The role of TGF‐β1/Smad2/3 pathway in platelet‐rich plasma in retarding intervertebral disc degeneration

Recent studies have suggested that platelet‐rich plasma (PRP) injections are an effective way to retard intervertebral disc degeneration, but the mechanism of action is unclear. Activated platelets release some growth factors, such as transforming growth factor‐β1 (TGF‐β1), which positively modulate the extracellular matrix of nucleus pulposus cells. The purpose of this study was to explore the mechanism underlying the PRP‐mediated inhibition of intervertebral disc degeneration. In an in vitro study, we found that the proliferation of nucleus pulposus cells was greatly enhanced with 2.5% PRP treatment. The TGF‐β1 concentration was much higher after PRP treatment. PRP administration effectively increased the collagen II, aggrecan and sox‐9 mRNA levels and decreased collagen X levels. However, Western blotting demonstrated that specifically inhibiting TGF‐β1 signalling could significantly prevent nucleus pulpous cellular expression of Smad2/3 and matrix protein. In a rabbit study, magnetic resonance imaging revealed significant recovery signal intensity in the intervertebral discs of the PRP injection group compared with the very low signal intensity in the control groups. Histologically, the PRP plus inhibitor injection group had significantly lower expression levels of Smad2/3 and collagen II than the PRP group. These results demonstrated that a high TGF‐β1 content in the platelets retarded disc degeneration in vitro and in vivo. Inhibiting the TGF‐β1/Smad2/3 pathway could prevent this recovery by inactivating Smad2/3 and down‐regulating the extracellular matrix. Therefore, the TGF‐β1/Smad2/3 pathway might play a critical role in the ability of PRP to retard intervertebral disc degeneration.     

TGF-β synergizes with ML264 to block IL-1β-induced matrix degradation mediated by Krüppel-like factor 5 in the nucleus pulposus

Intervertebral disc degeneration causes low back pain.Interleukin-1β (IL-1β) is a well-known inflammatory mediator that is involved in disc degeneration but its molecular mechanisms on catabolic and anabolic events in nucleus pulposus (NP) cells remain unclear. Krüppel-like factor 5 (KLF5) is associated with inflammation and was previously shown to cause cartilage degradation. In this study, we revealed that KLF5 is involved in IL-1β activated NF-kB cascade by enhancing both p65 phosphorylation and p65 acetylation. Moreover, the catabolic effect of KLF5 can be abolished by transforming growth factor-β (TGF-β) via promoting the proteasomal degradation of KLF5. Therefore, a KLF5 inhibitor ML264 was further proved to synergize with TGF-β to attenuate IL-1β-induced intervertebral disc degeneration. These results indicate the critical role of KLF5 in regulating intervertebral disc metabolism and suggest KLF5 inhibitor such as ML264 as potential compound for treatment of degenerative disc disease.     

Immunolocalization of type X collagen in human lumbar intervertebral discs during ageing and degeneration

 Type X collagen has so far not been reported to occur in human intervertebral discs. The objective of this study was therefore to investigate the occurrence of type X collagen in human lumbar intervertebral discs during ageing and degeneration. Ninety intervertebral discs with adjacent endplates were excised in toto from individuals (0–86 years) without known spinal disease and were processed for routine decalcified histology. Appropriate slices of each disc were processed for immunohistochemistry using a type-spec ific, monoclonal antibody raised against human type X collagen. Each intervertebral disc was examined for macroscopic and histomorphological features of disc degeneration. Immunohistochemically, a positive specific type X staining was observed in the hypertrophic zone of the growth plate and only in the interstitial matrix of juvenile (<2 years) nucleus pulposus. In adult discs, type X collagen could be localized in conjunction with advanced disc degeneration and first occurred in the disc matrix (i.e., pericellular region) of a 47-year-old specimen. Positive type X staining of the disc matrix was more frequently found in senile (>70 years) discs with end stages of disc degeneration. This study provides the first evidence for the occurrence of type X collagen in human lumbar intervertebral discs and it appears that type X collagen is re-expressed in late stages of disc degeneration. Accepted: 24 April 1997     

Annulus Fibrosus Cell Characteristics Are a Potential Source of Intervertebral Disc Pathogenesis

In the end stage of intervertebral disc degeneration, cartilage, bone, endothelial cells, and neurons appear in association with the worsening condition. The origin of the abnormal cells is not clear. This study investigated the properties of progenitor cells in the annulus fibrosus (AF) using one in vitro and two in vivo models. Cultivation of rabbit AF cells with chondrogenic media significantly increased expressions of collagen and aggrecan. Upon exposure to osteogenic conditions, the cultures showed increased mineralization and expression of osteopontin, runx2, and bmp2 genes. Two models were used in the in vivo subcutaneous implantation experiments: 1) rabbit AF tissue in a demineralized bone matrix (DBM) cylinder (DBM/AF), and, 2) rat intact and needle punctured lumbar discs. Bone formation in the AF tissue was detected and hypertrophic chondrocytes and osteoblasts were present 1 month after implantation of the DBM/AF to nude mice. In addition to collagen I and II, immunostaining shows collagen X and osteocalcin expression in DBM/AF specimens 4 months after implantation. Similar changes were detected in the injured discs. Almost the entire needle punctured disc had ossified at 6 months. The results suggest that AF cells have characteristics of progenitor cells and, under appropriate stimuli, are capable of differentiating into chondrocytes and osteoblasts in vitro as well as in vivo. Importantly, these cells may be a target for biological treatment of disc degeneration.     

