IVD Tissue Research Articles

EFFECT OF SPINAL FUSION ON BIOMECHANICS OF FACET JOINTS: SYNCHRONIZED SPATIO-TEMPORAL AND MECHANICAL ANALYSIS

ObjectivesUnderstanding lumbar facet joint involvement and biomechanical changes post spinal fusion is limited. This study aimed to establish an in vitro model assessing mechanical effects of fusion on human lumbar facet joints, employing synchronized motion, pressure, and stiffness analysis.Methods and ResultsSeven human lumbar spinal units (age 54 to 92, ethics 15/YH/0096) underwent fusion via a partial nucleotomy model mimicking a lateral cage approach with PMMA cement injection. Mechanical testing pre and post-fusion included measuring compressive displacement and load, local motion capture, and pressure mapping at the facet joints. pQCT imaging (82 microns isotropic) was carried out at each stage to assess the integrity of the vertebral endplates and quantify the amount of cement injected.Before fusion, relative facet joint displacement (6.5 ± 4.1 mm) at maximum load (1.1 kN) exceeded crosshead displacement (3.9 ± 1.5 mm), with loads transferred across both facet joints. After fusion, facet displacement (2.0 ± 1.2 mm) reduced compared to pre-fusion, as was the crosshead displacement (2.2 ± 0.6 mm). Post-fusion loads (71.4 ± 73.2 N) transferred were reduced compared to pre-fusion levels (194.5 ± 125.4 N). Analysis of CT images showed no endplate damage post-fusion, whilst the IVD tissue: cement volume ratio did not correlate with the post-fusion behaviour of the specimens.ConclusionAn in vitro model showed significant facet movement reduction with stand-alone interbody cage placement. This technique identifies changes in facet movement post-fusion, potentially contributing to subsequent spinal degeneration, highlighting its utility in biomechanical assessment.Conflicts of interestNoneSources of fundingThis work was funded by EPSRC, under grant EP/W015617/1.

Ex vivo intervertebral disc cultures: degeneration-induction methods and their implications for clinical translation

Because low back pain is frequently a result of intervertebral disc degeneration (IVDD), strategies to regenerate or repair the IVD are currently being investigated. Often, ex vivo disc cultures of non-human IVD organs or tissue explants are used that usually do not exhibit natural IVDD. Therefore, degenerative changes mimicking those reported in human IVDD need to be induced. To support researchers in selecting ex vivo disc cultures, a systematic search was performed for them and their potential use for studying human IVDD reviewed. Five degeneration induction categories (proinflammatory cytokines, injury/damage, degenerative loading, enzyme, and other) were identified in 129 studies across 7 species. Methods to induce degeneration are diverse and can induce mild to severe degenerative changes that progress over time, as described for human IVDD. The induced degenerative changes are model-specific and there is no "one-fits-all" IVDD induction method. Nevertheless, specific aspects of human IVDD can be well mimicked. Currently, spontaneously degenerated disc cultures from large animals capture human IVDD in most aspects. Combinatorial approaches of several induction methods using discs derived from large animals are promising to recapitulate pathological changes on several levels, such as cellular behaviour, extracellular matrix composition, and biomechanical function, and therefore better mimic human IVDD. Future disc culture setups might increase in complexity, and mimic human IVDD even better. As ex vivo disc cultures have the potential to reduce and even replace animal trials, especially during preclinical development, advancement of such models is highly relevant for more efficient and cost-effective clinical translation from bench-to-bedside.

Correlation between Adrenoceptor Expression and Clinical Parameters in Degenerated Lumbar Intervertebral Discs

Despite advanced knowledge of the cellular and biomechanical processes of intervertebral disc degeneration (IVDD), the trigger and underlying mechanisms remain unclear. Since the sympathetic nervous system (SNS) has been shown to exhibit catabolic effects in osteoarthritis pathogenesis, it is attractive to speculate that it also influences IVDD. Therefore, we explored the adrenoceptor (AR) expression profile in human IVDs and correlated it with clinical parameters of patients. IVD samples were collected from n = 43 patients undergoing lumbar spinal fusion surgery. AR gene expression was analyzed by semi-quantitative polymerase chain reaction. Clinical parameters as well as radiological Pfirrmann and Modic classification were collected and correlated with AR expression levels. In total human IVD homogenates α1A-, α1B-, α2A-, α2B-, α2C-, β1- and β2-AR genes were expressed. Expression of α1A- (r = 0.439), α2A- (r = 0.346) and β2-AR (r = 0.409) showed a positive and significant correlation with Pfirrmann grade. α1A-AR expression was significantly decreased in IVD tissue of patients with adjacent segment disease (p = 0.041). The results of this study indicate that a relationship between IVDD and AR expression exists. Thus, the SNS and its neurotransmitters might play a role in IVDD pathogenesis. The knowledge of differential AR expression in different etiologies could contribute to the development of new therapeutic approaches for IVDD.

The role of nerve fibers and their neurotransmitters in regulating intervertebral disc degeneration.

