with a focus on inflammatory cell infiltration, fibrosis, and capillary proliferation were also assessed.RESULTSThe number of CD16-positive cells was the highest in type 2 IVDs, and was suppressed following the infiltration of CD206-positive cells. The degree of chronic inflammation was significantly higher in type 2 and type 3 IVDs, and the number of nerve fibers was significantly higher in type 3 IVDs. The endothelial cells of small vessels were positive for nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 expression. Staining for tropomyosin receptor kinase (Trk)-A, Trk-B, and Trk-C was positive in aberrant fibers. In immunoblot analysis, the expression levels of these neurotrophic factors and receptors were significantly higher in type 2 and 3 IVDs.CONCLUSIONSThe polarity of macrophages around newly developed microvasculature might be altered with cervical IVD degeneration. A higher number of infiltrating M1 macrophages around the vessels was associated with chronic inflammation; however, their number got suppressed following the infiltration of M2 macrophages. The expression of neurotrophins in the capillaries of small vessels might contribute to neural ingrowth into degenerated IVDs.CLINICAL SIGNIFICANCEClarifying macrophages polarity change around new microvasculature associated with progression of IVD degeneration could enhance our understanding of the underlying mechanisms of neural ingrowth into degenerated IVDs and lead to development of a novel therapeutic target for prevention of IVD.     

Detrimental effects of discectomy on intervertebral disc biology can be decelerated by growth factor treatment during surgery: a large animal organ culture model

Background contextLumbar discectomies are common surgical interventions that treat radiculopathy by removing herniated and loose intervertebral disc (IVD) tissues. However, remaining IVD tissue can continue to degenerate resulting in long-term clinical problems. Little information is available on the effects of discectomy on IVD biology. Currently, no treatments exist that can suspend or reverse the degeneration of the remaining IVD.PurposeTo improve the knowledge on how discectomy procedures influence IVD physiology and to assess the potential of growth factor treatment as an augmentation during surgery.Study designTo determine effects of discectomy on IVDs with and without transforming growth factor beta 3 (TGFβ3) augmentation using bovine IVD organ culture.MethodsThis study determined effects of discectomy with and without TGFβ3 injection using 1-, 6-, and 19-day organ culture experiments. Treated IVDs were injected with 0.2 μg TGFβ3 in 20 μL phosphate-buffered saline+bovine serum albumin into several locations of the discectomy site. Cell viability, gene expression, nitric oxide (NO) release, IVD height, aggrecan degradation, and proteoglycan content were determined.ResultsDiscectomy significantly increased cell death, aggrecan degradation, and NO release in healthy IVDs. Transforming growth factor beta 3 injection treatment prevented or mitigated these effects for the 19-day culture period.ConclusionsDiscectomy procedures induced cell death, catabolism, and NO production in healthy IVDs, and we conclude that post-discectomy degeneration is likely to be associated with cell death and matrix degradation. Transforming growth factor beta 3 injection augmented discectomy procedures by acting to protect IVD tissues by maintaining cell viability, limiting matrix degradation, and suppressing NO. We conclude that discectomy procedures can be improved with injectable therapies at the time of surgery although further in vivo and human studies are required.     

Thermoreversible hyaluronan-based hydrogel supports in vitro and ex vivo disc-like differentiation of human mesenchymal stem cells

