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Study Design: Systematic review., Objective: The aim of this study was to investigate, quantify, compare, and compile the various mesenchymal stem cell (MSC) tissue sources within human spinal tissues to act as a compendium for clinical and research application., Summary of Background Data: Recent years have seen a dramatic increase in academic and clinical understanding of human MSCs. Previously limited to cells isolated from bone marrow, the past decade has illicited the characterization and isolation of human MSCs from adipose, bone marrow, synovium, muscle, periosteum, peripheral blood, umbilical cord, placenta, and numerous other tissues. As researchers explore practical applications of cells in these tissues, the absolute levels of MSCs in specific spinal tissue will be critical to guide future research., Methods: The PubMED, MEDLINE, EMBASE, and Cochrane databases were searched for articles relating to the harvest, characterization, isolation, and quantification of human MSCs from spinal tissues. Selected articles were examined for relevant data, categorized according to type of spinal tissue, and when possible, standardized to facilitate comparisons between sites., Results: Human MSC levels varied widely between spinal tissues. Yields for intervertebral disc demonstrated roughly 5% of viable cells to be positive for MSC surface markers. Cartilage endplate cells yielded 18,500 to 61,875 cells/0.8 mm thick sample of cartilage end plate. Ligamentum flavum yielded 250,000 to 500,000 cells/g of tissue. Annulus fibrosus fluorescence activated cell sorting treatment found 29% of cells positive for MSC marker Stro-1. Nucleus pulposus yielded mean tissue samples of 40,584 to 234,137 MSCs per gram of tissue., Conclusion: Numerous tissues within and surrounding the spine represent a consistent and reliable source for the harvest and isolation of human MSCs. Among the tissues of the spine, the annulus fibrosus and ligamentum flavum each offer considerable levels of MSCs, and may prove comparable to that of bone marrow., Level of Evidence: 5.

Study Design: Experimental animal study., Objective: This study investigated whether nitric oxide (NO) mediated protein nitration is involved in the pathogenesis of radiculopathy and whether the symptoms can be relieved by its suppression., Summary of Background Data: It has been reported that nitration of protein mediated by NO is involved in the degenerative neurological disorders, but its involvement is not clear in the radiculopathy., Methods: Two kinds of rat models of radiculopathy were used. Radiculopathy was induced either by ligation of spinal nerve roots or transplantation of autologous nucleus pulposus. In separate groups of rats, aminoguanidine, a potent nitric oxide synthetase inhibitor, was administered just before induction of radiculopathy, to suppress NO production and resultant nitration of protein. Sensation of the hind limb was evaluated by plantar stimulation test, and motor weakness was assessed by observation of gait pattern. Nitrotyrosine, product of protein nitration, was assayed quantitatively by Western immunoblotting., Results: Mechanical allodynia was observed in both compression and nucleus pulposus groups, but motor weakness was observed only in the compression group. Preoperative administration of aminoguanidine attenuated mechanical allodynia and motor weakness. Optical densities of nitrotyrosine bands increased significantly in radiculopathy groups, but they were lowered by administration of aminoguanidine., Conclusion: NO mediated protein nitration contributes to the development of both types of radiculopathies. Suppression of NO production can decrease protein nitration and relieve neural dysfunctions of radiculopathy., Level of Evidence: N/A.

Study Design: This longitudinal experimental study was conducted to investigate electrophysiologic characteristics, pain behavior, and histological changes in a rat model of lumbar disc herniation., Objective: To observe abnormal spontaneous activity (ASA) on needle electromyography (EMG) and to determine its relation with neuropathic pain behavior and histological changes longitudinally in a rat model of lumbar disc herniation., Summary of Background Data: Needle EMG is generally performed to determine the existence and the degree of radiculopathy caused by disc herniation. The local application of autologous nucleus pulposus to the spinal nerve has been shown to induce neuropathic pain. However, little is known about the relations between neuropathic pain and abnormal EMG findings and the manner in which they change with time in rat models of lumbar disc herniation., Methods: Twenty-five Sprague-Dawley rats were randomly assigned to either sham or experimental groups. In the experimental group, autologous nucleus pulposus was grafted on the left L5 dorsal root ganglion. All rats were evaluated for mechanical allodynia and thermal hyperalgesia and underwent needle EMG examinations before and on days 1, 5, 10, 20, 30, 40, and 50 after surgery. Morphologic changes of L5 spinal nerves and of sciatic nerves were assessed by toluidine blue staining on days 1, 5, and 50 after surgery., Results: A dramatic decrease in mechanical withdrawal threshold and in thermal withdrawal latency was observed on day 1 after surgery, and these changes persisted until day 50 after surgery. ASAs on needle EMG were observed on day 1 (33%), peaked on day 5 (93%), and gradually decreased from day 10 (69%) to day 40 (18%) after surgery. Pathologic findings of nerve fibers, such as swelling of myelin sheaths, demyelination, and degeneration of axoplasms were observed from day 1. These findings were exaggerated on day 5 and then diminished but were still evident on day 50., Conclusion: Neuropathic pain and pathologic changes in spinal nerve fibers probably remain even after ASAs in EMG have disappeared in our rat model of lumbar disc herniation. These results provide baseline data concerning the natural courses of electrophysiologic findings and of radicular pain in patients with intervertebral disc herniation.

Study Design: Behavior, mRNA and immunohistochemical assessment of the expression of fractalkine (CX3CL1) and its receptor (CX3CR1) in a rat model of disc herniation by autologous nucleus pulposus (NP) implantation., Objective: To evaluate the changes in expression of fractalkine and its receptor in the spinal cord and their association with pain behavior in a rat model of disc herniation., Summary of Background Data: Chemokines have recently been implicated in the pathophysiology of neuropathic pain. They mediate astrocytic migration and microglial proliferation, which are involved in the regulation of nociceptive transmission. Fractalkine is a chemokine, which participates in the mechanisms of neuropathic pain as a mediator of neuron-glia interactions., Methods: Sixty-six rats (NP-treated = 47, sham = 19) were implanted with autologous NP (approximately 3 mg) on the left L5 nerve root, just proximal to the dorsal root ganglion and tested for thermal hyperalgesia and mechanical allodynia before surgery and on days 1, 5, 10, 20, and 30 after surgery. The changes of expression of fractalkine and its receptor in the spinal cord were studied using real time PCR and immunohistochemistry., Results: Rats developed ipsilateral mechanical allodynia at day 1 and bilateral thermal hyperalgesia at day 5 after surgery, and these changes in sensitivity persisted throughout the observation period. The expression of mRNA for fractalkine in the spinal cord was increased by day 5 and remained upregulated for the duration of the experiment. The immunostaining for fractalkine increased in neurons and astrocytes and that for the fractalkine receptor increased in microglia in the dorsal horn ipsilateral to the disc herniation., Conclusion: Our results indicate that autologous implantation of NP induces thermal hyperalgesia and mechanical allodynia, and leads to an upregulation of fractalkine and its receptor in spinal neurons and glia, implicating fractalkine in association with radicular pain.

Study Design: Controlled, interventional, animal study., Objective: To examine the involvement of high-mobility group box 1 (HMGB1) in the neuropathic pain state induced by the application of nucleus pulposus onto the dorsal root ganglion (DRG) and to investigate the effect of HMGB1 neutralization antibody in the pathogenesis., Summary of Background Data: HMGB1 is a potent proinflammatory mediator when present extracellularly, and anti-HMGB1 neutralization antibody inhibits inflammation, cytokine expression, and macrophage activation., Methods: Thirty-nine adult female Sprague-Dawley rats (200-300 g) were used. The left L5/6 facet joint was removed, and the L5 DRG was exposed. Nucleus pulposus harvested from the tail was applied to the left L5 DRG. Then, 400 μg of anti-HMGB1 neutralization antibody was administered intraperitoneally after surgery. Behavioral testing using von Frey hairs was performed to investigate the mechanical withdrawal threshold. Neuronal damage was investigated by counting the number of activating transcription factor 3 (ATF3) neurons. The expressions of tumor necrosis factor-α (TNF-α) and HMGB1 were measured by double-labeled immunohistochemistry and immunoblotting., Results: Immunoblotting of harvested nucleus pulposus revealed HMGB1 in the nucleus pulposus. Double-labeled immunohistochemistry revealed that macrophages in the applied nucleus pulposus expressed HMGB1 and TNF-α. Administration of anti-HMGB1 neutralization antibody significantly reduced the TNF-α expression in the DRG and improved the pain-related behavior from day 2 to day 14., Conclusion: HMGB1 appears to play an important role in the development of pain-related behavior induced by the application of nucleus pulposus onto the DRG. HMGB1 from applied nucleus pulposus and actively secreted from macrophages would act as a proinflammatory mediator together with proinflammatory cytokines. HMGB1 blocking therapy might become a new treatment method for neuropathic pain.

