Cell sources proposed for nucleus pulposus regeneration.

Lower back pain (LBP) occurs in 80% of adults in their lifetime; resulting in LBP being one of the biggest causes of disability worldwide. Chronic LBP has been linked to the degeneration of the intervertebral disc (IVD). The current treatments for chronic back pain only provide alleviation of symptoms through pain relief, tissue removal, or spinal fusion; none of which target regenerating the degenerate IVD. As nucleus pulposus (NP) degeneration is thought to represent a key initiation site of IVD degeneration, cell therapy that specifically targets the restoration of the NP has been reviewed here. A literature search to quantitatively assess all cell types used in NP regeneration was undertaken. With key cell sources: NP cells; annulus fibrosus cells; notochordal cells; chondrocytes; bone marrow mesenchymal stromal cells; adipose-derived stromal cells; and induced pluripotent stem cells extensively analyzed for their regenerative potential of the NP. This review highlights: accessibility; expansion capability in vitro; cell survival in an IVD environment; regenerative potential; and safety for these key potential cell sources. In conclusion, while several potential cell sources have been proposed, iPSC may provide the most promising regenerative potential.

The use of auxetic materials in tissue engineering.

A number of biological tissues have been reported as behaving in an auxetic manner, defined by a negative Poisson's ratio. This describes the deformation of tissue which expands in the axial and the transverse directions simultaneously while under uniaxial tension; and contracts axially and transversely upon uniaxial compression. The discovery of auxetic behaviour within biological tissues has implications for the recreation of the auxetic loading environment within tissue engineering. Tissue engineers strive to recreate the natural properties of biological tissue and in order to recreate the unique loading environment of cells from auxetic tissue, an auxetic scaffold is required. A number of studies have used a variety of auxetic scaffolds within tissue engineering. Investigation into the effect of auxetic micro-environments created by auxetic scaffolds on cellular behaviour has demonstrated an increased cellular proliferation and enhanced differentiation. Here, we discuss studies which have identified auxetic behaviour within biological tissues, and where cells have been cultured within auxetic scaffolds, bringing together current knowledge of the potential use of auxetic materials in tissue engineering applications and biomedical devices.

In vivo safety and efficacy testing of a thermally triggered injectable hydrogel scaffold for bone regeneration and augmentation in a rat model.

Bone loss resulting from degenerative diseases and trauma is a significant clinical burden which is likely to grow exponentially with the aging population. In a number of conditions where pre-formed materials are clinically inappropriate an injectable bone forming hydrogel could be beneficial. The development of an injectable hydrogel to stimulate bone repair and regeneration would have broad clinical impact and economic benefit in a variety of orthopedic clinical applications. We have previously reported the development of a Laponite® crosslinked pNIPAM-co-DMAc (L-pNIPAM-co-DMAc) hydrogel delivery system, loaded with hydroxyapatite nanoparticles (HAPna), which was capable of inducing osteogenic differentiation of mesenchymal stem cells (MSCs) without the need for additional growth factors in vitro. However to enable progression towards clinical acceptability, biocompatibility and efficacy of the L-pNIPAM-co-DMAc hydrogel to induce bone repair in vivo must be determined. Biocompatibility was evaluated by subcutaneous implantation for 6 weeks in rats, and efficacy to augment bone repair was evaluated within a rat femur defect model for 4 weeks. No inflammatory reactions, organ toxicity or systemic toxicity were observed. In young male rats where hydrogel was injected, defect healing was less effective than sham operated controls when rat MSCs were incorporated. Enhanced bone healing was observed however, in aged exbreeder female rats where acellular hydrogel was injected, with increased deposition of collagen type I and Runx2. Integration of the hydrogel with surrounding bone was observed without the need for delivered MSCs; native cell infiltration was also seen and bone formation was observed within all hydrogel systems investigated. This hydrogel can be delivered directly into the target site, is biocompatible, promotes increased bone formation and facilitates migration of cells to promote integration with surrounding bone, for safe and efficacious bone repair.

Tissue Engineering Laboratory Models of the Small Intestine.

