No description available.

Background: Quantitative imaging surrogates of myelin and axonal integrity using magnetization transfer and diffusion tensor imaging may provide beneficial prognostic details on long-term post-surgical recovery in dogs with spinal cord injury (SCI) secondary to intervertebral disc herniation (IVDH). Hypothesis: Magnetization transfer ratio (MTR), mean diffusivity (MD), axial diffusivity (AD), radial diffusivity (RD), and fractional anisotropy (FA) will be significantly different in patients with a successful outcome compared to patients with an unsuccessful outcome. Animals: 61 dogs with SCI secondary to IVDH were included in the final analysis. All dogs had to undergo surgical correction for SCI secondary to IVDH and be followed out for 12 weeks. Methods: Prospective cohort study. MTR, MD, AD, RD, and FA were calculated in dogs with SCI secondary to IVDH. A Wilcoxon signed-rank test was used to compare MTR, MD, AD, RD, and FA values between patients with a successful outcome and patients with an unsuccessful outcome. Statistical significance was set at p<0.05. For quantitative imaging surrogates with a significant relationship with outcome, a receiver operator characteristic (ROC) curve analysis was performed and the sensitivity and specificity for predicting successful outcome. Results: MTR (p=0.0013) was significantly lower in patients with a successful outcome compared to patients with an unsuccessful outcome. FA (p=0.435) was not significantly between groups. MD (p=0.0006), AD (p=0.0008) and RD (p=0.0002) were significantly higher in patients with a successful outcome compared to patients with an unsuccessful outcome. ROC curves were performed for MTR, AD and RD. If MTR was ≤ 53, AD ≥ 1.7 × 10-3mm2/s or RD ≥ 0.37 × 10-3 mm2/s, this resulted in a sensitivity of 96.3% and specificity of 100 in predicting a successful outcome. Conclusion and clinical relevance: MTR, MD, AD, and RD were helpful in predicting successful outcome in canine patients with surgically treated SCI secondary to IVDH. A larger cohort is needed for further evaluation. / Master of Science / Background: Certain magnetic resonance imaging (MRI) techniques can provide information about the severity of spinal cord injury. The information obtained from these MRI techniques can be helpful in predicting prognosis in dogs with intervertebral disc disease (IVDD). Hypothesis: We hypothesized that the measurements obtained from these MRI techniques would be able to predict the patients who would be able to walk following surgery (good long-term outcome), versus the patients who did not regain the ability to walk following surgery (poor long-term outcome). Animals: 62 MRIs were performed on dogs with IVDD in our study and were followed out for 12 weeks following surgery to assess long-term outcome. Results: Of the 5 MRI techniques investigated, 4 of the techniques were found to be helpful in predicted long-term outcome. When these techniques were combined, the ability to predict long-term outcome improved. Using the combined technique, all 53 patients predicted to have a good long-term had a good long-term outcome. For patients with a poor long-term outcome, 9 were predicted to have a poor long-term outcome, but only 7 patients had a poor long-term outcome. Conclusion and clinical relevance: MRI can be helpful in predicting long-term outcome in dogs with IVDD following surgery. A larger population of dogs is needed for further evaluation.

No description available.