Background contextThe fate of human mesenchymal stem cells (hMSCs) supplied to the degenerating intervertebral disc (IVD) is still not fully understood and can be negatively affected by low oxygen, pH, and glucose concentration of the IVD environment. The hMSC survival and yield upon injection of compromised IVD could be improved by the use of an appropriate carrier and/or by predifferentiation of hMSCs before injection.PurposeTo optimize hMSC culture conditions in thermoreversible hyaluronan-based hydrogel, hyaluronan-poly(N-isopropylacrylamide) (HA-pNIPAM), to achieve differentiation toward the disc phenotype in vitro, and evaluate whether preconditioning contributes to a better hMSC response ex vivo.Study designIn vitro and ex vivo whole-organ culture of hMSCs.MethodsIn vitro cultures of hMSCs were conducted in HA-pNIPAM and alginate for 1 week under hypoxia in chondropermissive medium alone and with the supplementation of transforming growth factor β1 or growth and differentiation factor 5 (GDF-5). Ex vivo, hMSCs were either suspended in HA-pNIPAM and directly supplied to the IVDs or predifferentiated with GDF-5 for 1 week in HA-pNIPAM and then supplied to the IVDs. Cell viability was evaluated by Live-Dead assay, and DNA, glycosaminoglycan (GAG), and gene expression profiles were used to assess hMSC differentiation toward the disc phenotype.ResultsThe HA-pNIPAM induced hMSC differentiation toward the disc phenotype more effectively than alginate: in vitro, higher GAG/DNA ratio and higher collagen type II, SOX9, cytokeratin-19, cluster of differentiation 24, and forkhead box protein F1 expressions were found for hMSCs cultured in HA-pNIPAM compared with those cultured in alginate, regardless of the addition of growth factors. Ex vivo, direct combination of HA-pNIPAM with the disc environment induced a stronger disc-like differentiation of hMSCs than predifferentiation of hMSCs followed by their delivery to the discs.ConclusionsHyaluronan-based thermoreversible hydrogel supports hMSC differentiation toward the disc phenotype without the need for growth factor supplementation in vitro and ex vivo. Further in vivo studies are required to confirm the suitability of this hydrogel as an effective stem cell carrier for the treatment of IVD degeneration.     

Intervertebral disc cell- and hydrogel-supported and spontaneous intervertebral disc repair in nucleotomized sheep

Purpose

Regenerative repair is a promising new approach in treating damaged intervertebral discs. An experimental scheme was established for autologous and/or allogenic repair after massive disc injury.

Methods

Disc healing was promoted in 11 animals by injecting in vitro expanded autologous/homologous disc cells 2?weeks after stab injury of lumbar discs L1-2. The following control discs were used in our sheep injury model: L2-3, vehicle only; L3-4, injury only; L4-5, undamaged; and lumbar discs from four non-experimental animals. Disc cells were suspended in a biologically supportive albumin/hyaluronan two-component hydrogel solution that polymerizes when inserted in order to anchor cells at the injection site. The parameters studied were MRI, DNA, glycosaminoglycan, collagen content, histology, immunohistology for collagens type I, II and aggrecan, and mRNA expression of GAPDH, β-actin, collagen type I, II, X, aggrecan, lubricin, and IL-1β.

Results

All parameters demonstrated almost complete healing of the injured discs after 6?months, when compared with data from both the endogenous non-injured controls as well as from the healthy animals.

Conclusion

Sheep experience spontaneous recovery from disc injury. The process of endogenous repair can be enhanced by means of hydrogel-supported cells.     

E‐cadherin upregulates expression of matrix macromolecules aggrecan and collagen II in the intervertebral disc cells through activation of the intracellular BMP‐Smad1/5 pathway

E‐cadherin is a transmembrane protein that mediates cell–cell adhesion and cell–matrix interaction. Although the E‐cadherin has been shown to mediate a broad‐ranging cellular signals and functions, its effects on matrix metabolism of intervertebral discs (IVDs) are unknown. In this study, we investigated the effects of E‐cadherin on IVD matrix synthesis using pharmacological and molecular biology methods. We showed that high levels of the E‐cadherin are expressed in rabbits IVD cells. Our study indicates that the ectopic expression of E‐cadherin can stimulate matrix anabolism of the IVD cells, which was evidenced by increased expression of the matrix macromolecules aggrecan and collagen II. We found that E‐cadherin induces the expression of BMP‐4 and BMP‐7 genes and enhances Smad1/5 phosphorylation. Blocking BMP activity uses noggin suppressed E‐cadherin‐mediated upregulation of aggrecan and collagen II. Moreover, inhibition of Smad1/5 phosphorylation by dorsomorphin significantly repressed the E‐cadherin induced expression of aggrecan and collagen II at the both mRNA and protein levels. Together this study demonstrates that the E‐cadherin stimulates the synthesis of IVD matrix macromolecules aggrecan and collagen II through the induction of BMP genes and enhancement of the Smad1/5 phosphorylation. Thus E‐cadherin may have value in the treatment of degenerated discs. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1746–1752, 2012     

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.     