Intervertebral disc degeneration (IVDD) has been the major contributor to chronic lower back pain (LBP). Abnormal apoptosis, senescence, and pyroptosis of IVD cells, extracellular matrix (ECM) degradation, and infiltration of immune cells are the major molecular alternations during IVDD. Changes at tissue level frequently occur at advanced IVD tissue. Ectopic ingrowth of nerves within inner annulus fibrosus (AF) and nucleus pulposus (NP) tissue has been considered as the primary cause for LBP. Innervation at IVD tissue mainly included sensory and sympathetic nerves, and many markers for these two types of nerves have been detected since 1940. In fact, in osteoarthritis (OA), beyond pain transmission, the direct regulation of neuropeptides on functions of chondrocytes have attracted researchers' great attention recently. Many physical and pathological similarities between joint and IVD have shed us the light on the neurogenic mechanism involved in IVDD. Here, an overview of the advances in the nervous system within IVD tissue will be performed, with a discussion on in the role of nerve fibers and their neurotransmitters in regulating IVDD. We hope this review can attract more research interest to address neuromodulation and IVDD itself, which will enhance our understanding of the contribution of neuromodulation to the structural changes within IVD tissue and inflammatory responses and will help identify novel therapeutic targets and enable the effective treatment of IVDD disease.

Col9a2 gene deletion accelerates the degeneration of intervertebral discs.

As an essential component of the extracellular matrix (ECM) in cartilage, the α2 chain of type IX collagen (Col9a2), has been implicated in human intervertebral disc degeneration (IVDD). However, the precise role of the Col9a2 gene in the pathogenesis of IVDD has remained elusive. In the present study, the spines of Col9a2-deficient (Col9a2-/-) mice were systematically analyzed and compared with wild-type control mice using micro-CT (µCT), histomorphology, immunofluorescence, immunohistochemistry and reverse transcription-quantitative PCR (RT-qPCR). µCT analysis revealed that endplate (EP) osteochondral remodeling in the Col9a2-/- group was accompanied by a significant increase in EP porosity. Likewise, histopathological staining at 12 weeks revealed that the Col9a2-/- mice exhibited a marked early-stage IVDD phenotype, including EP sclerosis, calcification and annulus fibrosus rupture. The immunofluorescence results indicated that Col9a2 was extensively expressed in the IVDs, whereas it was barely detectable in Col9a2-/- mice. Immunohistochemical and RT-qPCR analyses demonstrated that the expression levels of Col2a1 and Aggrecan in the IVDs of Col9a2-/- mice were significantly decreased. In addition, the levels of Mmp13, ADAM metallopeptidase with thrombospondin type 1 motif 5, Col10a1 and Runx family transcription factor 2 were significantly elevated. These results suggested that deletion of the Col9a2 gene led to osteochondral remodeling of cartilage EP and suppressed ECM synthesis, accelerating matrix degradation and chondrocyte hypertrophy in the IVD tissue.

Aggressive strategies for regenerating intervertebral discs: stimulus-responsive composite hydrogels from single to multiscale delivery systems.

As our research on the physiopathology of intervertebral disc degeneration (IVD degeneration, IVDD) has advanced and tissue engineering has rapidly evolved, cell-, biomolecule- and nucleic acid-based hydrogel grafting strategies have been widely investigated for their ability to overcome the harsh microenvironment of IVDD. However, such single delivery systems suffer from excessive external dimensions, difficult performance control, the need for surgical implantation, and difficulty in eliminating degradation products. Stimulus-responsive composite hydrogels have good biocompatibility and controllable mechanical properties and can undergo solution-gel phase transition under certain conditions. Their combination with ready-to-use particles to form a multiscale delivery system may be a breakthrough for regenerative IVD strategies. In this paper, we focus on summarizing the progress of research on the stimulus response mechanisms of regenerative IVD-related biomaterials and their design as macro-, micro- and nanoparticles. Finally, we discuss multi-scale delivery systems as bioinks for bio-3D printing technology for customizing personalized artificial IVDs, which promises to take IVD regenerative strategies to new heights.

88. Cell stiffness decreases with severity of disc degeneration and inflammatory stimulation

88. Cell stiffness decreases with severity of disc degeneration and inflammatory stimulation

P14ARF inhibits regional inflammation and vascularization in intervertebral disc degeneration by upregulating TIMP3.