In recent years, three-dimensional (3D) cell culture models of the small intestine have gained much attention. These models support cell proliferation, migration, and differentiation and encourage tissue organization which is not possible in two-dimensional (2D) culture systems. Furthermore, the use of a wide variety of cell culture scaffolds and support substrates has revealed considerable differences in cell behavior and tissue organization. These systems have been used in combination with intestinal stem cells, organoid units, or human colonic adenocarcinoma cell lines such as Caco-2 and HT29-MTX to generate a number of in vitro and in vivo models of the intestine. In this study, we review the current 2D and 3D tissue engineering models of the intestine to determine the most effective sources of intestinal cells and current research on support scaffolds capable of inducing the morphological architecture and function of the intestinal mucosa.

Interleukin-1 receptor antagonist deficient mice provide insights into pathogenesis of human intervertebral disc degeneration.

OBJECTIVES: Interleukin 1 (IL-1) is potentially important in the pathogenesis of intervertebral disc (IVD) degeneration; increasing production of matrix degradation enzymes and inhibiting matrix synthesis. Although IL-1 polymorphisms have been linked to increased risk of IVD degeneration, it is still unclear whether IL-1 drives IVD degeneration in vivo or is a secondary feature of degeneration. Here, we investigated whether IVD degeneration could be induced spontaneously by the removal of the natural inhibitor of IL-1 (IL-1 receptor antagonist) in mice that lack a functional IL-1rn gene. METHODS: Histological staining and immunohistochemistry was performed on BALB/c IL-1rn(+/+) and IL-1rn(-/-) mice to examine degeneration and to localise and detect IL-1, matrix metalloproteinases (MMP)3, MMP7, a disintigrin and MMP with thrombospondin motifs (ADAMTS)4 protein production. In addition, IVD cells were isolated using collagenase and proliferation potential determined. RESULTS: IL-1rn(-/-) knockout mice displayed typical features of human disc degeneration: loss of proteoglycan and normal collagen structure and increased expression of matrix degrading enzymes: MMP3; MMP7 and ADAMTS4. Histological grade of degeneration increased in IL-1rn(-/-) mice which was more evident within older mice. In addition IVD cells isolated from IL-1rn(-/-) mice displayed reduced proliferation potential. CONCLUSIONS: Here, we show that IL-1rn(-/-) mice develop spinal abnormalities that resemble characteristic features associated with human disc degeneration. The current evidence is consistent with a role for IL-1 in the pathogenesis of IVD degeneration. The imbalance between IL-1 and IL-1Ra which is observed during human IVD degeneration could therefore be a causative factor in the degeneration of the IVD, and as such, is an appropriate pharmaceutical target for inhibiting degeneration.

Altered integrin mechanotransduction in human nucleus pulposus cells derived from degenerated discs.

OBJECTIVE: Several studies have demonstrated biologic responses of intervertebral disc (IVD) cells to loading, although the mechanotransduction pathways have not been elucidated. In articular chondrocytes, which have a phenotype similar to that of IVD cells, a number of mechanoreceptors have been identified, with alpha5beta1 integrin acting as a predominant mechanoreceptor. The purpose of this study was to investigate the role of integrin signaling in IVD cells during mechanical stimulation and to determine whether RGD integrins are involved. METHODS: Human nucleus pulposus (NP) cells derived from nondegenerated and degenerated discs were subjected to dynamic compressive loading in the presence of an RGD inhibitory peptide. Expression of the alpha5beta1 heterodimer in IVD tissue was examined by immunohistochemistry and possible alternative mechanoreceptors by real-time quantitative polymerase chain reaction. RESULTS: Aggrecan gene expression was decreased following loading of NP cells from nondegenerated and degenerated discs. This response was inhibited by treatment with an RGD peptide in cells from nondegenerated, but not degenerated, IVDs. Immunohistochemistry demonstrated that expression of the alpha5beta1 heterodimer was unaltered in degenerated IVD tissue as compared with normal IVD tissue. CONCLUSION: Our results indicate that the mechanotransduction pathways are altered in cells from degenerated IVDs. Mechanosensing in NP cells from nondegenerated discs occurs via RGD integrins, possibly via the alpha5beta1 integrin, while cells from degenerated discs show a different signaling pathway that does not appear to involve RGD integrins.