Study Design: this study was designed to examine the neuroprotective effects of asialo-erythropoietin (A-EPO) in a rat model of lumbar disc herniation., Objective: to investigate the effects of A-EPO on pain-related behavior, the expression of phosphorylated-p38 (p-p38) mitogen activated kinase, and the expression of tumor necrosis factor alpha (TNF-α) induced by nucleus pulposus (NP) application on the nerve root., Summary of Background Data: erythropoietin (EPO) has neuroprotective effects in a variety of models of central and peripheral nerve injuries. However, EPO is a hematopoietic growth factor and can therefore cause significant side effects such as thicker blood and promotion of blood clotting. A-EPO is a neuroprotective derivative of EPO that is not hematopoietic., Methods: female Sprague-Dawley rats (n = 149) were used in this study. NP harvested from the tail was applied to the left L5 nerve root and the rats were then divided into four groups: NP + nontreatment group, no further treatment; NP + A-EPO group, 13.4 microg/kg A-EPO; NP + EPO group, 13.4 microg/kg EPO; and NP + vehicle group, received vehicle. The substances were administered subcutaneously 1 day before surgery and daily for 2 weeks. In the sham group of animals, the L5 nerve root was exposed and NP was not applied. Withdrawal thresholds were determined by the von-Frey test 28 days after surgery. The expressions of p-p38 and TNF-α were assessed by immunohistochemical and immunoblotting analysis. Data were analyzed by unpaired Student t test and Dunnett t test (significance level, P < 0.05)., Results: in the NP + nontreatment and NP + vehicle groups, withdrawal thresholds were decreased significantly for 28 days compared with the sham group (P < 0.05). In the NP + A-EPO group, the thresholds were significantly increased on day 28, and in the NP + EPO group, the thresholds were significantly increased on days 21 and 28 (P < 0.05) compared with the NP + nontreatment and NP + vehicle groups. The expression of p-p38 in the NP + A-EPO group was significantly lower than that in the NP + vehicle group on day 1 (P < 0.05). The expression of TNF in the NP + A-EPO and NP + EPO groups was significantly lower than that in the NP + vehicle group on days 1 and 7 (P < 0.05)., Conclusions: A-EPO improved pain-related behavior and reduced the expression of p-p38 and TNF-α. The effect of A-EPO may be related to the inhibitory action of p-p38 and TNF-α in the dorsal root ganglion.

Study Design: A novel approach combining a rodent disc herniation model with electrophysiologic recordings of thalamic evoked responses., Objective: To assess short-term effects of nucleus pulposus (NP) application on dorsal root ganglions (DRG) on high threshold afferent fiber evoked activation in the thalamus., Summary of Background Data: Epidural application of NP in combination with mechanical compression induces pain related behavior in rats associated with enhanced activity of pain-processing neurons in the dorsal horn of the spinal cord. However, possible effects on neuronal activity in the pain processing ventral posterior lateral (VPL) thalamic nucleus following NP application on DRG have not been investigated., Methods: Responses in the contralateral VPL evoked by electrical stimulation of the sciatic nerve and of the fourth lumbar (L4) DRG were recorded in adult Sprague-Dawley rats. Records were obtained before and during application (5, 10, and 20 minutes) of NP or of adipose tissue (AT) to the L4 DRG. AT was used as control for mechanical effects of NP application., Results: Application of NP resulted in an increase of evoked thalamic responses to 138% +/- 10% of control after 20 minutes (P < 0.01), whereas AT application for 20 minutes resulted in a reduction of evoked responses to 77% +/- 4% (P < 0.05). Recordings in control animals (i.e., with no application) demonstrated stable evoked neuronal thalamic activity for up to 40 minutes., Conclusion: The study demonstrates that NP application onto DRG increases afferent fiber evoked responses in the thalamus and in view of the opposite effects of AT application suggests that these effects may be specific for NP. The results show that NP affects sensory transmitting pathways within a few minutes, possibly due to rapid and reversible alterations in the neuronal excitability. The study thus introduces a rodent model for studying sensory afferent evoked thalamic activity related to DRG injury which may be used to evaluate analgesics and anti-inflammatory drugs used for pain relief in disc herniation and neuropathic pain patients.

Study Design: An experimental animal study., Objective: To study if antitumor necrosis factor-alpha (TNF-alpha) antibody, which is administered at different times, reduces the pain behavior induced by application of nucleus pulposus (NP) to the nerve root., Summary of Background Data: Treatment with TNF-alpha inhibitor reduces the pain-related behavior induced by epidural application of NP in rats., Methods: Left L5 partial laminectomy was performed and NP was applied to the L5 nerve root in 24 rats. The rats were divided into 4 groups. In 3 groups, anti-rat TNF-alpha antibody was intravenously administered immediately after, or 6 or 20 days after NP application. The fourth group was not treated with anti-rat TNF-alpha antibody (untreated rats). The withdrawal threshold of the plantar surface was determined 1 day before up through 28 days after NP application., Results: The withdrawal threshold of rats that had been treated with anti-rat TNF-alpha antibody immediately after or 6 days after, but not 20 days after, NP application, was significantly higher than that of the untreated rats., Conclusions: Anti-TNF-alpha antibody reduced allodynia only when it was administered soon after the onset of allodynia. Late administration of anti-TNF-alpha antibody did not have an antiallodynic effect.

Study Design: Assessment of activated T and B cells in a subcutaneous chamber filled with autologous nucleus pulposus using flow cytometry and immunohistochemistry., Objectives: To examine if subcutaneously placed autologous nucleus pulposus may attract activated T and B cells in an animal model., Summary of Background Data: Nucleus pulposus has been suggested to trigger an autoimmune response if exposed to the immune system, for example, in association with disc herniation. T-cell activation represents a hallmark in the generation of an autoimmune response, subsequently leading to the differentiation of B cells, but a causal association between the exposure of nucleus pulposus to the systemic circulation and T and B cell activation is still lacking., Methods: Autologous nucleus pulposus was harvested from the intervertebral disc of 9 pigs and placed subcutaneously in perforated titanium chambers. In order to control for the effect of the titanium chamber, an additional empty chamber was placed subcutaneously in each pig. After 7 days, the pigs were killed and the chambers were harvested. Flow cytometry and immunohistochemistry were used for analysis of T-helper cells (CD4+), cytotoxic T cells (CD8+), and B cells (Igkappa) in the chamber exudates and T cells (CD45RC) in the remaining blood clot tissue of the chamber., Results: As compared with the empty chambers, the proportion of activated T cells (CD4+ and CD8+) was significantly higher in the exudate of the nucleus pulposus filled chamber. The proportion of activated B cells expressing immunoglobulin kappa (Igkappa) was also significantly elevated in the exudate of the nucleus pulposus chambers. The analysis of the remaining chamber tissue revealed a significantly higher amount of T cells (CD45RC) in the nucleus pulposus chambers than in the empty chambers., Conclusions: The present findings indicate that nucleus pulposus attracts activated T and B cells. However, since the cell population in the nucleus pulposus of young pigs may differ from that of adult humans, the obtained data may not be directly transferred to the human situation of a disc herniation. The observations in the present study may nevertheless explain some of the local tissue reactions occurring in association with disc herniation and nerve root involvement, thereby providing further insight into the pathophysiology of sciatica.

Study Design: Immunocytochemistry for acid-sensing ion channel 3 (ASIC3) in neurons of rat dorsal root ganglions (DRGs) from animals exposed to a model of lumbar disc herniation., Objective: To examine expression of ASIC3 in DRGs and the effect of a sodium channel blocker applied to the nerve root in a rat model of lumbar disc herniation., Summary of Background Data: Radicular pain is a common symptom of lumbar disc herniation in human beings. A depolarizing sodium channel gated by protons during tissue acidosis, ASIC3, is specifically expressed in sensory neurons. It has been associated with cardiac ischemic and inflammatory pain. We often perform spinal nerve root block for radicular pain using a sodium channel blocker, such as lidocaine; however, it has been unclear whether the effective period of this treatment is usually longer than the expected duration of efficacy., Methods: For the lumbar disc herniation model, nucleus pulposus was harvested from the tail and applied to the L5 nerve root, and the nerve roots were pinched. We evaluated mechanical allodynia in sham-operated animals and a disc herniation model. Immunohistochemistry was used to examine ASIC3 expression in L5 DRGs. Finally, the effect of lidocaine on pain and ASIC3 expression in the disc herniation model was examined., Results: Animals exposed to the lumbar disc herniation model showed allodynia for 8 days, and ASIC3 immunoreactivity was up-regulated in DRG neurons. After administration of lidocaine to spinal nerve roots affected by disc herniation, ASIC3 immunoreactivity was down-regulated in DRG neurons, and the level of mechanical allodynia was significantly decreased for 8 days., Conclusions: Our results suggest that ASIC3 in DRG neurons may play an important role in nerve root pain caused by lumbar disc herniation. Lidocaine decreased ASIC3 expression in DRG neurons and pain associated with the disc herniation model.

Study Design: This study was conducted to investigate the electromyographic changes and their pathophysiologic background in the animal model of nucleus pulposus-induced radiculopathy., Objectives: To observe the abnormal spontaneous activities in the electromyography (EMG) of rats with nucleus pulposus-induced radiculopathy and assess the role of nitric oxide in their development., Summary of Background Data: It has been shown that application of nucleus pulposus to nerve roots induces changes consistent with radiculopathy. However, to our knowledge, electromyographic findings and their background have never been studied in this model of radiculopathy., Methods: Autologous nucleus pulposus was harvested from the tails of Sprague-Dawley rats, and applied to L4 and L5 nerve roots. The rats were tested for mechanical allodynia, motor paresis, and needle EMG, before and after surgery. Specimens of nerve roots were stained histochemically for nitrotyrosine., Results: The rats had mechanical allodynia after surgery, but motor paresis was absent. EMG showed abnormal spontaneous activities after surgery, but only temporarily. Immunoreactivity for nitrotyrosine was detected in the cell bodies and axons of nerve roots., Conclusions: The data indicate that abnormal spontaneous activities can be observed in electromyographic examination of nucleus pulposus-induced radiculopathy. The development of these activities is considered related to nitric oxide-mediated protein nitration and resultant axonal dysfunction.