INTRODUÇÃO: A degeneração do disco intervertebral (DIV) é um processo crônico e apontado como o maior causador de cervicalgia e lombalgia. Esse processo geralmente conta com a degradação da matriz extracelular, expressão de citocinas inflamatórias e fatores angiogênicos e axonogênicos. Entretanto, muito pouco se sabe sobre esse processo em DIVs assintomáticos durante o envelhecimento, principalmente no segmento cervical. O objetivo desse estudo foi de delinear o perfil de moléculas relacionadas à degeneração discal em DIVs cervicais e lombares. MÉTODOS: Discos intervertebrais humanos cervicais e lombares (C4-C6 e L4-S1) foram coletados em autópsia de 30 indivíduos presumivelmente assintomáticos e divididos em grupos jovem (GJ < 35 anos, n=60) e idoso (GI > 65 anos, n=60). O nível de degeneração foi constatado pela escala de Thompson, e foi correlacionado com a detecção imuno-histoquímica das moléculas de MMP-1, -2, -3, TIMP-1, IL-1beta, TNF-alfa, VEGF, NGF-beta e BDNF. RESULTADOS: Todos os DIVs mostraram algum grau de degeneração, embora mais acentuadas no GI. As moléculas empenhadas no estudo foram identificadas em ambos grupos. A detecção imuno-histoquímica foi prevalente no citoplasma das células nativas do DIV e na região de interseção entre a placa vertebral e o arranjo fibro-colágeno. O envelhecimento propiciou, no disco cervical, maior expressão de MMP-2, -3, VEGF, NGF-beta e BDNF, enquanto que no disco lombar, a maior expressão foi de MMP-1, -2 -3, TIMP-1, TNF-alfa, VEGF e NGF-beta. DISCUSSÃO: O envelhecimento de DIVs cervicais e lombares caracterizou-se por exibir um processo catabólico e extensivo remodelamento da matriz extracelular, os quais podem ser interpretados como eventos que antecipam a doença degenerativa discal. Esse processo é capaz de levar a angiogênese e axonogênese de modo a ampliar o metabolismo aeróbio do DIV e captar informação nociceptiva como forma de defesa, uma vez que até nos discos lombares de indivíduos jovens essa última característica pôde ser observada. Discos assintomáticos também exibem moléculas relacionadas à doença degenerativa discal e talvez a inibição de parte dessas possa resultar em terapia preventiva / INTRODUCTION: Degeneration of the intervertebral disc (DIV) is a chronic process that pointed as a major cause of neck and low back pain. This process generally includes an extracellular matrix degradation, expression of inflammatory cytokines, angiogenesis and axonogenesis factors. However, there is a little known about this process in asymptomatic DIVs during aging, especially in the cervical region. The aim of this study was to delineate the profile of molecules related to disc degeneration in the cervical and lumbar discs. METHODS: Human cervical and lumbar intervertebral discs (C4-C6 e L4-S1) were harvested at autopsy from 30 asymptomatic individuals, and divided according to age with young (GJ < 35 years old, n=60) and elderly (GI > 65 years old, n=60) groups. Gross degeneration was graded according to the Thompson scale and this was correlated to the immunohistochemical detection of molecules of MMP-1, -2, -3, TIMP-1, IL-1beta, TNF-alfa, VEGF, NGF-beta e BDNF. RESULTS: Discs from GJ were significantly less degenerated than those of GI. The molecules involved in the study were identified in both groups. The immunohistochemical detection was prevalent in the cytoplasm of native disc cells and the region between the vertebral plate and fibrous collagen arrangement (intersection). Aging provided in cervical disc, increased expression of MMP-2, -3, VEGF, NGF and BDNF-beta, whereas in the lumbar disc the highest expression of MMP-1, -2, -3, TIMP-1, TNF-alfa, VEGF and NGF-beta was seen. DISCUSSION: The aging of cervical and lumbar DIV was marked by catabolic process and a extensive remodeling on extracellular matrix which can be interpreted as a predict event of the degenerative disc disease. This process can lead to angiogenesis and axonogenesis in order to expand the aerobic metabolism of the DIV and get nociceptive information as a defense, since even in the lumbar discs of young individuals this last feature can be observed. Asymptomatic discs also exhibit molecules related to degenerative disc disease and perhaps the inhibition some of these can result in preventive therapy

