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Aim:  To explore the effects of viable allogeneic disc tissue supplementation in younger patients with discogenic chronic low back pain (CLBP). Patients & methods: VAST was a randomized placebo-controlled trial of disc allograft supplementation in 218 patients with discogenic CLBP. We conducted a post hoc analysis of change from baseline to 12 months in Oswestry Disability Index (ODI) and visual analog scale for pain intensity scores stratified by patient age. Results: Patients aged <42 years receiving allograft experienced greater improvement in ODI (p = 0.042) and a higher ODI response rate (≥10-, ≥15- and ≥20-point reductions in ODI) than those receiving saline (p = 0.001, p = 0.002 and p = 0.021, respectively). Conclusion: Young patients with discogenic CLBP may have significant functional improvement following nonsurgical disc allograft supplementation.

The purpose of the present pilot study was to evaluate the effect of a hydrogel composed of hyaluronic acid (HA) and platelet-rich plasma (PRP) as a carrier for human mesenchymal stem cells (hMSCs) for intervertebral disc (IVD) regeneration using a disc organ culture model. HA was mixed with batroxobin (BTX) and PRP to form a hydrogel encapsulating 1 × 10 6 or 2 × 10 6 hMSCs. Bovine IVDs were nucleotomized and filled with hMSCs suspended in ~200 μL of the PRP/HA/BTX hydrogel. IVDs collected at day 0 and nucleotomized IVDs with no hMSCs and/or hydrogel alone were used as controls. hMSCs encapsulated in the hydrogel were also cultured in well plates to evaluate the effect of the IVD environment on hMSCs. After 1 week, tissue structure, scaffold integration, hMSC viability and gene expression of matrix and nucleus pulposus (NP) cell markers were assessed. Histological analysis showed a better preservation of the viability of the IVD tissue adjacent to the gel in the presence of hMSCs (~70%) compared to the hydrogel without hMSCs. Furthermore, disc morphology was maintained, and the hydrogel showed signs of integration with the surrounding tissues. At the gene expression level, the hydrogel loaded with hMSCs preserved the normal metabolism of the tissue. The IVD environment promoted hMSC differentiation towards a NP cell phenotype by increasing cytokeratin-19 (KRT19) gene expression. This study demonstrated that the hydrogel composed of HA/PRP/BTX represents a valid carrier for hMSCs being able to maintain a good cell viability while stimulating cell activity and NP marker expression.

In the last decade, intervertebral disc (IVD) decellularization has gained significant attention for tissue regenerative purposes as a successful therapeutic alternative for low back pain (LBP). We discuss the recent advances in IVD decellularization, repopulation, and sterilization procedures, highlighting the major challenges that need to be addressed for clinical translation., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Intervertebral disc (IVD) degeneration (IDD) is the main cause of low back pain in the clinic. In the advanced stage of IDD, both cell transplantation and gene therapy have obvious limitations. At this stage, tissue-engineered IVDs (TE-IVDs) provide new hope for the treatment of this disease. We aimed to construct a TE-IVD with a relatively complete structure. The inner annulus fibrosus (AF) was constructed using poly (butylene succinate-co-terephthalate) copolyester (PBST) electrospun fibers, and the outer AF consisted of solid PBST. The nucleus pulposus (NP) scaffold was constructed using a chitosan hydrogel, as reported in our previous research. The three components were assembled in vitro, and the mechanical properties were analyzed. AF and NP cells were implanted on the corresponding scaffolds. Then, the cell-seeded scaffolds were implanted subcutaneously in nude mice and cultured for 4 weeks; then they were removed and implanted into New Zealand white rabbits. After 4 weeks, their properties were analyzed. The PBST outer AF provided mechanical support for the whole TE-IVD. The electrospun film and chitosan hydrogel simulated the natural structure of the IVD well. Its mechanical property could meet the requirement of the normal IVD. Four weeks later, X-ray and MR imaging examination results suggested that the height of the intervertebral space was retained. The cells on the TE-IVD expressed extracellular matrix, which indicated that the cells maintained their biological function. Therefore, we conclude that the whole TE-IVD has biological and biomechanical properties to some extent, which is a promising candidate for IVD replacement therapies. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2305-2316, 2019., (© 2019 Wiley Periodicals, Inc.)

