Sesamin inhibits lipopolysaccharide-induced inflammation and extracellular matrix catabolism in rat intervertebral disc

Intervertebral disc (IVD) degeneration contributes to most spinal degenerative diseases, while treatment inhibiting IVD degeneration is still in the experimental stage. Sesamin, a bioactive component extracted from sesame, has been reported to exert chondroprotective and anti-inflammatory effects. Here, we analyzed the anti-inflammatory and anti-catabolic effects of sesamin on rat IVD in vitro and ex vivo. Results show that sesamin significantly inhibits the lipopolysaccharide (LPS)-induced expression of catabolic enzymes (MMP-1, MMP-3, MMP-13, ADAMTS-4, ADAMTS-5) and inflammation factors (IL-1β, TNF-α, iNOS, NO, COX-2, PGE2) in a dose-dependent manner in vitro. It is also proven that migration of macrophages induced by LPS can be inhibited by treatment with sesamin. Organ culture experiments demonstrate that sesamin protects the IVD from LPS-induced depletion of the extracellular matrix ex vivo. Moreover, sesamin suppresses LPS-induced activation of the mitogen-activated protein kinase (MAPK) pathway through inhibiting phosphorylation of JNK, the common downstream signaling pathway of LPS and IL-1β, which may be the potential mechanism of the effects of sesamin. In light of our results, sesamin protects the IVD from inflammation and extracellular matrix catabolism, presenting positive prospects in the treatment of IVD degenerative diseases.     

An injectable hydrogel incorporating mesenchymal precursor cells and pentosan polysulphate for intervertebral disc regeneration

Intervertebral disc (IVD) degeneration is one of the leading causes of lower back pain and a major health problem worldwide. Current surgical treatments include excision or immobilisation, with neither approach resulting in the repair of the degenerative disc. As such, a tissue engineering-based approach in which stem cells, coupled with an advanced delivery system, could overcome this deficiency and lead to a therapy that encourages functional fibrocartilage generation in the IVD. In this study, we have developed an injectable hydrogel system based on enzymatically-crosslinked polyethylene glycol and hyaluronic acid. We examined the effects of adding pentosan polysulphate (PPS), a synthetic glycosaminoglycan-like factor that has previously been shown (in vitro and in vivo) to this gel system in order to induce chondrogenesis in mesnchymal precursor cells (MPCs) when added as a soluble factor, even in the absence of additional growth factors such as TGF-β. We show that both the gelation rate and mechanical strength of the resulting hydrogels can be tuned in order to optimise the conditions required to produce gels with the desired combination of properties for an IVD scaffold. Human immunoselected STRO-1+ MPCs were then incorporated into the hydrogels. They were shown to retain good viability after both the initial formation of the gel and for longer-term culture periods in vitro. Furthermore, MPC/hydrogel composites formed cartilage-like tissue which was significantly enhanced by the incorporation of PPS into the hydrogels, particularly with respect to the deposition of type-II-collagen. Finally, using a wild-type rat subcutaneous implantation model, we examined the extent of any immune reaction and confirmed that this matrix is well tolerated by the host. Together these data provide evidence that such a system has significant potential as both a delivery vehicle for MPCs and as a matrix for fibrocartilage tissue engineering applications.     

Intervertebral disc regeneration using platelet-rich plasma and biodegradable gelatin hydrogel microspheres

This study evaluated the regenerative effects of platelet-rich plasma (PRP) for the degenerated intervertebral disc (IVD) in vivo. After induction of IVD degeneration in rabbits, we prepared PRP by centrifuging blood obtained from these rabbits. These PRP were injected into the nucleus pulposus (NP) of the degenerated IVDs after impregnation into gelatin hydrogel microspheres that can immobilize PRP growth factors physiochemically and release them in a sustained manner with the degradation of the microspheres. As controls, microspheres impregnated with phosphate-buffered saline (PBS) and PRP without microspheres were similarly injected. Histologically, notable progress in IVD degeneration with time courses was observed in the PBS control, PRP-only, and sham groups. In contrast, progress was remarkably suppressed over the 8-week period in the PRP group. Moreover, in immunohistochemistry, intense immunostaining for proteoglycan in the NP and inner layer of the annulus fibrosus was observed 8 weeks after administration of PRP-impregnated microspheres. Almost all microspheres were indistinct 8 weeks after the injection, and there were no apparent side effects in this study. Our results suggest that the combined administration of PRP and gelatin hydrogel microspheres into the IVD may be a promising therapeutic modality for IVD degeneration.     

