Posterior lumbar spinal dynamic stabilisation devices are intended to relieve the pain of spinal segments while prolonging the lifespan of adjacent intervertebral discs. This study focuses on the design of such a device, one that has the correct stiffness to stabilise the spinal segment by the correct amount. An initial literature survey covers contemporary topics related to the lumbar spine. Included topics are lumbar anatomy and kinematics, pathology of degenerative disc disease and treatment thereof, other spinal disorders such as spondylolisthesis and spinal stenosis, as well as the complications associated with lumbar dynamic stabilisation. The influence of factors such as fatigue and wear, as well as the properties of appropriate biomaterials are considered when determining the basis of the device design and development. Stabilising the spinal segment begins with correct material selection and design. Various designs and biomaterials are evaluated for their stiffness values and other user requirements. The simplest design, a U-shaped spring composed of carbon fibre-reinforced poly-ether-ether-ketone (CFR-PEEK) and anchored by polyaxial titanium pedicle screws, satisfies the most critical user requirements. Acceptable stiffness is achieved, fatigue life of the material is excellent and the device is very imaging-friendly. Due to financial constraints, however, a simpler concept that is cheaper and easier to rapid prototype was chosen. This concept involves a construct primarily manufactured from the titanium alloy Ti6Al4V extra-low interstitial (ELI) and cobalt-chrome-molybdenum (CCM) alloys. The first rapid prototype was manufactured using an additive manufacturing process (3D-printing). The development of the device was performed in three main stages: design, verification and validation. The main goal of the design was to achieve an acceptable stiffness to limit the spinal segmental range of motion (ROM) by a determined amount. The device stiffness was verified through simple calculations. The first prototype’s stiffness was validated in force-displacement tests. Further validation, beyond the scope of this study, will include fatigue tests to validate the fatigue life of the production-ready device. / MIng (Mechanical Engineering), North-West University, Potchefstroom Campus, 2014

Posterior lumbar spinal dynamic stabilisation devices are intended to relieve the pain of spinal segments while prolonging the lifespan of adjacent intervertebral discs. This study focuses on the design of such a device, one that has the correct stiffness to stabilise the spinal segment by the correct amount. An initial literature survey covers contemporary topics related to the lumbar spine. Included topics are lumbar anatomy and kinematics, pathology of degenerative disc disease and treatment thereof, other spinal disorders such as spondylolisthesis and spinal stenosis, as well as the complications associated with lumbar dynamic stabilisation. The influence of factors such as fatigue and wear, as well as the properties of appropriate biomaterials are considered when determining the basis of the device design and development. Stabilising the spinal segment begins with correct material selection and design. Various designs and biomaterials are evaluated for their stiffness values and other user requirements. The simplest design, a U-shaped spring composed of carbon fibre-reinforced poly-ether-ether-ketone (CFR-PEEK) and anchored by polyaxial titanium pedicle screws, satisfies the most critical user requirements. Acceptable stiffness is achieved, fatigue life of the material is excellent and the device is very imaging-friendly. Due to financial constraints, however, a simpler concept that is cheaper and easier to rapid prototype was chosen. This concept involves a construct primarily manufactured from the titanium alloy Ti6Al4V extra-low interstitial (ELI) and cobalt-chrome-molybdenum (CCM) alloys. The first rapid prototype was manufactured using an additive manufacturing process (3D-printing). The development of the device was performed in three main stages: design, verification and validation. The main goal of the design was to achieve an acceptable stiffness to limit the spinal segmental range of motion (ROM) by a determined amount. The device stiffness was verified through simple calculations. The first prototype’s stiffness was validated in force-displacement tests. Further validation, beyond the scope of this study, will include fatigue tests to validate the fatigue life of the production-ready device. / MIng (Mechanical Engineering), North-West University, Potchefstroom Campus, 2014