A biomaterial-based strategy is employed to regenerate the degenerated intervertebral disc, which is considered a major generator of neck and back pain. Although encouraging enhancements in the anatomy and kinematics of the degenerative disc have been gained by biomaterials with various formulations in animals, the number of biomaterials tested in humans is rare. At present, most studies that involve the use of newly developed biomaterials focus on regeneration of the degenerative disc, but not pain relief. In this review, we summarise the current state of the art in the field of biomaterial-based regeneration or repair for the nucleus pulposus, annulus fibrosus, and total disc transplantation in animals and humans, and we then provide essential suggestions for the development and clinical translation of biomaterials for disc regeneration. It is important for researchers to consider the commonly neglected issues instead of concentrating solely on biomaterial development and fabrication., (© 2018 John Wiley & Sons, Ltd.)

Biomaterials science has achieved significant advancements for the replacement, repair and regeneration of intervertebral disc tissues. However, the translation of this research to the clinic presents hurdles. The goal of this paper is to identify strategies to recapitulate the intrinsic complexities of the intervertebral disc, to highlight the unresolved issues in basic knowledge hindering the clinical translation, and finally to report on the emerging technologies in the biomaterials field. On this basis, we identify promising research directions, with the hope of stimulating further debate and advances for resolving clinical problems such as cervical and low back pain using biomaterial-based approaches., Statement of Significance: Although not life-threatening, intervertebral disc disorders have enormous impact on life quality and disability. Disc function within the human body is mainly mechanical, and therefore the use of biomaterials to rescue disc function and alleviate pain is logical. Despite intensive research, the clinical translation of biomaterial-based therapies is hampered by the intrinsic complexity of this organ. After decades of development, artificial discs or tissue replacements are still niche applications given their issues of integration and displacement with detrimental consequences. The struggles of biological therapies and tissue engineering are therefore understandable. However, recent advances in biomaterial science give new hope. In this paper we identify the most promising new directions for intervertebral disc biomaterials., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

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.

Purpose: Fresh frozen intervertebral disc allograft transplantation has been reported to be a viable treatment option for advanced degenerative disc diseases, but rapid degeneration of the postoperative allograft was found. Loss of nutrient supply is believed to be the most likely inducer because the disc allografts have to endure in an ischaemic environment until the nutrient pathway is re-established. The aim of this study was to focus on the revascularisation of the disc allograft after transplantation in goats., Methods: Twenty male goats were used in this study. Intervertebral disc allograft transplantation was performed at L4/L5. Groups of five goats were killed at 1.5, 6 and 12 m postoperatively, respectively. The transplanted segments were harvested, fixed, sagittally cut and decalcified for H&E staining and immunochemistry to observe the blood vessel formation at the endplates, anterior outer annulus, posterior outer annulus, inner annulus and the nucleus. The blood vessel density and the sectional vessel area were measured., Results: Blood vessels were first found in the marrow space of the bony endplate and the outer annulus at 1.5 month postoperatively. Then, they were able to penetrate to reach the cartilaginous endplate and the inner annulus after 6 months. Interestingly, the endplate area possessed the most abundant blood vessels, with the highest level of vessel density and area at the final follow-up. None of these newly formed vessels invaded the nucleus during the observation period., Conclusions: Revascularisation of the postoperative disc allograft has been determined, but its pattern was different from that in adult normal discs, suggesting that the typical nutrient diffusion pattern may be affected after transplantation.

Purpose: Fresh-frozen intervertebral disc (IVD) allograft transplantation has been successfully performed in the human cervical spine. Whether this non-fusion technology could truly decrease adjacent segment disease is still unknown. This study evaluated the long-term mobility of the IVD-transplanted segment and the impact on the adjacent spinal segments in a goat model., Methods: Twelve goats were used. IVD allograft transplantation was performed at lumbar L4/L5 in 5 goats; the other 7 goats were used as the untreated control (5) and for the supply of allografts (2). Post-operation lateral radiographs of the lumbar spine in the neutral, full-flexion and full-extension positions were taken at 1, 3, 6, 9 and 12 months. Disc height (DH) of the allograft and the adjacent levels was calculated and range of motion (ROM) was measured using the Cobb's method. The anatomy of the adjacent discs was observed histologically., Results: DH of the transplanted segment was decreased significantly after 3 months but no further reduction was recorded until the final follow-up. No obvious alteration was seen in the ROM of the transplanted segment at different time points with the ROM at 12 months being comparable to that of the untreated control. The DH and ROM in the adjacent segments were well maintained during the whole observation period. At post-operative 12 months, the ROM of the adjacent levels was similar to that of the untreated control and the anatomical morphology was well preserved., Conclusions: Lumbar IVD allograft transplantation in goats could restore the segmental mobility and did not negatively affect the adjacent segments after 12 months.