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No. of bitstreams: 2 Dissertação - Nara Lígia Leão Casa - 2015.pdf: 3128154 bytes, checksum: 290faf6f7e255eb06dcfd7b0baf48e03 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-12-02 / Introduction: Lumbar disc herniation (LDH) is the most common diagnosis among the degenerative changes in the lumbar spine and it can lead to functional limitations. Although several studies have shown a positive association between polymorphisms in the aggrecan gene and LDH, results are conflicting and vary across populations. Until now, such studies have not been carried out in Brazil. An understanding of this association can help in the prevention and intervention processes in patients with LDH. Objective: Investigate the association between polymorphisms in the aggrecan gene and LDH. Methods: This is a Case-Control study, paired by gender and age. A total of 119 male and female individuals from Goiania (Brazil) participated in the study; 39 individuals in the Case Group (CaG) and 80 in the Control Group (CtG). For each individual, sociodemographic and clinical data were collected as well as blood samples for genetic analysis. DNA was isolated and genotyped at the variable number of tandem repeats (VNTR) region of the aggrecan gene using polymerase chain reaction. Results: Both groups were homogeneous for socio-demographic, anthropometric, and life style variables. The sum of allele sizes at the VNTR region of the aggrecan gene were significantly lower in individuals with LDH, with allele A22 showing significantly higher frequency in this group. Comparison of allele size distribution indicated that shorter alleles (i.e., A13-A25) were more frequent in individuals with LDH, whereas alleles with a higher number of repeats were more frequent among healthy individuals. It should be noted, however, that differences were not significant. The most frequent alleles in both groups were A28, A27 and A29, respectively. Genotype A26/A26 was the most common among individuals in the CaG. Conclusions: An association between short alleles and LDH was observed in this study. This finding is in agreement with other reports in the literature, corroborating the hypothesis that individuals with shorter alleles have an aggrecan gene with fewer repeats. Therefore, a smaller number of chondroitin sulfate chains bind to the aggrecan in the intervertebral disc, leading to a decrease in its physiological function of hydration of the intervertebral disc, and thus to an increase in LDH susceptibility. / Introdução: A hérnia de disco lombar (HDL) é um diagnóstico frequente dentre as alterações degenerativas da coluna lombar e causa limitações funcionais importantes. Alguns estudos apontam para uma associação positiva entre polimorfismos no gene aggrecan e a HDL, porém os resultados ainda são conflitantes e variam entre as populações, não havendo pesquisas sobre o tema na população brasileira até o momento. Compreender essa associação pode auxiliar na prevenção e intervenção junto às pessoas com HDL. Objetivo: Investigar a associação entre polimorfismos no gene aggrecan com a hérnia de disco lombar. Métodos: Estudo caso-controle, pareado em quinquênio por idade e sexo. Participaram do estudo 119 pessoas de ambos os sexos, domiciliadas em Goiânia (Brasil), sendo 39 no Grupo Caso (Ca) e 80 no Controle (Ct). Foram coletados dados sociodemográficos e clínicos e amostras de sangue periférico; o DNA foi isolado para posterior genotipagem do número variável de repetições em Tandem (VNTR) do gene aggrecan (ACAN) através de PCR convencional. Resultados: Os grupos mostraram-se homogêneos quanto às variáveis sociodemográficas, antropométricas e hábitos de vida. O escore alélico do VNTR do gene aggrecan foi significativamente menor nos voluntários com HDL; o alelo A22 mostrou-se significativamente mais prevalente nesse mesmo grupo; no grupo Ca houve maior frequência dos alelos pequenos A13 - A25, enquanto no Ct maior frequência dos alelos grandes, porém, sem diferença estatisticamente significativa; em ambos os grupos os alelos mais frequentes foram A28, A27 e A29, respectivamente; o genótipo A26/A26 foi significativamente mais comum no Grupo Ca. Conclusões: Houve associação entre alelos pequenos com a HDL nos adultos pesquisados e compatibilidade com a literatura internacional, corroborando a hipótese de que indivíduos que possuem alelos pequenos possuem um aggrecan com menor número de repetições. Portanto, um número menor de cadeias de sulfato de condroitina se ligam no aggrecan do disco intervertebral, o que resulta na diminuição da sua função fisiológica de hidratação do disco e, consequentemente, aumenta a susceptibilidade individual à HDL.