Study Design: Assessment of the potential use of an immortalized human nucleus pulposus cell line as an alternative cell source in cell transplantation therapy for intervertebral disc degeneration., Objectives: To evaluate the effect of transplanting the human nucleus pulposus cell line into a disc degeneration model in rabbits and to define whether it is capable of becoming an alternative cell source for cell transplantation therapy for disc degeneration., Summary of Background Data: Interest in cell transplantation therapy for disc degeneration has been growing for several years, and a range of different cell types have been examined as possible donor cells. In addition, the establishment of a novel cell line that possesses some of the major characteristics of a normal human nucleus pulposus cells has been reported., Methods: Human nucleus pulposus cell line was established, and cells were transplanted into a rabbit disc degeneration model. At 4, 8, and 24 weeks after transplantation, inhibition of intervertebral disc degeneration was assessed by examining the disc height, macroscopic appearance, histologic findings, and immunohistochemistry. In addition, aggrecan, versican, and Type II collagen gene expression in the nucleus pulposus were measured semiquantitatively at the mRNA level. Furthermore, the survival of transplanted cells was examined using immunohistochemistry for Simian Virus 40 T antigen, and the presence of graft-versus-host reaction was assessed by immunohistochemistry for CD4 and CD58., Results: The disc height was significantly greater in the transplanted group than in the degenerative group's disc from 4 weeks' posttransplantation. Macroscopically, the nucleus pulposus was absent and there was loss of disc height in the degenerative group at 24 weeks after transplantation, whereas the nucleus pulposus was preserved in the transplanted group. Histologic examination showed that the structure of the inner anulus fibrosus was significantly preserved in the transplanted group, and the boundary between the nucleus and anulus could be clearly visualized. Expression of mRNAs of the nucleus pulposus matrix, aggrecan, and Type II collagen was significantly greater in the transplanted group than in the degenerative group. This indicates that transplantation of human nucleus pulposus cell line helped to preserve the matrix of the nucleus pulposus. Thus, transplantation of a human nucleus pulposus cell line was shown to delay disc degeneration in this rabbit model., Conclusion: The human nucleus pulposus cell line may become an alternative cell source for cell transplantation therapy of intervertebral disc degeneration.

Study Design: A prospective, nonblinded, multicenter study., Objective: To determine the patterns of allograft incorporation and new bone formation after ALIF using stand-alone threaded cortical allograft dowels with either autograft or rhBMP-2., Summary of Background Data: Accelerated healing patterns of allograft dowels have been reported in an animal study and a small human study of ALIF with rhBMP-2 and bone dowels., Methods: We enrolled and randomly assigned 131 patients with lumbar spondylosis who were undergoing single-level ALIF with allograft dowels to either the investigational group who received rhBMP-2 (79 patients) or the control group who received autologous bone graft (52 patients). Independent radiologists used plain radiographs and computed tomography scans to evaluate fusion. Allograft incorporation was assessed using a modified Bridwell-Lenke scale, and changes in bone density within the adjacent vertebral bodies were monitored., Results: At 12 months, all investigational patients had radiographic evidence of new bone formation and incorporation of the allografts into the adjacent vertebral endplates; there were no changes in fusion status at 24 months. Fusions were documented in 89% of patients in the control group at 12 months. The percentage declined to 81.5% at 24 months with 10% of the patients in the autograft group showing incomplete healing and 11% having no healing of the allograft dowels. On CT scans, 14 of the investigational patients (14 of 79; 18%) developed a transient, localized area of bone remodeling within the vertebral body adjacent to the allograft dowel. All were healed by 24 months. The transient appearance of these zones had no effect on clinical outcomes. No patient in the control group developed bone remodeling zones., Conclusion: We think that the use of rhBMP-2 is a promising method of facilitating allograft dowel incorporation and new bone formation in patients undergoing ALIF with allograft dowels.

Study Design: An experimental study in the pig with autologous transfer of nucleus pulpous and retroperitoneal fat to the subcutaneous space of the back., Objectives: To evaluate if there is neovascularization or neoinnervation of subcutaneously placed nucleus pulposus, in comparison to retroperitoneal fat, and under simultaneous treatment by certain antiangiogenetic drugs., Summary of Background Data: It has been suggested that intervertebral discs may be invaded by newly formed blood vessels and nerve fibers following injury of the anulus fibrosus. The nerve fibers have been considered to induce low back pain. However, it is still debated whether such ingrowth may occur and, if present, if this is based on the action of angiogenetic substances in the intervertebral disc or simply by normal would healing., Methods: In the first series, autologous nucleus pulposus and retroperitoneal fat was placed subcutaneously in 3 pigs. In the second series, autologous nucleus pulposus was placed subcutaneously with simultaneous treatment with methotrexate (n = 3), celecoxib (n = 3), doxycycline (n = 3), and infliximab (n = 3). After 7 days, the tissue was collected and processed immunohistochemically for the visualization of blood vessels and nerve fibers., Results: There was a number of blood vessels and nerve fibers in the nucleus pulposus samples, while no vessels were observed in the fat samples. Neither methotrexate nor celecoxib seemed to be able to reduce the ingrowth of blood vessels (neovascularization) or nerve fibers (neoinnervation). Treatment by doxycycline and infliximab markedly reduced both neovascularization and neoinnervation., Conclusions: Subcutaneously placed autologous nucleus pulposus displays an ingrowth of newly formed blood vessels and nerve fibers within 7 days, in contrast to retroperitoneal fat. Such ingrowth seems to be reduced by doxycycline and infliximab, 2 cytokine inhibitors. The data suggest that the ingrowth may be induced by bioactive substances within the nucleus pulposus. The clinical importance of these data has yet to be elucidated.

Study Design: By the technique of tissue engineering, composite intervertebral disc implants were fabricated as novel materials for disc replacement, implanted into athymic mice, and removed at times up to 12 weeks., Objectives: The goal of this study was to construct composite intervertebral disc structures consisting of anulus fibrosus cells and nucleus pulposus cells seeded on polyglycolic acid and calcium alginate matrices, respectively., Summary of Background Data: Previous work has documented the growth of anulus fibrosus cells on collagen matrices and nucleus pulposus cells cultured on multiple matrices, but there is no documentation of composite disc implants., Methods: Lumbar intervertebral discs were harvested from sheep spine, and the nucleus pulposus was separated from surrounding anulus fibrosus. Each tissue was digested in collagenase type II. After 3 weeks in culture, cells were seeded into implants. The shape of the anulus fibrosus scaffold was fabricated from polyglycolic acid and polylactic acid, and anulus fibrosus cells were pipetted onto the scaffold and allowed to attach for 1 day. Nucleus pulposus cells were suspended in 2% alginate and injected into the center of the anulus fibrosus. The disc implants were placed in the subcutaneous space of the dorsum of athymic mice and harvested at 4, 8, and 12 weeks. At each time point, 4 samples were stored in -70 degrees C for collagen typing and analysis of proteoglycan, hydroxyproline, and DNA. Other samples were fixed in 10% formalin for Safranin-O staining., Results: The gross morphology and histology of engineered discs strongly resembled those of native intervertebral discs. Biochemical markers of matrix synthesis were present, increasing with time, and were similar to native tissue at 12 weeks. Tissue-engineered anulus fibrosus was rich in type I collagen but nucleus pulposus contained type II collagen, similar to the native disc., Conclusion: These results demonstrate the feasibility of creating a composite intervertebral disc with both anulusfibrosus and nucleus pulposus for clinical applications.

Study Design: Work presented here used a small animal model to demonstrate the feasibility and usefulness of in vivo bioluminescent imaging to studying degenerative disc disease., Objectives: To determine the utility of in vivo bioluminescent imaging to monitor the temporal and spatial expression of genetically modified cells within the intervertebral disc of a rodent model., Summary of the Background Data: Noninvasive imaging of genetically engineered cells in the spine has the advantage of allowing events to be tracked without killing the animal and can be used to follow the time course of a particular therapy. Results are presented on the use of Sprague-Dawley rats in experimental studies in which the luciferase reporter gene was delivered to the lumbar intervertebral disc through adenovirus-mediated or cell-based transfer techniques to demonstrate the feasibility to monitor gene expression noninvasively over time., Methods: Tissue culture, disc surgery, and in vivo bioluminescent imaging were used. The intervertebral disc of the rat was either injected in situ with an adenovirus containing the luciferase reporter gene or implanted with fat, bone marrow or intervertebral disc cells transduced ex vivo and contained in a bioresorbable carrier. Results were assessed with in vivo bioluminescent imaging at several time points., Conclusion: Data from 11 animals were obtained with imaging up to 14 days. To our knowledge, this is the first description of in vivo bioluminescence imaging to study spinal conditions. We have characterized the relative expression of three cell types transduced with the Ad-luc virus by ex vivo transfection followed by cell implantation in the rat spine and compared them to one another and to direct infection of Ad-luc adenovirus in situ. Our results demonstrate the feasibility of tracing genetically altered cells in the spine. This technique has the potential to be used to noninvasively track the fate and expression of therapeutic genes within the spine of small animals used in disc research.