Previous human study suggested that fresh-frozen intervertebral disc allograft transplantation can relieve neurological symptoms and restore segmental kinematics. Before wide clinical application, research into the pathophysiology of the postoperative disc allograft is needed. One important question that remains to be answered in disc allografting is the healing process of the host-graft interface and the subsequent change of the endplates. With the goat model for lumbar disc allografting, histology, micro-computed tomography analysis, scanning electron microscopy and energy-dispersive X-ray spectroscopy mapping were applied to evaluate the healing of the host-graft interfaces, the remodelling of subchondral bone, and the changes of the bony and cartilaginous endplates after transplantation. It was found that healing of the host-graft interfaces started at 1.5 months and was completed at 6 months by natural remodelling. This bony remodelling was also noted in the subchondral bone area after 6 months. The bony endplate was well preserved initially, but was gradually replaced by trabecular bone afterwards; on the other hand, the cartilaginous endplate became atrophic at 6 months and nearly disappeared at the final follow-up. Collectively, after intervertebral disc allograft transplantation, bony healing and remodelling were seen which ensured the stability and mobility of the disc-transplanted segment, but the integrity of bony and cartilaginous endplates was gradually lost and nearly disappeared finally.

Objective: To discuss the effect of three different ways of annulus fibrosus incision on the biomechanical strength of intervertebral disc., Methods: A total of 30 goats underwent intervertebral disc nucleus pulposus extraction at L3,4 and 45 by the working channel in group A (n=10), by circular incision in group B (n=10), and by square incision in group C (n=10). The body weight, male and female ratio, age, intraoperative blood loss, and wound healing time were recorded and compared among 3 groups. The survival rate and wound healing situation were observed after operation. At 24 weeks after operation, the goats were sacrificed, MRI images were taken to observe the signal intensity of nucleus pulposus. The disc height of L(3,4) and L(4,5) was measured to calculate the loss of disc height; biomechanical test was used to assess the strength of the disc and anulus. Histological staining was also conducted to observe the repair effect at L(4,5)., Results: There was no significant difference in body weight, male to female ratio, age, intraoperative blood loss, and wound healing time among groups (P>0.05). All goats survived to the end of the experiment. MRI examination showed decreased signal intensity in 3 groups, indicating intervertebral disc degeneration. According to modified Thompson classification method, the degree of intervertebral disc degeneration of group A was significantly higher than that of groups B and C (P<0.05), but no significant difference was found between groups B and C (P>0.05). Difference was not significant in intervertebral space height before operation among 3 groups (P>0.05). But after 24 weeks, the intervertebral space height in group A was significantly higher than that in groups B and C (P<0.05), and the intervertebral space height loss in group A was significantly lower than that in groups B and C (P<0.05). The biomechanical strength in group A was also significantly higher than that in groups B and C (P<0.05), but no significant difference was found between group B and group C (P>0.05). HE and Masson staining showed good continuity of annulus fibrosus and clear layers in group A; poor continuity of annulus fibrosus and obvious scar tissues were observed in groups B and C., Conclusion: Application of working channel may have less destruction of annulus fibrosus, it plays a positive role in the maintenance of biomechanical strength and repair of annulus fibrosus.

Purpose: Fresh-frozen intervertebral disc transplantation was determined to be an effective treatment for degenerative disc diseases in rhesus monkeys and in humans. Further research in improving different aspects of disc allografts transplantation is needed and will be investigated in large animal models. This study reports the detailed surgical technique of intervertebral disc transplantation without internal fixation and the important notes to ensure success in goats., Methods: Fifty-one male goats were used in this study. Ten goats were used as intervertebral disc allograft donors; the remaining forty-one goats were used to develop the surgical technique for intervertebral disc allograft transplantation. Radiographs, ex vivo MRI and gross observation were used to monitor the stability and healing of the disc allografts at 3 months, postoperatively., Results: Size matching of the disc allograft, preservation of the anterior longitudinal ligament and an appropriate portion of the annulus fibrosus at the recipient site were crucial for stable graft retention. Additionally, a slightly reduced height of the disc allograft compared to that of the recipient slot may avoid graft endplate fracture., Conclusions: Lumbar intervertebral disc transplantation without internal fixation can be successfully performed in goats.

