Nucleus replacement has recently come into favor as a possible treatment for Degenerative Disc Disease. Replacing degenerative nucleus tissue with a synthetic material that mimics healthy nucleus tissue may restore normal function and biomechanics to the disc and delay or obviate the need for more invasive procedures such as total disc replacement and fusion. This thesis evaluated a novel protein polymer hydrogel composed of silk and elastin as a nucleus replacement material. There are three experimental components; one in vivo and two in vitro portions. In the first experimental portion, a large animal model was developed to evaluate the biocompatibility of the material as well as the effect on surrounding boney and soft tissues. Three discs were evaluated in each animal; sham, discectomy and discectomy treated with hydrogel. Discs were evaluated at 4, 26 and 52 weeks. The hydrogel group showed a quiet cellular response, as well as decreased boney remodeling and fewer degenerative changes when compared to the discectomy group. The second experimental portion evaluated the biomechanics of 9 cadaveric motion segments loaded in axial rotation, lateral bending, flexion/extension (FE) and compression. Specimens were tested sequentially in the intact state, following annulotomy, discectomy and after hydrogel treatment. Range of Motion (ROM) in FE was shown to increase from the intact state (8.50+/-1.44˚) to the discectomy state (9.86+/-1.77˚) and decrease following hydrogel treatment (8.66+/-0.76˚) to be similar to the intact ROM. The third experimental portion investigates the effect of three commonly applied testing conditions on the mechanical properties of spinal segments. 27 motion segments were tested at 18˚C wrapped with Phosphate Buffered Saline (PBS), at 37˚C in a PBS bath, and at 37˚C and 100% humidity. Specimens were tested hourly for 6 hours. The heated conditions were shown to have lower stiffness and increased range of motion when compared 18 ˚C tests. Repeated testing with time increased neutral zone and ROM for all modes of bending. As tests are repeated over time, tissue properties change and may mask the ability of a nucleus replacement to restore biomechanics.

Mechanical low back pain is a disabling condition often associated with degenerative disc disease (DDD). Treatment for DDD includes non-operative pain management, total disc arthroplasty and nucleus pulposus (NP) replacement. A thiol-modified hyaluronan elastin-like polypeptide (TMHA/EP) composite has been under consideration as a NP replacement and has shown promise in vitro. This thesis aims to determine the effects of TMHA/EP composite augmentation on spinal motion segment mechanics in healthy and induced early stage DDD porcine intervertebral discs (IVD). Healthy IVD augmentation on average increased axial compressive stiffness, while bending and rotational stability decreased. Early stage DDD porcine IVD had compromised mechanical integrity in comparison to healthy controls. TMHA/EP augmentation of the mechanically compromised IVDs through two injection techniques worked to restore spinal stability, exhibiting mechanical properties similar to healthy IVDs. This work demonstrates the potential of the injectable TMHA/EP composite in providing initial structural stabilization in early stages of DDD.

Mechanical low back pain is a disabling condition often associated with degenerative disc disease (DDD). Treatment for DDD includes non-operative pain management, total disc arthroplasty and nucleus pulposus (NP) replacement. A thiol-modified hyaluronan elastin-like polypeptide (TMHA/EP) composite has been under consideration as a NP replacement and has shown promise in vitro. This thesis aims to determine the effects of TMHA/EP composite augmentation on spinal motion segment mechanics in healthy and induced early stage DDD porcine intervertebral discs (IVD). Healthy IVD augmentation on average increased axial compressive stiffness, while bending and rotational stability decreased. Early stage DDD porcine IVD had compromised mechanical integrity in comparison to healthy controls. TMHA/EP augmentation of the mechanically compromised IVDs through two injection techniques worked to restore spinal stability, exhibiting mechanical properties similar to healthy IVDs. This work demonstrates the potential of the injectable TMHA/EP composite in providing initial structural stabilization in early stages of DDD.

Degenerative disc disease (DDD) is a common medical issue among human adults, leading to back pain and potentially, disability, decreasing an individual’s quality of life. In the United States alone, huge economic impacts are apparent with an estimated $50- 100 billion attributed to lost productivity and medical costs related to DDD. Spinal degeneration occurs in the intervertebral disc (IVD) and once damaged, the IVD is incapable of adequate self-repair. A regenerative therapy incorporating nucleus pulposus (NP) tissue engineering may provide an answer to spinal degeneration. The objective of this in vitro study was to evaluate the potential of a thiol-modified hyaluronan (TMHA) and elastin-like polypeptide (ELP) as a hydrogel scaffold for nucleus pulposus tissue engineering. Two materials, one composed of TMHA only and one a 3:1 TMHA/ELP, crosslinked with polyethylene glycol diacrylate (PEGDA), were seeded with cultured human NP cells and cyclic hydrostatic loading was applied at 1MPa for 3 hours a day for 3 consecutive days. Cell viability and gene expression were analyzed. A decreasing trend in cell viability with time and cyclic hydrostatic pressure loading was observed and statistically significant differences were observed between the TMHA unloaded treatment group at day 0 and the TMHA loaded treatment group at day 4 and between the TMHA unloaded treatment group at day 0 and the 3:1 TMHA/ELP loaded group at day 4. Comparisons between TMHA only and 3:1 TMHA/ELP hydrogels for the same treatment indicate similar trends and no statistically significant differences in biological effects were observed. Gene expression analysis indicated low frequency expression of NP extracellular matrix (ECM) molecules regardless of time point or cyclic hydrostatic pressure application. These results are revealing in that the 3:1 TMHA/ELP hydrogel did not support NP cells significantly better than the TMHA hydrogel, though cell source and hydrostatic pressure generation issues may have impacted this finding. Additional studies with alternative cell type and a refined hydrostatic pressure application method may better illuminate the efficacy of a 3:1 TMHA/ELP hydrogel as for NP tissue engineering. / Thesis (Master, Chemical Engineering) -- Queen's University, 2011-03-17 14:16:28.83