Study Design: This study analyzed the effects of autografted nucleus pulposus on nerve root axon morphology, neurophysiologic function, and sodium channel expression., Objectives: To investigate the chronic effects of the epidural implantation of nucleus pulposus on nerve root morphology, neural activity, ectopic discharge, mechanosensitivity, and sodium channel expression., Summary of Background Data: It has been reported that ectopic discharges were recorded antidromically from sural nerve on compressing nucleus pulposus exposed spinal nerves. However, it is not clear what the effects of nucleus pulposus are on ectopic discharges recorded directly from the spinal nerve roots. It is also not clear what the effects of nucleus pulposus are on the threshold pressure to provoke ectopic discharges in the spinal nerves. Sodium channel content increases in remodeling axons after nerve injury, but it is not clear what the effects of nucleus pulposus are on sodium channel expression in spinal nerve., Methods: Forty-six male Sprague-Dawley rats were used, 20 in a nucleus pulposus-implanted group, 18 in a fat-implanted group, and 8 in a normal group. Fresh autografted nucleus pulposus or fat tissue was implanted into the dorsal epidural space at the L4-L5 disc level. On the 7th, 21st, or 42nd day, neurophysiologic recordings were made to determine nerve root response to compression. Nerve roots were then harvested to determine sodium channel protein concentration and histologic changes in the nerve root. The correlations between sodium channel density and neural activity and mechanosensitivity of dorsal root were analyzed statistically., Results: Ectopic discharge rate was higher in nucleus pulposus 7-day group. Threshold pressure to evoke ectopic discharges was lower in the nucleus pulposus 7-day group, and higher in the nucleus pulposus 42-day group compared to the normal group. Sodium channel protein density increased in the nucleus pulposus 7-day and nucleus pulposus 21-day group compared to normal nerve. Sodium channel density changes were not correlated to threshold pressure. Ectopic discharge rate increased with increase of sodium channel density in the nerve roots. The number of axons with neuropathy increased in the nucleus pulposus 7-day and 21-day groups., Conclusions: Acute exposure of nerve root to nucleus pulposus resulted in increased number of axons with neuropathy, higher intensity of ectopic discharges on compression, and nerve mechanosensitization. Chronic exposure resulted in mechanical desensitization. Changes of sodium channel density were correlated to ectopic discharge rate.

Study Design: An in vivo experimental study to examine the possibility of using fresh frozen intervertebral disc allograft in disc transplantation., Objectives: To investigate the long-term radiographic, pathologic, biochemical, and biomechanical changes of fresh frozen disc allograft in a bipedal animal model., Summary of Background Data: It has been shown that intervertebral disc autograft is able to survive and maintain some degree of tissue metabolism and segmental mobility after transplantation in a bipedal animal model. However, the long-term results of disc allografting and the associated problems of graft rejection are unknown., Methods: Seventeen rhesus monkeys (15 male, 2 female) between 5 and 8 years of age and weighing between 6.7 and 11.8 kg were used in this study. Of these 17 subjects, two were used as intervertebral disc donors and three were used as controls for the biomechanical testing. The remaining 12 monkeys were randomly divided into a short-term group (n = 4, followed up for 2, 4, 6, and 8 weeks, respectively), a midterm group (n = 6, 6 months), and a long-term group (n = 2, 24 months). Radiologic, histologic, biochemical, and biomechanical changes were investigated., Results: Radiography and macro- and microhistologic examination showed severe disc degeneration at 24 months of follow-up. Disc height decreased mainly in the early postoperative stage. Decreased water, proteoglycan, and hydroxyproline contents of the allograft were observed at 6 and 24 months of follow-up. The biomechanical properties of the transplanted allograft were similar to those of control., Conclusion: Fresh frozen disc allografts can survive and maintain some degree of cell metabolism and segmental mobility at 24 months after transplantation. However, severe disc degeneration is also observed at this stage.

Study Design: Cultured annulus fibrosus cells within an atelocollagen honeycomb-shaped scaffold with a membrane seal were allografted into the lacunas of intervertebral discs of which the nucleus pulposus had been vaporized using an indocyanine green dye-enhanced laser. Regeneration of the intervertebral disc was assessed based on the viability and histologic status of the allografted annulus fibrosus cells, as well as the prevention of narrowing disc space., Objectives: To study the regeneration of intervertebral disc after laser discectomy using tissue-engineering methods., Summary of Background Data: Intervertebral disc is the most avascular tissue in the human body, and its ability to regenerate is as low as that of articular cartilage. When nucleotomy is carried out, little regeneration of the annulus fibrosus is observed; consequently, intervertebral disc degeneration is inevitable., Methods: Annulus fibrosus cells isolated from 20 Japanese white rabbits were labeled with a PKH-26 fluorescent dye and seeded within an atelocollagen honeycomb-shaped scaffold with a membrane seal. Annulus fibrosus cells cultured in atelocollagen honeycomb-shaped scaffold with a membrane seal for 1 week were allografted into the lacunas of intervertebral discs of recipient rabbit of which the nucleus pulposus had been vaporized using an ICG dye-enhanced laser. Soft radiograph photographs of the lumbar spine of these anesthetized rabbits were taken, the disc space measured, and the lumbar spine extracted 2, 4, 8, and 12 weeks after the operation. The proliferation of allografted annulus fibrosus cells with 5-bromo-2'-deoxyuridine/PKH-26 fluorescent labels was assessed using consecutive frozen sections, and safranin-O staining carried out for histologic evaluation., Results: The allografted annulus fibrosus cells were viable and showed proliferation activity with a hyaline-like cartilage being produced. The narrowing of the intervertebral disc space of the cell translation group was significantly prevented, as shown, up to 12 postoperative weeks., Conclusion: The annulus fibrosus cells cultured in an ACHMS-scaffold were allografted into the lacunae of nucleus pulposus (obtained using laser vaporization), as well as the hole of annulus fibrosus (obtained by laser fiber insertion) of rabbit intervertebral discs. These cells were viable and showed cell proliferation in the disc tissues of recipients.

Study Design: Work presented here used a small animal model to illustrate the feasibility of autologous disc cell implantation., Objectives: To develop a small animal model for autologous disc cell implantation., Summary of the Background Data: The use of autologous disc cells in the potential treatment of disc degeneration offers attractive possibilities for novel therapies. Results are presented on the use of the sand rat (Psammomys obesus), a small rodent that spontaneously develops disc degeneration during aging, in experimental studies in which cells were harvested from a lumbar intervertebral disc, expanded in monolayer tissue culture, labeled with agents that allow subsequent immunolocalization of these cells, and implanted in a second disc site of the donor animal., Methods: Tissue culture, disc surgery, histology, and immunocytochemistry were used. Cells were either engrafted in a bioresorbable carrier tested for cell compatibility or injected into the recipient disc. Results were assessed with radiographic examination of the implantation site and with histology and immunocytochemistry., Conclusion: Data from 15 animals were obtained with engraftment resident in the animal for up to 33 weeks. Immunocytologic identification of engrafted cells showed that they integrated into the disc and were surrounded by normal matrix at time points up to 8 months postengraftment. Engrafted cells exhibited either a spindle-shaped morphology in the annulus or a rounded chondrocyte-like morphology in the nucleus. Although technically challenging, the authors' experience showed that autologous disc cell implantation can be successful and that the sand rat is a valuable model for autologous disc cell studies.

Study Design: To analyze the effects of tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma on cauda equina function and to define if any of these cytokines could induce nerve root dysfunction comparable with the situation with application of nucleus pulposus., Summary of Background Data: Proinflammatory cytokines derived from the intervertebral disc have been suggested to mediate the nucleus pulposus-induced nerve root injury following local application of nucleus pulposus. However, it is not known if such cytokines may induce similar injury if applied separately., Methods: A total of 29 pigs were used. Nucleus pulposus was harvested from lumbar discs and applied to the sacrococcygeal cauda equina following laminectomy of the first coccygeal vertebra in seven pigs. Five pigs received 1.66 microg of tumor necrosis factor-alpha, five pigs received 0.85 microg of interleukin-1beta, and five pigs received 1.66 microg of interferon-gamma. Seven pigs received autologous fat for control. Nerve conduction velocity was studied by local electrical stimulation and recordings in the tail muscles 7 days after the application., Results: Application of nucleus pulposus and fat induced similar effects as seen in previous studies, with normal nerve conduction velocity for fat and a significant reduction for nucleus pulposus. Application of both interleukin-1beta and IFN-gamma induced slight reductions of nerve conduction velocity compared with fat, but they were not statistically significant. Tumor necrosis factor-alpha, however, induced a reduction of the velocity that was even more pronounced than for nucleus pulposus., Conclusion: Based on previous observations and the data of the present study, one may conclude that tumor necrosis factor-alpha from nucleus pulposus cells seems to be intimately involved with the basic pathophysiologic events leading to both nerve root dysfunction and pain after local, epidural application of nucleus pulposus. One may therefore also suspect that pharmacologic inhibition of tumor necrosis factor-alpha may at least theoretically be considered in the clinical situation with disc herniation and sciatica.