Study Design: An animal experiment about intervertebral disc allograft., Objective: To explore the feasibility to decellularize disc allografts treated by 6°Co Gamma Irradiation, and simultaneously, to assess the possibility to make use of the decellularized natural disc scaffold for disc degeneration biotherapy., Summary of Background Data: Studies of both animal and human disc allograft transplantation indicated that the disc allograft may serve as a scaffold to undertake the physiological responsibility of the segment., Methods: Experiment in vitro: 48 discs of beagles were harvested and divided randomly into four groups including a control group and three irradiated groups. Immediate cell viability and biomechanical properties of the discs were checked and comparisons were made among these groups. Experiment in vivo: 24 beagles accepted single-level allografted disc treated with different doses of gamma irradiation. Plain X-rays and MRIs were taken before and after surgery. Then, the spinal columns were harvested en bloc from the sacrificed beagles and were examined morphologically., Results: There were significant differences of both the annulus fibrosus and nucleus pulposus immediate cell viabilities among the various groups. There were no obvious differences of the biomechanical properties among the four groups. The disc height and range of motion decreased significantly in all groups as time went on. The observed indexes in irradiated groups were much smaller than those in the control group, but the indexes in 18-kGy group were larger than those in 25-kGy and 50-kGy groups. Both MRI and macroscopic findings showed that the segmental degeneration in the control and 18-kGy group was less severe than that in 25-kGy and 50-kGy groups., Conclusion: Gamma Irradiation can decellularize disc allograft successfully to provide natural scaffold for the study of degenerative disc disease therapy, and also can be used as an effective method to produce adjustable animal models.

Object: Tissue-engineered intervertebral discs (TE-IVDs) represent a new experimental approach for the treatment of degenerative disc disease. Compared with mechanical implants, TE-IVDs may better mimic the properties of native discs. The authors conducted a study to evaluate the outcome of TE-IVDs implanted into the rat-tail spine using radiological parameters and histology., Methods: Tissue-engineered intervertebral discs consist of a distinct nucleus pulposus (NP) and anulus fibrosus (AF) that are engineered in vitro from sheep IVD chondrocytes. In 10 athymic rats a discectomy in the caudal spine was performed. The discs were replaced with TE-IVDs. Animals were kept alive for 8 months and were killed for histological evaluation. At 1, 5, and 8 months, MR images were obtained; T1-weighted sequences were used for disc height measurements, and T2-weighted sequences were used for morphological analysis. Quantitative T2 relaxation time analysis was used to assess the water content and T1ρ-relaxation time to assess the proteoglycan content of TE-IVDs., Results: Disc height of the transplanted segments remained constant between 68% and 74% of healthy discs. Examination of TE-IVDs on MR images revealed morphology similar to that of native discs. T2-relaxation time did not differ between implanted and healthy discs, indicating similar water content of the NP tissue. The size of the NP decreased in TE-IVDs. Proteoglycan content in the NP was lower than it was in control discs. Ossification of the implanted segment was not observed. Histological examination revealed an AF consisting of an organized parallel-aligned fiber structure. The NP matrix appeared amorphous and contained cells that resembled chondrocytes., Conclusions: The TE-IVDs remained viable over 8 months in vivo and maintained a structure similar to that of native discs. Tissue-engineered intervertebral discs should be explored further as an option for the potential treatment of degenerative disc disease.