Interleukin-10 promoter polymorphisms associated with susceptibility to lumbar disc degeneration in a Chinese cohort

We investigated a possible association between interleukin (IL)-10 single nucleotide polymorphisms (SNPs) and susceptibility to and severity of lumbar disc degeneration (LDD) in a Chinese cohort of 320 patients with LDD and 269 gender- and age-matched controls. The degree of disc degeneration was determined by magnetic resonance imaging using Schneiderman's classification. Genetic analysis of IL-10 promoter polymorphisms (at -1082 A/G, -819 T/C, and -592 A/C) was carried out by PCR-RFLP. A total of 134 herniated lumbar intervertebral discs were collected during surgery for IL-10 mRNA detection. For SNPs at -592, the A allele and AA genotype frequencies were significantly higher in LDD patients than in controls. Similarly, the AA genotype and A allele frequencies at -1082 were significantly higher in cases than in controls. Among the LDD subjects, carriers of AA at -592 and GG at -1082 had significantly lower mean IL-10 mRNA expression than the other two genotypes. The SNPs at each locus were not significantly associated with severity grade in the LDD patients. Logistic regression analyses showed that the AA at -1082, AA at -592, and IL-10 mRNA expression level were independent risk factors for LDD. We conclude that the IL-10 SNPs at -1082 A/G and -592 A/C as well as IL-10 mRNA in the herniated lumbar intervertebral discs are associated with susceptibility to LDD in this Chinese cohort, but not with disease severity.     

Sox9治疗兔椎间盘退变的效果及机制研究

目的:探究Sox9用于治疗椎间盘退变的效果及调控机制。方法:将Ad-sox9和Ad-GFP各20μL分别转染至椎间盘退变兔的髓核组织中,转染后3、7、30、60天取材,采用免疫组化、免疫荧光和MRI等研究方法检测椎间盘髓核组织中II型胶原、蛋白多糖的表达情况,并分析对椎间盘退变的改善情况。结果:免疫组化染色显示sox9组中椎间盘髓核组织中II型胶原、蛋白多糖的表达明显升高,MRI显示sox9组椎间盘T2像信号有明显改善(P<0.05)。结论:体内转染腺病毒介导的sox9基因能够增加椎间盘内II型胶原和蛋白多糖的表达,并抑制椎间盘的退变进程。     

Chondrocyte‐like nested cells in the aged intervertebral disc are late‐stage nucleus pulposus cells

Aging is a major risk factor of intervertebral disc degeneration and a leading cause of back pain. Pathological changes associated with disc degeneration include the absence of large, vacuolated and reticular‐shaped nucleus pulposus cells, and appearance of smaller cells nested in lacunae. These small nested cells are conventionally described as chondrocyte‐like cells; however, their origin in the intervertebral disc is unknown. Here, using a genetic mouse model and a fate mapping strategy, we have found that the chondrocyte‐like cells in degenerating intervertebral discs are, in fact, nucleus pulposus cells. With aging, the nucleus pulposus cells fuse their cell membranes to form the nested lacunae. Next, we characterized the expression of sonic hedgehog (SHH), crucial for the maintenance of nucleus pulposus cells, and found that as intervertebral discs age and degenerate, expression of SHH and its target Brachyury is gradually lost. The results indicate that the chondrocyte‐like phenotype represents a terminal stage of differentiation preceding loss of nucleus pulposus cells and disc collapse.     

Upregulation of P53 promotes nucleus pulposus cell apoptosis in intervertebral disc degeneration through upregulating NDRG2

P53 is an apoptosis marker which is involved in determining nucleus pulposus (NP) cell fate. Little is known about P53 interaction with N-Myc downstream-regulated gene 2 (NDRG2) in intervertebral disc degeneration (IVDD). Here, we studied the role of the P53-NDRG2 axis in IVDD. We found that NDRG2 was expressed in NP tissue obtained from patients with IVDD. The level of NDRG2 was positively related to the severity of IVDD, as determined by Pfirrmann grading. Subsequently, we overexpressed NDRG2 in human NP cells by adenoviral transfection and studied the effects of increased levels of NDRG2 on the viability and apoptosis of these cells. NDRG2 overexpression induced NP cell apoptosis and reduced viability in NP cells obtained from patient with IVDD. We also found that the level of P53 was elevated in NP cells from patients with IVDD and treatment with exogenous P53 upregulated NDRG2 in NP cells. Last, IVDD model was established in P53 knockout mice and the pathological changes in the intervertebral discs and NDRG2 expression were examined. P53 knockout can reduce the damage of NP tissues after IVDD surgery to some extent. Restoration of NDRG2 antagonized the effect of P53 knockout on IVDD. Collectively, this study suggests that elevated P53 in NP cells stimulates apoptosis of the cells by upregulating NDRG2 expression, thereby exacerbating IVDD.