In this study, we identified P14 alternate reading frame (P14ARF) as a novel regulator of inflammation and vascularization in intervertebral disk degeneration (IVDD). We collected IVD tissues from IVDD patients and normal individuals for analysis of P14ARF expression. We also induced experimental IVDD by needle puncture injuries in the caudal intervertebral disks of Sprague-Dawley (SD) rats and achieved recombinant adenovirus-mediated P14ARF overexpression in experimental IVDD rats. Regulation relationships between P14ARF and tissue inhibitors of metalloproteinases-3 (TIMP3) were confirmed in P14ARF-overexpressed and TIMP3-depleted nucleus pulposus (NP) cells. Tube formation in vitro was evaluated in coculture systems of human umbilical vein endothelial cells (HUVECs) and rat degenerated NP cells (DNPCs). Inflammatory response was assessed from levels of TNF-α, IL-1β, and IL-6 and neovascularization from expression of endothelial growth factor (VEGF). The P14ARF and TIMP3 were downregulated in degenerated IVD tissue derived from patients and experimental IVDD rats. Overexpressed P14ARF suppressed inflammatory cytokine levels and vascularization. There was decreased in vitro tube formation in response to P14ARF overexpression and TIMP3 elevation. Finally, attenuated inflammatory responses and suppression of VEGF were achieved by P14ARF-mediated promotion of TIMP3 in rat DNPCs. Taken together, the present study reveals that P14ARF/TIMP3 modulation of inflammatory response and vascularization in the context of IVDD highlights a potential target for future therapeutic strategies.

Investigation of the Effects of Methylphenidate, an Amphetamine Derivative, on Intervertebral Disc Tissue Cell Cultures and Matrix Structures.

To investigate the effects of methylphenidate (MPH), on intervertebral disc tissue (IVD) cell cultures and extracellular matrix structures. Changes in the expression of some important marker genes involved in anabolic and catabolic mechanisms of IVD extracellular matrix formation were also evaluated. Primary cultures of nucleus pulposus cells (NPCs) and annulus fibrosus cells (AFCs) were isolated from tissues obtained from the operated patients. Cell viability and proliferation were tested, and the cell surface morphologies were evaluated by microscopy. The expressions of the chondroadherin (CHAD), cartilage oligomeric matrix protein (COMP), interleukin-1 beta (IL-1β) and matrix metalloproteinase (MMP) -7 and MMP-19 genes were evaluated using the quantitative real-time polymerase chain reaction (qRT-PCR). A value of p < 0.05 was considered statistically significant. The viability and proliferation of intervertebral disc tissue cells decreased in response to MPH treatment and the expression of the investigated genes also changed. The data obtained from in-vitro studies may not directly adaptable to clinical applications. However, the fact that the central nervous system stimulant MPH can suppress proliferation of cells derived from IVD tissue should be considered carefully by clinicians.

Wednesday, September 26, 2018 7:35 AM–9:00 AM ePosters: P6. Chondrogenic differentiation of adipose tissue for nucleus pulposus regeneration

BACKGROUND CONTEXT Despite tremendous in vitro achievements, the clinical breakthrough towards intervertebral disc tissue engineering is still missing. The reasons for this are multiple: current protocols are based on the isolation of nucleus pulposus cells from herniated IVD tissue. The subsequent in vitro expansion is time and cost consuming and no consensus on the optimal scaffold material has been reached. Adipose tissue contains stem cells capable of differentiating towards the chondrocytic lineage and an extracellular matrix consisting of collagen and glycosaminoglycans. PURPOSE Aim of the presented study is to test the possibility of differentiating total adipose tissue towards tissue of a fibrocartilginous phenotype suitable for nucleus pulposus regeneration. STUDY DESIGN/SETTING Laboratory study. PATIENT SAMPLE Fat tissue biopsies from 10 patients harvested from surgical waste material were used with informed consent. OUTCOME MEASURES Histologic evaluation, durometry and quantification of collagen and glycosaminoglycan content. METHODS One half of the tissue was placed in standard cell culture medium while the other half was placed in a commercially available chondrogenic differentiation medium. Tissue samples were incubated at 37°C, 5% CO2 and 90% humidity for 3-6 weeks followed by histologic evaluation, durometry and quantification of collagen and glycosaminoglycan content. RESULTS Chondrogenic transdifferentiated fat tissue demonstrated a smooth surface remodelling and a significant increase in tissue density. Durometric rigidity increased from Shore 0.25 (control) to 5 (p CONCLUSIONS Total adipose tissue can be successfully differentiated towards tissue of a NPP phenotype and shows a potential to create a fully autologous, scaffold free nucleus pulposus transplant.

IAPP modulates cellular autophagy, apoptosis, and extracellular matrix metabolism in human intervertebral disc cells

