Abstract Background: The process that involves the migration of the nucleus pulposus from the innermost annular layers and culminates with the final extrusion of the nucleus has been limited to a few studies. This investigation was directed towards a better understanding of the herniation process. The architecture of the annulus fibrosus and the mechanism of progressive disc herniation were analyzed, using a controlled porcine model. Microscopic and histochemical techniques were employed. <br /><br /> Methodology: Two studies were performed. In the first stage, the macroscopic and microscopic structures of twelve cervical intervertebral discs were compared with young human disc data from studies reported in the literature. Important structural features were studied such as annulus fibrosus thickness, number of lamellae, lamellae thickness, orientation of the lamellae fibers and blood supply. In the second study, sixteen fresh-frozen functional spine units were submitted to repetitive flexion?extension motions combined with a low compressive load in a servo-hydraulic dynamic testing system. Discograms, dissections and histochemical techniques were applied to characterize the cumulative damage. The experiment produced eight complete herniations, four partial herniations and four specimens without any microscopic detectable annular damage. <br /><br /> Results and Discussion: The structure of the cervical porcine disc resembles the lumbar human disc. Some differences are evident. The size of the annulus is smaller, the thickness of the lamellae is narrower and the number of layers is fewer in the pig. It is hypothesized that the flexion-extension motion combined with a low-level load produced an increased hydraulic pressure in the inner wall of the posterior annulus. This pressure and repetitive motion first produced a small cleft, spreading the collagen bundles inside the first layer. The nuclear material was "pumped" through the small cleft to the first layer filling the layer creating a fluid-filled pocket between the collagen fibers. Once the "pocket" acquired enough pressure a new cleft was produced in the weakest part of the layer allowing the nuclear material to create a new "pocket" in the second layer. This was the first stage of damage and disc herniation production. This mechanism was repeated until the nucleus traveled along the annulus reaching the posterior longitudinal ligament. At this point a complete extrusion herniation was produced. <br /><br /> Conclusion: The porcine model appears to be suitable as a model to understand the mechanism of disc herniation when the spine is subjected to flexion-extension motions combined with a low-level load. The first cumulative injury appears to be a cleft between the lamellae bundles produced by the nuclear hydraulic pressure. A cumulative load/cumulative injury model approach was used to create the damage that was quantified in the study.

This study investigates the changes due to crosslinking treatment in stiffness, permeability, and glycosaminoglycan (GAG) content of bovine intervertebral discs. The objective of this study was to determine the mechanical and biochemical effects of crosslinking treatment on lumbar bovine tissue. Previous studies have found that crosslinking can increase stiffness and permeability in the intervertebral disc. These changes have not yet been investigated by confined compression, stress-relaxation tests of young bovine tissue. Eleven lumbar motion segments were harvested from calf spines and soaked in a saline solution or one of four crosslinking treatments (genipin, methylglyoxal, proanthrocyanidin, and EDC). Five mm diameter samples were removed from the midannulus region at anterior / anterior-lateral locations, confined in a saline bath, swelled to equilibrium, and tested in confined compression stress-relaxation to 15% strain in 5% increments. Radial samples were also harvested, treated with saline solution and EDC, and tested in the same manner. The aggregate modulus and hydraulic permeability were calculated using the nonlinear biphasic theory. Swelling pressure was calculated as the load at swelling equilibrium. GAG content was measured using the dimethylmethylene blue assay. Differences with P value < 0.05 were considered significant. In the axial orientation, all crosslinking treatments except methyglyoxal at least doubled the aggregate modulus relative to soaked controls (P less than 0.05). Genipin treatment resulted in 78% lower axial permeability, proanthrocyanidin (PA) 50% lower, and EDC treatment 84% lower relative to soaked controls (P &lt; 0.05). GAG content measured in the methyglyoxal treatment group was 25% lower than in soaked control group. Genipin (G), proanthrocyanidin (PA), and EDC treatment increased the swelling pressure by at least 65% (P less than 0.05). In the radial orientation, EDC treatment increased the stiffness by 75%, and did not significantly affect the permeability or swelling pressure. Some crosslinking treatments proved effective in increasing the stiffness and swelling pressure of the disc. The increased swelling pressure in G, PA, and EDC treatment groups relative to soaked controls suggests reduced GAG leaching during soaking treatment, further confirmed by the reduction in permeability in these groups.

