Low back pain represents a significant concern in the United States, with 70% of individuals experiencing symptoms at some point in their lifetime. Although the specific cause of low back pain remains unclear, symptoms have been strongly associated with degeneration of the intervertebral disc. Insufficient nutritional supply to the disc is believed to be a major mechanism for tissue degeneration. Understanding nutrients' transport in intervertebral disc is crucial to elucidate the mechanisms of disc degeneration, and to develop strategies for tissue repair (in vivo), and tissue engineering (in vitro). Transport in intervertebral disc is complex and involves a series of electromechanical, chemical and biological coupled events. Despite of the large amount of studies performed in the past, transport phenomena in the disc are still poorly understood. This is partly due to the limited number of available experimental techniques for investigating transport properties, and the paucity of theoretical or numerical methods for systematically predicting the mechanisms of solute transport in intervertebral disc. In this dissertation, a theoretical and experimental approach was taken in order to investigate the mechanisms of solute transport and binding interactions in intervertebral disc. New imaging techniques were developed for the experimental determination of diffusive and binding parameters in biological tissues. The techniques are based on the principle of fluorescence recovery after photobleaching, and allow the determination of the anisotropic diffusion tensor, and the rates of binding and unbinding of a solute to the extracellular matrix of a biological tissue. When applied to the characterization of transport properties of intervertebral disc, these methods allowed the establishment of a relationship between solute anisotropic and inhomogeneous diffusivity and the unique morphology of human lumbar annulus fibrosus. A mixture theory for charged hydrated soft tissues was presented as a framework for theoretical investigations on solute transport and binding interactions in cartilaginous tissues. Based on this theoretical framework and on experimental observations, a finite element model was developed to predict solute diffusive-convective-reactive transport in cartilaginous tissues. The numerical model was applied to simulate the effect of mechanical loading on solute transport and binding interactions in cartilage explants and intervertebral disc.

Crosslinking soft tissue has become more common in tissue engineering applications, and recent studies have demonstrated that soft tissue mechanical behavior can be directly altered through crosslinking, but increased understanding of how crosslinking affects intervertebral disc mechanical behavior is needed. In vitro testing of bovine disc and motion segments was used to characterize several important aspects of disc behavior in response to crosslinking after both soaking and injection treatment. The first study was a comparison of different crosslinkers to determine the effect on tensile properties of disc tissue. Circumferential specimens were taken from bovine annulus and then soak treated with an optimized crosslinking formulation or sham solution. A non-contacting laser micrometer was used to measure cross sectional area, after which tension testing until failure was performed to determine yield strain, yield stress, ultimate stress, peak modulus, and resilience. The crosslinkers were observed to produce different changes in the properties, with the measured properties generally increasing. The second study used bilateral annular injections to simulate a clinically relevant delivery method. The dose response of the motion segment’s neutral zone stability metrics against injection concentration was mapped. Concentrations of 20 mM and less had no significant effects on the stability metrics. 40mM demonstrated a change in neutral zone stiffness, while at least 80mM was required to significantly affect neutral zone length. Thus, meaningful changes in joint neutral zone stability were demonstrated using clinically relevant injection and chemical formulations. The third study used combinations of biochemical and accelerated mechanical cyclic loading to degrade gelatin and annulus fibrosus specimens with and without genipin treatment. Genipin crosslinking attenuated changes during cyclic loading to specimen geometry and compliance relative to control samples. Full recovery of genipin treated samples appeared to be hampered, at least partially from continued crosslinking during the accelerated testing. The fourth study tested the effect of genipin crosslinking to resist interlamellar shearing of the annulus lamella. Using a recently reported test method that shears adjacent lamella, crosslinked specimens were noted to have significantly higher yield force, peak force, and resilience compared to sham treated controls, supporting the hypothesis that crosslinking would increase the load bearing ability of the interface.