Human cells derived from degenerate intervertebral discs respond differently to those derived from non-degenerate intervertebral discs following application of dynamic hydrostatic pressure.

The intervertebral disc (IVD) is one of the body's most important load-bearing structures with the major mechanical force experienced in the nucleus pulposus (NP) being hydrostatic pressure (HP). Physiological levels of HP have an anabolic effect on IVD matrix metabolism in cells derived from non-degenerate animal and herniated IVD while excessive HP has a catabolic effect. However, no studies have investigated the response of non-degenerate and degenerate human disc cells derived from non-herniated discs to HP. Here we investigate the effect of physiological HP on such cells using a novel loading rig. Human IVD cells (both NP and AF) cultured in alginate were subjected to dynamic HP (0.8-1.7 MPa 0.5 Hz) for 2 h. Cell viability was assessed, RNA extracted and qRT-PCR for 18 s, c-fos, Sox-9, collagen type II, aggrecan and MMP-3 performed. Cell viability was unaffected by the loading regime. In non-degenerate NP cells, HP increased c-fos, aggrecan, Sox-9 and collagen type II (significantly so in the case of c-fos and aggrecan), but not MMP-3 gene expression. In contrast, application of HP to AF or degenerate NP cells had no effect on target gene expression. Our data shows that cells obtained from the healthy NP respond to dynamic HP by up-regulating genes indicative of healthy matrix homeostasis. However, responses differed in degenerate NP cells suggesting that an altered mechanotransduction pathway may be operational.

Caveolin-1 expression and stress-induced premature senescence in human intervertebral disc degeneration.

INTRODUCTION: Chronic and debilitating low back pain is a common condition and a huge economic burden. Many cases are attributed to age-related degeneration of the intervertebral disc (IVD); however, age-related degeneration appears to occur at an accelerated rate in some individuals. We have previously demonstrated biomarkers of cellular senescence within the human IVD and suggested a role for senescence in IVD degeneration. Senescence occurs with ageing but can also occur prematurely in response to stress. We hypothesised that stress-induced premature senescence (SIPS) occurs within the IVD and here we have investigated the expression and production of caveolin-1, a protein that has been shown previously to be upregulated in SIPS. METHODS: Caveolin-1 gene expression in human nucleus pulposus (NP) cells was assessed by conventional and quantitative real-time polymerase chain reaction (PCR), and caveolin-1 protein expression was examined within human IVDs using immunohistochemistry. The correlation between caveolin-1 and p16INK4a (biomarker of cellular senescence) gene expression was investigated using quantitative real-time PCR. RESULTS: Caveolin-1 gene expression and protein expression were demonstrated within the human IVD for the first time. NP cells from degenerate discs exhibited elevated levels of caveolin-1 which did not relate to increasing chronological age. A negative correlation was observed between gene expression for caveolin-1 and donor age, and no correlation was found between caveolin-1 protein expression and age. A positive correlation was identified between gene expression of caveolin-1 and p16INK4a. CONCLUSION: Our findings are consistent with a role for caveolin-1 in degenerative rather than age-induced changes in the NP. Its expression in IVD tissue and its association with the senescent phenotype suggest that caveolin-1 and SIPS may play a prominent role in the pathogenesis of IVD degeneration.

Catabolic cytokine expression in degenerate and herniated human intervertebral discs: IL-1beta and TNFalpha expression profile.