A biomechanical study of intervertebral foraminal narrowing during simulated automotive head-forward and head-turned rear impacts. The objective of this study was to quantify foraminal width, height and area narrowing during head-forward and head-turned rear impacts, and evaluate the potential for nerve root and ganglion impingement. Muscle weakness and paresthesias, documented in whiplash patients, have been associated with neural compression within the cervical intervertebral foramen. Rotated head posture at the time of rear impact has been correlated with increased frequency and severity of chronic radicular symptoms, as compared to facing forward. No studies have quantified dynamic changes in foramen dimensions during head-forward or head-turned rear impacts. Six whole cervical spine specimens with muscle force replication and surrogate head underwent simulated whiplash at 3.5, 5, 6.5 and 8 g, following non-injurious baseline 2 g acceleration. Continuous dynamic foraminal width, height and area narrowing were recorded, and the peaks were determined during each impact and statistically compared to baseline narrowing. During head-forward rear impacts, significant increases (P<0.05) in average peak foraminal width narrowing above baseline were observed at C5-C6 beginning with 3.5 g impact. No significant increases in average peak foraminal height narrowing were observed, while average peak foraminal areas were significantly narrower than baseline at C4-C5 at 3.5, 5 and 6.5 g. During head-turned rear impacts, significant increases (P<0.05) in average peak foraminal width narrowing above baseline of up to 1.8 mm in the left C5-C6 foramen at 8 g were observed. Average peak dynamic foraminal height was significantly narrower than baseline at right C2-C3 foramen at 5 g and 6.5 g, while no significant increases in foraminal area were observed. Extrapolation of the present head-forward rear impact results indicated that the greatest potential for ganglia compression injury was at the lower cervical spine, C5-C6 and C6-C7. The present head-turned rear impact results indicated that the greatest potential ganglia compression injury exists at C5-C6 and C6-C7. Greater potential for ganglia compression injury exists at C3-C4 and C4-C5 due to head-turned rear impact, as compared to head-forward rear impact. Acute ganglia compression may produce a sensitized neural response to repeat compression leading to chronic radiculopathy following head-forward and head-turned rear impacts. Dynamic ganglion or nerve root compression may also lead to chronic radiculopathy.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 149-163). Also available online.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 149-163). Also available in print.

published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

Upon aging, the intervertebral disc (IVD) inevitably undergoes degeneration characterized by biochemical and morphologic changes. IVD degeneration can lead to multiple clinical disorders such as back and neck pain, and myelopathy. Low back pain can disable up to 85% of the adult population and results in a significant restriction of social activities and inability to work. Such disorder incurs billions of dollars in medical expenditures each year. Despite advances in the detection and treatment of the degeneration, the regeneration of the IVD remains low because current therapies are limited by exogenous curing approaches. New strategies for the reversal of IVD degeneration, including gene, cytokine, and stem cell therapies that can influence the anabolic and catabolic pathways in disc cells have been under investigation. These therapies aim to rejuvenate or replace diminished nucleus pulposus cells in the degenerative IVD. Recent reports have put forth a proposal of “endogenous disc stem cells”, suggesting that cells derived from the degenerative IVD tissue possess stem cell properties. These putative stem cells are believed to regulate the development and homeostasis of the IVD tissue. In this study, we identified and characterized a stem cell population from the IVD of healthy Rhesus monkey, termed disc stem/progenitor cells (DSCs). We show that the DSCs possess clonogenicity, multipotency and self-renewal capacity. The DSCs are phenotypically similar to bone marrow mesenchymal stem cells (BMSCs) but they are not identical. The DSCs show a faster growth rate under hypoxia than normoxia. DSCs derived from nucleus pulposus (DSCNP) show a stable expression level of hypoxia inducible factor-1 alpha (Hif-1ａ) in response to hypoxia. DSCs derived from annulus fibrosus (DSCAF) are more resistant to apoptosis under hypoxia than DSCNP. More importantly, small leucine-rich proteoglycans (SLRPs) are identified as important DSC niche components. We show that biglycan (bgn) and decorin (dcn) reduce the susceptibility of DSCs to hypoxia-induced apoptosis by promoting the expression of hypoxia inducible factors (HIFs). Our findings suggest that DSCs rely on the unique niche components for survival. In summary, our findings propose the existence of endogenous stem cells in IVD. Further study of the DSCs may provide new insights into the biology of IVD and facilitate the design of new strategies to treat disc degeneration in future. / published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