abstract: Intervertebral Disc Degeneration (IVDD) is a complex phenomenon characterizing the desiccation and structural compromise of the primary joint in the human spine. The intervertebral disc (IVD) serves to connect vertebral bodies, cushion shock, and allow for flexion and extension of the vertebral column. Often presenting in the 4th or 5th decades of life as low back pain, this disease was originally believed to be the result of natural “wear and tear” coupled with repetitive mechanical insult, and as such most studies focus on patients between 40 and 50 years of age. Research over the past two decades, however, has demonstrated that environmental factors have only a modest effect on disc degeneration, with genetic influences playing a much more substantial role. Extensive research has focused on this process, though definitive risk factors and a clear pathophysiology have proven elusive. The aim of this study was to assemble a cohort of patients exhibiting definitive signs of degeneration who were well below the average age of presentation, with minimal or no exposure to suspected environmental risk factors and to conduct a targeted genome analysis in an attempt to elucidate a common genetic component. Through whole genome sequencing and analysis, the results corroborated findings in a previous study, as well as demonstrated a potential connection and influence between mutations found in IVD structural or functional genes, and the provocation of IVDD. Though the sample size was limited in scale and age, these findings suggest that further IVDD research into the association of variants in collagen, aggrecan and the insulin-like growth factor receptor genes of young patients with an early presentation of disc degeneration and minimal exposure to suspected risk factors is merited. / Dissertation/Thesis / Masters Thesis Biology 2016

Introduction: A computer-based classification system is proposed for the characterization of cervical intervertebral disc degeneration from saggital magnetic resonance images. Materials and methods: Cervical intervertebral discs from saggital magnetic resonance images where assessed by an experienced orthopaedist as normal or degenerated (narrowed) employing Matsumoto’s classification scheme. The digital images where enhanced and the intervertebral discs which comprised the regions of interest were segmented. First and second order statistics textural features extracted from thirty-four discs (16 normal and 16 degenerated) were used in order to design and test the classification system. In addition textural features were calculated employing Laws TEM images. The existence of statistically significant differences between the textural features values that were generated from normal and degenerated discs was verified employing the Student’s paired t-test. A subset with the most discriminating features (p<0.01) was selected and the Exhaustive Search and Leave-One-Out methods were used to find the best features combination and validate the classification accuracy of the system. The proposed system used the Least Squares Minimum Distance Classifier in combination with four textural features with comprised the best features combination in order to classify the discs as normal or degenerated. Results: The overall classification accuracy was 93.8% misdiagnosing 2 discs. In addition the system’s sensitivity in detecting a narrow disc was 93.8% and its specificity was also 93.8%. Conclusion: Further investigation and the use of a larger sample for validation could make the proposed system a trustworthy and useful tool to the physicians for the evaluation of degenerative disc disease in the cervical spine. / Σκοπός: Η στένωση των μεσοσπονδύλιων δίσκων της αυχενικής μοίρας, ως κύρια έκφραση εκφυλιστικής νόσου, είναι μια από τις σημαντικότερες αιτίες πρόκλησης πόνου στην περιοχή του αυχένα. Στην κλινική πράξη η αξιολόγηση της στένωσης γίνεται μέσω μέτρησης του μεσοσπονδύλιου διαστήματος, σε διάφορες απεικονίσεις της αυχενικής μοίρας του ασθενούς. Στην παρούσα εργασία προτείνεται μια υπολογιστική μέθοδος ανάλυσης εικόνας, για την αυτοματοποιημένη εκτίμηση της στένωσης από εικόνες μαγνητικής τομογραφίας. Υλικό και Μέθοδος: Μελετήθηκαν 34 μεσοσπονδύλιοι δίσκοι από οβελιαίες τομές μαγνητικής τομογραφίας της αυχενικής μοίρας, οι οποίες ελήφθησαν με χρήση Τ2 ακολουθίας. Η στένωση των μεσοσπονδύλιων δίσκων αξιολογήθηκε από έμπειρο ορθοπαιδικό βάσει της κλίμακας Matsumoto. Οι δίσκοι χωρίστηκαν σε δύο κατηγορίες: (α) 16 φυσιολογικοί και (β) 16 δίσκοι που παρουσίαζαν στένωση. Με χρήση διαδραστικού περιβάλλοντος επεξεργασίας εικάνας καθορίστηκε το περίγραμμα των μεσοσπονδύλιων δίσκων οι οποίοι αποτελούν τις προς ανάλυση περιοχές ενδιαφέροντος (Π.Ε.). Σε κάθε Π.Ε. εφαρμόστηκαν αλγόριθμοι εξαγωγής χαρακτηριστικών υφής. Συγκεκριμένα υπολογίστικαν χαρακτηριστικά υφής από στατιστικά πρώτης και δεύτερης τάξης καθώς και χαρακτηριστικά από τα μέτρα ενέργειας υφλης κατλα Laws. Τα παραπάνω χαρακτηριστικά, ποσοτικοποιούν διαγνωστικές πληροφορίες της έντασης του σήματος της Π.Ε. και συσχετίζονται με τη βιοχημική σύσταση των απεικονιζόμενων δομών. Τα εξαχθέντα χαρακτηριστικά υφής αξιοποιήθηκαν για τη σχεδίαση του ταξινομητή ελάχιστης απόστασης ελαχίστων τετραγώνων, ο οποίος χρησιμοποιήθηκε για το διαχωρισμό μεταξύ φυσιολογικών δίσκων και δίσκων που παρουσίαζαν στένωση (εκφυλισμένων). Αποτελέσματα: Η ακρίβεια της ταξινόμησης φυσιολογικών και εκφυλισμένων μεσοσπονδύλιων δίσκων ανήλθε σε 93.8%. Η ευαισθησία καθώς και η ειδικότητα της μεθόδου, σε ότι αφορά την ανίχνευση εκφυλισμένων δίσκων, είναι επίσης 93.8%. Συμπέρασμα: Με δεδομένο το μικρό μέγεθος του δείγματος που χρησιμοποιήθηκε για το σχεδιασμό της μεθόδου, απαιτούνται περετέρω εργασίες πιστοποίησης της ακρίβειας ταξινόμησης, προκειμένου η μέθοδος αυτή να αξιοποιηθεί από ακτινολόγους και ορθοπαιδικους, ως βοηθητικό διαγνωστικό εργαλείο.