Study Design: An electrophysiologic study to examine responses of dorsal horn neurons in the rat L5 spinal cord to noxious stimuli after disc herniation or application of the nucleus pulposus to the L5 nerve root., Objectives: To investigate the pathogenic role of nucleus pulposus in the neural mechanism underlying sciatica and low back pain., Summary of Background Data: Application of the nucleus pulposus to the lumbar nerve root induced morphologic, vascular, and functional changes in the nerve root, suggesting that some factors in the nucleus pulposus may be implicated in the pathogenesis of chronic changes. However, it has not been studied whether the epidural application of nucleus pulposus enhances nociresponses of pain-processing neurons in the superficial dorsal horn of the spinal cord., Methods: Extracellular recordings were made from the L5 dorsal horn neurons in 20 Wistar rats. The wide-dynamic-range (WDR) neurons activated by electric stimulation of the ipsilateral footpad of hindpaw were selected, and their responses to noxious and innocuous stimulation were examined after L5-L6 disc herniation onto the L5 nerve root (Group A) and application of the autologous nucleus pulposus (Group B) or fat tissues (Groups C and D)., Results: The herniation of the nucleus pulposus (Group A) and application of the autologous nucleus pulposus (Group B) to the nerve root remarkably enhanced responses of WDR neurons to noxious stimuli for hours, whereas application of fat tissue scarcely enhanced nociresponses (Groups C and D)., Conclusions: Results suggest that somewhat pathogenic factors in the nucleus pulposus may have a crucial role in the induction of hyperalgesia. This may help to elucidate the reason why a severe pain is sometimes induced without a visually identified protrusion.

Study Design: This study analyzed the effects of the autologous nucleus pulposus (NP) on nerve root function using electrophysiologic methods., Objectives: To investigate the chronic effects of the NP applied to nerve roots and dorsal root ganglions (DRGs) on ectopic firing and nerve conduction velocity., Summary of Background Data: It was reported that ectopic firing in DRGs is involved in abnormal sensation such as sciatica and that ectopic firing was increased shortly after application of the NP to nerve roots. It was also reported that various inflammatory reactions occur around herniated NP clinically, which may affect nerve roots. However, the chronic action of NP on ectopic firing in the stage of inflammatory reaction is unknown., Methods: Forty-six rats were divided into three groups: nucleus pulposus (NP) group, sham group, and control group. In the NP group the autologous NP was applied to L4 and L5 nerve roots. At 1, 2, 4, and 8 weeks after treatment, antidromically propagated ectopic firing from nerve roots was recorded from the sural nerve, and the nerve conduction velocity between the cauda equina and sciatic nerve was analyzed. The frequencies of ectopic firing in the three groups were compared. The ratios of the nerve conduction velocities on the treatment side and opposite side were compared between the NP and sham groups., Results: The frequency was significantly higher in the NP group than in the sham group at 4 weeks. There were no significant differences among the three groups at 8 weeks. There was no significant difference in the treatment side/opposite side ratio of nerve conduction velocity between the NP and sham groups at any time point., Conclusions: Increased ectopic firing in the stage of inflammatory reaction to NP may be related to abnormal sensation.

Study Design: An experimental study was conducted to evaluate the effects of lidocaine on nucleus pulposus-induced pathophysiologic changes., Objectives: To investigate the effects of lidocaine on blood flow in the hind paws and endoneurial fluid pressure in the dorsal root ganglia in a rat model of herniated nucleus pulposus, and to clarify the therapeutic mechanisms of nerve root infiltration., Summary of Background Data: It has been shown experimentally that application of nucleus pulposus to the nerve roots increases endoneurial fluid pressure and decreases blood flow in the dorsal root ganglia and the corresponding hind paw. These changes are thought to be an important pathogenic mechanism associated with sciatica caused by disc herniation. Nerve root infiltration is one of the nonoperative effective therapies for radiculopathy caused by disc herniation. However, the therapeutic mechanisms still are unknown., Methods: For this study, 21 Sprague-Dawley rats were used. Autologous nucleus pulposus was applied to the nerve root with a piece of Spongel containing lidocaine (lido group) or physiologic saline solution (control group). In Series 1 of this study (Blood Flow in the Hind Paw), blood flow in the corresponding hind paws was monitored continuously using a laser Doppler flowmeter before application of the test solutions, and every 5 minutes thereafter for an additional 3 hours in both the control (n = 5) and lido (n = 5) groups. In Series 2 of this study (Endoneurial Fluid Pressure in the Dorsal Root Ganglion), endoneurial fluid pressure was recorded with a servo-null micropipette system using glass micropipettes before and 3 hours after application of the test solutions in both the control (n = 6) and lido (n = 5) groups. After measurements, dorsal root ganglia were assessed for histology., Results: In Series 1, blood flow in the corresponding hind paw in the control group showed significant reduction as compared with that of the Lido group, starting about 90 minutes after application (P < 0.01-0.05). Hind paw blood flow in the lido group did not show any reduction during measurements. In Series 2, the value of endoneurial fluid pressure in the lido group 3 hours after application was significantly lower than in the control group (P < 0.01). Interstitial (endoneurial) edema in the dorsal root ganglion in the lido group appeared to be qualitatively less than in the control group., Conclusions: The data indicate that lidocaine reduces the pathophysiologic changes in the dorsal root ganglion and hind paws induced by nucleus pulposus. These effects of lidocaine may relate to the mechanisms underlying the therapeutic effects of nerve root infiltration.

Study Design: Nerve conduction velocity was measured in the pig cauda equina after local application of anulus fibrosus or in vitro/postmortem degenerated nucleus pulposus from the same pig., Objectives: To analyze the effects of anulus fibrosus and degenerated nucleus pulposus on nerve conduction velocity., Summary of Background Data: Previous studies on nucleus pulposus-induced effects on nerve roots have used normal, nondegenerated nucleus pulposus. Because both anulus fibrosus and degenerated nucleus pulposus are commonly seen in the clinical situation of disc herniation, the value of the previous work could be questioned., Methods: Anulus fibrosus and nucleus pulposus were harvested using a retroperitoneal approach. The nucleus pulposus was degenerated artificially either by addition of sodium lactate with HCl added to form a pH of either 6.0 or 3.5 (in vitro degeneration), or by storing the nucleus pulposus at 4 C until a pH of 6.0 (postmortem degeneration) was reached. After epidural application, the nerve conduction velocity was determined at 7 days (anulus fibrosus) or 3 days (degenerated nucleus pulposus)., Results: Application of anulus fibrosus did not induce any reduction of nerve conduction velocity. In vitro and postmortem degenerated nucleus pulposus induced a reduction of nerve conduction velocity similar to that of normal nucleus pulposus., Conclusions: Although only nerve function and not pain was assessed, it seems likely that previous experiments using normal nucleus pulposus may be relevant for evaluating the pathophysiologic mechanisms behind the nucleus pulposus-induced nerve root injury, also in a clinical perspective.

Study Design: We have developed a new artificial intervertebral disc consisting of triaxial three-dimensional fabric for the sheep lumbar spine. To clarify the characteristics of the new implant, a series of biomechanical tests and morphologic evaluations were conducted., Objectives: To investigate the static, viscoelastic, and fatigue properties of the three-dimensional fabric disc in comparison with natural sheep disc and to evaluate their biomechanical and morphologic alteration in vivo., Summary of Background Data: In its human dimensions the three-dimensional fabric disc revealed mechanical properties similar to a natural human disc., Methods: The disc-body units from sheep spine and the sheep three-dimensional fabric discs underwent tensile-compressive (200 N), torsional (5 Nm), and creep-recovery tests (30 minutes-30 minutes, 200 N). After fatigue loading (2 million, compressive 200 N) the biomechanical changes and the debris were investigated. For in vivo evaluation after placing in the sheep psoas muscles for 6 months, the surface of the three-dimensional fabric disc was evaluated using macroscopy and scanning electron microscopy, followed by previous biomechanical tests., Results: The behavior of the sheep three-dimensional fabric disc was similar to that of natural sheep disc in tensile-compressive and creep-recovery tests. In torsional testing the behavior of natural sheep disc was more rigid than that of the sheep three-dimensional fabric disc. After fatigue loading there was no biomechanical change and no debris detected. Six months after surgery no morphologic deterioration was observed nor were there changes in biomechanical parameters., Conclusions: The sheep three-dimensional fabric disc exhibited biomechanical and morphologic biostability, appropriate viscoelasticity, and excellent fatigue properties. The three-dimensional fabric disc has a potential for clinical application of human intervertebral disc replacement.

Study Design: An experimental study to clarify the effects of nucleus pulposus on blood flow in the dorsal root ganglion and hindpaws., Objectives: To investigate the effects of application of nucleus pulposus to nerve root on blood flow in the dorsal root ganglion and the corresponding hindpaw., Summary of Background Data: It has been reported experimentally that application of nucleus pulposus into the epidural space induces morphologic and functional changes in the nerve roots and induces compartment syndrome in the dorsal root ganglia. However, it has not been clarified which of these changes induces symptoms in the lower limbs., Methods: Sixteen adult, female Sprague-Dawley rats had the left L5 nerve root and associated dorsal root ganglions exposed. Autologous nucleus pulposus was applied to the L5 nerve root, just proximal to the dorsal root ganglion (NP group). For control, the same volume of muscle tissue was applied similarly to the neural tissue (control group). Blood flow in the dorsal root ganglion, corresponding hindpaw, and the contralateral hindpaw was continuously monitored by two-channel laser Doppler flowmeter for 3 hours. After measurement of blood flow, the nerve root and dorsal root ganglion were processed for histology and evaluated by light microscope., Results: Blood flow in the NP group was reduced, not only in the dorsal root ganglion, but also in the corresponding hindpaw. These reductions were statistically significant compared with the control group (P < 0.01). Edema was the principal pathologic finding seen consistently in the nerve roots and in many of the associated dorsal root ganglia from nucleus pulposus-treated animals., Conclusion: Application of nucleus pulposus to nerve root decreased blood flow in the dorsal root ganglion and corresponding hindpaw. These basic pathophysiologic changes are associated with compression injuries caused by herniated discs and are accepted neuropathologic mechanisms of injury associated with painful neuropathies. These acute observations in the dorsal root ganglion and the hindpaw may be important initial factors in the pathogenesis of radicular leg pain (sciatica) due to disc herniation.

