Rozprawy doktorskie na temat „Nucleus Pulposus Regeneration”

Thesis (Ph.D.)--University of California, San Francisco with the University of California, Berkeley, 2009.
Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3639. Adviser: Jeffrey C. Lotz.

Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2008.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 27-28).
In this study biomaterial scaffolds for regeneration of nucleus pulposus were developed by freeze drying slurries with different proportions of collagen II (CII), chondroitin-6-sulfate (CS), and hyaluronic acid (HA). The scaffolds were analyzed using biochemical assays to determine final composition. Chemically cross-linked scaffolds were analyzed to determine pore size and cross-link density. It was determined that every material type contained large enough pore size (275 gm) to seed nucleus pulposus cells and mesenchymal stem cells. The addition of CS to the scaffold increased pore size. It was also found that increasing levels of CS and HA resulted in lower cross-link density. These materials will be used next in In Vitro studies to determine their viability as regenerative tissue engineering constructs.
by Jacob A. Simson.
S.B.

Back pain is a highly prevalent condition whose pathogenesis is associated with intervertebral disc (IVD) degeneration. Degeneration is driven by abnormal cell biology, particularly within the IVD’s inner core, the nucleus pulposus (NP). In recent years, there has been an ever-increasing search for cell-based therapies aimed at correcting the cell biology and thus repairing/regenerating the degenerate IVD. The success of these novel therapies, however, requires a thorough understanding of IVD development and of the phenotype of its cells. The embryonic, foetal and juvenile NP is populated by large vacuolated notochordal cells that with skeletal maturity are replaced by smaller NP cells. Since notochordal cells have been shown to display protective and anabolic roles in the IVD their loss in humans has often been suggested to initiate the degenerative process. As such, a detailed understanding of notochordal cells and their regulatory pathways may help identify factors involved in IVD homeostasis and aid the development of novel cell-based therapies targeting IVD degeneration. The study of human notochordal cells has, however, been hindered by ethical, logistical and technical difficulties in obtaining suitable samples and, as such, the human notochordal cell phenotype is, to date, unknown, constituting a major limitation in the field. The work presented here was conducted with the objective of developing a methodology to isolate human developing notochordal cells (NP progenitors) from adjacent sclerotomal cells (annulus fibrosus and vertebral body progenitors), to characterise the notochordal cell phenotype and identify potential factors involved in notochordal cell biology. Initially, human embryonic and foetal spines were characterised to assess their suitability as a source of notochordal cells and to identify a notochord-specific marker that could be used to isolate notochordal cells for microarray studies. The human developing spine contained large vacuolated notochordal cells in all stages analysed (3.5-18 weeks post-conception (WPC)) that specifically expressed KRT8, KRT18 and KRT19 at all stages and CD24 between 5.5-18 WPC. KRT18 and CD24 were independently used to label notochordal cells (7.5-14 weeks post-conception) and separate them from sclerotomal cells. Methodologies were developed to allow extraction of RNA of sufficient quality for microarray analysis from fixed, permeabilised (in the case of KRT18) and/or, labelled and sorted cells (CD24). Microarray analysis identified and real-time qPCR and, for some markers, immunohistochemistry, validated GRB14, SLC19A1, FGF10, ADORA3, TBXA2R, CDH6, ANPEP, CD69, CD24, RTN1, PRPH, MAP1B, ISL1 and CLDN1 as human notochordal cell markers. Ingenuity pathway analysis was performed to investigate the pathways/networks and upstream regulators and downstream effectors of notochordal cells. Inhibition of inflammation and angiogenesis were identified as relevant to notochordal cell biology, function and, possibly, to the known protective and anabolic role notochordal cells display in the IVD. Notochordal marker gene expression was identified in adult NP tissue, and negatively correlated with degeneration. Proteins encoded by ADORA3 and MAP1B were expressed by a proportion of adult NP cells, suggesting the presence of notochord-derived cells in the adult NP.Importantly, this is the first study to detail a methodology and successfully isolate human notochordal cells. Such methodology has the potential to be used to culture and investigate the biology of viable human notochordal cells (CD24+ve). Future studies aimed at developing cell-based therapies for IVD degeneration could also use these identified markers to assess appropriate stem cell differentiation to notochordal cells.