INTRODUÇÃO: A degeneração do disco intervertebral (DIV) é um processo crônico e apontado como o maior causador de cervicalgia e lombalgia. Esse processo geralmente conta com a degradação da matriz extracelular, expressão de citocinas inflamatórias e fatores angiogênicos e axonogênicos. Entretanto, muito pouco se sabe sobre esse processo em DIVs assintomáticos durante o envelhecimento, principalmente no segmento cervical. O objetivo desse estudo foi de delinear o perfil de moléculas relacionadas à degeneração discal em DIVs cervicais e lombares. MÉTODOS: Discos intervertebrais humanos cervicais e lombares (C4-C6 e L4-S1) foram coletados em autópsia de 30 indivíduos presumivelmente assintomáticos e divididos em grupos jovem (GJ < 35 anos, n=60) e idoso (GI > 65 anos, n=60). O nível de degeneração foi constatado pela escala de Thompson, e foi correlacionado com a detecção imuno-histoquímica das moléculas de MMP-1, -2, -3, TIMP-1, IL-1beta, TNF-alfa, VEGF, NGF-beta e BDNF. RESULTADOS: Todos os DIVs mostraram algum grau de degeneração, embora mais acentuadas no GI. As moléculas empenhadas no estudo foram identificadas em ambos grupos. A detecção imuno-histoquímica foi prevalente no citoplasma das células nativas do DIV e na região de interseção entre a placa vertebral e o arranjo fibro-colágeno. O envelhecimento propiciou, no disco cervical, maior expressão de MMP-2, -3, VEGF, NGF-beta e BDNF, enquanto que no disco lombar, a maior expressão foi de MMP-1, -2 -3, TIMP-1, TNF-alfa, VEGF e NGF-beta. DISCUSSÃO: O envelhecimento de DIVs cervicais e lombares caracterizou-se por exibir um processo catabólico e extensivo remodelamento da matriz extracelular, os quais podem ser interpretados como eventos que antecipam a doença degenerativa discal. Esse processo é capaz de levar a angiogênese e axonogênese de modo a ampliar o metabolismo aeróbio do DIV e captar informação nociceptiva como forma de defesa, uma vez que até nos discos lombares de indivíduos jovens essa última característica pôde ser observada. Discos assintomáticos também exibem moléculas relacionadas à doença degenerativa discal e talvez a inibição de parte dessas possa resultar em terapia preventiva / INTRODUCTION: Degeneration of the intervertebral disc (DIV) is a chronic process that pointed as a major cause of neck and low back pain. This process generally includes an extracellular matrix degradation, expression of inflammatory cytokines, angiogenesis and axonogenesis factors. However, there is a little known about this process in asymptomatic DIVs during aging, especially in the cervical region. The aim of this study was to delineate the profile of molecules related to disc degeneration in the cervical and lumbar discs. METHODS: Human cervical and lumbar intervertebral discs (C4-C6 e L4-S1) were harvested at autopsy from 30 asymptomatic individuals, and divided according to age with young (GJ < 35 years old, n=60) and elderly (GI > 65 years old, n=60) groups. Gross degeneration was graded according to the Thompson scale and this was correlated to the immunohistochemical detection of molecules of MMP-1, -2, -3, TIMP-1, IL-1beta, TNF-alfa, VEGF, NGF-beta e BDNF. RESULTS: Discs from GJ were significantly less degenerated than those of GI. The molecules involved in the study were identified in both groups. The immunohistochemical detection was prevalent in the cytoplasm of native disc cells and the region between the vertebral plate and fibrous collagen arrangement (intersection). Aging provided in cervical disc, increased expression of MMP-2, -3, VEGF, NGF and BDNF-beta, whereas in the lumbar disc the highest expression of MMP-1, -2, -3, TIMP-1, TNF-alfa, VEGF and NGF-beta was seen. DISCUSSION: The aging of cervical and lumbar DIV was marked by catabolic process and a extensive remodeling on extracellular matrix which can be interpreted as a predict event of the degenerative disc disease. This process can lead to angiogenesis and axonogenesis in order to expand the aerobic metabolism of the DIV and get nociceptive information as a defense, since even in the lumbar discs of young individuals this last feature can be observed. Asymptomatic discs also exhibit molecules related to degenerative disc disease and perhaps the inhibition some of these can result in preventive therapy