Cell delivery to the pathological intervertebral disc (IVD) has significant therapeutic potential for enhancing IVD regeneration. The development of injectable biomaterials that retain delivered cells, promote cell survival, and maintain or promote an NP cell phenotype in vivo remains a significant challenge. Previous studies have demonstrated NP cell - laminin interactions in the nucleus pulposus (NP) region of the IVD that promote cell attachment and biosynthesis. These findings suggest that incorporating laminin ligands into carriers for cell delivery may be beneficial for promoting NP cell survival and phenotype. Here, an injectable, laminin-111 functionalized poly(ethylene glycol) (PEG-LM111) hydrogel was developed as a biomaterial carrier for cell delivery to the IVD. We evaluated the mechanical properties of the PEG-LM111 hydrogel, and its ability to retain delivered cells in the IVD space. Gelation occurred in approximately 20 min without an initiator, with dynamic shear moduli in the range of 0.9-1.4 kPa. Primary NP cell retention in cultured IVD explants was significantly higher over 14 days when cells were delivered within a PEG-LM111 carrier, as compared to cells in liquid suspension. Together, these results suggest this injectable laminin-functionalized biomaterial may be an easy to use carrier for delivering cells to the IVD., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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.

Objectives: To investigate whether the intervention of nucleus pulposus (NP) cells or human telomerase reverse transcriptase (hTERT) gene-transfected NP cells can prevent the degeneration process after allograft total disc transplantation., Methods: Eighteen canine lumbar intervertebral discs were obtained from five canines and cryopreserved in liquid nitrogen. Canine nucleus pulposus cells were isolated and transduced with recombinant adeno-associated virus (rAAV)-hTERT. The cells were injected into the discs to construct a tissue-engineered allograft disc (group A). NP cells and DMEM/F12 were used for positive control (group B) and blank control (group C). 18 beagle dogs received the three groups of allograft intervertebral disc (IVD) composites implantation, respectively. Radiographic examinations were performed at 4, 8, and 12 weeks postimplantation. At 12 weeks after operation, all dogs were sacrificed and the lumbar spines were harvested for the biomechanical analysis, and then the allografts underwent histological analysis, ectogenic NP cell tracing, and hTERT mRNA analysis., Results: Bony fusion between the intervertebral disc allograft and the adjacent host intervertebral body were observed in all animals. The disc height and T2 signal intensity preservation in group A and B was better than group C. Magnetic resonance images (MRI) showed typical degenerative changes in group C. In group A, the normalized grayscale of the transplanted disc on MRI was significant higher compared with the controls at 12 weeks. A biomechanical test showed a poor stability preservation in group C compared to group A and B. PKH-26-positive cells were identified within the allograft discs in group A at 12 weeks, providing evidence of cell survival. Histological analysis showed the NP cell morphology, cell number, and distribution of the allograft discs was better preserved in group A and B compared to group C at a 12-week follow-up., Conclusion: The present study demonstrated that NP cells or hTERT-loaded NP cells intervention could effectively resist the degeneration of the allogenic transplanted intervertebral discs in a beagle model. The hTERT-loaded NP cells had a better antidegeneration effect on the transplanted disc than NP cells. This modified disc regeneration technique through NP cell injection or manipulation may have the potential to ensure the long-term function preservation of allograft disc transplantation.

The sheep is becoming increasingly used as a large animal model for preclinical spine surgery studies. Access to the ovine lumbar intervertebral discs has traditionally been via an anterior or anterolateral approach, which requires larger wound incisions and, at times, significant abdominal retraction. We present a new minimally invasive operative technique for a far-lateral approach to the ovine lumbar spine that allows for smaller incisions, excellent visualisation of intervertebral discs, and minimal abdominal retraction and is well tolerated by animals with minimal morbidity.

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.

Even though human nucleus pulposus (HNP) cells have a limited replicative lifespan in vitro culture, the lifespan of ovine nucleus pulposus cells transferred by lipofectamine with human telomerase reverse transcriptase (hTERT) gene could be prolonged. However, the lipofectamine is a transient expression system with an expressed duration of hTERT for less than 73 days. Here we present a viral vector system to investigate whether recombinant adenoassociated virus vector-mediated hTERT gene (rAAV-hTERT) could safely prolong the activities of HNP cells. Our hypothesis was that the activities of HNP cells could be extended with rAAV-hTERT gene transfer. Therefore, we designed an in vitro and in vivo evaluation of a HNP cells gene transfectant. Second-generation HNP cells were transfected with rAAV-hTERT and the expression of hTERT determined by reverse transcription-polymerase chain reaction (RT-PCR) and western blot. Cumulative population doubling, senescence assay, real-time RT-PCR, and enzyme-linked immunosorbent assay were used to determine cellular activities. Genetic phenotype was validated by karyotypic analysis and ontogenicity by nude mice injected in vivo. The continuing expression of hTERT gene and production of extra cellular matrix for 120 days were found in the transfected HNP cells, and karyotypic instability was detected, but there was no evidence of polyploidy or oncogenicity in nude mouse. The results showed that this transfection model might provide a resource cells necessary for the biological treatment of degenerative disc disease., (© Mary Ann Liebert, Inc.)