Low back pain is a common and debilitating disorder. Current evidence implicates intervertebral disc (IVD) degeneration and herniation as major causes, although the pathogenesis is poorly understood. While several cytokines have been implicated in the process of IVD degeneration and herniation, investigations have predominately focused on Interleukin 1 (IL-1) and tumor necrosis factor alpha (TNFalpha). However, to date no studies have investigated the expression of these cytokines simultaneously in IVD degeneration or herniation, or determined which may be the predominant cytokine associated with these disease states. Using quantitative real time PCR and immunohistochemistry we investigated gene and protein expression for IL-1beta, TNFalpha and their receptors in non-degenerate, degenerate and herniated human IVDs. IL-1beta gene expression was observed in a greater proportion of IVDs than TNFalpha (79% versus 59%). Degenerate and herniated IVDs displayed higher levels of both cytokines than non-degenerate IVDs, although in degenerate IVDs higher levels of IL-1beta gene expression (1,300 copies/100 ng cDNA) were observed compared to those of TNFalpha (250 copies of TNFalpha/100 ng cDNA). Degenerate IVDs showed ten-fold higher IL-1 receptor gene expression compared to non-degenerate IVDs. In addition, 80% of degenerate IVD cells displayed IL-1 receptor immunopositivity compared to only 30% of cells in non-degenerate IVDs. However, no increase in TNF receptor I gene or protein expression was observed in degenerate or herniated IVDs compared to non-degenerate IVDs. We have demonstrated that although both cytokines are produced by human IVD cells, IL-1beta is expressed at higher levels and in more IVDs, particularly in more degenerate IVDs (grades 4 to 12). Importantly, this study has highlighted an increase in gene and protein production for the IL-1 receptor type I but not the TNF receptor type I in degenerate IVDs. The data thus suggest that although both cytokines may be involved in the pathogenesis of IVD degeneration, IL-1 may have a more significant role than TNFalpha, and thus may be a better target for therapeutic intervention.

Accelerated cellular senescence in degenerate intervertebral discs: a possible role in the pathogenesis of intervertebral disc degeneration.

Current evidence implicates intervertebral disc degeneration as a major cause of low back pain, although its pathogenesis is poorly understood. Numerous characteristic features of disc degeneration mimic those seen during ageing but appear to occur at an accelerated rate. We hypothesised that this is due to accelerated cellular senescence, which causes fundamental changes in the ability of disc cells to maintain the intervertebral disc (IVD) matrix, thus leading to IVD degeneration. Cells isolated from non-degenerate and degenerate human tissue were assessed for mean telomere length, senescence-associated beta-galactosidase (SA-beta-gal), and replicative potential. Expression of P16INK4A (increased in cellular senescence) was also investigated in IVD tissue by means of immunohistochemistry. RNA from tissue and cultured cells was used for real-time polymerase chain reaction analysis for matrix metalloproteinase-13, ADAMTS 5 (a disintegrin and metalloprotease with thrombospondin motifs 5), and P16INK4A. Mean telomere length decreased with age in cells from non-degenerate tissue and also decreased with progressive stages of degeneration. In non-degenerate discs, there was an age-related increase in cellular expression of P16INK4A. Cells from degenerate discs (even from young patients) exhibited increased expression of P16INK4A, increased SA-beta-gal staining, and a decrease in replicative potential. Importantly, there was a positive correlation between P16INK4A and matrix-degrading enzyme gene expression. Our findings indicate that disc cell senescence occurs in vivo and is accelerated in IVD degeneration. Furthermore, the senescent phenotype is associated with increased catabolism, implicating cellular senescence in the pathogenesis of IVD degeneration.

The role of interleukin-1 in the pathogenesis of human intervertebral disc degeneration.

In this study, we investigated the hypotheses that in human intervertebral disc (IVD) degeneration there is local production of the cytokine IL-1, and that this locally produced cytokine can induce the cellular and matrix changes of IVD degeneration. Immunohistochemistry was used to localize five members of the IL-1 family (IL-1alpha, IL-1beta, IL-1Ra (IL-1 receptor antagonist), IL-1RI (IL-1 receptor, type I), and ICE (IL-1beta-converting enzyme)) in non-degenerate and degenerate human IVDs. In addition, cells derived from non-degenerate and degenerate human IVDs were challenged with IL-1 agonists and the response was investigated using real-time PCR for a number of matrix-degrading enzymes, matrix proteins, and members of the IL-1 family. This study has shown that native disc cells from non-degenerate and degenerate discs produced the IL-1 agonists, antagonist, the active receptor, and IL-1beta-converting enzyme. In addition, immunopositivity for these proteins, with the exception of IL-1Ra, increased with severity of degeneration. We have also shown that IL-1 treatment of human IVD cells resulted in increased gene expression for the matrix-degrading enzymes (MMP 3 (matrix metalloproteinase 3), MMP 13 (matrix metalloproteinase 13), and ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin motifs)) and a decrease in the gene expression for matrix genes (aggrecan, collagen II, collagen I, and SOX6). In conclusion we have shown that IL-1 is produced in the degenerate IVD. It is synthesized by native disc cells, and treatment of human disc cells with IL-1 induces an imbalance between catabolic and anabolic events, responses that represent the changes seen during disc degeneration. Therefore, inhibiting IL-1 could be an important therapeutic target for preventing and reversing disc degeneration.