<p>Intervertebral disc (IVD) disorders are a major contributor to disability and health costs. Disc disorders and resulting pain may be preceded by changes which first occur in the nucleus pulposus (NP) region of the IVD, where significant alterations in tissue cellularity, composition, and structure begin early in human life and continue with increasing age and degeneration. These changes coincide with the loss of a distinct cell population, notochordally-derived immature NP cells, which may play a key role in the generation and maintenance of this tissue. These cells reside in a gelatinous, highly-hydrated extracellular matrix (ECM) environment and exhibit in situ cell-matrix and cell-cell interactions which are quite distinct from cells in other regions of the disc or in other cartilagenous, including expression of laminin cell-matrix receptors and cell-associated laminin proteins. The ECM environment is known to be a key regulator of cellular behaviors, with ECM ligands and elasticity modulating cell adhesion, organization, differentiation, and phenotype. The primary motivating hypothesis of this thesis is that the unique ECM environment of the NP plays a key role in modulating immature NP cell behaviors, and that laminin ligands, in combination with ECM elasticity, modulate immature NP cell behaviors including adhesion, organization, and phenotype.</p><p>To investigate this hypothesis, flow cytometric analyses were performed to examine IVD cell integrin receptor expression over time in culture, including assessment of key laminin-binding integrin subunits. The roles of specific integrin receptors modulating NP cell adhesion to ECM proteins were identified in studies utilizing function-blocking antibodies. NP cell adhesion, spreading, and relative cell adhesion strength was assessed on various ECM constituents, including specific isoforms of laminin. Additionally, studies were performed to examine the roles of ECM ligand and substrate stiffness in modulating NP cellular organization, utilizing polyacrylamide gel substrates with tunable mechanical properties and functionalized with different ECM ligands. Finally, the role of ECM environment was examined on one key measure of NP cell function, proteoglycan production, over time in culture.</p><p>NP cells isolated from immature NP tissues were found to express high levels of the laminin-binding integrin subunit alpha 6 ex situ and maintain this expression over time in culture. Function blocking studies revealed this receptor to be a key regulator of NP cell adhesion to laminin, in contrast to cells from the adjacent AF region, which did not express this receptor nor adhere to laminin. Cell adhesion studies demonstrated that NP cells preferentially interact with two laminin isoforms, LM-511 and LM-332, in comparison to other ECM proteins, with enhanced cell attachment, spreading, and adhesion strength on surfaces coated with these ligands. These findings correspond with laminin isoform and receptor expression patterns identified in immature NP tissues. Additionally, NP cell-cell interactions were found to be modulated by both ECM ligand and substrate stiffness, with soft, laminin-functionalized substrates promoting self-assembly of NP cells into cell clusters with morphologies similar to those identified in immature NP tissues. Finally, culture of immature NP cells on soft, laminin-rich substrates was found to promote a key measure of NP cell function, proteoglycan synthesis.</p><p>The studies presented here demonstrate that immature NP cells interact uniquely with laminin extracellular matrix proteins, and that laminin ligands and matrix elasticity are two key regulators of NP cell organization and phenotype in the IVD. These findings suggest that alterations in one or both of these factors during IVD aging or degeneration may contribute to the differentiation or loss of this unique cell population. Additionally, these results indicate that soft, laminin-functionalized biomaterials may be appropriate for in vitro culture and expansion of immature NP cells, as well as for use in NP tissue engineering strategies.</p> / Dissertation

<p>Intervertebral disc (IVD) disorders can cause pain and disability for affected individuals. A subset of IVD disorders are thought to originate in the nucleus pulposus (NP) region of the IVD, due to alterations in tissue structure and composition with age and injury. Cells of the NP undergo a phenotypic and behavioral shift with age, changes that are thought to disrupt tissue homeostasis and lead to disc degeneration. There is significant interest in developing biomaterials and strategies to revert adult degenerate NP cells to healthy, young NP cell phenotypes with increased biosynthesis and cell clustering. Studies demonstrate that healthy porcine NP cells interact with laminin proteins through specific integrin subunits on soft substrates in a process that regulates cell morphology and biosynthesis. The central hypothesis of this dissertation is that the engagement of cell-surface adhesion receptors, using laminin-mimetic peptides on a controlled stiffness material, can revert adult degenerate NP cellular phenotype and behaviors to their healthy, biosynthetically active form.</p><p>In the first part of this dissertation, integrin subunits used by adult degenerate human NP cells to attach to laminin were revealed using flow cytometric analyses, function blocking antibodies, and immunohistochemistry. Secondly, NP cell recognition peptides were identified by screening laminin-mimetic peptides for cell attachment. Finally, human NP cells were cultured on peptide functionalized polyacrylamide gels to examine the effect of ligand and substrate stiffness in regulating adult human NP cell phenotype and biosynthesis. </p><p>Findings reveal that adult human NP cells express and utilize integrin subunits α3, α5, and β1 to attach to laminins, in contrast to integrin α6β1 found previously for healthy porcine NP cells. This difference between current and previous findings likely arises from aging-associated difference in NP cells between human and porcine tissues. Select laminin-mimetic peptides, chosen from the literature and informed by NP cell integrin expression, were found to promote significant NP cell attachment in a concentration dependent manner. Finally, a subset of laminin mimetic peptides were found to promote a young NP cell phenotype by increasing cell clustering on soft (0.3 kPa) and stiff (14 kPa) substrates as well as increasing proteoglycan synthesis on soft substrates. </p><p>The studies presented in this dissertation demonstrate that adult degenerate human NP cells attach to laminin proteins in an integrin dependent manner. Furthermore, laminin-mimetic peptides are able to mediate human NP cell attachment at levels comparable to full-length laminin, increase cell clustering when presented on soft and stiff substrates, and can increase NP cell biosynthesis when presented on soft substrates. Utilizing laminin-mimetic peptides may allow for the design of biomaterials that promote a healthy young NP phenotype for a variety of disc therapies.</p> / Dissertation