Nucleus replacement has recently come into favor as a possible treatment for Degenerative Disc Disease. Replacing degenerative nucleus tissue with a synthetic material that mimics healthy nucleus tissue may restore normal function and biomechanics to the disc and delay or obviate the need for more invasive procedures such as total disc replacement and fusion. This thesis evaluated a novel protein polymer hydrogel composed of silk and elastin as a nucleus replacement material. There are three experimental components; one in vivo and two in vitro portions. In the first experimental portion, a large animal model was developed to evaluate the biocompatibility of the material as well as the effect on surrounding boney and soft tissues. Three discs were evaluated in each animal; sham, discectomy and discectomy treated with hydrogel. Discs were evaluated at 4, 26 and 52 weeks. The hydrogel group showed a quiet cellular response, as well as decreased boney remodeling and fewer degenerative changes when compared to the discectomy group. The second experimental portion evaluated the biomechanics of 9 cadaveric motion segments loaded in axial rotation, lateral bending, flexion/extension (FE) and compression. Specimens were tested sequentially in the intact state, following annulotomy, discectomy and after hydrogel treatment. Range of Motion (ROM) in FE was shown to increase from the intact state (8.50+/-1.44˚) to the discectomy state (9.86+/-1.77˚) and decrease following hydrogel treatment (8.66+/-0.76˚) to be similar to the intact ROM. The third experimental portion investigates the effect of three commonly applied testing conditions on the mechanical properties of spinal segments. 27 motion segments were tested at 18˚C wrapped with Phosphate Buffered Saline (PBS), at 37˚C in a PBS bath, and at 37˚C and 100% humidity. Specimens were tested hourly for 6 hours. The heated conditions were shown to have lower stiffness and increased range of motion when compared 18 ˚C tests. Repeated testing with time increased neutral zone and ROM for all modes of bending. As tests are repeated over time, tissue properties change and may mask the ability of a nucleus replacement to restore biomechanics.

Back pain is one of the most common physical conditions in the United States, for which approximately 15% of the population will visit a doctor every year. The most common type of back pain is low back pain (LBP) and millions of dollars are spent every year healthcare are a due to LBP. Although poorly understood, low back pain has been associated to interveterbral disc (IVD) degeneration. The IVD is an important structure that helps maintaining normal skeletal support. It is composed of three different tissues called the annulus fibrosus (AF), and the nucleus pulposus (NP), attached to a cartilage endplate (CEP) at its top and bottom surfaces. The AF tissue is composed of chondrocyte-like cells, while the NP tissue is composed of notochordal cells at a young age, which are replaced by mature NP cells later in life. Common signs of degeneration are the inability to maintain extracellular matrix integrity and calcification of the cartilage endplate. Extracellular matrix synthesis and cartilage endplate calcification are closely related to production of adenosine triphosphate (ATP) or energy metabolism of the cells. AF and NP tissues are known to be structurally and compositionally different; therefore it is believed that their metabolic pathways are also distinct. The objective of this study was to determine the differences between AF and NP cells, specifically in their energy metabolism with and without dynamic loading.

<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.

A cysteine-containing elastin-like polypeptide (ELP2cys) was successfully synthesized and purified, and was shown to behave in a similar fashion to other well-characterized ELPs. Incorporating the ELP2cys as a crosslinking agent into a solution of sulphated hyaluronan (CMHA-S) not only decreased the gelation time of the solution but also increased the crosslinking density of the resultant hydrogel, in turn increasing both the resiliency and stiffness of the construct. Preliminary in vitro work involved culture of human disc cells, followed by their encapsulation within the hydrogel. Unfortunately the results were inconclusive, although it appeared as though the addition of ELP2cys to the matrix did not negatively affect the viability of the cells, as compared to hydrogels with CMHA-S only. This study showed that ELP2cys is a valuable addition to the family of recombinant elastin-like polypeptides, and shows promise as a crosslinking agent in the formation of hyaluronan hydrogels.

<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