<p>Intervertebral disc (IVD) disorders and age-related degeneration are believed to contribute to low back pain. There is significant interest in cell-based strategies for regenerating the nucleus pulposus (NP) region of the disc; however, few scaffolds have been evaluated for their ability to promote or maintain an immature NP cell phenotype. Additionally, while cell delivery to the pathological IVD has significant therapeutic potential for enhancing NP 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 NP region of the IVD that promote cell attachment and biosynthesis. These findings suggest that incorporating laminin ligands into biomaterial scaffolds for NP tissue engineering or cell delivery to the disc may be beneficial for promoting NP cell survival and phenotype. In this dissertation, laminin-111 (LM111) functionalized poly(ethylene glycol) (PEG) hydrogels were developed and evaluated as biomaterial scaffolds for cell-based NP regeneration. </p><p>Here, PEG-LM111 conjugates with functional acrylate groups for crosslinking were synthesized and characterized to allow for protein coupling to both photocrosslinkable and injectable PEG-based biomaterial scaffolds. PEG-LM111 conjugates synthesized using low ratios of PEG to LM111 were found support NP cell attachment and signaling in a manner similar to unmodified LM111. A single PEG-LM111 conjugate was conjugated to photocrosslinkable PEG-LM111 hydrogels, and studies were performed to evaluate the effects of hydrogel formulation on immature NP cell phenotype in vitro. When primary immature porcine NP cells were seeded onto PEG-LM111 hydrogels of varying stiffnesses, softer LM111 presenting hydrogels were found to promote cell clustering and increased levels of sGAG production as compared to stiffer LM111 presenting and PEG-only gels. When cells were encapsulated in 3D gels, hydrogel formulation was found to influence NP cell metabolism and expression of proposed NP phenotypic markers, with higher expression of N-cadherin and cytokeratin 8 observed for cells cultured in softer (<1 kPa) PEG-LM111 hydrogels. </p><p>A novel, injectable PEG-LM111 hydrogel was developed as a biomaterial carrier for cell delivery to the IVD. PEG-LM111 conjugates were crosslinked via a Michael-type addition reaction upon the addition of PEG-octoacrylate and PEG-dithiol. Injectable PEG-LM111 hydrogel gelation time, mechanical properties, and ability to retain delivered cells in the IVD space were evaluated. Gelation occurred in approximately 20 minutes 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 hydrogel carrier, as compared to cells in liquid suspension. </p><p>The studies presented in this dissertation demonstrate that soft, LM111 functionalized hydrogels may promote or maintain the expression of specific markers and cell-cell interactions characteristic of an immature NP cell phenotype. Furthermore, these findings suggest that this novel, injectable laminin-functionalized biomaterial may be an easy to use and biocompatible carrier for delivering cells to the IVD.</p> / Dissertation

This thesis comprises three phases with a combined aim which was to investigate the effect of position on the lumbar intervertebral disc (IVD). The effect of position on the lumbar IVD in asymptomatic subjects and subjects with discogenic low back pain (DLBP) was explored using positional Magnetic Resonance Imaging (pMRI). Convenience samples of 11 asymptomatic and 34 DLBP subjects were recruited to have sagittal and axial pMRI scans performed in sitting (Neutral, Flexed and Extended), standing and lying (Supine and Prone extension) positions. The sagittal plane migration of the nucleus pulposus (NP) of each lumbar IVD in each position was measured from the sagittal and axial pMRI scans. Within and between group inferential analysis was performed using nonparametric tests. Both the asymptomatic and DLBP subjects’ demonstrated that position had statistically significant effects on the sagittal plane NP migration. Both groups demonstrated significantly greater posterior sagittal plane NP migration in Neutral and Flexed sitting positions compared to the other positions. However, between group comparisons identified that the asymptomatic subjects also demonstrated significantly greater posterior sagittal plane NP migration than the DLBP subjects. This pattern was more common in the upper lumbar IVDs (L1/2 and L2/3) between positions and less common in the lower IVDs (L4/5 and L5/S1) between positions. New knowledge regarding the behaviour of the lumbar IVD emerged from this research. The differences detected between the asymptomatic and DLBP subjects suggest that some current theories regarding DLBP may be incorrect. The results also support imaging of DLBP subjects in sitting positions as opposed to current supine positions. Although the limitations of the study reduce generalisation of the results, the implications for clinical practice, imaging and suggestions for further research from this work are important to improve understanding and conservative management of DLBP.