Physiological testosterone levels enhance chondrogenic extracellular matrix synthesis by male intervertebral disc cells in vitro,but not by mesenchymal stem cells

Background contextTestosterone (T) is a hormone and regulator involved in the processes of development of the organism (ie, promoting development of bone and muscle mass). Although T effects on the mesenchyme-derived muscle, bone, and adipose tissues are well studied, T effects on intervertebral disc (IVD) have not been reported.PurposeThe aim was to test the following hypothesis: if a physiological concentration of T (～30 nM) can improve in vitro chondrogenesis of human IVD cells and mesenchymal stem cells (MSCs).Study design/settingHuman IVD cells and MSCs were differentiated to chondrogenic lineage on gelatin scaffolds for 4 weeks, in the presence or absence of T.MethodsChondrogenesis was assessed by cell viability, by measuring gene expression with quantitative polymerase chain reaction and extracellular matrix (ECM) accumulation with immunoblotting, immunohistochemical, and biochemical methods.ResultsSupplementation of T to chondrogenic culture did not affect viability. In male IVD cells, T had a beneficial impact on chondrogenesis, particularly in nucleus pulposus cells, demonstrated by an increased expression of aggrecan, collagen type I, and especially collagen type II. Conversely, T had no effects on chondrogenesis of female IVD cells or MSCs from both genders. A gene expression array of transforming growth factor β/bone morphogenetic protein signaling cascade showed that in male IVD cells, T promoted a stable general but nonsignificant increase in gene expression. Furthermore, aromatase inhibitor anastrazole repressed the effect of T on ECM expression by IVD cells. The results suggest that T increased ECM accumulation in male IVD cells in combination with its conversion to estradiol by the enzyme aromatase.ConclusionsWe demonstrated that T effectively enhances in vitro chondrogenesis in male IVD cells, rising the interest in the possible role of sex hormones in IVD degeneration. Nevertheless, T does not affect chondrogenic differentiation of female IVD cells and MSCs from both genders.     

Computed tomography–guided sub–end plate injection of pingyangmycin for a novel rabbit model of slowly progressive disc degeneration

Background context

Different animal models are used in disc degenerative disease research by now. To our knowledge, a functional animal model that mimics ischemic and slowly progressive disc degeneration of humans does not exist.

Expression and regulation of metalloproteinases and their inhibitors in intervertebral disc aging and degeneration

Background contextDestruction of extracellular matrix (ECM) leads to intervertebral disc degeneration (IDD), which underlies many spine-related disorders. Matrix metalloproteinases (MMPs), and disintegrins and metalloproteinases with thrombospondin motifs (ADAMTSs) are believed to be the major proteolytic enzymes responsible for ECM degradation in the intervertebral disc (IVD).PurposeTo summarize the current literature on gene expression and regulation of MMPs, ADAMTSs, and tissue inhibitors of metalloproteinases (TIMPs) in IVD aging and IDD.MethodsA comprehensive literature review of gene expression of MMP, ADAMTS, and TIMP in human IDD and reported studies on regulatory factors controlling their expressions and activities in both human and animal model systems.ResultsUpregulation of specific MMPs (MMP-1, -2, -3, -7, -8, -10, and -13) and ADAMTS (ADAMTS-1, -4, and -15) were reported in human degenerated IVDs. However, it is still unclear from conflicting published studies whether the expression of ADAMTS-5, the predominant aggrecanase, is increased with IDD. Tissue inhibitors of metalloproteinase-3 is downregulated, whereas TIMP-1 is upregulated in human degenerated IVDs relative to nondegenerated IVDs. Numerous studies indicate that the expression levels of MMP and ADAMTS are modulated by a combination of many factors, including mechanical, inflammatory, and oxidative stress, some of which are mediated in part through the p38 mitogen-activated protein kinase pathway. Genetic predisposition also plays an important role in determining gene expression of MMP-1, -2, -3, and -9.ConclusionsUpregulation of MMP and ADAMTS expression and enzymatic activity is implicated in disc ECM destruction, leading to the development of IDD. Future IDD therapeutics depends on identifying specific MMPs and ADAMTSs whose dysregulation result in pathological proteolysis of disc ECM.     

