published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

Current treatments to intervertebral disc degeneration alter spine biomechanics and have complications. Tissue engineering offers an approach to regenerate a biological disc that provides flexibility and stability to, and integrates with the spine. To date, a scaffold that mimics the extracellular matrix composition and mechanical strength of a native disc is lacked. In this project, a biphasic scaffold was fabricated using glycosaminoglycan (GAG) and collagen, the prevalent ma-trix components in a native disc. It also adapted the structure of the disc, with la-mellae of collagen surrounding a collagen-GAG (CG) core. The first part of this project studied chemical modification of CG and evaluated the physiochemical and biological properties of modified CGs. As only loosely bound by GAG under physiological environment, collagen was modified by deamination, methylation and amination, and yielded Deaminated, Methylated and Aminated CGs upon co-precipitation with GAG. While GAG was mostly lost within 1 day in Untreated and Deaminated CGs, 20% and 40% GAG was retained after 6 days in Methylated and Aminated CGs respectively. In cell-seeded Aminated CG, over 60% GAG was retained after 8 days. Aminated CG, having the highest GAG/HYP of 4.5, best simulated the GAG-rich nucleus pulposus tissue. In ultrastructural analysis, Aminated CG consisted of abundant granular sub-stances that resembled the nucleus pulposus. Despite the differential initial number adhered to the CG scaffolds, human mesenchymal stem cells (hMSCs) had over 90% viability at all time points. Cell morphology was distinct, being round in Untreated and Methylated CGs but elongated in Deaminated and Aminated ones. The adhesion of hMSCs via collagen receptor, integrin alpha2beta1, was observed in all CG scaffolds, while adhesion via general matrix receptor, integrin alphaV, was extensive in all but Aminated CG. Based on improved GAG incor-poration and retention, which approximate the matrix composition of nucleus pulposus, Aminated CG was chosen as the core of the biphasic scaffold. The second part of this project studied lamination in biphasic disc scaffold and evaluated its mechanical properties in creep, recovery and dynamic loadings. A process was optimized to encapsulate a CG under physiological condition whilst producing an intact collagen gel, which allowed the CG to retain more GAGs and to be confined by the annulus structurally as was in the disc. This encasing approach was repeated for multiple lamellae, one lamella per day. Scaffolds with more lamellae had increased viscous compliance in creep and recovery, which was explained by the less laminated scaffolds being overloaded. Another lamination approach replaced most encasing lamellae with coiling ones. Despite low sample size, it was shown that this combined approach produced scaffolds with lower elastic and viscous compliances and longer equilibrating time in both creep and recovery, and higher complex modulus under dynamic loading. Full recovery was not achieved by any scaffold. This study demonstrated that a biphasic disc scaffold, made of GAG and collagen, contained similar matrix components to native disc, was almost mechanically comparable to the disc, and was cyto-compatible. It paved way towards tissue engineering of intervertebral disc and the intervertebral disc motion segment. / published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