The pathogenic process of intervertebral disc degeneration (IDD) is characterized by imbalance in the extracellular matrix (ECM) metabolism. Nucleus pulposus (NP) cells have important roles in maintaining the proper structure and tissue homeostasis of disc ECM. These cells need adequate supply of glucose and oxygen. Islet amyloid polypeptide (IAPP) exerts its biological effects by regulating glucose metabolism. The purpose of this study was to investigate the expression of IAPP in degenerated IVD tissue, and IAPP modulation of ECM metabolism in human NP cells, especially the crosstalk mechanism between apoptosis and autophagy in these cells. We found that the expression of IAPP and Calcr-RAMP decreased considerably during IDD progression, along with the decrease in the expression of AG, BG, and Col2A1. Induction of IAPP in NP cells by transfection with pLV-IAPP enhanced the synthesis of aggrecan and Col2A1 and attenuated the expression of pro-inflammatory factors, tumor necrosis factor (TNF)-α, and interleukin (IL)-1. Upregulation of IAPP also affected the expression of the catabolic markers—matrix metalloproteinases (MMPs) 3, 9 and 13 and ADAMTS 4 and 5. Downregulation of IAPP by siRNA inhibited the expression of anabolic genes but increased the expression of catabolic genes and inflammatory factors. The expressions of autophagic and apoptotic markers in NP cells transfected with pLV-IAPP were upregulated, including BECLIN1, ATG5, ATG7, LC3 II/I and Bcl-2, while significantly increase in the expression of Bax and Caspase-3 in NP cells transfected with pLV-siIAPP. Mechanistically, PI3K/AKT-mTOR and p38/JNK MAPK signal pathways were involved. We propose that IAPP might play a pivotal role in the development of IDD, by regulating ECM metabolism and controlling the crosstalk between apoptosis and autophagy in NP, thus potentially offering a novel therapeutic approach to the treatment of IDD.

Inhibiting IL-1 signaling pathways to inhibit catabolic processes in disc degeneration.

Intervertebral disc degeneration is characterized by an imbalance between catabolic and anabolic signaling, with an increase in catabolic cytokines particularly IL-1β, a key regulator of IVD degeneration. This study aimed to investigate intracellular signaling pathways activated by IL-1β, and GDF-5 in the degenerate IVD to identify potential new therapeutic targets. Human NP cells were cultured in alginate beads to regain in vivo phenotype prior to stimulation with IL-1β or GDF-5 for 30 min, a proteasome profiler array was initially utilized to screen activation status of 46 signaling proteins. Immunoflourescence was used to investigate activation of the NFκB pathway. Cell-based ELISAs were then deployed to confirm results for ERK1/2, p38 MAPK, c-jun, and IκB signaling. IHC was utilized to investigate native activation status within human IVD tissue between grades of degeneration. Finally, cells were stimulated with IL-1β in the absence or presence of p38 MAPK, c-jun, JNK, and NFκB inhibitors to investigate effects on MMP3, MMP13, IL-1β, IL-6, and IL-8 mRNA expression. This study demonstrated three key signaling pathways which were differentially activated by IL-1β but not GDF-5; namely p38 MAPK, c-jun, and NFκB. While ERK 1/2 was activated by both GDF-5 and IL-1. Immunohistochemistry demonstrated p38 MAPK, c-jun, and NFκB were activated during human IVD degeneration and inhibition of these pathways reduced or abrogated the catabolic effects of IL-1β, with inhibition of NFκB signaling demonstrating most widespread inhibition of IL-1β catabolic effects. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:74-85, 2017.

Qualitative grading of disc degeneration by magnetic resonance in the lumbar and cervical spine: lack of correlation with histology in surgical cases

Background: Clinically, magnetic resonance (MR) imaging is the most effective non-invasive tool for assessing IVD degeneration. Histological examination of the IVD provides a more detailed assessment of the pathological changes at a tissue level. However, very few reports have studied the relationship between these techniques. Identifying a relationship may allow more detailed staging of IVD degeneration, of importance in targeting future regenerative therapies. Objectives: To investigate the relationship between MR and histological grading of IVD degeneration in the cervical and lumbar spine in patients undergoing discectomy. Methods: Lumbar (N = 99) and cervical (N = 106) IVD samples were obtained from adult patients undergoing discectomy surgery for symptomatic IVD herniation and graded to ascertain a histological grade of degeneration. The pre-operative MR images from these patients were graded for the degree of IVD (MR grade) and vertebral end-plate degeneration (Modic Changes, MC). The relationship between histological and MR grades of degeneration were studied. Results: In lumbar and cervical IVD the majority of samples (93%) exhibited moderate levels of degeneration (ie MR grades 3-4) on pre-operative MR scans. Histologically, most specimens displayed moderate to severe grades of degeneration in lumbar (99%) and cervical spine (93%). MR grade was weakly correlated with patient age in lumbar and cervical study groups. MR and histological grades of IVD degeneration did not correlate in lumbar or cervical study groups. MC were more common in the lumbar than cervical spine (e.g. 39 versus 20% grade 2 changes; p < 0.05), but failed to correlate with MR or histological grades for degeneration. Conclusions: In this surgical series, the resected IVD tissue displayed moderate to severe degeneration, but there is no correlation between MR and histological grades using a qualitative classification system. There remains a need for a quantitative, non-invasive, pre-clinical measure of IVD degeneration that correlates with histological changes seen in the IVD.