The current study characterizes the mechanical and biodegradation properties of a polycarbonate polyurethane (PU) electrospun nanofiber scaffold intended for use in the growth of a tissue engineered annulus fibrosus (AF) intervertebral disc component. Both the tensile strength and initial modulus of aligned scaffolds were higher than those of random scaffolds and remained unaffected during a 4 week biodegradation study, suggesting a surface-mediated degradation mechanism. The resulting degradation products were non-toxic. Confined compressive mechanical force of 1kPa, was applied at 1Hz to in vitro bovine AF tissue grown on the scaffolds to investigate the influence of mechanical force on AF tissue production, which was found to decrease significantly at 72 hours relative to 24 hours, independent of any effects from mechanical forces. Overall, the consistent rate of PU degradation, along with mechanical properties comparable to those of native AF tissue, and the absence of cytotoxic effects, make this polymer suitable for further investigation for use in tissue-engineering the AF.

The current study characterizes the mechanical and biodegradation properties of a polycarbonate polyurethane (PU) electrospun nanofiber scaffold intended for use in the growth of a tissue engineered annulus fibrosus (AF) intervertebral disc component. Both the tensile strength and initial modulus of aligned scaffolds were higher than those of random scaffolds and remained unaffected during a 4 week biodegradation study, suggesting a surface-mediated degradation mechanism. The resulting degradation products were non-toxic. Confined compressive mechanical force of 1kPa, was applied at 1Hz to in vitro bovine AF tissue grown on the scaffolds to investigate the influence of mechanical force on AF tissue production, which was found to decrease significantly at 72 hours relative to 24 hours, independent of any effects from mechanical forces. Overall, the consistent rate of PU degradation, along with mechanical properties comparable to those of native AF tissue, and the absence of cytotoxic effects, make this polymer suitable for further investigation for use in tissue-engineering the AF.

Degenerative Veränderungen der Lendenwirbelsäule beginnen bereits im Alter von unter 20 Jahren und betreffen vor allem die unteren 3 Bewegungssegmente. Die degenerativen Veränderungen an der Bandscheibe gehen mit einer Kalzifizierung der Grund- und Deckplatten der Wirbelkörper einher, was zu einer Reduktion der Nährstoffversorgung der Bandscheibe und damit zu einem Untergang der matrixbildenden Zellen und konsekutiv zu einem Flüssigkeitsverlust in der Bandscheibe führt. Als Folge nimmt die Belastung der Bandscheibe weiter ab. Die aktuellen Therapieoptionen umfassen sowohl die konservative als auch die operative Therapie, wobei allerdings nur die Folgen der Degeneration behandelt werden. Ziel einer Zelltransplantation ist es, der Bandscheibe wieder matrixbildende Zellen zur Verfügung zu stellen, damit die nutritiven Veränderungen auszugleichen und eventuell reversibel zu machen. Dieser Effekt konnte im Tierversuch nachgewiesen werden, woraufhin eine klinische Studie initiiert wurde. Im Rahmen der publizierten klinischen prospektiven, randomisierten Studie konnte gezeigt werden, dass die Transplantation autologer Chondrozyten, die bei einer notwendigen operativen Therapie eines sequestrierten Bandscheibenvorfalls gewonnen wurden, einen sowohl klinisch als auch bildmorphologisch positiven Effekt auf die degenerierten Bandscheiben hat. Es kam zu einer signifikanten Reduktion der Schmerzsymptomatik und einer Steigerung der Lebensqualität in der Gruppe der transplantierten Patienten. Die Bandscheibenhöhe zeigt sich stabil über den Beobachtungszeitraum von 2 Jahren.