The Quantitative Structural and Compositional Analyses of Degenerating Intervertebral Discs Using Magnetic Resonance Imaging and Contrast-Enhanced Micro-Computed Tomography

The intervertebral disc (IVD) is susceptible to degenerative changes that are associated with low back pain. Murine models are often used to investigate the mechanistic changes in the development, aging, and diseased states of the IVD, yet the detection of early degenerative changes in structure is challenging because of the minute size of the murine IVDs. Histology is the gold standard for examining the IVD structure, but it is susceptible to sectioning artifacts, spatial biases, and requires the destructive preparation of the sample. We have previously demonstrated the feasibility of using Ioversol for the contrast-enhanced micro-computed tomography (microCT) to visualize and quantitate the intact healthy murine IVD. In this work, we demonstrate utility of this approach to monitor the longitudinal changes of in vitro nucleolytic- and mechanical injury- degeneration models of the murine discs and introduce novel quantitative metrics to characterize the structure and composition of the IVD. Moreover, we compared the imaging quality and quantitation of these in vitro models to magnetic resonance imaging (MRI) and histology. Stab puncture, trypsin injection, and collagenase injection all induced detectable and significant changes in structure and composition of the discs overtime. Compared to MRI and histology, contrast-enhanced microCT produced superior images that capture the degenerative progression in these models. Contrast-enhanced microCT was also capable of monitoring the structural deteriorations via the changes in disc height and volume, and novel the nucleus pulposus intensity/disc intensity (NI/DI) parameter provides a surrogate measure of proteoglycan composition (R = 0.96). Overall, this approach allows for the nondestructive monitoring of the structure and composition of the IVD at very high resolutions.     

Fibrin promotes proliferation and matrix production of intervertebral disc cells cultured in three-dimensional poly(lactic-co-glycolic acid) scaffold

Previously, we have proven that fibrin and poly(lactic-co-glycolic acid) (PLGA) scaffolds facilitate cell proliferation, matrix production and early chondrogenesis of rabbit articular chondrocytes in in vitro and in vivo experiments. In this study, we evaluated the potential of fibrin/PLGA scaffold for intervertebral disc (IVD) tissue engineering using annulus fibrosus (AF) and nucleus pulposus (NP) cells in relation to potential clinical application. PLGA scaffolds were soaked in cells–fibrin suspension and polymerized by dropping thrombin–sodium chloride (CaCl₂) solution. A PLGA–cell complex without fibrin was used as control. Higher cellular proliferation activity was observed in fibrin/PLGA-seeded AF and NP cells at each time point of 3, 7, 14 and 7 days using the MTT assay. After 3 weeks in vitro incubation, fibrin/PLGA exhibited a firmer gross morphology than PLGA groups. A significant cartilaginous tissue formation was observed in fibrin/PLGA, as proven by the development of cells cluster of various sizes and three-dimensional (3D) cartilaginous histoarchitecture and the presence of proteoglycan-rich matrix and glycosaminoglycan (GAG). The sGAG production measured by 1,9-dimethylmethylene blue (DMMB) assay revealed greater sGAG production in fibrin/PLGA than PLGA group. Immunohistochemical analyses showed expressions of collagen type II, aggrecan core protein and collagen type I genes throughout in vitro culture in both fibrin/PLGA and PLGA. In conclusion, fibrin promotes cell proliferation, stable in vitro tissue morphology, superior cartilaginous tissue formation and sGAG production of AF and NP cells cultured in PLGA scaffold. The 3D porous PLGA scaffold–cell complexes using fibrin can provide a vehicle for delivery of cells to regenerate tissue-engineered IVD tissue.     