Temos aktualumas: 95% žmonių, kurių amžius 25–55 metai turi juosmeninės stuburo dalies tarpslankstelinę disko išvaržą L4–L5 arba L5–S1 segmentuose (Jordon et al., 2009). McKenzie tiesimo judesiai padeda daugumai pacientų, kurie skundžiasi juosmeninės stuburo dalies skausmais (Richmond, 2012). Tai saugus, nebrangus ir patikimas metodas, kuris naudojamas, kai norima išspręsti juosmeninės stuburo dalies skausmus su skausmo plitimu į koją (McKenzie, 2011). Darbo objektas: kineziterapijos ir McKenzie pratimų poveikis skausmui ir stuburo paslankumui esant juosmeninės stuburo dalies išvaržai. Tikslas: nustatyti McKenzie pratimų poveikį skausmui bei stuburo paslankumui esant juosmeninės stuburo dalies išvaržai. Uždaviniai: 1.Įvertinti ir palyginti juosmeninės stuburo dalies skausmo pokyčius taikant kineziterapiją ir McKenzie pratimus. 2.Įvertinti ir palyginti stuburo dalies paslankumą taikant kineziterapiją ir McKenzie pratimus. 3.Įvertinti ir palyginti funkcinę negalią klausimynais taikant kineziterapiją ir McKenzie pratimus. Hipotezė: manome, kad taikant McKenzie pratimus kartu su tradicine kineziterapija labiau sumažės skausmas, pagerės stuburo paslankumas juosmeninėje stuburo dalyje bei pagerės funkcinė būklė. Išvados: 1.McKenzie pratimai labiau sumažino juosmeninės stuburo dalies skausmą nei tradicinė kineziterapija. 2.McKnezie pratimai ir tradicinė kineziterapija pagerino juosmeninės stuburo dalies paslankumą. 3.McKenzie pratimai ir tradicinė kineziterapija sumažino... [toliau žr. visą tekstą] / Importance of the study: relevance of the topic: 95% of people, whos age is 25–55 years, have lumbar spine disc herniation in L4–L5 or L5–S1 segments (Jordon et al., 2009). McKenzie laying movements help for the most of the patients, who are complaining about lumbar spine part pains (Richmond, 2012). It is a safe, cheap and reliable method, which is used to solve lumbar spine part pains with pain spreading to the leg (McKenzie, 2011). Object of the research: effect of physical therapy and McKenzie exercises on pain and spine mobility in the lumbar spine hernia. The aim of the research: to determine the effect of McKenzie exercises regarding pain and mobility of spine in lumbar herniated disc. The task of the research: 1.To evaluate and compare the changes in pain of lumbar spine through physical therapy and McKenzie exercises. 2.To evaluate and compare changes in the mobility of lumbar spine through physical therapy and McKenzie exercises. 3.To evaluate and compare the functional disability after using physical therapy and McKenzie exercises. Hypothesis: the expected outcome is that physical therapy along with McKenzie exercises will reduce the pain, improve spinal mobility in the lumbar spine part and improves functional status Conclusions: 1.McKenzie exercises greater reduced the pain in lower back than conventional physical therapy. 2.McKnezie exercises and conventional physiotherapy improved the mobility of the lumbar spine. 3.McKenzie exercises and... [to full text]