Intervertebral disc (IVD) degeneration is associated with low back pain (LBP). It has been suggested that changes in the IVD physio-chemical microenvironment (i.e. hypoxia, reduced nutrient and acidic conditions) may lead to disc degeneration. Studying the response of human nucleus pulposus (NP) cells to these conditions could establish the causal relationship between IVD microenvironment and aberrant cellular behaviour, characteristic of disc degeneration. Human bone marrow mesenchymal stem cells (BM-MSCs) are a promising cell population for disc regeneration. However, knowledge of their survival and functioning in the microenvironment of the IVD is still lacking. Moreover, in vitro co-culture model studies that are used to study MSC/disc cell interaction, also need to consider the effect of the microenvironment on cellular responses. BM-MSCs and degenerate NP cells were cultured alone or co-cultured in monolayer under hypoxia (2%O2), reduced nutritional (2% serum or/and 5mM glucose) and acidic (moderate pH 6.8 or severe pH 6.5) conditions alone or in combination for 7 days. Cell viability, proliferation, gene and protein expression was assessed. Degenerate NP cells and BM-MSCs maintained good cell viability under all conditions. Both cell types demonstrated overall similar proliferation and gene and protein responses under the majority of the conditions and combinations studied. Hypoxia promoted aggrecan and versican matrix biosynthesis in both cell types. Nutrient deprived and moderate acidic conditions (pH 6.8) inhibited proliferation of both cell types. Interestingly the combination of hypoxia with these conditions showed a protective effect in modulating cell proliferation. These results imply that hypoxia may be beneficial in some instances. Nutrient deprived conditions had a relatively minor effect on degenerate NP cell gene and protein expression but these conditions specifically inhibited VCAN expression in BM-MSCs. The combination of hypoxia with these conditions increased or restored VCAN expression. Interestingly the combination of hypoxia with reduced glucose conditions increased aggrecan and versican matrix biosynthesis in both NP cells and BM-MSCs. The combination of hypoxia and complete nutrient deprived conditions (both reduced serum and reduced glucose) impaired ACAN, VCAN and PAX-1 gene and aggrecan and versican protein expression in degenerate NP cells implicating disc hypoxia and complete nutrient deprived combined microenvironment in accelerating degenerate changes in NP cells. In contrast, these conditions showed no such detrimental effects on BM-MSC gene and protein expression. pH 6.5 was critical for both cell types proliferation and ACAN and VCAN gene expression suggesting that severe acidic conditions may exacerbate degenerative changes and be inhibitory for implanted MSCs. Finally, a combination of hypoxia, complete nutrient deprived and moderate acidic conditions, reduced cell proliferation without affecting the gene expression profile of both cell types. IVD-like physio-chemical microenvironmental conditions also appeared to influence differentiation of BM-MSC and modulation of degenerate NP cell phenotype observed during co-culture. Noticeably hypoxia, reduced serum or reduced glucose conditions stimulated BM-MSC differentiation and modulation of degenerate NP cell phenotype. Hypoxia also increased or recovered changes at gene expression level in both BM-MSCs and degenerate NP cells under nutrient deprived (reduced serum or/and reduced glucose) conditions during co-culture. Degenerate NP cell and BM-MSC co-culture also showed noticeable increase in aggrecan and versican biosynthesis under hypoxia and reduced glucose combine conditions, implicating these in improving the co-culture responses. Severe pH condition alone, pH 6.8 in combination with hypoxia and finally all IVD-like physio-chemical conditions together compromised co-culture responses. Such results imply that IVD-like physio-chemical microenvironmental conditions may influence MSC based regenerative outcomes. This work has increased our understanding about the influence of disc harsh microenvironment on degeneration and regeneration processes.

Debilitating chronic back pain caused by severe spinal disc degeneration leads to loss of mobility, affecting quality of life, a significant loss of productivity for the employee and the employer. Currently available surgical intervention options, such as spinal fusion and total disc replacement, seeking only to alleviate pain, are not only invasive, but fail to address the underlying biological causes of spinal disc degeneration, or restore normal physiological spinal motion. Recently proposed tissue engineering approaches focus on stopping and reversing the degenerative cascade, which has a promising regenerative effect, though not without significant challenges before a clinical application is made available, including tumourigenesis risks and proof of efficacy. A minimally invasive nucleus pulposus replacement option, which preserves the competent annulus fibrosis, while replacing the removed degenerated nucleus tissue with a prosthesis, provides an alternative for early disc degeneration, though most commercially available types are at clinical trial stages. There is an opportunity for developing a minimally invasive nucleus pulposus replacement type spinal implant system that restores disc biomechanics and addresses biological degenerative causes. This body of work details the design, development, fabrication, prototyping, verification and validation of this novel implant system. The implant system consisted of a configuration of scaffold and hydrogel interpenetrating polymer network composite delivered minimally invasively via a cannula system, after the nucleus pulposus is removed in a nucleotomy with a set of specialised tools. Implantation of the novel prosthesis was shown to be successful in various spinal disc models, in meeting identified design and functional requirements, including biomechanical loading, resistance to expulsion and radiopacity.