Assessment of glycosaminoglycan distribution in human lumbar intervertebral discs using chemical exchange saturation transfer at 3 T: feasibility and initial experience

Recent studies have proposed that glycosaminoglycan chemical exchange saturation transfer (gagCEST) is associated with a loss of glycosaminoglycans (GAGs), which may be an initiating factor in intervertebral disc (IVD) degeneration. Despite its promising potential, this application has not been reported in human in vivo IVD studies because of the challenges of B₀ magnetic field inhomogeneity in gagCEST. This study aimed to evaluate the feasibility of quantifying CEST values in IVDs of healthy volunteers using a clinical 3 T scanner. A single‐slice turbo spin echo sequence was used to quantify the CEST effect in various GAG phantoms and in IVDs of 12 volunteers. The phantom results indicated high correlation between gagCEST and GAG concentrations (R² = 0.95). With optimal B₀ inhomogeneity correction, in vivo CEST maps of IVDs showed robust contrast between the nucleus pulposus (NP) and the annulus fibrosus (AF) (p < 0.01), as well as higher signal in the central relative to the peripheral NP. In addition, a trend of decreasing CEST values from upper to lower disc levels was evident in NP. Our results demonstrate that in vivo gagCEST quantification in human lumbar IVDs is feasible at 3 T in combination with successful B₀ inhomogeneity correction, but without significant hardware modifications. Our initial findings suggest that it would be worthwhile to perform direct correlation studies between CEST and GAGs using cadaver samples, and to extend this novel technique to studies on patients with degenerative discs to better understand its distinct imaging features relative to conventional techniques. Copyright © 2011 John Wiley & Sons, Ltd.     

Reliable chemical exchange saturation transfer imaging of human lumbar intervertebral discs using reduced‐field‐of‐view turbo spin echo at 3.0 T

The reduced field‐of‐view (rFOV) turbo‐spin‐echo (TSE) technique, which effectively suppresses bowel movement artifacts, is developed for the purpose of chemical exchange saturation transfer (CEST) imaging of the intervertebral disc (IVD) in vivo. Attempts to quantify IVD CEST signals in a clinical setting require high reliability and accuracy, which is often compromised in the conventionally used technique. The proposed rFOV TSE CEST method demonstrated significantly superior reproducibility when compared with the conventional technique on healthy volunteers, implying it is a more reliable measurement. Phantom study revealed a linear relation between CEST signal and glycosaminoglycan (GAG) concentration. The feasibility of detecting IVD degeneration was demonstrated on a healthy volunteer, indicating that the proposed method is a promising tool to quantify disc degeneration. Copyright © 2013 John Wiley & Sons, Ltd.     

BMP-2 enhances TGF-beta3-mediated chondrogenic differentiation of human bone marrow multipotent mesenchymal stromal cells in alginate bead culture

This study addresses synergistic effects of bone morphogenetic protein-2 (BMP-2) and transforming growth factor-beta3 (TGF-beta3) in the induction of chondrocytic differentiation of bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro for potential use in intervertebral disc (IVD) repair. Human BM MSCs encapsulated in alginate beads were induced to differentiate in serum-free medium containing BMP-2 and TGF-beta3. The expression of chondrocytic genes and proteins was analyzed by real-time PCR, western blot, histological, and immunohistochemical assays. This differentiation system showed a potent induction of chondrocytic phenotypes. The expression of chondrocytic markers, such as aggrecan (ACAN) and type II collagen (COL2A1), was upregulated at higher levels than using TGF-beta3 alone. Blocking BMP-2 by noggin completely suppressed BMP-2-enhanced gene and protein expression, confirming a crucial input of BMP-2 signaling in this differentiation process. Inhibition of extracellular signal-regulated kinases 1 and 2 signaling resulted in an increase in ACAN and COL2A1 gene expression, suggesting a negative regulatory role of this pathway. In conclusion, BMP-2 enhances TGF-beta3-mediated chondrogenesis of MSCs. The combination of BMP-2 and TGF-beta3 in alginate culture is superior to the standard differentiation method using TGF-beta alone. This potent induction system may provide an alternative cell source for IVD and cartilage regeneration in clinical practice.     