Intervertebral disc(IVD) degeneration is associated with low back pain (LBP).Despite the endplates are located adjacent to the IVD, the phenotypes of endplate and vertebral bone marrow changes remain unclear. The objective of this study is to assess the prevalence and the associated determinants of these phenotypes of the lumbar spine. 2449 Southern Chinese subjects (aged 10 –88 years) undergone sagittal T2-weighted magnetic resonance imaging (MRI)examination to assess the presence of endplate and vertebral bone marrow changes including Schmorl’s nodes (SN) and Modic changes (MC),respectively, and scored for additional radiographic features over the lumbar spine. Subjects’ demographics, clinical profile, and functional status were assessed by means of standardized questionnaires. Anovel6-domain SN morphological classification based on MRI was developed to further analyze the characteristics of SN and its association with disc degeneration (DD). The prevalence of SN was 16.4%. Males, taller and heavier individuals had a significantly increased likelihood of SN. Overall presence of SN was age-independent, but was significantly associated with DD, and linearly correlated with increase in severity of DD. SN were particularly associated with severe DD at the upper two lumbar levels (L1/2 – L2/3). Based on the SN classification system, specific SN characteristics and endplate linkage patterns were found. Of these, two SN types were identified:“Typical SN” and “Atypical SN”. “Typical SN” were those smaller size SN with various shapes that were frequently located at caudal endplates of L1/2–L3/4disc levels. One variant of “Atypical SN” were those rectangular shape SN, which predominantly located at the posterior region of the rostral endplates of L1/2–L2/3disc levels. The other variant of “Atypical SN” was larger size SN with irregular shape that frequently presented at the L4/5endplates, they were also likely to be associated with marrow changes. Despite “Atypical SN” only entailed 8.3% of all identified SN, they were associated with increased severity of DD than “Typical SN”. Although the overall prevalence of MC among Southern Chinese was low (5.8%), after adjustments for other confounding factors, the presence of MC was associated with the presence and severity of LBP. Interestingly, the determinants of MC at upper (L1/2 – L3/4) and lower (L4/5 – L5/S1) lumbar showed distinct difference. The presence of MC at upper lumbar levels was only associated with the disc integrity only (i.e. the presence of disc displacement, and DD score), while the presence of MC at the lower levels, apart from the disc integrity, was also associated with increasing age, the presence of SN, smoking and obesity status. This study is the largest MRI study assessing the phenotypes of endplate and vertebral bone marrow changes in-vivo. The prevalence and distribution of SN and MC vary throughout the lumbar spine, and there are level and region-specific variations regarding these phenotypes. Determinants of SN and MC may be similar but do vary, suggesting distinct etiological factors. Findings of this study broaden the understanding of the various phenotypes of the lumbar spine and its association with DD. / published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

Thesis (MScIng)--University of Stellenbosch, 2007. / Bibliography. Also available via the Internet.

published_or_final_version / abstract / Orthopaedics and Traumatology / Master / Master of Philosophy

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

Low back pain is highly prevalent in the developed world, with 80% of the population being affected at some point in their lives. Herniation, a common injury to the intervertebral disc, is characterized as the posterior migration of the nucleus pulposus through the layers of the annulus fibrosus. Various risk factors have been associated with the development of disc herniation, but the mechanisms are largely not understood. For example, exposure to vibration has been linked to the occurrence of herniation, yet our understanding of this association is not clear. It is hypothesized that vibration cyclically loads the tissues of the intervertebral disc until failure occurs as a result of fatigue. Tissues at risk of fatigue failure may include the intra-lamellar matrix, the connective tissue found between collagen fibres within a single lamella, and the inter-lamellar matrix, the connective tissue found between adjacent lamellae. In order to determine the mechanistic link between vibration and herniation, a firm understanding of the properties of the intervertebral disc as well as the intra and inter-lamellar matrices are of utmost importance. Further, it is important to determine these properties under physiological loading scenarios. This thesis consists of five studies, which have each provided a unique piece to the intervertebral disc herniation puzzle in order to better understand this mechanistic link. First, it was discovered that annular tissue is subject to significantly higher stresses and is stiffer under biaxial strain as compared to uniaxial strain. Biaxial strain is more representative of the in vivo loading scenario and provides more accurate information regarding scenarios that the annulus can tolerate and those that can result in injury. It was also revealed that, when strained at physiological strain rates (up to 4%/sec), these mechanical properties do not change such that they are independent of strain rate. Therefore, when strained at varying rates akin to voluntary movement, the annulus is not subject to higher stresses or altered stiffness. Second, the effect of vibration, an acknowledged risk factor for herniation, was examined on the mechanical properties of the intra and inter-lamellar matrices. It was discovered that vibration altered these matrices such that they were more extensible and strained to greater magnitudes, yet did not reach higher stresses before failing. It was hypothesized that this increased extensibility was due to damage to elastin, as elastin assists in minimizing tissue deformation and helps tissues recover from tensile strain. The final study assessed the effect of exposure to vibration on the development of disc herniation. The initiation of herniation was observed in a significantly greater number of intervertebral discs exposed to vibration as compared to a control condition. Although epidemiological studies had documented a correlation between exposure to vibration and herniation, this was the first study to conclude that exposure to vibration is in fact a mechanical risk factor for the development of herniation and increases the incidence of herniation. Further, based on the findings of the mechanical properties of the intra and inter-lamellar matrices, and in particular the observed 15-20 times greater failure strength of the intra as compared to inter-lamellar matrix, it would appear that the inter-lamellar matrix, and thus delamination, may be the weakest link in the herniation pathway. This thesis has uncovered new information regarding physiological mechanical properties of the annulus. Further, new information regarding the intra and inter-lamellar matrices was obtained, improving our understanding of the healthy disc. Last, by subjecting the disc to a known risk factor for herniation, hypotheses were generated regarding the initiation and progression of disc herniation, specifically related to the roles of the intra and inter-lamellar matrices.