The adolescent idiopathic scoliotic IVD displays advanced aggrecanolysis and a glycosaminoglycan composition similar to that of aged human and ovine IVDs

Purpose

The present study was designed to ascertain how altered biomechanics in adolescent idiopathic scoliotic (AIS) intervertebral discs (IVDs) affected tissue compositions and aggrecan processing compared to age matched and aged human IVDs. Newborn, 2- and 10-year-old ovine IVDs were also examined.

Methods

Aggrecan populations were separated by Sepharose CL2B chromatography, composite agarose polyacrylamide gel electrophoresis (CAPAGE) and identified by immunoblotting. The KS and CS content of IVD tissue extracts from AIS IVDs were compared with age-matched normal adolescent IVDs and with old human IVDs. Extracts from newborn, 2- and 10-year-old ovine IVDs were also examined in a similar manner.

Results

Adolescent idiopathic scoliotic IVD Aggrecan populations shared similar levels of polydispersity and aggregatability with hyaluronan as old IVD proteoglycans. CAPAGE demonstrated three aggrecan populations in AIS, aged human and ovine IVDs increased polydispersity and mobility in CAPAGE. AIS IVDs had GAG compositions similar to aged human and ovine IVDs. Sulphated KS (5-D-4) and chondroitin-6-sulphate, 3-B-3(+) were markers of tissue maturation, and chondroitin-4-sulphate, 2-B-6(+) was prominent in immature IVDs but its levels were lower in mature IVDs.

Discussion

Sulphated KS and 3-B-3(+) CS were prominently associated with IVD maturation and AIS IVDs, while the 2-B-6(+) CS isomer was associated with immature IVD tissues. The polydispersity of aggrecan in AIS IVDs, which was similar to in old human and ovine IVDs, reflected altered processing in the AIS IVDs in response to the biomechanical microenvironments the disc cells were exposed to in AIS IVDs.

Graphical abstract

These slides can be retrieved under Electronic Supplementary Material.

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Patterns of lumbar disc degeneration are different in degenerative disc disease and disc prolapse magnetic resonance imaging analysis of 224 patients

Background contextExisting research on lumbar disc degeneration has remained inconclusive regarding its etiology, pathogenesis, symptomatology, prevention, and management. Degenerative disc disease (DDD) and disc prolapse (DP) are common diseases affecting the lumbar discs. Although they manifest clinically differently, existing studies on disc degeneration have included patients with both these features, leading to wide variations in observations. The possible relationship or disaffect between DDD and DP is not fully evaluated.PurposeTo analyze the patterns of lumbar disc degeneration in patients with chronic back pain and DDD and those with acute DP.Study designProspective, magnetic resonance imaging–based radiological study.MethodsTwo groups of patients (aged 20–50 years) were prospectively studied. Group 1 included patients requiring a single level microdiscectomy for acute DP. Group 2 included patients with chronic low back pain and DDD. Discs were assessed by magnetic resonance imaging through Pfirmann grading, Schmorl nodes, Modic changes, and the total end-plate damage score for all the five lumbar discs.ResultsGroup 1 (DP) had 91 patients and group 2 (DDD) had 133 patients. DP and DDD patients differed significantly in the number, extent, and severity of degeneration. DDD patients had a significantly higher number of degenerated discs than DP patients (p<.000). The incidence of multilevel and pan-lumbar degeneration was also significantly higher in DDD group. The pattern of degeneration also differed in both the groups. DDD patients had predominant upper lumbar involvement, whereas DP patients had mainly lower lumbar degeneration. Modic changes were more common in DP patients, especially at the prolapsed level. Modic changes were present in 37% of prolapsed levels compared with 9.9% of normal discs (p<.00). The total end-plate damage score had a positive correlation with disc degeneration in both the groups. Further the mean total end-plate damage score at prolapsed level was also significantly higher.ConclusionThe results suggest that patients with disc prolapse, and those with back pain with DDD are clinically and radiologically different groups of patients with varying patterns, severity, and extent of disc degeneration. This is the first study in literature to compare and identify significant differences in these two commonly encountered patient groups. In patients with single-level DP, the majority of the other discs are nondegenerate, the lower lumbar spine is predominantly involved and the end-plate damage is higher. Patients with back pain and DDD have larger number of degenerate discs, early multilevel degeneration, and predominant upper lumbar degeneration. The knowledge that these two groups of patients are different clinically and radiologically is critical for our improved understanding of the disease and for future studies on disc degeneration and disc prolapse.     