La dégénérescence discale lombaire (DDL) est caractérisée par un vieillissement prématuré du disque intervertébral (DIV) et une déshydratation progressive du nucleus pulposus (NP) entrainant in fine des lombalgies. L'objectif général de ce travail est d'établir des données précliniques afin de régénérer le DIV en cas de DDL modérée. Dans un premier chapitre, nous avons déterminé une association de facteur de croissance et un mode de culture visant à obtenir une prédifférentiation nucléopulpogénique de cellules stromales mésenchymateuses (CSMs) humaines issues de la moelle osseuse. Nos résultats montrent que la culture tridimensionnelle des CSMs en billes d'alginate en présence de TGF-β3, GDF-5 et BMP-7 les oriente vers un phénotype cartilagineux. Dans un deuxième chapitre, nous avons élaboré un modèle porcin de DDL induite par cryolésion et nous l'avons comparé aux techniques de référence. L'évaluation de l'importance de la DDL a été effectuée par scanner, IRM et histologiquement. Un score histologique de DDL porcine a été décrit et validé. La cryolésion a permis d'obtenir une DDL plus importante que les autres techniques. Dans un troisième chapitre, nous avons injecté les CSMs préorientées dans les DIV lésés. L'analyse IRM a montré une amélioration de l'intensité du signal et de la surface du NP après injection des cellules. L'analyse immunohistologique a montré une survie des CSMs dans les DIV porcins à 2 mois. Dans un quatrième chapitre, nous avons comparé les taux de fusion et de complication pour la RhBMP-2 et la greffe spongieuse autologue dans les arthrodèses lombaires par voie antérieure dans une même cohorte de patients. La RhBMP-2 était associée à un taux de fusion inférieur et un taux de complications radiologiques supérieur à l'autogreffe spongieuse. / Degenerative disc disease (DDD) is characterized by premature aging of the intervertebral disc (IVD) and gradual dehydration of the nucleus pulposus (NP), ultimately causing back pain. The general objective of this work is to establish preclinical data to regenerate the IVD in moderate DDD. In the first chapter, we have identified a growth factor association and a culture method to achieve nucleopulpogenic prédifférentiation of mesenchymal stromal cells (MSCs) derived from human bone marrow. Our results show that the three-dimensional culture of MSCs in alginate beads in the presence of TGF-β3, GDF-5 and BMP-7 directs them to a cartilaginous phenotype. In the second chapter, we developed a porcine model of DDD, induced by cryoinjury, and compared it to reference techniques. Assessing the importance of DDD was performed by CT, MRI and histologically. A histological score of porcine DDD has been described and validated. Cryoinjury yielded a higher DDD that other techniques. In a third chapter, we injected preoriented MSCs in cryo-injured IVDs. MRI analysis showed an improvement in the signal intensity and the surface of the NP after the injection. The immunohistological analysis showed a survival of the MSCs in the porcine IVD 2 months after injection. In a fourth chapter, we compared the rate of fusion and complication for rhBMP-2 and autologous cancellous graft in the anterior lumbar interbody fusion, in the same cohort of patients. RhBMP-2 was associated with a lower fusion rate and a higher rate of radiological complications than the cancellous autograft.