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.

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.

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.

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.

Dissertation

碩士
國立臺灣大學
醫學工程學研究所
96
Disc degeneration is strongly associated with back pain and herniation resulting in the cost of health care decreasing. Some evidence shows that disc degeneration originates in the nucleus pulposus (NP). However, current treatments such as physical therapy, pharmaceutical treatment, spinal fusion, artificial disc replacement and discectomy attempt to reduce pain rather than repair the degenerated disc. Cell–based therapies are aimed at repairing the degenerated disc, which is a potential treatment of disc degeneration. Therefore, the purpose of this study was to prepare the thermosensitive hydrogel as cell carrier of NP cells. In this study, the thermosensitive chitosan/gelatin/β- glycerol phosphate disodium salt hydrogels (C/G/GP hydrogels) was investigated. Nucleus pulposus cells which were harvested from the intervertebral discs of the adult New Zealand white rabbits were encapsulated in C/G/GP hydrogels. The gelation temperature, gelation time and gel strength were evaluated by rheometer. Compared with the formulations using only C/GP, the gel strength of C/G/GP was increased at 37°C. In the C/G/GP system, raising the concentration of gelatin resulted in a decrease in the gelation temperature and gelation time. The results of the in vitro cytotoxicity showed that the C/GP and the C/G/GP hydrogel are biocompatible. In the degradation test, raising the concentration of gelatin seems to increase the percentage of weight loss. The ratio of sulfated glycosaminoglycan (GAG) to DNA of NP cells cultured in hydrogels showed significantly higher than monolayer-cultured at the end of 3-week. Compared with monolayer-cultured, the mRNA expression of type II Collagen, Aggrecan, MMP-3, MMP-9, IGF-1, BMP-7 and TGF-β in hydrogel-cultured NP cells was significantly enhanced. The results showed that the C/G/GP solution remains liquid at room temperature but form the hydrogel at approximate body temperature under a neutral pH. The formed hydrogel is biocompatible and biodegradable. As three-dimensional carrier for NP cell culture, these results suggest the C/G/GP hydrogel is a suitable scaffold for the cell culture. These features make the C/G/GP hydrogel potential application as an injectable cell carrier for NP regeneration.

博士
國立臺灣大學
醫學工程學研究所
93
【Study Design】A bioactive material was developed with the composition of gelatin, chondoitin-6-sulfate, and hyaluronan. Human nucleus pulposus cells were 3- dimensionally cultured in this tri-copolymer scaffold in vitro, and then their cell proliferation and matrix productivity were measured by ELISA assays, immuno- histochemical staining, and real-time reverse-transcriptase polymerase chain reaction (RT-PCR). 【Objective】To evaluate the feasibility of gelatin/chondoitin-6-sulfate/hyaluronan tri-copolymer serving as a bioactive scaffold for regeneration of human nucleus pulposus. 【Summary of Background Data】Degeneration of the intervertebral disc starts from the nucleus pulposus and finally leads to various associated spinal disorders. Symptomatic intervertebral disc degeneration results in substantial pain and increased health cost. Current treatment options for degenerative disc disease may alleviate clinical symptoms but are not able to stop and sometimes even would accelerate the degeneration in the intervertebral disc. Tissue engineering approaches for treating degenerative intervertebral discs aim to promote tissue regeneration then retard or even reverse degenerative process. Combination of cells and bioactive scaffold is one of the promising tissue engineering methods for regeneration of the intervertebral disc including the nucleus pulposus. 【Materials and Methods】The gelatin/chondroitin-6-sulfate/hyaluronan tri-copolymer was fabricated into scaffold discs and was cross-linked by glutaraldehyde. Nucleus pulposus cells were isolated from human nucleus pulposus tissues and expanded in monolayer culture. Each scaffold disc was seeded with 1x106 cells and then cultured in vitro for 4 weeks. The cell-scaffold hybrids as well as the control scaffolds were then analyzed on their cell viability/proliferation, content of sulfated glycos- aminoglycans, mRNA expression of selected genes, histological and immuno- histochemical studies. 【Results】The cell-scaffold hybrids demonstrated active cell viability/proliferation and their net increases of sulfated glycosaminoglycans were progressively higher during a 4-week cultivation. In comparison to monolayer cells, scaffold-cultured cells showed significantly higher mRNA expression in type II collagen, aggrecan, Sox9, TGF-β1, and TIMP-1. Expressions of mRNA were significantly suppressed in type I collagen, type X collagen, IL-1 and Fas-associating death domain protein. Histological studies showed newly synthesized matrices containing glycos- aminoglycans deposits and type II collagen in the cell-scaffold hybrids while the scaffold substrate lost its originally entrapped glycosaminoglycans during the in vitro cultivation. 【Conclusions】The gelatin/chondroitin-6-sulfate/hyaluronan tri-copolymer scaffold has positive effects on cell proliferation and matrix production to human nucleus pulposus cells. The tri-copolymer scaffold is promising bioactive material that warrants further investigation into their application in regeneration of nucleus pulposus. 【Future Works】After a successful in vitro experiment on culturing human nucleus pulposus cells in the tri-copolymer scaffold, an animal study is a mandatory step before proceeding to clinical application. In addition, searching for healthy and viable nucleus pulposus cells is also important for promoting cell proliferation and matrix productivity. Combination of mesenchymal stem cells and current/future bioactive scaffolds is also worthwhile being investigated.