Transplantation of mesenchymal stem cells enhances axonal outgrowth and cell survival in an organotypic spinal cord slice culture

Mesenchymal stem cells (MSCs) have demonstrated a measurable therapeutic effect following transplantation into animal models of spinal cord injury. However, the mechanism(s) by which transplanted cells promote nerve regeneration and/or functional recovery remains indeterminate. Several studies have suggested that MSCs promote tissue repair via secretion of trophic factors, but delineating the effect of such factors is difficult due to the complexity of the in vivo systems. Therefore, we developed an organotypic spinal cord slice culture system that can be sustained for sufficient periods of time in vitro to evaluate nerve regeneration as an ex vivo model of spinal cord injury. Using this model, we demonstrate that treatment of lumbar slices of spinal cord with lysolecithin induced a significant degree of cell death and demyelination of nerve fibers, but that these effects were ameliorated to a significant extent following co-culture of slices with human MSCs (hMSCs). The results indicate that transplanted hMSCs alter the tissue microenvironment in a way that promotes survival of endogenous cells, including injured neurons, immature oligodendrocytes and oligodendrocyte progenitor cells. This ex vivo culture system represents a useful tool to further dissect the mechanism(s) by which MSCs promote regeneration of injured nervous tissue.     

Chondrogenesis of Synovium-Derived Mesenchymal Stem Cells in Photopolymerizing Hydrogel Scaffolds

Recently, tissues adjacent to the wound sites are regarded as a promising therapeutic cell source for curing and repairing purpose. Specifically, therapeutic stem cells have been identified in synovial tissue, a tissue adjacent to articular cartilage. The purpose of this study was to explore therapeutic chondrogenesis with rabbit synovium-derived mesenchymal stem cells (SMSCs) encapsulated in photopolymerized hydrogels. A non-degradable poly(ethylene glycol) diacrylate (PEGDA)-based hydrogel and biodegradable phosphoester–poly(ethylene glycol) (PhosPEG)-based hydrogel were both applied as 3-D scaffolds mediating SMSC chondrogenesis in vitro. The viability of SMSCs in both hydrogels was assessed by fluorescent Live/Dead assay and WST-1 assay. Levels of genes and proteins specific to SMSC chondrogenesis were evaluated by real-time RT-PCR, biochemical analysis and immunohistochemical analysis, respectively. The results demonstrated that SMSCs continue to have a high viability when encapsulated in the hydrogel. By treatment with transforming growth factor (TGF)-β1 or TGF-β3, positive SMSC chondrogenesis was successfully achieved in both gels, with the best outcome in the PEGDA system. It can be concluded that both PEGDA and PhosPEG hydrogels are appropriate cell-delivery vehicles for SMSC chondrogenesis. Especially as a biodegradable material, PhosPEG hydrogel displayed great potentials in future applications for articular cartilage regeneration coupling with SMSCs.     

Roles of large aggregating proteoglycans in human intervertebral disc degeneration

Degeneration of the intervertebral discs, a natural progression of the aging process, is strongly implicated as a cause of low back pain. Aggrecan is the major structural proteoglycan in the extracellular matrix of the intervertebral disc. It is large, possessing numerous glycosaminoglycan chains and the ability to form aggregates in association with hyaluronan. The negatively charged glycosaminoglycan side chains in aggrecan in the nucleus pulposus of the intervertebral discs can bind electrostatically to polar water molecules, which are crucial for maintaining the well-hydrated state that enables the discs to undergo reversible deformation under compressive loading. A more in-depth understanding of the molecular basis of disc degeneration is essential to the design of therapeutic solutions to treat degenerative discs. Within this scope, we discuss the current knowledge concerning the structure and function of aggrecan in intervertebral disc degeneration. These data suggest that aggrecan plays a central role in the function and degeneration of the intervertebral disc, which may suggest potential aggrecan-based therapies for disc regeneration.     

Current Challenges for the Advancement of Neural Stem Cell Biology and Transplantation Research

Transplantation of neural stem cells (NSC) is hoped to become a promising primary or secondary therapy for the treatment of various neurodegenerative disorders of the central nervous system (CNS), as demonstrated by multiple pre-clinical animal studies in which functional recovery has already been demonstrated. However, for NSC therapy to be successful, the first challenge will be to define a transplantable cell population. In the first part of this review, we will briefly discuss the main features of ex vivo culture and characterisation of NSC. Next, NSC grafting itself may not only result in the regeneration of lost tissue, but more importantly has the potential to improve functional outcome through many bystander mechanisms. In the second part of this review, we will briefly discuss several pre-clinical studies that contributed to a better understanding of the therapeutic potential of NSC grafts in vivo. However, while many pre-clinical animal studies mainly report on the clinical benefit of NSC grafting, little is known about the actual in vivo fate of grafted NSC. Therefore, the third part of this review will focus on non-invasive imaging techniques for monitoring cellular grafts in the brain under in vivo conditions. Finally, as NSC transplantation research has evolved during the past decade, it has become clear that the host micro-environment itself, either in healthy or injured condition, is an important player in defining success of NSC grafting. The final part of this review will focus on the host environmental influence on survival, migration and differentiation of grafted NSC.     

