Effect of age on outcomes after allogeneic disc tissue supplementation in patients with chronic discogenic low back pain in the VAST trial.

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 VAST trial evaluated a new treatment for patients with chronic back pain resulting from one or two degenerated spinal discs. The treatment consists of a single injection of disc tissue supplement. A total of 218 adults participated in the study; most received the active treatment, while a smaller number (39 patients) received an injection of saline. In this paper we explain what happened over the 12 months after the injections. Patients who were younger (<42 years old) experienced more functional benefits (i.e., ability to perform daily tasks) after active treatment compared with those who received the saline injection, as measured by disability score. In contrast, older patients (≥42 years old) experienced functional benefits with both active and saline treatments, with no differences between the groups. There were more side effects in both age groups in those who received the active treatment compared with those who received saline, but almost all of the side effects were temporary and not serious. Clinical Trial Registration number: NCT03709901 (ClinicalTrials.gov).

A Hyaluronan and Platelet-Rich Plasma Hydrogel for Mesenchymal Stem Cell Delivery in the Intervertebral Disc: An Organ Culture Study.

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 × 106 or 2 × 106 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.

Decellularized Scaffolds for Intervertebral Disc Regeneration.

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.

The establishment and biological assessment of a whole tissue-engineered intervertebral disc with PBST fibers and a chitosan hydrogel in vitro and in vivo.

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.

Captação e preservação de disco intervertebral humano em banco de tecidos / Capture and preservation of human intervertebral disc in a tissue bank

Doença degenerativa da coluna vertebral, sobretudo, o acometimento do disco intervertebral, é frequente na população mundial. O tratamento cirúrgico apresenta indicações precisas constituindo o padrão-ouro, a artrodese. Esta modalidade, entretanto, altera a mobilidade e a biomecânica do segmento funcional. A criopreservação de disco intervertebral em banco de tecidos e sua posterior utilização como substituto do disco degenerado objetivam manter a fisiologia local. Propor técnica cirúrgica de ressecção de bloco de coluna lombar e avaliar a possibilidade da manutenção das características histológicas dos discos intervertebrais submetidos ao congelamento com criopreservante. Foram obtidos espécimes de 6 doadores cadáveres humanos após avaliação e autorização de captação de tecido musculoesquelético de acordo com as normas do Sistema Nacional de Transplantes por meio da Portaria nº 2600 de 21 de outubro de 2009 do Ministério da Saúde. Após a realização de uma técnica de ressecção no bloco da coluna lombar, o material foi transportado para o banco de tecidos do Instituto Nacional de Traumatologia e Ortopedia Jamil Haddad em um recipiente específico com soro fisiológico 0,9% à temperatura de 4°C. Cada bloco de coluna lombar gerou, após o processamento do tecido realizado até 8 horas da captação, 3 discos intervertebrais. Dois discos foram armazenados em meio crioprotetor e congelados de forma gradual até -80°C. Cada disco foi descongelado no 7° e 15° dia, seccionados longitudinal e transversalmente e então, confeccionadas lâminas coradas com hematoxilina-eosina para análise histológica; Um disco controle, não-congelado, foi preservado em formol 10% e sofreu o mesmo processo de secção e preparo de lâminas, coloração. O estudo histológico das lâminas observou a presença ou não de alterações morfoestruturais (ânulo fibroso, núcleo pulposo) e celularidade, pontuada e classificada em muito alterada (0-3 pontos), moderadamente alterada (4-7 pontos) e inalterada (8-10 pontos). A técnica cirúrgica de ressecção en bloc da coluna vertebral foi reproduzida em todos os doadores cadáveres. A análise histológica revelou que poucas alterações foram observadas após 7 dias de congelamento do disco em meio criopreservante. Observamos que, embora tecnicamente demandante, a ressecção de bloco de coluna lombar é reprodutível. A preservação das propriedades do disco intervertebral por, no mínimo, 7 dias é possível e torna-se uma possibilidade de material para transplantação em futuras artroplastias biológicas

Degenerative disease of the vertebral column, especially the involvement of the intervertebral disc, is frequent in the world population. The surgical treatment presents precise indications constituting the gold standard, the arthrodesis. This modality, however, alters the mobility and biomechanics of the functional segment. The cryopreservation of intervertebral disk in a tissue bank and its posterior use as a substitute for the degenerate disc aim to maintain the local physiology. To propose surgical technique of lumbar spine block resection and to evaluate the possibility of maintaining the histological characteristics of the intervertebral discs submitted to the freezing with cryopreservant. Specimens of 6 human cadaveric donors were obtained after evaluation and authorization of the capture of musculo-skeletal tissue in accordance with the norms of the National System of Transplants through Ordinance No. 2600 of October 21, 2009 of the Ministry of Health. After performing a resection technique in the lumbar spine block, the material was transported to the tissue bank of Jamil Haddad National Institute of Traumatology and Orthopedics in a specific container with 0.9% saline solution at 4 ° C. Each lumbar spine block generated, after processing the tissue performed up to 8 hours of the capture, 3 intervertebral discs. Two discs were stored in cryopreservation and frozen gradually to -80 ° C. Each disc was thawed on day 7 and day 15, sectioned longitudinally and transversely, and then, blades stained with hematoxylin-eosin for histological analysis; One disc control, non-frozen, was preserved in 10% formaldehyde and underwent the same process of sectioning and preparation of blades staining. The histological study of the slides observed the presence or absence of morphostructural alterations (fibrous annulus, nucleus pulposus) and cellularity, scored and classified as highly altered (0-3 points), moderately altered (4-7 points) and unchanged (8-10 points). The surgical technique of en bloc resection of the spine was reproduced in all cadaveric donors. Histological analysis revealed that few changes were observed after 7 days of cryopreservant disk freezing. We observed that, although technically demanding, lumbar spine block resection is reproducible. The preservation of the intervertebral disc properties for at least 7 days is possible and becomes a possibility of material for transplantation in future biological arthroplasties

Biomaterials for intervertebral disc regeneration: Current status and looming challenges.

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.

Lessons to be learned and future directions for intervertebral disc biomaterials.

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.

Mesenchymal Stem Cell Levels of Human Spinal Tissues.

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.

Lumbar intervertebral disc allograft transplantation: the revascularisation pattern.

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.

Lumbar intervertebral disc allograft transplantation: long-term mobility and impact on the adjacent segments.

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.

Lumbar intervertebral disc allograft transplantation: healing and remodelling of the bony structure.

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.

[EXPERIMENTAL STUDY ON EFFECT OF THREE DIFFERENT OPERATIVE WAYS OF ANNULUS FIBROSUS INCISION ON INTERVERTEBRAL DISC BIOMECHANICAL STRENGTH].

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.

Surgical technique for lumbar intervertebral disc transplantation in a goat model.

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.

The effect of gamma irradiation on the biological properties of intervertebral disc allografts: in vitro and in vivo studies in a beagle model.

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.

Tissue-engineered intervertebral discs: MRI results and histology in the rodent spine.

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.

Injectable laminin-functionalized hydrogel for nucleus pulposus regeneration.

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.

Inhibition of nitric oxide mediated protein nitration: therapeutic implications in experimental radiculopathy.

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.

Tissue-engineered allograft intervertebral disc transplantation for the treatment of degenerative disc disease: experimental study in a beagle model.

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.

Lateral surgical approach to lumbar intervertebral discs in an ovine model.

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.

Extending the activities of human nucleus pulposus cells with recombinant adeno-associated virus vector-mediated human telomerase reverse transcriptase gene transfer.

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.