Degenerative disc disease (DDD) is a medical condition whereby the intervertebral discs (IVD) of the human spine degenerates and may cause pain which significantly affects the quality of one掇 life. Its prevalence has sparked off much research in deciphering its causes and developing new treatments. Recently, attempts to treat this degenerative problem have turned to seeking answers from regenerative medicine. One approach is to deliver mesenchymal stem cells (MSCs) with or without carriers to the nucleus pulposus (NP) in degenerative disc to restore both its structural and functional properties. However, the optimal conditions and signals for inducing MSCs differentiation toward NP-like phenotype have not been identified. This work aimed to develop injectable microspheres with matrix microenvironment mimicking that of native NP tissue for MSCs delivery. Firstly, it was aimed to establish a collagen microencapsulation based 3D culture system for maintenance of the phenotype of nucleus pulposus cells (NPCs) and remodeling of the collagen matrix to one that was similar to the native NP. Secondly, it was aimed to optimize a decellularization protocol for complete removal of the encapsulated NPCs with minimal loss of remodeled extracellular matrix. Thirdly, it was aimed to investigate whether this acellular matrix produced by NPCs was inductive for MSCs discogenic differentiation. Finally, it was aimed to evaluate the efficacy of the MSC-seeded acellular matrix microspheres in a pilot rabbit disc degeneration model. It demonstrated that NPCs maintained their phenotype, survived within the collagen microspheres and produced NP-like ECM such as glycosaminoglycan (GAG) and collagen type II. GAG production of NPCs was found to positively correlate with the dosage of TGF-? within a short period, initial collagen concentration and cell seeding density. An optimized decellularization protocol with 50mM SB-10, 0.6mM SB-16 and 0.14% Triton X-200 was established to completely remove the encapsulated NPCs with partial retention of the GAG-rich matrix. The decellularized microspheres were able to be repopulated with human MSCs (hMSCs) or rabbit MSCs (rMSCs). Within the NPC-derived acellular matrix, the repopulated hMSCs were able to partially exhibit NPC phenotype with upregulated expression of a few NPC markers and NP-like ECM according to histological, biochemical, immunohistological and real-time PCR results. In the pilot in vivo evaluation study, preliminary results showed that intra-discal injection of MSCs reseeded acellular NPC-matrix microspheres maintained a better water content than the control MSC-microspheres without the NPC-derived acellular matrix. This work reconstituted in vitro a NP-like 3D matrix and provided preliminary evidence on discogenic differentiation of MSCs in such a matrix environment. This work laid down the foundation to future development of stem cell-based therapies for DDD. Further studies should focus on deciphering the soluble and insoluble composition of such a NP-like matrix environment and understanding the molecular mechanism of the cell-matrix interactions involved. / published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