A Feasibility Study of Cell Injection Therapy in Human Intervertebral Discs Cultured under Dynamic Load

Introduction Painful IVD degeneration affects the quality of life for millions of people, and it presents a major problem for direct and indirect global health care costs. A preclinical ex vivo screening platform is needed to study the relationship between mechanobiology, disc matrix composition, and metabolism in the context of degenerative disease. We therefore developed an ex vivo organ culture model that facilitates culture of intact human discs in a controlled dynamically loaded environment. The bioreactor is used in combination with a previously reported harvesting method, which maintains the integrity of the intervertebral discs by preserving the noncalcified part of the cartilage end plate and the entire annulus. Here, we determine IVD tissue integrity and cell viability in the system, and investigate the suitability toward cell supplementation for tissue repair. Methods A total of nine lumbar IVDs from seven individual donors were obtained through organ donations via Transplant Québec. The spines were assessed by X-ray to evaluate degree of degeneration and six discs were isolated from four donors as previously described. Overall, 106 primary fluorescently labeled allogeneic NP cells from three separate donors were seeded per 1 mL of thermogelling hyaluronic acid hydrogel (HA-pNIPAM). Approximately, 250 µL of cell/gel mixtures were injected laterally into the NP region of isolated discs. IVDs with the newly transplanted cells were transferred to the bioreactors and were loaded with 0.1 to 0.6 MPa cyclic loads for two periods of 2 hours/day. The dynamic compressive load periods were interrupted by recovery periods of 6 hours and 14 hours, respectively, under 0.1 MPa static loads. The scheme was repeated for 3 or 7 or 14 consecutive days. After loading, portions of nucleus pulposus and annulus fibrosus were excised and analyzed via LIVE/DEAD assay, and smaller sections were visualized using a laser-scanning confocal microscope. The proportion of viable cells was quantified. Results Our culture system maintains &gt; 80% cell viability of the resident IVD cells and shows no signs of adverse effects in the extracellular matrix after more than 14 days of dynamic culture. Allogeneic NP cells were successfully transplanted in freshly isolated human discs. The transplanted cells were found dispersed throughout the nuclear and annular regions and displayed maintained viability after up to 14 days of dynamic culture. Conclusion This study shows that it is feasible to culture human IVDs under dynamic loading for more than 14 days while maintaining cell viability, tissue homeostasis, and integrity. It also presents a preclinical ex vivo screening platform where cell-based therapies, utilizing isolated disc cells or stem cells can be tested in combination with various injectable hydrogels. In addition, the effect of load magnitude and frequency, growth factors, O2 tension, glucose levels, and bioactive therapeutics can be evaluated. The bioreactor can also be used to evaluate loss or gain of mechanical function as a result of biological changes. Such knowledge is important for determining the value of cell/hydrogel injections and patient advisement on physical activities following a biological repair procedure.

Intervertebral Disc Tissue Engineering with Natural Extracellular Matrix-Derived Biphasic Composite Scaffolds

Tissue engineering has provided an alternative therapeutic possibility for degenerative disc diseases. However, we lack an ideal scaffold for IVD tissue engineering. The goal of this study is to fabricate a novel biomimetic biphasic scaffold for IVD tissue engineering and evaluate the feasibility of developing tissue-engineered IVD in vitro and in vivo. In present study we developed a novel integrated biphasic IVD scaffold using a simple freeze-drying and cross-linking technique of pig bone matrix gelatin (BMG) for the outer annulus fibrosus (AF) phase and pig acellular cartilage ECM (ACECM) for the inner nucleus pulposus (NP) phase. Histology and SEM results indicated no residual cells remaining in the scaffold that featured an interconnected porous microstructure (pore size of AF and NP phase 401.4±13.1 μm and 231.6±57.2 μm, respectively). PKH26-labeled AF and NP cells were seeded into the scaffold and cultured in vitro. SEM confirmed that seeded cells could anchor onto the scaffold. Live/dead staining showed that live cells (green fluorescence) were distributed in the scaffold, with no dead cells (red fluorescence) being found. The cell—scaffold constructs were implanted subcutaneously into nude mice and cultured for 6 weeks in vivo. IVD-like tissue formed in nude mice as confirmed by histology. Cells in hybrid constructs originated from PKH26-labeled cells, as confirmed by in vivo fluorescence imaging system. In conclusion, the study demonstrates the feasibility of developing a tissue-engineered IVD in vivo with a BMG- and ACECM-derived integrated AF-NP biphasic scaffold. As well, PKH26 fluorescent labeling with in vivo fluorescent imaging can be used to track cells and analyse cell—scaffold constructs in vivo.

Progranulin knockout accelerates intervertebral disc degeneration in aging mice.

Intervertebral disc (IVD) degeneration is a common degenerative disease, yet much is unknown about the mechanisms during its pathogenesis. Herein we investigated whether progranulin (PGRN), a chondroprotective growth factor, is associated with IVD degeneration. PGRN was detectable in both human and murine IVD. The levels of PGRN were upregulated in murine IVD tissue during aging process. Loss of PGRN resulted in an early onset of degenerative changes in the IVD tissue and altered expressions of the degeneration-associated molecules in the mouse IVD tissue. Moreover, PGRN knockout mice exhibited accelerated IVD matrix degeneration, abnormal bone formation and exaggerated bone resorption in vertebra with aging. The acceleration of IVD degeneration observed in PGRN null mice was probably due to the enhanced activation of NF-κB signaling and β-catenin signaling. Taken together, PGRN may play a critical role in homeostasis of IVD, and may serve as a potential molecular target for prevention and treatment of disc degenerative diseases.