Downregulated interleukin 37 expression associated with aggravation of intervertebral disc degeneration

Interleukin 37 (IL-37) is an anti-inflammatory cytokine which was proven to be associated with several diseases characterized with excessive-inflammation. The pathologic process of Intervertebral disc degeneration (IVDD) is also companied by uncurbed inflammation, many cytokines were reported presenting in the process. However, there is little IL-37 related knowledge in IVDD up to now. The aim of this study was to investigate whether IL-37 expression in degenerative intervertebral disc (IVD) is different from that in non-degenerative disc and to evaluate the relationship between IL-37 expression, overexpression of pro-inflammatory cytokines and development of degeneration. Human nucleus pulposus samples were obtained from patients with disc degenerative disease and vertebra fractures undergoing discectomy and fusion. Subsequently, expression of IL-37 was assessed by real-time quantitative polymerase chain reaction (RT-PCR) and western blotting. Gene expression level was measured for IL-1α, IL-1β, IL-6, IL-16, TNF-α, TGF-β1 and Smad3. Degree of degeneration was evaluated for MRI with modified Pfirrmann grading system. The results showed that IL-37 had a decreased expression in degenerative samples compared to that in normal samples both at mRNA and protein level. Instead, significant elevated gene expression of IL-1β, IL-16, TNF-α, TGF-β1 and Smad3 were detected in degenerative samples. High correlations were observed between IL-37, IL-1β, IL-16, TGF-β1, Smad3 and degeneration degree of IVD. Downregulation of IL-37 expression appeared to result in overexpression of pro-inflammatory cytokines, such as IL-1β and IL-16, in degenerative IVD and may be a contributor involved in the progression of IVDD.     

MicroRNA-16 inhibits the lipopolysaccharide-induced inflammatory response in nucleus pulposus cells of the intervertebral disc by targeting TAB3

IntroductionA variety of inflammatory mediators are produced by the degenerative human intervertebral disc (IVD) tissues spontaneously, suggesting their role in the development of intervertebral disc degeneration (IDD). Our present study was designed to investigate the regulatory effect of microRNA-16 (miR-16) on the lipopolysaccharide (LPS)-induced inflammatory response in nucleus pulposus (NP) cells of the IVD.Material and methodsNP cells were treated with LPS to induce inflammatory responses. The expression of miRNA and genes was determined by qRT-PCR. Western blot and an ELISA kit were used to detect the proteins and protein expression, respectively. A dual luciferase reporter assay was applied to identify the correlation between a miRNA and a gene, and to test nuclear factor-κB (NF-κB) activity.ResultsThe results suggested that miR-16 positively regulated the mRNA and protein expression of extracellular matrix (ECM) genes (including aggrecan and collagen II) in NP cells, while it was negatively correlated with ECM degrading enzymes (including MMP3, MMP13, ADAMTS4, ADAMTS5) and related genes of nitric oxide (NO) reaction. Further studies revealed that miR-16 could oppositely regulate NF-κB and MAPK pathways by directly mediating their upstream gene TAB3.ConclusionsOur study suggested that, in NP cells of the IVD, miR-16 could exert inhibitory effects on the LPS-induced inflammatory response through NF-κB and MAPK pathways by directly mediating TAB3. In this way, miR-16 would play a protective role against LPS-induced IDD and inflammation. Therefore, miR-16 may be a novel therapeutic target for the inhibit of the ECM in the IVD.     