Σκοπός: Ο νεοεννευρωμένος μεσοσπονδύλιος δίσκος (ΜΔ), κλινικά θεωρείται μια σημαντική πηγή πόνου στους ασθενείς με οσφυαλγία. Η ταυτοποίηση των νεοσχηματιζόμενων νεύρων, που εκφράζουν τους υποδοχείς των νευροτροφινών, στην εκφύλιση του ΜΔ, σπονδυλικών επιπέδων που αναφέρονται ως πηγή δισκογενή πόνου και η συσχέτισή τους με το ενδοκανναβινοειδές σύστημα αναλγησίας, αποτελεί το αντικείμενο αυτής της μελέτης. Υλικό και μέθοδος: Συλλέχθηκαν 50 ανθρώπινοι ΜΔ ή τμήματα αυτών, μετά από δισκεκτομή σε χειρουργεία κήλης και εκφυλιστικών παθήσεων της Οσφυϊκής Μοίρας της Σπονδυλικής Στήλης (ΟΜΣΣ). Εφαρμόσθηκε ανοσοϊστοχημεία για την ανίχνευση νευρικών ινών, στους ΜΔ, αφού διαπιστώθηκε η εκφύλισή τους. Συσχετίσθηκε η ανάπτυξη νευρικών ινών των εκφυλισμένων ΜΔ με την έκφραση των υψηλής και χαμηλής συγγένειας υποδοχέων των νευροτροφινών, TrkA, TrkB, TrkC και p75, αντίστοιχα. Στα περιστατικά με ανάπτυξη νευρικών ινών ανιχνεύθηκε ανοσοϊστοχημικά η έκφραση του υποδοχέα CB1 των ενδοκανναβινοειδών. Αποτελέσματα: Η εκτεταμένη νεύρωση στον ΜΔ που φέρεται στον έσω ινώδη δακτύλιο (ΙΔ) και μπορεί να φτάνει μέχρι τον πηκτοειδή πυρήνα (ΠΠ), φαίνεται να είναι εξαρτώμενη από τους υποδοχείς των νευροτροφινών TrkA, TrkB, TrkC και p75. Μικρά αιμοφόρα αγγεία συνοδεύουν τις νευρικές ίνες στα σπονδυλικά επίπεδα του ΜΔ που δίνουν δισκογενή πόνο και εκφράζουν επίσης τους υποδοχείς των νευροτροφινών. Ο CB1 υποδοχέας των ενδοκανναβινοειδών ταυτοποιήθηκε σε όλα τα περιστατικά που εμφάνιζαν ανάπτυξη νευρικών ινών. Βρέθηκε να εκφράζεται στα αγγεία που συνοδεύουν τις νευρικές ίνες και σε μερικά νεύρα. Δεν ταυτοποιήθηκε ανάπτυξη νευρικών ινών σε εκφυλισμένους ΜΔ σπονδυλικών επιπέδων που δεν αποτελούσαν αιτία δισκογενούς οσφυαλγίας. Συμπέρασμα: Η αλγοϊδιοδεκτική ανάπτυξη νευρικών ινών στον ΜΔ συνδέεται με την έκφραση των υποδοχέων των νευροτροφινών στις νεοσχηματιζόμενες νευρικές ίνες, δείχνοντας ότι όχι μόνο ο NGF, αλλά και άλλες νευροτροφίνες εμπλέκονται στην ανάπτυξη νευρικών ινών. Η έκφραση του CB1 υποδοχέα των ενδοκανναβινοειδών αποτελεί πεδίο έρευνας για τη θεραπεία του δισκογενή πόνου. / The current study was designed in order to detect immunohistochemically the expression of cannabinoid CB1 receptor and high and low affinity neurotrophin receptors TrkA, TrkB, TrkC and p75 respectively in human painful intervertebral disc (IVD). Previous data have shown an association between nerve ingrowth in human degenerated IVD and patient’s experience of pain. The nociceptive nerve ingrowth into painful IVD is causally linked with Nerve Growth Factor (NGF) production by blood vessels growing into the IVD. NGF causes further innervation and is involved in processes that result in an inflammatory hyperalgesia. There is evidence that cannabinoids show a neuronal CB1 receptor-mediated antihyperalgesic action and a separate inhibition of proinflammatory neuroimmune processes. A total of 50 IVD specimens after lumbar discectomy were included in the present study. The degeneration of IVD was identified and connected with clinical background of patients. An immunoperoxidase method on formalin-fixed, paraffin-embedded tissue sections and specific antibodies against CB1 and neurotrophin receptors (TrkA, TrkB, TrkC and p75) were used. Immunohistochemistry was also performed to detect neural and vascular markers: tubulin (βIII) and CD31. Neurotrophin receptors (TrkA, TrkB, TrkC and p75) expression was detected only in innervated degenerated IVD. Blood microvessels accompanied nerve fibers growing into IVD expressed also neurotrophin receptors. Furthermore, in some nerve fibers CB1 cannabinoid receptor expression was identified. Interestingly, CB1 receptor expression was detected in the majority of vessels accompanying nerve fibers. Nociceptive nerve ingrowth into degenerated IVD was strongly associated with all neurotrophin receptors expression indicating that not only NGF but also and others neurotrophins are involved in nerve ingrowth. Expression of CB1 cannabinoid receptor in innervated IVD suggests a possible therapeutic site of analgesic action. Additional studies would be necessary to clarify the functional role of CB1 cannabinoid receptor in degenerated intervertebral disc.

The need for the development of a rigorous analytical model of the lumbar spine to clarify the role of mechanical factors in low-back disorders has long been recognized. In response to this need, a general three dimensional nonlinear finite element program has been developed as part of this work and has been applied to the analysis of a lumbar L(,2-3) joint including the posterior elements. The analysis accounts for both the material and geometric nonlinearities and is based on a representation of the nucleus as an incompressible inviscid fluid and of the annulus as a composite of collagenous fibres embedded in a matrix of ground substance. The facet articulation has been accounted for by treating it as a general moving contact problem. The ligaments have been modelled as a collection of nonlinear axial elements. The geometry of the finite element model is based on in-vitro measurements. / The response of the joint under single compression, single flexion, single extension and also under flexion or extension combined with compression and sagittal shear has been analyzed for both the normal and degenerated states of the nucleus. Validity of the model has then been established by a comparison of those predictions which are also amenable to direct measurements. The states of strain and stress in different components of the lumbar joint have been thoroughly studied under all the foregoing loading conditions. / Those elements of the joint predicted to be vulnerable to mechanical failure or damage under the above types of loading have been identified. These results have been correlated with the lumbar joint injuries reported clinically. Furthermore, some joint injury mechanisms and degeneration processes have been proposed and the supporting clinical evidences have been presented.