Study Design: After autotransplanting the nucleus pulposus into the epidural space of rabbits, chondroitinase ABC was administered, and the effect of chondroitinase ABC was examined., Objectives: To examine whether chondroitinase ABC accelerates resolution of the nucleus pulposus transplanted into the epidural space., Summary of Background Data: No previous reports exist on the effect of chondroitinase ABC on the nucleus pulposus in the epidural space., Methods: In Study 1, autotransplantation of the nucleus pulposus into the epidural space was performed in rabbits. Histologic change was observed. In Study 2, dry weight, deoxyribonucleic acid content, and the amount of glycosaminoglycans of autotransplanted nucleus pulposus were quantified and compared with the respective values in the control group, chondroitinase-ABC-injected group, and phosphate-buffered saline-injected group. In Study 3, granulocytes obtained from the blood of a rabbit treated with chondroitinase ABC or phosphate-buffered saline were added to the nucleus pulposus taken from the same rabbit., Results: In chondroitinase ABC group, inflammatory cells tended to infiltrate earlier than those in the control group (Study 1). The dry weight of recollected nucleus pulposus in the chondroitinase ABC group was significantly less than in the other groups. Deoxyribonucleic acid content in the nucleus pulposus tended to be larger in the chondroitinase ABC group, although no significant difference in content compared with that in the other groups was found. Regarding the residual glycosaminoglycans in the transplanted nucleus pulposus, the amount of chondroitin sulfate markedly decreased in the chondroitinase ABC group (Study 2). The number of granulocytes infiltrating the nucleus pulposus was distinctly large in the chondroitinase ABC group (Study 3)., Conclusion: Chondroitinase ABC can enhance resolution of the nucleus pulposus in the epidural space.

Study Design: Histologic examination was performed on the autologous intervertebral disc material that was removed from the intervertebral space at L1-L2 and then relocated to the L4 posterior epidural space after the addition of basic fibroblast growth factor (bFGF) in a rabbit., Objectives: To evaluate whether basic fibroblast growth factor influences the resorption process of the herniated intervertebral disc through the promotion of angiogenesis and chemotaxis., Summary of Background Data: It has been reported that newly formed vessels, inflammatory cells, and their products may play an important role in the spontaneous resorption process of herniated intervertebral discs. In a rabbit model that mimics the sequestration type of intervertebral disc herniation, it has been reported that the autologous intervertebral disc material that relocated into the epidural space was penetrated by newly formed vessels originating from the epidural fat tissue. Therefore, it is possible that promotion of angiogenesis may influence the resorption of herniated intervertebral discs. Basic fibroblast growth factor is well known as an angiogenesis stimulation factor in vivo., Methods: Thirty-six adult rabbits were divided into three groups. The L1-L2 intervertebral disc was partially incised through a retroperitoneal approach in each rabbit. The harvested disc material, which contained nucleus pulposus and anulus fibrosus, was immersed in one of three kinds of solution before relocation into the posterior epidural space at L4. In the control group, the harvested intervertebral disc was immersed in physiologic saline for 2 hours before relocation. In the group receiving 5 micrograms bFGF, the disc was immersed in 5 micrograms/mL bFGF for 2 hours before the relocation. In the group receiving 20 micrograms bFGF, the disc was immersed in 20 micrograms/mL bFGF for 2 hours before the relocation. Rabbits of each group were killed for histologic examination 1, 2, 4, and 8 weeks after surgery., Results: In the bFGF-treated groups, newly formed vessels were observed to be in more numerous than those in the control group, 1 and 2 weeks after surgery. The number of inflammatory cells, including macrophages, lymphocytes, and fibroblasts, also increased in the bFGF-treated groups. The period from the surgery to the degradation of the intervertebral disc in the bFGF-treated groups was shorter than that in the control group, although the resorption process of the relocated discs was also observed in the control group. The size of relocated intervertebral discs in the bFGF-treated groups decreased at a higher rate than in the control group as time progressed. The rate of decrease in the size of discs in the group treated with 20 micrograms bFGF was more than that in the group treated with 5 micrograms., Conclusions: Epidural injection of bFGF facilitated the resorption of the intervertebral disc relocated to the epidural space.

Study Design: The effects of nucleus pulposus and various treatments to block tumor necrosis factor alpha activity were evaluated in an experimental set-up using immunohistochemistry and nerve conduction velocity recordings., Objectives: To assess the presence of tumor necrosis factor alpha in pig nucleus pulposus cells, and to see if block of tumor necrosis factor alpha also blocks the nucleus-pulposus-induced reduction of nerve root conduction velocity., Summary and Background Data: A meta-analysis of observed effects induced by nucleus pulposus revealed that these effects might relate to one specific cytokine-tumor necrosis factor alpha., Methods: Series-1: Cultured nucleus pulposus cells were stained immunohistologically with a monoclonal antibody for tumor necrosis factor alpha. Series-2: Nucleus pulposus was harvested from lumbar discs and applied to the sacrococcygeal cauda equina in 13 pigs autologously. Four pigs received 100 mg of doxycycline intravenously; five pigs had a blocking monoclonal antibody to tumor necrosis factor alpha applied locally in the nucleus pulposus, and four pigs remained nontreated, forming a control group. Three days after the application, the nerve root conduction velocity was determined over the application zone by local electrical stimulation., Results: Series-1: Tumor necrosis factor alpha was found to be present in the nucleus pulposus cells. Series-2: The selective antibody to tumor necrosis factor alpha limited the reduction of nerve conduction velocity, although in comparison with the control group this was not statistically significant. However, treatment with doxycycline significantly blocked the nucleus-pulposus-induced reduction of conduction velocity., Conclusion: For the first time, a specific substance, tumor necrosis factor alpha, has been linked to the nucleus-pulposus-induced effects of nerve roots after local application. Although the effects of this substance may be synergistic with those of other similar substances, the data of the current study may be of significant importance for the continued understanding of nucleus pulposus' biologic activity, and of possible potential use for future strategies in managing sciatica.

Study Design: Application of autologous nucleus pulposus on nerve roots and treatment with local application of lidocaine in the pig., Objectives: Studies of the effects of lidocaine on nucleus pulposus-exposed nerve roots., Summary of Background Data: Nerve root infiltration may improve radicular symptoms beyond the pharmacologic duration of local anesthetics, but the mechanisms for this effect are not known., Methods: Nucleus pulposus was harvested from a lumbar disc and placed onto the sacrococcygeal cauda equina in pigs. In Series 1, early lidocaine treatment of nucleus pulposus-induced nerve root injury, pigs received 2% lidocaine (n = 5) or saline (n = 5) before and after surgery. Nerve conduction velocity and histologic appearance were studied after 3 days. In Series 2, delayed lidocaine treatment of nucleus pulposus-induced nerve root injury, after 7 days 2% lidocaine was administered epidurally to nucleus pulposus-exposed (n = 4) and -nonexposed (n = 4) nerve roots. Nerve conduction velocity, muscle action potentials, and histologic appearance were assessed., Results: In Series 1, early treatment with lidocaine limited the reduction in nerve conduction velocity. The epidural inflammation was less in lidocaine treated animals. In Series 2, nerve conduction velocity was lower in nucleus pulposus-exposed animals than in nonexposed animals. The initial reduction of nerve conduction velocity and muscle action potential was similar between the groups, but the recovery of muscle action potential was slower and less complete in nucleus pulposus-exposed nerve roots. There was minimal histologic nerve injury in both series and in both protocols., Conclusions: Early treatment with lidocaine may reduce nucleus pulposus-induced nerve root injury. Lidocaine induced a delayed recovery in nerve roots exposed to nucleus pulposus. Further studies are needed to clarify the therapeutic effects of nerve root infiltration and the pathophysiology of nucleus pulposus-induced nerve root injury.

Study Design: Nerve conduction velocity in pig nerve roots was assessed after application of various preparations of nucleus pulposus and control., Objective: To study whether cultured nucleus pulposus cells could reduce nerve conduction velocity after epidural application., Summary of Background Data: It is known that nucleus pulposus applied epidurally may reduce the nerve conduction velocity of the adjacent nerve roots and that this reduction seems to be related to the cells of the nucleus pulposus., Methods: Nucleus pulposus cells and fibroblasts were cultured for 3 weeks, and various preparations were applied to the cauda equina in 29 pigs. The cells were always from the same animals from which they had been harvested. After 1 week, nerve conduction velocity was determined by local electrical stimulation., Results: Application of live fibroblasts and conditioned culture medium from the nucleus pulposus cell culture dishes did not induce significant reduction of conduction velocity, compared with application of dead fibroblasts, which served as control. However, application of live and dead nucleus pulposus cells induced significant reductions., Conclusions: Application of nucleus pulposus cells reproduced the previously seen reduction in nerve conduction velocity induced by nonmodified nucleus pulposus. Because membranes of the nucleus pulposus cells had similar effects, it can be assumed that the effects are related to membrane-bound substances or structures.