Back pain is a massive global public health problem with multiple contributing factors including obesity. Obesity is thought to be linked to back pain through mechanical factors. However, obesity also causes a systemic low-grade inflammatory milieu. This would suggest a possible biochemical link between obesity, intervertebral disc degeneration, and back pain. Furthermore, the relationship between obesity and the clinical presentation of spine patients is unclear. This thesis aims to examine the effect of and relationship between obesity, the intervertebral discs, and back pain from biochemical, clinical, and epidemiological perspectives. In this thesis, an in vitro study assessed the effect of leptin, a fat-specific cytokine, upon the intervertebral disc. The bovine intervertebral disc was used as a model in a cell culture system. An ex vivo study examined leptin and pro-inflammatory cytokines produced by paraspinal adipose tissue taken during routine surgical procedures from spinal patients. Plasma taken from patients presenting with low back pain was analysed by mass spectrometry and multiplex immunoassay to identify possible protein biomarkers. At an epidemiological level, statistical modelling of the Genodisc patient population was conducted. This was a pan-European study of 2636 patients presenting to tertiary spinal units. Analyses were performed to examine relationships between obesity, quantified by body mass index (BMI), and pain, clinical diagnosis, and spinal degeneration identified on magnetic resonance imaging (MRI). Leptin was shown to increase the production of and expression of degradative and pain-generating molecules by disc cells. A pro-inflammatory environment, especially IL-6, potentiated this response. Leptin and pro-inflammatory cytokines produced by paraspinal fat were unrelated to clinical symptoms. However, levels of the pro-inflammatory cytokines, TNF-&alpha; and IL-6, were raised in the plasma of patients with greater pain or those with spinal stenosis. Furthermore, clusterin and complement were identified, by mass spectrometry, as potential biomarkers for spine patients. Epidemiological analyses revealed that obesity was associated with greater back pain, although the magnitude of this association was small. Similarly, obesity was associated with a diagnosis of spinal stenosis. Finally, increased BMI was found to be an independent predictor of disc degeneration, spinal stenosis, and disc herniation on MRI. In summary, this thesis has furthered the clinical understanding of lumbar spine pathology and back pain. It will provide clinicians with a better framework to assess spine patients. These results show that obesity is associated with lumbar spine degeneration and pain. Leptin could be a factor mediating this relationship. Further studies should concentrate on clarifying the mechanism of action of leptin upon the intervertebral disc and assessing the longitudinal effect of obesity upon the lumbar spine. In this thesis, an in vitro study assessed the effect of leptin, a fat-specific cytokine, upon the intervertebral disc. The bovine intervertebral disc was used as a model in a cell culture system. An ex vivo study examined leptin and pro-inflammatory cytokines produced by paraspinal adipose tissue taken during routine surgical procedures from spinal patients. Plasma taken from patients presenting with low back pain was analysed by mass spectrometry and multiplex immunoassay to identify possible protein biomarkers. At an epidemiological level, statistical modelling of the Genodisc patient population was conducted. This was a pan-European study of 2636 patients presenting to tertiary spinal units. Analyses were performed to examine relationships between obesity, quantified by body mass index (BMI), and pain, clinical diagnosis, and spinal degeneration identified on magnetic resonance imaging (MRI). Leptin was shown to increase the production of and expression of degradative and pain-generating molecules by disc cells. A pro-inflammatory environment, especially IL-6, potentiated this response. Leptin and pro-inflammatory cytokines produced by paraspinal fat were unrelated to clinical symptoms. However, levels of the pro-inflammatory cytokines, TNF-α and IL-6, were raised in the plasma of patients with greater pain or those with spinal stenosis. Furthermore, clusterin and complement were identified, by mass spectrometry, as potential biomarkers for spine patients. Epidemiological analyses revealed that obesity was associated with greater back pain, although the magnitude of this association was small. Similarly, obesity was associated with a diagnosis of spinal stenosis. Finally, increased BMI was found to be an independent predictor of disc degeneration, spinal stenosis, and disc herniation on MRI. In summary, this thesis has furthered the clinical understanding of lumbar spine pathology and back pain. It will provide clinicians with a better framework to assess spine patients. These results show that obesity is associated with lumbar spine degeneration and pain. Leptin could be a factor mediating this relationship. Further studies should concentrate on clarifying the mechanism of action of leptin upon the intervertebral disc and assessing the longitudinal effect of obesity upon the lumbar spine.