Localization of degradative enzymes and their inhibitors in the degenerate human intervertebral disc.

The histological and biochemical changes that occur in the extracellular matrix of the intervertebral disc (IVD) during ageing and degeneration have been investigated extensively. However, the mechanisms behind these changes are not fully understood. A number of studies have suggested the involvement of matrix metalloproteinases (MMPs) and ADAMTS in IVD degeneration, but few have localized the site of production of these enzymes to the cells of the degenerate disc. This study uses immunohistochemical techniques to localize and quantify the production of degrading enzymes (MMPs 1, 3, and 13, and ADAMTS 4) and their inhibitors (TIMPS 1, 2, and 3) within non-degenerate and degenerate discs of varying severity of degeneration. In all discs investigated, the cells that produced the enzymes and their inhibitors were the chondrocyte-like cells of the nucleus pulposus and inner annulus fibrosus (AF), with little immunopositivity in the outer AF. Non-degenerate discs showed low numbers of cells expressing the degradative enzymes MMP 1 and ADAMTS 4, suggesting a role for these enzymes in normal homeostasis. No MMP 3 or MMP 13 immunopositivity was observed in non-degenerate discs. In degenerate discs, the number of cells immunopositive for MMPs 1, 3, 13 and ADAMTS 4 increased with the severity of degeneration. This increase in degrading enzymes was also accompanied by increases in the number of cells immunopositive for TIMPs 1 and 2 but not TIMP 3. This study highlights that although the expression of a number of MMPs increases with degeneration, this is accompanied by an increase in their inhibitors. However, the increase in the number of cells immunoreactive for ADAMTS 4 with increasing degeneration was not paralleled by a rise in its inhibitor TIMP 3. This finding indicates that the aggrecanases, rather then the MMPs, are a possible therapeutic target for the inhibition of disc degeneration.

Studies of human intervertebral disc cell function in a constrained in vitro tissue culture system.

STUDY DESIGN: This is a laboratory-based study examining a novel in vitro culture system for intervertebral disc tissue. OBJECTIVES: Address the hypothesis that "the novel culture system will preserve intervertebral disc tissue matrix and cell function and prevent cellular apoptosis for periods up to 21 days." SUMMARY OF BACKGROUND DATA: Studies of cell function in human intervertebral disc tissue are scarce. In vivo study of human intervertebral disc cells remains impracticable; in situ molecular biology in histologic sections lacks a dynamic dimension; and as for in vitro studies, cell culture often lacks physiologic relevance and explant cultures are subject to loss of tissue integrity and altered cell behavior. There is a biologic and therapeutic need for a satisfactory explant culture system for studying human intervertebral disc tissue in a controlled environment. METHODS: Samples of human intervertebral disc tissue, obtained at surgery, were examined for a number of tissue and cell parameters immediately after excision (controls) and following culture of tissue samples either in a plastic ring or unconstrained in tissue culture medium for up to 3 weeks. Data were compared between cultured tissue and controls. RESULTS: By comparison with control tissue, unconstrained explants swelled, tissue structure was disturbed, and there were profound changes in cell function. By contrast, tissue cultured in plastic rings maintained tissue structure, and after 3 weeks, the cellular parameters were the same as in controls. CONCLUSIONS: This is the first reported system to preserve cell function of human discal explants for long periods in tissue culture. It will be a useful tool for a wide range of investigations of intervertebral disc biology that have not hitherto been possible.