Developing computational models of the human spine has been a hot topic in biornechanical research for a couple of decades in order to have an understanding of the behaviour of the whole spine and the individual spinal parts under various loading conditions. The objectives of this thesis are to develop a biofidefic multi-body model of the whole human spine especially for dynamic analysis of impact situations, such as frontal impact in a car crash, and to generate finite element (FE) models of the specific spinal parts to investigate causes of injury of the spinal components. As a proposed approach, the predictions of the multi-body model under dynamic impact loading conditions, such as reaction forces at lumbar motion segments, were utilised not only to have a better understanding of the gross kinetics and kinematics of the human spine, but also to constitute the boundary conditions for the finite element models of the selected spinal components. This novel approach provides a versatile, cost effective and powerful tool to analyse the behaviour of the spine under various loading conditions which in turn helps to develop a better understanding of injury mechanisms.

Degenerative disc disease is a common ailment with enormous medical, psychosocial and economic ramifications. This study was designed to investigate the utility of a thiol-modified hyaluronan(TMHA) and elastin-like polypeptide(EP) composite material as a potential tissue engineering scaffold to reconstitute the nucleus pulposus in early degenerative disc disease. TMHA and EP were combined in various concentrations and cross-linked using poly(ethylene glycol)diacrylate. Resulting materials were evaluated biomechanically and biologically. Confined compression testing revealed that the addition of EP to TMHA-based gels resulted in a stiffer construct, but remained an order of magnitude less stiff than native nucleus. The in vitro cell culture experiments with human intervertebral disc cells demonstrated 70% cell viability at three weeks with apparent maintenance of phenotype. The addition of EP did not have a significant biologic effect. An in vivo pilot study demonstrated biocompatibility of the TMHA-based hydrogels; additional power is required to adequately assess treatment effect.

Tissue engineering the annulus fibrosus (AF) for use in a functional intervertebral disc replacement is a promising alternative to current treatments of degenerative disc disease. Polycarbonate urethane (PU) scaffolds have demonstrated the ability to support AF cell attachment and matrix synthesis and are suitable for tissue engineering the AF. The present study investigates the effects of the physical and biochemical environment on AF cells grown on aligned nanofibrous PU scaffolds. First, the effect of dynamic spinner flask culture and fibronectin pre-coating on tissue formation was analyzed and then the role of scaffold fibre tension on annulus fibrosus cells was examined using a tailored culture system. The results of these studies demonstrated that AF cells are sensitive to differences in biochemical cues at the scaffold surface and their physical environment and respond by altering their cellular responses and, potentially by manipulating their microenvironments, including the physical characteristics of the PU-ADO scaffolds.

Degenerative disc disease is a common ailment with enormous medical, psychosocial and economic ramifications. This study was designed to investigate the utility of a thiol-modified hyaluronan(TMHA) and elastin-like polypeptide(EP) composite material as a potential tissue engineering scaffold to reconstitute the nucleus pulposus in early degenerative disc disease. TMHA and EP were combined in various concentrations and cross-linked using poly(ethylene glycol)diacrylate. Resulting materials were evaluated biomechanically and biologically. Confined compression testing revealed that the addition of EP to TMHA-based gels resulted in a stiffer construct, but remained an order of magnitude less stiff than native nucleus. The in vitro cell culture experiments with human intervertebral disc cells demonstrated 70% cell viability at three weeks with apparent maintenance of phenotype. The addition of EP did not have a significant biologic effect. An in vivo pilot study demonstrated biocompatibility of the TMHA-based hydrogels; additional power is required to adequately assess treatment effect.

Tissue engineering the annulus fibrosus (AF) for use in a functional intervertebral disc replacement is a promising alternative to current treatments of degenerative disc disease. Polycarbonate urethane (PU) scaffolds have demonstrated the ability to support AF cell attachment and matrix synthesis and are suitable for tissue engineering the AF. The present study investigates the effects of the physical and biochemical environment on AF cells grown on aligned nanofibrous PU scaffolds. First, the effect of dynamic spinner flask culture and fibronectin pre-coating on tissue formation was analyzed and then the role of scaffold fibre tension on annulus fibrosus cells was examined using a tailored culture system. The results of these studies demonstrated that AF cells are sensitive to differences in biochemical cues at the scaffold surface and their physical environment and respond by altering their cellular responses and, potentially by manipulating their microenvironments, including the physical characteristics of the PU-ADO scaffolds.