Study Design: An analysis of the histologic changes in intervertebral discs after percutaneous reinsertion of the nucleus pulposus in rats., Objective: To devise a way to delay further disc degeneration resulting from spinal deformity and the adverse effects of various treatments., Summary of Background Data: The role of the nucleus pulposus of the intervertebral disc described by many investigators has not been fully clarified., Methods: Disc herniation was induced in the tails of 112 Wistar rats, using a fixation device between the 5th and 8th coccygeal vertebrae. After percutaneous nucleotomy at coccygeal vertebrae 5-6 and 6-7, fresh nucleus pulposus, cryopreserved nucleus pulposus, or an artificial substitute was inserted into the intervertebral disc at coccygeal vertebrae 5-6. Two, 4, or 8 weeks after reinsertion, disc sections from each coccygeal level were studied histopathologically., Results: In the groups with reinsertion of fresh or cryopreserved nucleus pulposus, degenerative changes of the disc with the reinserted nucleus at coccygeal vertebrae 5-6 were milder than those of the disc without reinsertion at coccygeal vertebrae 6-7. However, no apparent benefit from reinsertion was observed in the group with artificial substitutes., Conclusions: Early reinsertion of the nucleus pulposus (fresh or cryopreserved) delays degeneration of such disc materials as the anulus fibrosus, endplate, and remaining nucleus pulposus.

Study Design: Monocyte chemoattractant protein-1 was investigated in an experimental rat model using immunohistochemistry., Objective: To ascertain the precise mechanism of macrophage recruitment in the early phase of disc resorption., Summary of Background Data: In previous studies, many investigators reported that disc herniation was resorbed by monocytic phagocytosis. However, how the recruitment of monocytes was triggered is still unknown., Methods: The autologous intervertebral discs from tails of Wistar rats were subcutaneously implanted into the abdomen. These discs were obtained on days 2, 3, 7, and 14 after implantation and were used for immunohistochemical study and for quantitative analysis of monocyte chemoattractant protein-1 by sandwich enzyme-linked immunosorbent assay., Results: Monocyte chemoattractant protein-1-positive granulocytes and macrophages were observed surrounding the intervertebral disc, and monocyte chemoattractant protein-1-positive disc chondrocytes were observed in the nucleus pulposus and the inner anulus fibrosus on day 3. By day 7, monocyte chemoattractant protein-1-positive and TRPM-3-positive macrophages appeared in the granulation tissue, and some of these cells invaded the nucleus pulposus and inner anulus fibrosus. The concentration of monocyte chemoattractant protein-1 was highest on day 3., Conclusion: Intervertebral disc chondrocytes have chemotactic properties and play an active role in the recruitment of monocytes involved in disc resorption.

Study Design: A study of neovascularization with respect to the anulus and the endplate., Objectives: To determine the ability of the anulus and the endplate to induce neovascularization and to study the effect of the endplate on the neovascularizing activity of the anulus., Summary of Background Data: Previous studies showed that herniations contain varying amounts of nucleus pulposus, anulus fibrosus, and endplate. Results of recent studies have shown preferential neovascularization around the herniated anulus rather than the endplate., Methods: Human L4-L5 discs were harvested from three specimens. In the left cornea of New Zealand White rabbits, an anulus (AF group) or an endplate (EP group) was implanted 4 mm from the superior corneoscleral limbus. In a third group (AF and EP group), an endplate was implanted between the anulus and the limbus. Twelve assays were done for each group. Sham surgeries were done on a fourth group as a control. Neovascularization was monitored by stereomicroscopy and scored from 0 (no neovascularization) to +5 (capillaries growing around the implant). Histologic studies were done at weekly intervals., Results: In the AF group, the anulus induced neovascularization in 11 or 12 corneas. By the third week, the anulus appeared thinner, and the newly formed vessels regressed. In the EP group, only two corneas showed neovascularization; 10 corneas showed no vascularization. In the AF and EP group, three corneas showed no neovascularization, seven had sustained neovascularization, and two had vascularization. In the corneas containing the endplates, the implants remained unchanged. Control corneas did show vascularization. Histologic sections showed that neovascularization and cell infiltration were most abundant in the AF group, followed by the AF&EP and the EP groups., Conclusion: The reactions to anulus fibrosus and to cartilage endplate differ. Anulus fibrosus may contain soluble substances that induce neovascularization, leading to its degradation and absorption. Cartilage endplate fails to induce neovascularization or diminishes the neovascular response, preventing its degradation. Disc herniations that contain a significant amount of endplate may not be degraded nor absorbed, leading to failure in spontaneous remission of symptoms.

Study Design: This study was designed to localize the cells that produce nitric oxide in a lumbar disc herniation by histochemical method, including in situ hybridization., Objective: To clarify which cells in herniated lumbar discs produce nitric oxide., Summary of Background Data: It was reported that herniated lumbar intervertebral disc specimens in culture are capable of producing nitric oxide., Methods: Surgical specimens from lumbar disc herniation were examined to determine nitric oxide synthase histologically using nicotinamide adenine dinucleotide phosphate diaphorase histochemistry. Allografts of intervertebral disc materials were placed on the epidural space at L6 level in the rat. Nitric oxide synthase was examined in the applied tissues using nicotinamide adenine dinucleotide phosphate diaphorase histochemistry and in situ hybridization histochemistry., Results: Nicotinamide adenine dinucleotide phosphate diaphorase (nitric oxide synthase) positive cells were observed in 2 (40%) of 5 herniated disc materials in patients. The positive cells were mainly in granulation tissue around intervertebral disc materials. In animal models, nitric oxide synthase-positive cells were observed in all specimens at 1 and 2 weeks postoperatively. Newly formed vessels and small round cells in granulation tissue around the grafted intervertebral disc showed positive reaction. In situ hybridization demonstrated the expression of inducible isoform of nitric oxide synthase messenger RNA (mRNA) identical to small round cells around the applied intervertebral disc., Conclusion: Nitric oxide in a lumbar disc herniation is mainly produced by cells in granulation tissue around the herniated intervertebral disc.

Study Design: To elucidate the pathomechanisms of radicular pain secondary to lumbar disc herniation., Objectives: To evaluate whether intervertebral disc material applied to the sciatic nerve produces hyperalgesia, and if the hyperalgesia in influenced by inhibitors of phospholipase A2 and nitric oxide synthase., Summary of Background Data: Previously, the authors reported that application of nucleus pulposus and anulus fibrosus material to the lumbar epidural space produces different forms of hyperalgesia (mechanical versus thermal), with different and distinct histologic changes. Additional pharmacologic studies showed that phospholipase A2 and nitric oxide are involved in the mechanisms that produce the mechanical and thermal hyperalgesia, respectively, N omega-nitro-L-arginine methyl ester and mepacrine are relatively selective inhibitors of nitric oxide synthase and phospholipase A2, respectively. However, it is not known what the relation is between the hyperalgesia produced and the activation and involvement of phospholipase A2 and production of nitric oxide, or why the application of nucleus pulposus and nucleus pulposus with anulus fibrosus produces different types of hyperalgesia., Methods: Experiments were performed in five groups of rats: The control group (no treatment), the sham group (exposure of the sciatic nerve only), the fat group (allografted fat on the sciatic nerve), the nucleus pulposus group (allografted nucleus pulposus) and the nucleus pulposus + anulus fibrosus group (allografted nucleus pulposus and anulus fibrosus). Withdrawal threshold and latency from mechanical pressure and a radiant heat to hind paws were measured preoperatively and postoperatively. After local sciatic nerve administration of N theta-nitro-L-arginine methyl ester or mepacrine into the operated site, sensitivities to noxious stimuli were reevaluated after treatment., Results: Only rats in the nucleus pulposus group showed evidence of mechanical hyperalgesia. However, injection of N theta-nitro-L-arginine methyl ester resulted in evidence of mechanical hyperalgesia in the nucleus pulposus + anulus fibrosus group. Mechanical hyperalgesia was produced in the nucleus pulposus group and after injection of N theta-nitro-L-arginine methyl ester in the nucleus pulposus+anulus fibrosus group, both of which returned to normal after mepacrine injection. There were no significant changes in sensitivity to thermal stimuli in any of the experimental groups., Conclusion: It appears that phospholipase A2 and nitric oxide play important but different roles in pathomechanisms of radicular pain in lumbar disc herniation.

Study Design: Autologous nucleus pulposus was modified and applied to the cauda equina in pigs. Histology and neurophysiology were assessed after 7 days., Objectives: To assess if alterations of the nucleus pulposus would change the degree and distribution of the nerve injury induced by autologous nucleus pulposus., Summary of Background Data: It was reported recently that nucleus pulposus may induce structural and functional changes in nerve roots after epidural application. The basic mechanisms causing these changes are not fully understood., Methods: Nucleus pulposus was harvested from lumbar discs and submitted to either of three treatments; 37 C for 24 hours (n = 5), -20 C for 24 hours (n = 5), or digestion by hyaluronidase for 24 hours (n = 6). In two additional pigs, nucleus pulposus was applied just after harvest as a control to verify previous observations. After 7 days, nerve conduction velocity was recorded, and specimens were processed for blinded light microscopic assessment., Results: When nucleus pulposus was applied just after harvest, or when it had been kept at 37 C or digested by hyaluronidase for 24 hours, there was a significant reduction in nerve conduction velocity similar to previous observations. When nucleus pulposus had been kept at -20 C for 24 hours, however, there was no reduction in conduction velocity. There were no apparent differences between the groups at the histologic assessment. Staining of the nucleus pulposus showed that the cells in the nucleus pulposus exposed to -20 C were lysed, whereas the cells in the nucleus pulposus treated by the two other methods were mainly unaffected., Conclusions: Because freezing of the nucleus pulposus probably kills the cells but does not affect other components, one may assume that the biologic effects induced by the nucleus pulposus may be related to its cell population.