Low back pain is highly prevalent in the developed world, with 80% of the population being affected at some point in their lives. Herniation, a common injury to the intervertebral disc, is characterized as the posterior migration of the nucleus pulposus through the layers of the annulus fibrosus. Various risk factors have been associated with the development of disc herniation, but the mechanisms are largely not understood. For example, exposure to vibration has been linked to the occurrence of herniation, yet our understanding of this association is not clear. It is hypothesized that vibration cyclically loads the tissues of the intervertebral disc until failure occurs as a result of fatigue. Tissues at risk of fatigue failure may include the intra-lamellar matrix, the connective tissue found between collagen fibres within a single lamella, and the inter-lamellar matrix, the connective tissue found between adjacent lamellae. In order to determine the mechanistic link between vibration and herniation, a firm understanding of the properties of the intervertebral disc as well as the intra and inter-lamellar matrices are of utmost importance. Further, it is important to determine these properties under physiological loading scenarios. This thesis consists of five studies, which have each provided a unique piece to the intervertebral disc herniation puzzle in order to better understand this mechanistic link. First, it was discovered that annular tissue is subject to significantly higher stresses and is stiffer under biaxial strain as compared to uniaxial strain. Biaxial strain is more representative of the in vivo loading scenario and provides more accurate information regarding scenarios that the annulus can tolerate and those that can result in injury. It was also revealed that, when strained at physiological strain rates (up to 4%/sec), these mechanical properties do not change such that they are independent of strain rate. Therefore, when strained at varying rates akin to voluntary movement, the annulus is not subject to higher stresses or altered stiffness. Second, the effect of vibration, an acknowledged risk factor for herniation, was examined on the mechanical properties of the intra and inter-lamellar matrices. It was discovered that vibration altered these matrices such that they were more extensible and strained to greater magnitudes, yet did not reach higher stresses before failing. It was hypothesized that this increased extensibility was due to damage to elastin, as elastin assists in minimizing tissue deformation and helps tissues recover from tensile strain. The final study assessed the effect of exposure to vibration on the development of disc herniation. The initiation of herniation was observed in a significantly greater number of intervertebral discs exposed to vibration as compared to a control condition. Although epidemiological studies had documented a correlation between exposure to vibration and herniation, this was the first study to conclude that exposure to vibration is in fact a mechanical risk factor for the development of herniation and increases the incidence of herniation. Further, based on the findings of the mechanical properties of the intra and inter-lamellar matrices, and in particular the observed 15-20 times greater failure strength of the intra as compared to inter-lamellar matrix, it would appear that the inter-lamellar matrix, and thus delamination, may be the weakest link in the herniation pathway. This thesis has uncovered new information regarding physiological mechanical properties of the annulus. Further, new information regarding the intra and inter-lamellar matrices was obtained, improving our understanding of the healthy disc. Last, by subjecting the disc to a known risk factor for herniation, hypotheses were generated regarding the initiation and progression of disc herniation, specifically related to the roles of the intra and inter-lamellar matrices.

Thesis (Ph. D.)--Drexel University, 2004. / Includes abstract and vita. Includes bibliographical references (leaves 168-182).