The role of the ERK1/2 pathway as an alternative to the aging-diminished cyclic AMP pathway in calcitonin-mediated chondrogenesis in human nucleus pulposus

Human disc degeneration initiated by aging in the central nucleus pulposus (hNP) is an irreversible process and the recovery has become seriously emerging. In this study, the related mechanisms of calcitonin on the regeneration of hNP and the effects of calcitonin on the age-related alterations were examined. The harvested hNP population was designated as YhNP (from young donor, age <50) and OhNP (from old donor, age >50). Primary OhNP cells showed more hypertrophic phenotypes than YhNP. However, calcitonin (10(-8)-10(-6)?M) was able to induce the same chondrogenesis in both YhNP and OhNP by elevating chondrogenic specific-mRNA and protein expressions. Their cell viabilities were increased with calcitonin treatment. No significant differences of calcitonin receptor (CTR) were expressed between YhNP and OhNP cells. Interestingly, in calcitonin-induced pathways for chondrogenesis, highly increased cyclic AMP (cAMP) was detected in YhNP but was strongly diminished by aging in OhNP after calcitonin treatment. However, to maintain the chondrogenesis, calcitonin-induced an alterative phosphorylated ERK1/2 (p-ERK) in both cells. After inhibiting ERK1/2 by PD98059, calcitonin-induced chondrogenesis in OhNP was almost restrained while YhNP cells were not affected. Our results demonstrated that the regeneration of calcitonin on hNP was maintained with aging which was satisfied by an alternative signaling pathway. Therefore, calcitonin shows great potential for clinical therapy for disc regeneration without aging considerations.     

Expression of receptors for putative anabolic growth factors in human intervertebral disc: implications for repair and regeneration of the disc

Low back pain (LBP) is a common, debilitating and economically important disorder. Current evidence implicates loss of intervertebral disc (IVD) matrix consequent upon 'degeneration' as a major cause of LBP. Degeneration of the IVD involves increases in degradative enzymes and decreases in the extracellular matrix (ECM) component in a process that is controlled by a range of cytokines and growth factors. Studies have suggested using anabolic growth factors to regenerate the normal matrix of the IVD, hence restoring disc height and reversing degenerative disc disease. However, for such therapies to be successful it is vital that the target cells (i.e. the disc cells) express the appropriate receptors. This immunohistochemical study has for the first time investigated the expression and localization of four potentially beneficial growth factor receptors (i.e. TGFbetaRII, BMPRII, FGFR3 and IGFRI) in non-degenerate and degenerate human IVDs. Receptor expression was quantified across regions of the normal and degenerate disc and showed that cells of the nucleus pulposus (NP) and inner annulus fibrosus (IAF) expressed significantly higher levels of the four growth factor receptors investigated. There were no significant differences between the four growth factor expression in non-degenerate and degenerate biopsies. However, expression of TGFbetaRII, FGFR3 and IGFRI, but not BMP RII, were observed in the ingrowing blood vessels that characterize part of the disease aetiology. In conclusion, this study has demonstrated the expression of the four growth factor receptors at similar levels in the chondrocyte-like cells of the NP and IAF in both non-degenerate and degenerate discs, implicating a role in normal disc homeostasis and suggesting that the application of these growth factors to the degenerate human IVD would stimulate matrix production. However, the expression of some of the growth factor receptors on ingrowing blood vessels might be problematic in a therapeutic approach.     

Decellularized bovine intervertebral disc as a natural scaffold for xenogenic cell studies

Low back pain that is associated with disc degeneration contributes to a huge economic burden in the worldwide healthcare system. Traditional methods, such as spinal fusion, have been adopted to relieve mechanical back pain, but this is compromised by decreased spinal motion. Tissue engineering has attracted much attention, and aims to correct the changes fundamentally occurring in the discs by a combination of cell biology, molecular biology and engineering. Synthetic materials including poly(l-lactic acid) or poly(glycolic acid) and biomolecules like hyaluronic acid or collagen have been adopted in the development of disc scaffolds for studying therapeutic approaches. Nevertheless, the complex biological and mechanical environment of the intervertebral disc (IVD) makes the synthesis of an artificial IVD with biomaterials a difficult task. Thus the aim of this study was to develop a natural disc scaffold for culturing disc cells for future development of biological disc constructs. We adopted a combination of currently used decellularization techniques to decellularize bovine IVD to create a complete endplate-to-endplate IVD scaffold. By altering the chemical and physical decellularization parameters, we reported the removal of up to 70% of the endogenous cells, and were able to preserve the glycosaminoglycan content, collagen fibril architecture and mechanical properties of the discs. The reintroduction of nucleus pulposus cells into the scaffold indicated a high survival rate over 7 days, with cell penetration. We have shown here that conventional methods used for decellularizing thin tissues can also be applied to large organs, such as IVD. Our findings suggest the potential of using decellularized IVD as a scaffold for IVD bioengineering and culturing of cells in the context of the IVD niche.     