Functional impact of integrin α5β1 on the homeostasis of intervertebral discs: a study of mechanotransduction pathways using a novel dynamic loading organ culture system

Background contextIntervertebral disc (IVD) degeneration, a major cause of low back pain, is considered to be induced by daily mechanical loading. Mechanical stress is widely known to affect cell survival and extracellular matrix metabolism in many cell types. Although the involvement of integrin α5β1 transmembrane mechanoreceptor in IVD degeneration has been reported, the precise function of integrin α5β1 remains obscure. PurposeTo reflect IVD tissue response to mechanical stress using a dynamic loading organ culture system and elucidate the functional impact of integrin α5β1 on the pathomechanism of IVD degeneration. Study designAn ex vivo study using a dynamic loading organ culture system. MethodsNinety-six rat IVD explants were examined. Intervertebral discs were subjected to 1.3 MPa, 1.0 Hz dynamic compressive load in the presence or absence of an Arg-Gly-Asp (RGD) peptide with affinity to the fibronectin binding-site of integrin α5β1. Cell viability and histomorphology were assessed. The localization of integrin α5β1 in the IVD was assessed by immunohistochemistry. Gene expression levels of IVD cells were evaluated using real-time reverse transcription-polymerase chain reaction. ResultsIn the nucleus pulposus (NP), cell density and viability were reduced by dynamic compressive load. Histologic degenerative alterations, mainly seen in the NP, were the morphologic changes of NP cells. In both NP and annulus fibrosus (AF), immunohistochemistry revealed localization of integrin α5β1 and that the messenger-RNA expression of integrin α5β1 was increased by dynamic load. Dynamic load induced a catabolic effect, the stimulation of matrix metalloproteinase-3 and -13 gene expressions by NP and AF cells. The RGD peptide partially blocked the histologic alterations and the catabolic effect. ConclusionsThe dynamic loading organ culture system simulated cellular responses to mechanical loading of the IVD. Our results suggest that IVD cells recognize the mechanical stress through RGD integrins, particularly the α5β1 subtype that is highly expressed in NP and AF cells. Further experiments using this system will provide information about pathomechanisms of IVD degeneration through the mechanotransduction pathways.

Intervertebral disc and stem cells cocultured in biomimetic extracellular matrix stimulated by cyclic compression in perfusion bioreactor

Background contextIntervertebral disc (IVD) degeneration often causes back pain. Current treatments for disc degeneration, including both surgical and nonsurgical approaches, tend to compromise the disc movement and cannot fully restore functions of the IVD. Instead, cell-based IVD tissue engineering seems promising as an ultimate therapy for IVD degeneration. PurposeTo tissue-engineer an IVD ex vivo as a biological substitute to replace degenerative IVD. Study designAn extracellular matrix (ECM) structure-mimetic scaffold, cocultured human IVD cells and human mesenchymal stem cells (hMSCs), and mechanical stimulation were used to biofabricate a tissue-engineered IVD. MethodsAn optimal ratio of human annulus fibrosus (hAF) cells to hMSCs for AF generation within aligned nanofibers, and that of human nucleus pulposus (hNP) cells to hMSCs for NP generation within hydrogels were first determined after comparing different coculture ratios of hAF or hNP cells to hMSCs. Nanofibrous strips seeded with cocultured hAF cells/hMSCs were constructed into multilayer concentric rings, enclosing an inner core of hydrogel seeded with hNP cells/hMSCs. A piece of nonwoven nanofibrous mat seeded with hMSC-derived osteoblasts was assembled on the top of the cellular nanofiber/hydrogel assembly, as an interface layer between the cartilagenous end plate and vertebral body. The final assembled construct was then maintained in an osteochondral cocktail medium and stimulated with compressive loading to further enhance the hAF and hNP cells differentiation and increase the IVD ECM production. ResultsAmong all cocultured groups, hAF cells and hMSCs in the ratio of 2:1 cultured in nanofibers showed the closest mRNA expression levels of AF-related markers to positive control hAF cells, whereas hNP cells and hMSCs in the ratio of 1:2 cultured in hydrogels showed the closest expression levels of NP-related markers to positive control hNP cells. The effects of compressive loading on chondrogenesis of hAF or hNP cell and hMSC coculture were dependent on the scaffold structure; the expression of cartilage-related markers in AF nanofibers was downregulated, whereas that in NP hydrogel was upregulated. Interestingly, we found that hMSC-derived osteogenic cells in the interface layer were turned into chondrogenic lineage cells, with decreased expression of osteogenic markers and increased expression of chondrogenic markers. ConclusionsWe demonstrate a unique approach using a biomimetic scaffold, IVD and stem cell coculture, and mechanical stimulation to tissue-engineer a biological IVD substitute. The results show that our approach provides both favorable physical and chemical cues through cell-matrix and cell-cell interactions and mechanobiological induction to enhance IVD generation ex vivo. Our findings may lead to viable tissue engineering applications of generating a functional biological IVD for the treatment of disc degeneration.