The intervertebral disc (IVD), situated between adjoining vertebrae, consists of the gelatinous nucleus pulposus (NP) in the centre surrounded by the tougher annulus fibrosus (AF). Its main roles are to distribute loads and to act as joints. With aging, degenerative disc disease (DDD) occurs due to an imbalance in anabolic and catabolic events in the IVD, which results in a loss of function. Lower back pain (LBP) affects 84% of people at some point in their lifetime and is strongly associated with DDD. Current LBP treatments have limited long term efficacy and are symptomatic rather than curative. Cell-based therapies are regarded to hold great potential for the treatment of DDD as it has been hypothesised that they could regenerate the damaged tissue and alleviate LBP. A number of natural and synthetic biomaterials have been investigated as NP tissue engineering scaffolds with varying results. In this study, a self assembling peptide hydrogel (SAPH) was investigated for its potential as a cell carrier and/or scaffold for NP tissue engineering. SAPHs display the advantages of natural polymer hydrogels such as biocompatibility and biodegradability whilst combining the advantages of synthetic materials such as controlled structural and mechanical propertiesCharacterisation determined that the SAPH nanofibrous architecture had features that were of similar scale to extracellular matrix (ECM) components of the human NP. The mechanical properties of the SAPH could be optimised to closely match the native tissue. The system could shear thin and self-heal making the system ideally suited to delivery via minimally invasive procedure. The three dimensional (3D) culture of bovine NP cells (bNPCs) in the SAPH demonstrated that the NP phenotype could be restored after de-differentiation during monolayer culture. Gene expression results demonstrated that ‘traditional’ and ‘novel’ NP markers were highly expressed throughout in vitro culture. Cell viability was high, cell population remained stable and bNPCs adopted the characteristic rounded morphology of native NPCs. Finally, type II collagen and aggrecan, the main ECM components of the NP, were deposited with increasing production over culture period. Growth differentiation factor 6 (GDF-6) has been identified as the most promising current growth factor for inducing discogenic differentiation from human bone marrow mesenchymal stem cell (h-BMMSCs). After samples were stimulated with GDF-6, gene expression results confirmed that a NP-like phenotype could be induced with high expression of ‘traditional’ and ‘novel’ NP markers. Cell viability was high, cell population remained stable and NP associated ECM components were deposited with cells displaying a rounded morphology. Interestingly, when h-BMMSCs were cultured without GDF-6, it was strongly suggested that spontaneous discogenic differentiation occurred after culture in the SAPHs as ‘traditional’ and ‘novel’ NP markers were highly expressed, morphology was comparable to native NPCs and type II collagen and aggrecan were deposited extracellularly. If these findings were accurate then this is the first study to demonstrate that a NP-like phenotype could be induced from MSCs without use of an exogenous growth factor or a discogenic bioactive motif. Despite exciting and novel results, further work is required to confirm the potential of SAPHs for NP tissue engineering scaffolds.

Degenerative Veränderungen der Lendenwirbelsäule beginnen bereits im Alter von unter 20 Jahren und betreffen vor allem die unteren 3 Bewegungssegmente. Die degenerativen Veränderungen an der Bandscheibe gehen mit einer Kalzifizierung der Grund- und Deckplatten der Wirbelkörper einher, was zu einer Reduktion der Nährstoffversorgung der Bandscheibe und damit zu einem Untergang der matrixbildenden Zellen und konsekutiv zu einem Flüssigkeitsverlust in der Bandscheibe führt. Als Folge nimmt die Belastung der Bandscheibe weiter ab. Die aktuellen Therapieoptionen umfassen sowohl die konservative als auch die operative Therapie, wobei allerdings nur die Folgen der Degeneration behandelt werden. Ziel einer Zelltransplantation ist es, der Bandscheibe wieder matrixbildende Zellen zur Verfügung zu stellen, damit die nutritiven Veränderungen auszugleichen und eventuell reversibel zu machen. Dieser Effekt konnte im Tierversuch nachgewiesen werden, woraufhin eine klinische Studie initiiert wurde. Im Rahmen der publizierten klinischen prospektiven, randomisierten Studie konnte gezeigt werden, dass die Transplantation autologer Chondrozyten, die bei einer notwendigen operativen Therapie eines sequestrierten Bandscheibenvorfalls gewonnen wurden, einen sowohl klinisch als auch bildmorphologisch positiven Effekt auf die degenerierten Bandscheiben hat. Es kam zu einer signifikanten Reduktion der Schmerzsymptomatik und einer Steigerung der Lebensqualität in der Gruppe der transplantierten Patienten. Die Bandscheibenhöhe zeigt sich stabil über den Beobachtungszeitraum von 2 Jahren.