辛伐他汀对兔髓核细胞的影响

目的 观察辛伐他汀对兔髓核细胞Ⅱ型胶原(ColⅡ)及聚集蛋白聚糖(Agg)表达的影响.方法 取兔髓核细胞进行原代培养,传至第3代行ColⅡ免疫组织化学鉴定后随机分为5组,以不同浓度辛伐他汀处理:A组:空白对照组;B、C、D、E组分别为0.1、0.2、0.4、0.8 μmol/L辛伐他汀组.运用半定量逆转录-聚合酶链反应(RT-PCR)检测ColⅡ、Agg含量的变化,并行细胞活力检测.结果 辛伐他汀浓度超过0.2 μmol/L时ColⅡ及Agg的表达增加,0.4 μmoL/L时达到高峰(P＜0.05),0.8 μmol/L时ColⅡ及Agg表达下降.0.8 μmol/L处理组影响细胞活力,0.1～0.4 μmol/L范围时细胞活性无明显影响(P＜0.05).结论 辛伐他汀可促进兔髓核细胞ColⅡ及Agg的表达,改善椎间盘退变进程;在＜0.4 μmol/L的较低浓度内对细胞活力无明显影响.     

Organ culture stability of the intervertebral disc: Rat versus rabbit

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

Bone morphogenetic proteins and degenerative disk disease

Bone morphogenetic proteins (BMPs) are involved not only in osteogenesis but also in chondrogenesis. They play an important role in the development and maintenance of the intervertebral disk (IVD). For this reason, an increasing amount of research has been performed to examine the relationship between BMPs and degenerative disk disease (DDD). Moreover, researchers are examining the safe use of BMPs as a potential treatment for diskogenic back pain. We performed a literature search using databases from the US National Library of Medicine and the National Institutes of Health to identify studies relating BMPs to DDD. According to in vitro and in vivo studies in different animal and human IVDs, BMP-2 and BMP-7 are upregulated with aging and with induced disk injury; this represents an anabolic response. Direct administration of BMP-2 to IVD cells results in increased production of components of the extracellular matrix. Upregulation of the BMP pathway via other agents, namely simvastatin and LIM mineralization protein-1, has resulted in similar outcomes. Adenoviruses loaded with BMPs, transfected either directly to IVD cells or via articular chondrocytic vectors, also resulted in reversal of the typical findings in DDD. We conclude that the use of BMPs to treat DDD has a promising future. Further studies are indicated to determine optimal delivery and efficacy in humans.     

Abundance of calpain and aggrecan-cleavage products of calpain in degenerated human intervertebral discs

Objective

To assess the expression of calpains and calpain-induced aggrecan fragmentation in early and advanced stages of degeneration of human intervertebral discs (IVDs).

Design

Disc tissue samples of 55 patients (mean age, 51.2 ± 22.3 years) who underwent intervertebral fusion were divided into groups with early and advanced degeneration based on the Thompson magnetic resonance imaging (MRI) scale. In advanced degeneration group, five patients (mean age, 35.5 ± 11.4 years) of lumbar disc herniation (LDH) were included. Protein levels of m- and μ-calpains and their inhibitor calpastatin were assayed, and immunohistochemical techniques were used to localize and quantify the production of the enzymes. To investigate calpain activity, we assayed purified aggrecan fragmentation in disc tissue by Western blotting and immunohistochemistry with VPGVA antibody, which recognizes the m-calpain generated neo-epitope GVA.

Results

Discs at early stages of degeneration expressed low levels of m- and μ-calpains and calpastatin, and few cells expressed degenerative enzymes. At more advanced stages of degeneration, the expression and number of cells immunopositive for m-calpain, μ-calpain and calpastatin were significantly higher. Further finding showed that anti-GVA-reactive aggrecan fragments were significantly higher in discs at advanced compared with early stages of degeneration. Herniated disc samples showed stronger expression and more cells immunopositive for calpains, calpastatin and GVA in the nucleus pulposus than in the annulus fibrous.

Conclusions

The expression of calpains, together with m-calpain-induced degradation products of extracellular matrix, was correlated with the degree of disc degeneration in human IVD tissue. These findings suggest that calpains may be involved in IVD degeneration via proteoglycan (PG) cleavage.