Upstream Upregulation of Glycosaminoglycans Production by Targeting Synthesizing Enzymes Using A Nonviral Gene Delivery Method for Intervertebral Disk Regeneration

IntroductionProteoglycans, described as ground substance of the intervertebral disk tissue, are proteins rich in glycosaminoglycans (GAGs) providing stiffness and elasticity to the IVD. During IVD degeneration, a decrease of GAG is observed. This decrease results in loss of water content which alters its mechanical properties1. Glucuronyltransferase I (GT-I) and Xylosyltransferase I (XT-I) are two key enzymes involved in the synthesis of GAGs. Both enzymes have been shown to be downregulated in degenerated cartilage2,3. While GT-I has been reported to have a crucial role in GAG synthesis in the IVD, the implication of XT-I has not yet been demonstrated. Nonviral-based gene therapy is an attractive therapeutic approach because of its relative safety compared to viral therapies. The hypothesis of this study is that nonviral mediated delivery of GT-I and XT-I plasmids will increase the GAG production in IVD cells. The specific objectives of this study were1 to investigate the expression of GT-I and XT-I in relation to aging within the IVD tissue and2 to upregulate GT-I and XT-I expression using a polymeric transfecting agent for gene delivery of p GT-I and p XT-I.Materials and Methods(1)Expression of GAGs, GT-I and XT-I in relation to aging IVD tissues were harvested from 6-month-old, 1-, 2-, and 8-year-old bovine tails. One half of the IVD tissue was fixed by 4% paraformaldehyde. After sucrose treatment and sectioning, safranin O/Fast green staining and immunostaining of XT-I and GT-I were carried out. The other half was digested by 0.5 mg/mL of proteinase K for biochemical (DMMB assay) and HPLC analyses.(2)Polymeric transfection-mediated gene delivery of pGT-I and pXT-I Nucleus pulposus and annulus fibrosus cells (passage 1) and adipose derived stem cells (ADSCs) (passage 3), extracted from 6-month-old bovine caudal disks and rabbit adipose tissue, respectively, were seeded at a density of 50,000 cells/cm2. After 24 hours, cells were treated with XT-I and/or GT-I TOPO TA plasmids DNA complexed with commercial partial degraded polyamidoamine dendrimer (SuperFect, Qiagen) for 30 minutes before addition to cells (1 µg of plasmid DNA final concentration). p Luc was used as the nontargeting plasmid and control for transfection (luciferase activity) (empty vector (EV)). Medium was then changed daily until the completion of the study. Cellular metabolic activity (AlamarBlue) and proliferation (Picogreen), GAG quantification (DMMB), gene expression (XT-I, GT-I and aggrecan), and enzyme activity analyses were conducted on day 2 and 4 posttransfection.Results(1)Expression of GAGs, XT-I, and GT-I with aging A decrease of the intensity of GAG staining associated with a disorganization of the tissue was observed with aging. GAG downregulation was confirmed by a significant decrease of sulfated GAGs (6092 ± 393 µg GAG/µg DNA for 6-month-old IVD to 1033 ± 530 µg GAG/µg DNA for 8-year-old IVD) within the tissue (Fig. 1). Associated with this phenomenon, a decrease of XT-I and GT-I expression was noted by immunohistochemistry. Indeed, the high expression of XT-I and GT-I observed within 6-month-old bovine IVD decreased with aging to become nondetectable within 8-year-old bovine IVD.(2)Nonviral mediated pXT-I and pGT-I delivery After 2 and 4 days posttreatment, using polymer and pDNA complexes (polyplexes), over 80% cell viability was seen regardless of the plasmid and cell types. ADSCs, NP, and AF cells showed luciferase activity 2 and 4 days posttransfection. The expression of both plasmids GT-I and XT-I was observed in the three cell types.DownloadOpen in new tabDownload in PowerPointFigure 1 I-sGAG content within NP and AF tissues with aging. p < 0.05, Anova one-way test. II-GAG distribution in 6-month-old, 1-, 2-, and 8-year-old bovine IVD tissues (Safranin O (GAG, orange), Fast Green (collagen fibers, green) staining) (A-D) NP tissue (E-H) AF tissue.ConclusionA downregulation of GAGs was observed within bovine IVDs with aging. This decrease is associated with a downregulation of XT-I and GT-I expression known to be implicated in the GAG production. The use of a nonviral system shows promising results for gene transfer by their ability to transfect IVD cells.Acknowledgments Science Foundation Ireland, Research Frontiers Programme (07/RFP/ENMF482), Science Foundation Ireland, 07/SRC/B1163, AO Foundation (S-09-7P), 07/RFP/ENMF482 STTF 09.I confirm having declared any potential conflict of interest for all authors listed on this abstractNoDisclosure of InterestNone declaredHalloran D, et al. Tissue Engineering 2007;13:1927–1954Venkatesan N, et al. Proc Natl Acad Sci USA 2004;101(52):18087–18092Venkatesan N, et al. FASEB J 2009;23(3):813–822

