BackgroundThe current surgical procedure of choice for lumbar intervertebral disc (IVD) herniation is discectomy. However, defects within IVD produced upon discectomy may impair tissue healing and predispose patients to subsequent IVD degeneration. This study aimed to investigate whether the use of an acellular bioresorbable ultra-purified alginate (UPAL) gel implantation system is safe and effective as a reparative therapeutic strategy after lumbar discectomy.MethodsHuman IVD cells were cultured in a three-dimensional system in UPAL gel. In addition, lumbar spines of sheep were used for mechanical analysis. Finally, the gel was implanted into IVD after discectomy in rabbits and sheep in vivo.FindingsThe UPAL gel was biocompatible with human IVD cells and promoted extracellular matrix production after discectomy, demonstrating sufficient biomechanical characteristics without material protrusion.InterpretationThe present results indicate the safety and efficacy of UPAL gels in a large animal model and suggest that these gels represent a novel therapeutic strategy after discectomy in cases of lumbar IVD herniation.FundGrant-in-Aid for the Ministry of Education, Culture, Sports, Science, and Technology of Japan, Japan Agency for Medical Research and Development, and the Mochida Pharmaceutical Co., Ltd.

Intervertebral disk (IVD) degeneration causes debilitating low back pain in much of the worldwide population. No efficient treatment exists because of an unclear pathogenesis. One characteristic event early in such degeneration is the apoptosis of nucleus pulposus (NP) cells embedded in IVDs. Excessive biomechanical loading may also be a major etiology of IVD degeneration. The present study used in vitro and in vivo models of compressive loading to elucidate the underlying mechanism of IVD degeneration. In addition, we investigated whether the inhibition of apoptosis is a potential clinical therapeutic strategy for the treatment of IVD degeneration induced by biomechanical stress. A TUNEL assay showed that NP cell-agarose three-dimensional composite cultures subjected to uniaxial, unconfined, static, compressive loading exhibited a time-dependent increase in apoptosis. Western blot analysis revealed the up-regulation of several extracellular matrix-degrading enzymes and down-regulation of tissue inhibitor of metalloproteinase 1. These responses to compressive loading were all significantly inhibited by caspase 3 siRNA. In the in vivo model of compressive loading-induced IVD degeneration, a single local injection of caspase 3 siRNA significantly inhibited IVD degeneration by magnetic resonance imaging, histological findings, IHC, and TUNEL assay. The present study suggests that caspase 3 siRNA attenuates overload-induced IVD degeneration by inhibiting NP cell apoptosis and the expression of matrix-degrading enzymes.

Although human intervertebral disc degeneration can lead to several spinal diseases, its pathogenesis remains unclear. This study aimed to create a new histological classification applicable to an in vivo mouse intervertebral disc degeneration model induced by needle puncture. One hundred six mice were operated and the L4/5 intervertebral disc was punctured with a 35- or 33-gauge needle. Micro-computed tomography scanning was performed, and the punctured region was confirmed. Evaluation was performed by using magnetic resonance imaging and histology by employing our classification scoring system. Our histological classification scores correlated well with the findings of magnetic resonance imaging and could detect degenerative progression, irrespective of the punctured region. However, the magnetic resonance imaging analysis revealed that there was no significant degenerative intervertebral disc change between the ventrally punctured and non-punctured control groups. To induce significant degeneration in the lumbar intervertebral discs, the central or dorsal region should be punctured instead of the ventral region.

BackgroundIntervertebral disc degeneration is a significant cause of degenerative spinal diseases. Nucleus pulposus (NP) cells reportedly fail to survive in large degenerated discs with limited nutrient availability. Therefore, understanding the regulatory mechanism of the molecular response of NP cells to nutrient deprivation may reveal a new strategy to treat disc degeneration. This study aimed to identify genes related to nutrient deprivation in NP cells on a global scale in an experimental nutrient deprivation model.Methodology/Principal FindingsRat NP cells were subjected to serum starvation. Global gene expression was profiled by microarray analysis. Confirmation of the selected genes was obtained by real-time polymerase chain reaction array analysis. Western blotting was used to confirm the expression of selected genes. Functional interactions between p21Cip1 and caspase 3 were examined. Finally, flow cytometric analyses of NP cells were performed. Microarray analysis revealed 2922 differentially expressed probe sets with ≥1.5-fold changes in expression. Serum starvation of NP cells significantly affected the expression of several genes involved in DNA damage checkpoints of the cell cycle, including Atm, Brca1, Cdc25, Gadd45, Hus1, Ppm1D, Rad 9, Tp53, and Cyclin D1. Both p27Kip1 and p53 protein expression was upregulated in serum-starved cells. p21Cip1 expression remained in NP cells transfected with short interfering RNA targeting caspase 3 (caspase 3 siRNA). Both G1 arrest and apoptosis induced by serum starvation were inhibited in cells transfected with caspase 3 siRNA.Conclusions/SignificanceNutrient deprivation in NP cells results in the activation of a signaling response including DNA damage checkpoint genes regulating the cell cycle. These results provide novel possibilities to improve the success of intervertebral disc regenerative techniques.

BackgroundAnalgesic discography (discoblock) can be used to diagnose or treat discogenic low back pain by injecting a small amount of local anesthetics. However, recent in vitro studies have revealed cytotoxic effects of local anesthetics on intervertebral disc (IVD) cells. Here we aimed to investigate the deteriorative effects of lidocaine and bupivacaine on rabbit IVDs using an organotypic culture model and an in vivo long-term follow-up model.MethodsFor the organotypic culture model, rabbit IVDs were harvested and cultured for 3 or 7 days after intradiscal injection of local anesthetics (1% lidocaine or 0.5% bupivacaine). Nucleus pulposus (NP) cell death was measured using confocal microscopy. Histological and TUNEL assays were performed. For in vivo study, each local anesthetic was injected into rabbit lumbar IVDs under a fluoroscope. Six or 12 months after the injection, each IVD was prepared for magnetic resonance imaging (MRI) and histological analysis.ResultsIn the organotypic culture model, both anesthetic agents induced time-dependent NP cell death; when compared with injected saline solution, significant effects were detected within 7 days. Compared with the saline group, TUNEL-positive NP cells were significantly increased in the bupivacaine group. In the in vivo study, MRI analysis did not show any significant difference. Histological analysis revealed that IVD degeneration occurred to a significantly level in the saline- and local anesthetics-injected groups compared with the untreated control or puncture-only groups. However, there was no significant difference between the saline and anesthetic agents groups.Conclusions/SignificanceIn the in vivo model using healthy IVDs, there was no strong evidence to suggest that discoblock with local anesthetics has the potential of inducing IVD degeneration other than the initial mechanical damage of the pressurized injection. Further studies should be performed to investigate the deteriorative effects of the local injection of analgesic agents on degenerated IVDs.