Study Design: This study was designed to evaluate whether allografts of intervertebral disc materials produce hyperalgesia in the rat and whether an immune response, pH, or chemicals correlate with the induced hyperalgesia., Objective: To elucidate the pathomechanisms of radicular pain secondary to lumbar disc herniation., Summary of Background Data: It has been reported that a low pH, an autoimmune reaction, or chemical radiculitis is likely responsible for radicular pain associated with lumbar disc herniation. In animal studies, it has been shown that hyperalgesia (an increased sensitivity to painful stimuli) involves activation of phospholipase A2 and nitric oxide synthase., Methods: Fat, nucleus pulposus, and anulus fibrosus were allografted into the epidural space at L6 in the rat. Withdrawal response thresholds to mechanical stimuli and withdrawal response latencies to thermal stimuli on the tail and pH in the applied tissues were measured after surgery. Interleukin-1, phospholipase A2, and nitric oxide synthase were examined in the applied tissues using immunohistochemistry, nicotineamide adenine dinucleotide phosphate-diaphorase histochemistry, and in situ hybridization., Results: Allografted fat did not produce hyperalgesia. Allografts of nucleus pulposus and nucleus pulposus plus anulus fibrosis showed evidence of mechanical and thermal hyperalgesia, respectively. There were no observed changes in pH over time. Although interleukin-1 was demonstrated in all applied tissues, phospholipase A2 was only observed around the applied nucleus A2 was only observed around the applied nucleus pulposus and nucleus pulposus plus anulus fibrosus. Nitric oxide synthase was only markedly increased around the applied tissues., Conclusion: The nucleus pulposus and anulus fibrosus produce different forms of hyperalgesia (mechanical vs. thermal) associated with different and distinct immunohistochemical changes. It is possible that radicular pain of a lumbar disc herniation results from chemicals, such as phospholipase A2 and nitric oxide.

Study Design: Ultrastructural changes were analyzed by transmission electron microscopy in nerve roots exposed to autologous nucleus pulposus experimentally., Objectives: To assess if ultrastructural changes were present in areas with no light microscopic changes in nerve roots exposed to autologous nucleus pulposus in a pig model., Summary of Background Data: Previous analyses have shown that there is focal nerve fiber damage in nerve roots exposed to autologous nucleus pulposus in the pig. These changes could not fully explain the reduction in nerve conduction velocity seen in the same nerve roots. In the present study, the parts of the nerve roots that did not display breakdown of axons or myelin sheaths at the light microscopic level were analyzed regarding ultrastructural changes., Methods: In a previous study, nucleus pulposus was harvested from a lumbar disc and placed epidurally onto the cauda equina at the sacrococcygeal level in pigs. Retroperitoneal fat was used as control. After 1, 3, and 7 days, the nerve roots were excised and processed for light microscopy. Parts of the nerve roots that appeared normal at the light microscopic level were further processed for the present electron microscopic examination., Results: Significant ultrastructural changes, such as expansion of the Schwann cell cytoplasm and intracellular edema with vesicular swelling of the Schmidt-Lanterman incisures, were observed in nerve fibers with normal axons. Although present after nucleus pulposus and control application, the changes were more pronounced after the application of nucleus pulposus., Conclusions: Epidural application of autologous nucleus pulposus without any pressure may induce not only nerve function impairment but also axonal injury and significant primary Schwann cell damage with vesicular swelling of Schmidt-Lanterman incisures. However, because axonal and Schwann cell changes affected only part of the nerve fibers, further causes of the impaired nerve conduction need to be determined.

Study Design: A lumbar disc is a tissue that is often a cause of low back pain. We conducted experimental allografting of intervertebral discs in dogs., Objectives: To evaluate the clinical potential for allografting of intervertebral disc., Summary of Background Data: There have been few reports on the allografting of intervertebral discs. The allografting of discs frozen at -196 C has never been documented. In the present experiments, we studied the effect of freezing at -80 C and -196 C on disc allografts to 25 hybrid adult dogs., Methods: A vertebral disc was obtained from donor dogs with parts of the adjoining vertebral bodies to serve as the allograft disc unit. After soaking in 10% dimethyl sulfoxide at 4 C, the allograft discs were frozen and stored at either -80 C or -196 C. After storage for approximately 4 weeks, they were transplanted peritoneally into the recipient lumbar vertebrae and fixed using plates and screws to preserve mobility of the grafted disc. At the same time, cells from allograft discs were incubated to examine their ability to synthesize proteoglycan and collagen. Radiologic examination of the changes after surgery in the intervertebral space was followed serially., Results: Radiographs revealed a complete bone union of the vertebral bodies at 5 months and gradual narrowing of the intervertebral space beginning at 6 months. Histologically, the anulus fibrosus was well preserved, especially in disc units stored at -196 C. Cellular synthetic activity was seriously diminished., Conclusions: Our results indicate that allograft of intervertebral discs has clinical potential because it can serve as a dynamic disc spacer for a certain period of time, but its long-term merits and demerits have to be established.

Study Design: An investigation of the use of intervertebral disc allografting as a potential alternative to intervertebral fusion was performed. In 13 adult mongrel dogs, intervertebral disc units cryopreserved after slow freezing were grafted., Objectives: To evaluate the morphologic and biosynthetic damage caused by cryopreserving the canine intervertebral disc before allografting., Summary of Background Data: Cryopreservation did not alter the normal histologic appearance. The metabolic activity, measured by 35S-sulfate incorporation, was observed to be 44% of the fresh control., Methods: Transplanted intervertebral discs were examined histologically and radiographically at 12, 24, and 48 weeks after surgery., Results: The allografted disc had preserved anular and nuclear architecture with moderate loss of chondrocytes 12 weeks after grafting. However, the intervertebral disc showed evidence of progressive disc degeneration 1 year after transplantation., Conclusions: More investigation of storage methods is needed to enhance the viability of the intervertebral disc for successful intervertebral disc allografting.

Study Design: Degenerative lumbar disc disease has been implicated as a cause of low back pain. Current treatment options for low back pain involve nonphysiologic fusion of the involved segments and have variable success rates. This is an experimental study of lumbar intervertebral disc transplantation using a canine surgical model., Objectives: This study evaluated the feasibility of lumbar disc transplantation and its effects on disc metabolism and morphology., Method: Eight mature mongrel dogs underwent disc transfer surgeries, in which the L2-L3 and L4-L5 intervertebral discs, with a small segment of adjacent superior and inferior vertebral body, were removed and transposed. The transplanted disc were stabilized by plates or by a flexible cable wire construct using Songer cables (DANEK, Inc., Memphis, TN). Unrestricted activity was allowed postoperatively. At 4 months, the spines were harvested, and the transplanted discs were evaluated biochemically and histologically. Intervening nontransplanted discs served as viable controls and thrice-frozen discs served as nonviable controls. Cell viability was assessed by measuring proteoglycan synthesis and DNA content., Results: Proteoglycan synthesis (35S uptake normalized to DNA content) was maintained in transplanted anulus fibrosus tissue, but was decreased in nucleus pulposus samples (P < 0.05). DNA content was not altered significantly in the transplanted discs. Histologic analysis of the transplanted discs showed revascularization and remodeling of the bone adjacent to the disc and preservation of the lamellar architecture of the anulus fibrosus. The transplanted nucleus pulposus samples had chondrocyte-like cells present, but the staining characteristics of the nucleus material was variable. The contour of the transplanted disc endplates was irregular in all specimens., Conclusions: The structure and function of autograft intervertebral discs were maintained after disc transfer surgery; the transplant discs, however, were not completely normal in either their morphology or their metabolic functioning.

Vertebral column allografts, with their intervertebral discs, were implanted into thoracic spine defects (T7-T9) in 11 dogs in an attempt to re-establish spinal stability and preserve spinal biomechanics. Before implantation, the allografts were harvested under sterile conditions from similar-sized dogs and deep frozen at -80 C. The animals were followed for 18 months postoperatively. Radiographs demonstrated gradual loss of intervertebral disc height. Biomechanical analysis showed that the dogs with allografts had no significant difference in spine stiffness compared with normal spines in compression, flexion, and extension testing. Control spines that had been fused were significantly stiffer than the allograft spines in all modes tested (P less than 0.05). Histologic analysis showed incorporation of the allograft but with incomplete revascularization of the allograft's eighth thoracic body. This investigation found that vertebral body allografts with intervertebral discs can function successfully for 18 months in a canine model. This research may assist in the development of physiologic treatment for spinal deficiencies in humans.

The transplantation of spinal allografts to correct defects that include disc, body, or segments of both is currently under experimental investigation. A method of graft preservation that will least compromise the biomechanical integrity of the bodies or discs is required. Using a five-mode biomechanical analysis, the authors compared the change in stiffness of ten preserved canine spines. The freeze-dried specimens lost a significantly greater amount of stiffness in compression, flexion, extension, and torsion than did the frozen specimens. Therefore, from a biomechanical viewpoint, deep-freezing is superior to freeze-drying for spinal allograft preservation.