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.

The aim of this study was to construct tissue-engineered composite intervertebral disc (IVD) consisting of demineralized bone matrix gelatin (DBMG) and collagen II/hyaluronate/chondroitin-6-sulfate (CII/HyA-CS) scaffolds seeded with anulus fibrosus (AF) and nucleus pulposus (NP) cells, respectively. The cell-scaffold hybrids were implanted in the subcutaneous space of the dorsum of athymic mice and harvested at 4, 8, and 12 weeks. At each time point, the gross and histological morphology and biochemical properties were evaluated. Our results are as following: the gross morphology and histology of the composite resembled those of native IVD. Morphological studies revealed progressive tissue formation and junction integration between AF and NP regions. Biochemical composition detection indicated that the content of DNA, proteoglycan and hydroxyproline increased with time, and were similar to native tissue at 12 weeks. All these results demonstrated the feasibility of creating a tissue-engineered composite IVD with similar morphological and biochemical properties to the native tissue., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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.

Severe intervertebral disc (IVD) degeneration often requires disc excision and spinal fusion, which leads to loss of spinal segment mobility. Implantation of an allograft disc or tissue engineered disc construct emerges as an alternative to artificial disc replacement for preserving the motion of the degenerated level. Establishment of a bank of cadaveric or engineered cryopreserved discs enables size matching, and facilitates clinical management. However, there is a lack of understanding of the behaviour of disc cells during cryopreservation, as well as how to maximize their survival, such that disc graft properties can be preserved. Here, we report on the effect of alterations in cooling rates, cryoprotective agents (CPAs), and duration of pre-cryopreservation incubation in CPA on cellular activity in whole porcine lumbar discs. Our results indicated that cooling rates of -0.3 degrees C/min and -0.5 degrees C/min resulted in the least loss of metabolic activity in nucleus pulposus (NP) and annulus fibrosus (AF) respectively, while metabolic activity is best maintained by using a combination of 10% dimethylsulphoxide (DMSO) and 10% propylene-glycol (PG) as CPA. By the use of such parameters, metabolic activity of the NP and the AF cells could be maintained at 70% and 45%, respectively, of that of the fresh tissue. Mechanical testing and histological evaluation showed no significant differences in mechanical properties or alterations in disc structure compared to fresh discs. Despite the limitations of the animal model, our findings provide a framework for establishing an applicable cryopreservation protocol for human disc allografts or tissue-engineered disc constructs.

This study was performed in 36 rabbits to investigate the role of midkine (MK) in the resorption of herniated intervertebral discs. The L(1-2) disc was excised and immersed in one of three kinds of solution for two hours before relocation into the L4 epidural space. In the MK-treated group, the weight of relocated intervertebral discs decreased more over time than in the control group. Newly formed vessels and inflammatory cells were more frequently observed in the MK-treated group than in the control group two weeks after surgery. The degradation of matrix was more significant in the MK-treated group than in the control group four weeks after surgery. Larger areas were replaced by fibrous tissues in the MK-treated group eight weeks after surgery. Thus, MK can accelerate the resorption of the intervertebral disc relocation to the epidural space. Epidural injection of MK may contribute to the therapy of lumber disc herniation.

Although intervertebral disc herniation and associated sciatica is a common disease, its molecular pathogenesis is not well understood. Immune responses are thought to be involved. This study provides direct evidence that even non-degenerated nucleus pulposus (NP) cells elicit immune responses. An in vitro colony forming inhibition assay demonstrated the suppressive effects of autologous spleen cells on NP cells and an in vitro cytotoxicity assay showed the positive cytotoxic effects of natural killer (NK) cells and macrophages on NP cells. Non-degenerated rat NP tissues transplanted into wild type rats and immune-deficient mice demonstrated a significantly higher NP cell survival rate in immune-deficient mice. Immunohistochemical staining showed the presence of macrophages and NK cells in the transplanted NP tissues. These results suggest that even non-degenerated autologous NP cells are recognized by macrophages and NK cells, which may have an immunological function in the early phase of disc herniation. These findings contribute to understanding resorption and the inflammatory reaction to disc herniation.