<p>Juvenile nucleus pulposus (NP) cells of the intervertebral disc (IVD) are large, vacuolated cells that form cell clusters with numerous cell-cell interactions. With maturation and aging, NP cells lose their ability to form these cell clusters, with associated changes in NP cell phenotype, morphology and proteoglycan synthesis that may contribute to IVD degeneration. Studies demonstrate healthy, juvenile NP cells exhibit potential for preservation of multi-cell clusters and NP cell phenotype when cultured upon soft, laminin-containing substrates; however, the mechanisms that regulate metabolism and phenotype of these NP cells are not understood. N-cadherin is a cell adhesion molecule that is present in juvenile NP cells, but disappears with age. The goal of this dissertation was to reveal the role of N-cadherin for NP cells in multi-cell clusters that contribute to the maintenance of the juvenile NP cell morphology and phenotype in vitro, and to evaluate the potential for laminin- functionalized poly(ethylene glycol) (PEG-LM) hydrogels to promote human NP cells towards a juvenile NP cell phenotype. </p><p>In this dissertation, juvenile porcine IVD cells were promoted to form cell clusters in vitro, and analyzed for preservation of the juvenile NP phenotype on soft, laminin-rich hydrogels. In the first part of this dissertation, preservation of the porcine juvenile NP cell phenotype and presence of N-cadherin was analyzed by culturing porcine NP cells on soft, laminin-rich or PEG-LM hydrogels. Secondly, cadherin-blocking experiments were performed to prevent cluster formation in order to study the importance of cluster formation in NP cell signaling. Finally, human IVD cells were cultured on PEG-LM hydrogels to investigate the potential to revert degenerate, human NP cells toward a juvenile NP cell phenotype and morphology. </p><p>Findings reveal soft (<500 Pa), laminin-rich substrates promote NP cell clustering, a key feature of the juvenile NP cell that is associated with N-cadherin positive expression. Additionally, N-cadherin-mediated cell-clustering regulates NP cell matrix production and gene expression of NP-specific and NP-matrix related markers. Inhibition of N-cadherin-mediated contacts resulted in decreased expression of juvenile NP cell features. Finally, juvenile human NP cells are also able to form N-cadherin positive cell clusters on soft, PEG-LM hydrogels with higher expression of juvenile NP cell features compared to culturing on stiff PEG-LM hydrogels. Some degenerate, human NP cells are also able to form N-cadherin positive cell clusters with some features of the juvenile NP cell. </p><p>The studies presented in this dissertation support the proposed hypothesis and establish the importance of soft, laminin-rich substrates in promoting NP cell clustering behaviors with associated features of a juvenile cell phenotype and morphology. Additionally, these studies establish a regulatory role for N-cadherin in juvenile NP cells and suggest that preservation of N-cadherin-mediated cell-cell contacts is important for preserving the juvenile NP cell phenotype and morphology. Furthermore, findings from this dissertation reveal the ability to promote degenerate, mature human NP cells towards a juvenile NP cell phenotype, demonstrating the potential to use PEG-LM hydrogels as a means for autologous cell delivery for the restoration of healthy IVD.</p> / Dissertation