Upstream Upregulation of Glycosaminoglycans Production by Targeting Synthesizing Enzymes Using a Nonviral Gene Delivery Method for Intervertebral Disk Regeneration

Introduction Proteoglycans, described as ground substance of the intervertebral disk tissue, are proteins rich in glycosaminoglycans (GAGs) providing stiffness and elasticity to the IVD. During IVD degeneration, a decrease of GAG is observed. This decrease results in loss of water content which alters its mechanical properties1. Glucuronyltransferase I (GT-I) and Xylosyltransferase I (XT-I) are two key enzymes involved in the synthesis of GAGs. Both enzymes have been shown to be downregulated in degenerated cartilage2,3. While GT-I has been reported to have a crucial role in GAG synthesis in the IVD, the implication of XT-I has not yet been demonstrated. Nonviral-based gene therapy is an attractive therapeutic approach because of its relative safety compared to viral therapies. The hypothesis of this study is that nonviral mediated delivery of GT-I and XT-I plasmids will increase the GAG production in IVD cells. The specific objectives of this study were1 to investigate the expression of GT-I and XT-I in relation to aging within the IVD tissue and2 to upregulate GT-I and XT-I expression using a polymeric transfecting agent for gene delivery of p GT-I and p XT-I. Materials and Methods (1)Expression of GAGs, GT-I and XT-I in relation to aging IVD tissues were harvested from 6-month-old, 1-, 2-, and 8-year-old bovine tails. One half of the IVD tissue was fixed by 4% paraformaldehyde. After sucrose treatment and sectioning, safranin O/Fast green staining and immunostaining of XT-I and GT-I were carried out. The other half was digested by 0.5 mg/mL of proteinase K for biochemical (DMMB assay) and HPLC analyses. (2)Polymeric transfection-mediated gene delivery of pGT-I and pXT-I Nucleus pulposus and annulus fibrosus cells (passage 1) and adipose derived stem cells (ADSCs) (passage 3), extracted from 6-month-old bovine caudal disks and rabbit adipose tissue, respectively, were seeded at a density of 50,000 cells/cm2. After 24 hours, cells were treated with XT-I and/or GT-I TOPO TA plasmids DNA complexed with commercial partial degraded polyamidoamine dendrimer (SuperFect, Qiagen) for 30 minutes before addition to cells (1 µg of plasmid DNA final concentration). p Luc was used as the nontargeting plasmid and control for transfection (luciferase activity) (empty vector (EV)). Medium was then changed daily until the completion of the study. Cellular metabolic activity (AlamarBlue) and proliferation (Picogreen), GAG quantification (DMMB), gene expression (XT-I, GT-I and aggrecan), and enzyme activity analyses were conducted on day 2 and 4 posttransfection. Results (1)Expression of GAGs, XT-I, and GT-I with aging A decrease of the intensity of GAG staining associated with a disorganization of the tissue was observed with aging. GAG downregulation was confirmed by a significant decrease of sulfated GAGs (6092 ± 393 µg GAG/µg DNA for 6-month-old IVD to 1033 ± 530 µg GAG/µg DNA for 8-year-old IVD) within the tissue ( Fig. 1 ). Associated with this phenomenon, a decrease of XT-I and GT-I expression was noted by immunohistochemistry. Indeed, the high expression of XT-I and GT-I observed within 6-month-old bovine IVD decreased with aging to become nondetectable within 8-year-old bovine IVD. (2)Nonviral mediated pXT-I and pGT-I delivery After 2 and 4 days posttreatment, using polymer and pDNA complexes (polyplexes), over 80% cell viability was seen regardless of the plasmid and cell types. ADSCs, NP, and AF cells showed luciferase activity 2 and 4 days posttransfection. The expression of both plasmids GT-I and XT-I was observed in the three cell types. [Figure: see text] Conclusion A downregulation of GAGs was observed within bovine IVDs with aging. This decrease is associated with a downregulation of XT-I and GT-I expression known to be implicated in the GAG production. The use of a nonviral system shows promising results for gene transfer by their ability to transfect IVD cells. Acknowledgments Science Foundation Ireland, Research Frontiers Programme (07/RFP/ENMF482), Science Foundation Ireland, 07/SRC/B1163, AO Foundation (S-09-7P), 07/RFP/ENMF482 STTF 09. I confirm having declared any potential conflict of interest for all authors listed on this abstract No Disclosure of Interest None declared Halloran D, et al. Tissue Engineering 2007;13:1927–1954 Venkatesan N, et al. Proc Natl Acad Sci USA 2004;101(52):18087–18092 Venkatesan N, et al. FASEB J 2009;23(3):813–822