The nucleus pulposus (NP) of the intervertebral disc in cattle and humans shows the most dramatic changes with aging of any cartilaginous tissue. In humans, notochordal cells disappear from the NP and are replaced with chondrocytic cells by adolescence. However, notochordal cells of the NP persist into adult life in some species, such as rats and rabbits. Therefore, comparison of the metabolic activity of notochordal and nonnotochordal cells is considered to be important for determining the type of cell to use for transplantation to regenerate intervertebral discs. In this study, we investigated the notochordal NP cells of rats and rabbits, as well as nonnotochordal (chondrocyte-like) bovine NP cells, in a three-dimensional culture system to examine whether proteoglycan metabolism varied among these three cell types. As a result, bovine NP cells produced around 0.18 mg/mL of glycosaminoglycan after culture for 5 days, while rat and rabbit NP cells produced about four and two times more glycosaminoglycan than bovine cells, respectively. In conclusion, this study demonstrated marked differences of energy metabolism and production of matrix components between notochordal and nonnotochordal NP cells. Animals with notochordal cells in the NP, such as rats and rabbits, may not provide good models for investigation of biological repair and tissue engineering for human disc disorders.

Intervertebral disc transplantation was developed in a bipedal animal model through the stages of autograft, fresh allograft and fresh frozen allograft. Results showed that the allografts were able to survive through a deep freezing protocol and maintain cell viability after transplantation without significant immunoreaction. Although degeneration of the allograft appeared to be inevitable, it was able to maintain stability and mobility of the functional spinal unit. These findings were similarly reproduced in the human clinical trial with excellent mid-term clinical results at 5 years. The process of evolution and findings were summarized in this review.

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.

Background: Spinal fusion can be complicated by accelerated degeneration of the adjacent segments. Artificial disc replacements have been developed, but results are variable. Successful transplantations of intervertebral disc autografts, fresh allografts, and fresh-frozen allografts-ie, a non-fusion strategy-in which the mobility and stability of the spinal segment were preserved have been done in a primate model. Our aim was to determine the feasibility, safety, and long-term clinical results of disc transplantation in human beings., Methods: Five patients, average age 47 years, with cervical disc herniation underwent transplantation of fresh-frozen composite disc allografts after disc excision. Serial MRI and static and dynamic radiographs were used to monitor the status of the grafts and the sagittal stability and mobility of the segment., Findings: Good union of the graft endplates was seen by the end of 3 months after surgery in all patients. At a minimum follow-up of 5 years, the neurological symptoms of all patients had improved from before surgery levels. No immunoreaction was encountered. There was no olisthesis and only mild degenerative changes of the transplanted discs. All except one of the discs showed preservation of 7.0-11.3 degrees of sagittal motion at the final follow-up. MRI at 5 years showed preservation of hydration in at least two discs., Interpretation: Despite signs of mild disc degeneration, the motion and stability of the spinal unit was preserved after transplantation of fresh-frozen allogenic intervertebral discs in our patients. With further refinements, such transplantations could be an effective treatment for degenerative disc disease.

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.

The normal IVD clinically acts to support and dissipate loads while permitting multiaxial motions of the spine. Its demanding mechanical function is provided by a well-defined microstructural organization and biochemical composition. IVD degeneration is a complex process that disrupts this well-defined organization and biochemical balance. One hallmark of IVD degeneration is the loss of proteoglycan and water in the NP. Because of the central role of proteoglycans in the function of the IVD, restoration of normal proteoglycan production may be critical. Many different biological strategies have been developed, including the use of cells, scaffolds, and molecules. The molecules used to treat disc degeneration include anticatabolics and growth factors, which may influence the cell proliferation rate and phenotypic expression of the cells. Delivery of the molecules may include direct injection into the disc and also in vivo and ex vivo gene therapy using a viral vector. Although many of the in vitro and in vivo studies have exhibited promise in reversing the observed changes of disc degeneration, the unanswered question is whether these efforts will translate to the relief of patients' symptoms, the most common of which is back pain.