博士
國立臺灣大學
醫學工程學研究所
100
Disc degeneration is strongly associated with back pain and herniation that increase the costs of health care. The degeneration of intervertebral disc (IVD) could be divided into 5 stages. In the first and second stages, there are no significant symptoms but could be traced by magnetic resonance imaging or computed tomography-scan. Generally, no aggressive treatment would be processed in the clinics. Recent studies indicated that overproduction of reactive oxygen species (ROS) may accelerate the degenerative process of IVD and associate with apoptosis of nucleus pulposus (NP) cells and degradation of extracellular matrix. Ferulic acid (FA) is an excellent antioxidant and relatively stable in air. FA has been proven to have ability to prevent ROS-induced diseases. The object of the study was aimed to evaluate the possible therapeutic effect of FA on hydrogen peroxide (H2O2)-induced oxidative stress NP cells and the feasibility of use the thermosensitive chitosan/gelatin/glycerophosphate (C/G/GP) hydrogel as a sustained release system of FA for early treatment in IVD degeneration. In the study, NP cells were harvested from the IVD of New Zealand rabbits. The results showed that 500 μM of FA might be the threshold to treat NP cells without cytotoxicity. Post-treatment of FA on H2O2-induced oxidative stress NP cells significantly up regulated the expression of aggrecan, type II collagen and BMP-7 and down regulated the expression of MMP-3 in mRNA level. Post-treatment of FA on H2O2-induced oxidative stress NP cells could restore the production of sulfated glycosaminoglycans (GAGs) and inhibit the apoptosis caused by H2O2. The results showed that the release of FA from C/G/GP hydrogel could decrease the H2O2-induced oxidative stress. Post-treatment of FA-incorporated C/G/GP hydrogel on H2O2-induced oxidative stress NP cells showed up-regulation of aggrecan and type II collagen and down-regulation of MMP-3 in mRNA level. The results of sulfated GAGs to DNA ratio and alcian blue staining revealed that the GAGs production of H2O2-induced oxidative stress NP cells could reach to normal level. The results of caspase-3 activity and TUNEL staining indicated that FA-incorporated C/G/GP hydrogel decreased the apoptosis of H2O2-induced oxidative stress NP cells. The results showed that FA was successfully immobilized on C/G/GP hydrogel by N-(3-dimethylaminopropyl)-N''-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) crosslinking method. The gelation temperature of the FA-immobilized C/G/GP hydrogel was 31.80 degree celsius under neutral pH. Post-treatment of FA-immobilized C/G/GP hydrogel on H2O2-induced oxidative stress NP cells showed down-regulation of MMP-3 and up-regulation aggrecan and type II collagen in mRNA level. The sulfated GAGs production of H2O2-induced oxidative stress NP cells could be increased to the normal level in the post-treatment of FA-immobilized C/G/GP hydrogel group. The results of caspase-3 activity and TUNEL staining showed that the apoptosis of H2O2-induced oxidative stress NP cells could be inhibited by post-treatment of FA-immobilized C/G/GP hydrogel. From the results of the study, FA could be used as a therapeutic molecule for NP regeneration and FA-incorporated C/G/GP hydrogel might be potentially applied as a long-term release system. The immobilization of FA on C/G/GP hydrogel could significantly prolong the release period of FA. These results suggest that combination of FA and thermosensitive C/G/GP hydrogel can treat NP cells from the damage caused by oxidative stress and may apply in minimally invasive surgery for NP regeneration in the future.

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.