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.

This study investigated cytokine expression, behavioral and neurophysiologic changes in Lewis rats whose lumbar nerve roots were exposed to nucleus pulposus (NP). Allografted NP or fat was implanted over the left L5 nerve root. Sham rats had no NP or fat implantation. Control rats had no surgery. Rats were allowed to survive for 7 days and were tested daily for hind-paw mechanical and thermal withdrawal response (TWR). Granulation tissue was processed by immunohistochemistry for cytokines--interleukin 1 beta (IL-1beta), interleukin 6 (IL-6) and tumor necrosis factor (TNF). Neurophysiological response from the L5 nerve roots was also characterized after 7 days. Significant staining density for IL-1beta, IL-6 and TNF was observed in NP granulation tissue compared with fat and sham (p<0.05). However, there were no significant thermal and mechanical behavioral changes. TWR data computed as percentage-difference scores indicated no significant changes in withdrawal response between the four groups, although NP-treated group showed a trend of decreasing withdrawal latency. Comparison of combined percentage-difference scores revealed increased sensitivity in the NP group on days 4, 5 and 6, 7 when compared with control rats only, with no significant changes in the percentage-difference scores of fat and sham rats when compared to control. Neurophysiologically, the percentage increase in discharge rate in NP-treated rats was higher than control (p<0.05) but not higher than fat and sham rats. These results support the inflammatory nature of NP but offer limited support to NP-mediated thermal behavioral changes.

Herniation of the nucleus pulposus (NP) from lumbar intervertebral discs commonly results in radiculopathic pain possibly through a neuroinflammatory response. NP sensitizes dorsal horn neuronal responses, but it is unknown whether this reflects a central or peripheral sensitization. To study central sensitization, we tested if NP enhances windup--the progressive increase in the response of a nociceptive spinal neuron to repeated electrical C-fiber stimulation--a phenomenon that may partly account for temporal summation of pain. Single-unit recordings were made from wide dynamic range (WDR; n = 36) or nociceptive-specific (NS; n = 8) L5 dorsal horn neurons in 44 isoflurane-anesthetized rats. Subcutaneous electrodes delivered electrical stimuli (20 pulses, 3 times the C-fiber threshold, 0.5 ms) to the receptive field on the hindpaw. Autologous NP was harvested from a tail disc and placed onto the L5 dorsal root ganglion after recording of baseline responses (n = 22). Controls had saline applied similarly (n = 22). Electrical stimulus trains (0.1, 0.3, and 1 Hz; 5-min interstimulus interval) were repeated every 30 min for 3-6 h after each treatment. The total number of evoked spikes (summed across all 20 stimuli) to 0.1 Hz was enhanced 3 h after NP, mainly in the after-discharge (AD) period (latency > 400 ms). Total responses to 0.3 and 1.0 Hz were also enhanced at > or = 60 min after NP in both the C-fiber (100- to 400-ms latency) and AD periods, whereas the absolute windup (C-fiber + AD - 20 times the initial response) increased at > or = 90 min after treatment. In saline controls, windup was not enhanced at any time after treatment for any stimulus frequency, although there was a trend toward enhancement at 0.3 Hz. These results are consistent with NP-induced central sensitization. Mechanical responses were not significantly enhanced after saline or NP treatment. We speculate that inflammatory agents released from (or recruited by) NP affect the dorsal root ganglion (and/or are transported to cord) to enhance primary afferent excitation of nociceptive dorsal horn neurons.

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.

A parametric study was conducted to evaluate axial stiffness of the interbody fusion, compressive stress, and bulging in the endplate due to changes in the spacer position with/without fusion bone using an anatomically accurate and validated L2-L3 finite-element model exercised under physiological axial compression. The results show that the spacer plays an important role in initial stability for fusion, and high compressive force is predicted at the ventral endplate for the models with the spacer and fusion bone together. By varying the positioning of the spacer anteriorly along anteroposterior axis, no significant change in terms of axial stiffness, compressive stress, and bulging of the endplate are predicted for the implant model. The findings suggest that varying the spacer position in surgical situations does not affect the mechanical behavior of the lumbar spine after interbody fusion.

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.