Arterial Wall Adaptation under Elevated Longitudinal Stretch in Organ Culture

Arteries in vivo are subjected to large longitudinal stretch which may change significantly due to vascular disease and surgery. However, little is known about the effect of longitudinal stretch on vascular function and wall remodeling, although the effects of tensile and shear stress from blood pressure and flow have been well documented. To study the effect of longitudinal stretch on vascular function and wall remodeling, porcine carotid arteries were longitudinally stretched 20% more than in vivo for 5 days while being maintained in an ex vivo organ culture system under conditions of pulsatile flow at physiologic pressure. Vessel viability was demonstrated by strong vasomotor responses to norepinephrine (NE, 10^-6M), carbachol (10^-6M), and sodium nitroprusside (10^-5M), as well as by dense staining for mitochondrial activity and a low occurrence of cell necrosis. Cell proliferation was examined by incorporation of bromodeoxyuridine (BrdU). Results showed that arteries maintain normal structure and viability after 5 days in organ culture. Both the stretched and control arteries demonstrated significant contractile responses. For example, both stretched and control arteries showed approximately 10% diameter contraction in response to NE. Stretched arteries contained 8% BrdU-positive cells compared to 5% in controls (p < 0.05). These results indicate that longitudinal stretch promotes cell proliferation in arteries while maintaining arterial function. © 2003 Biomedical Engineering Society. PAC2003: 8719Rr, 8717Ee, 8719Uv     

Contractile Responses in Arteries Subjected to Hypertensive Pressure in Seven-Day Organ Culture

Early stage changes in hypertensive arteries have a significant effect on the long-term adaptation of the arteries. Compared to the long-term adaptation, little is known about the early dimensional and functional changes in hypertensive arteries in the first few days of hypertension. To study the early stage changes in hypertensive arteries, porcine common carotid arteries were cultured for seven days in a simplified ex vivo artery organ culture system with pulsatile flow under hypertensive (200±30 mm Hg) or normotensive (100±20 mm Hg) pressure conditions while maintaining a physiological mean wall shear stress of 15 dyn/cm².Vessel viability was demonstrated by contractile diameter responses to norepinephrine (NE), carbachol (CCh), and sodium nitroprusside (SNP) as well as staining for mitochondrial activity and cell apoptosis/necrosis. The results show that arteries demonstrated strong contractile responses to NE, CCh, and SNP, basal tone, and viable mitochondria in the organ culture system for seven days. Hypertensive arteries demonstrated a stronger contractile response than normotensive arteries (p < 0.05). Diameter enlargement was observed in hypertensive arteries as compared to arteries cultured under normotensive conditions. In conclusion, the pulsatile culture system can maintain arteries viable with active vasomotion tone for up to seven days. Hypertensive pressure causes arterial adaptation by significantly increasing arterial diameter and contractile response within the first seven days. © 2001 Biomedical Engineering Society. PAC01: 8719Uv, 8719Rr, 8780Rb     

TLR2 activation in corneal stromal cells by Staphylococcus aureus‐induced keratitis

The Toll‐Like Receptor 2 (TLR2) plays an active and important role in Staphylococcus aureus‐induced chronic ocular inflammation. The aim of this study was to investigate the expression and function of TLR2 of corneal stromal cells in ex vivo rabbit model of S. aureus keratitis. Corneal buttons with sclera rims placed in an ex vivo air‐interface organ culture were assigned to two groups: corneas with epithelial and stromal abrasions. Each group was then divided into two sub‐groups exposed to UV‐killed S. aureus ATCC 6538P and S. aureus ATCC 29213, respectively. TLR2 and IL‐8 mRNA expressions were analyzed by quantitative real‐time RT‐PCR. TLR2 localization was visualized by immunofluorescence analysis. The results demonstrated that TLR2 and IL‐8 mRNA were significantly expressed in the stromal cells of the groups exposed to S. aureus strains. Moreover, it has been demonstrated that, after corneal injury, keratocytes differentiated into myofibroblasts became able to express TLR2 only when exposed to S. aureus. Identification of mechanisms regulation of corneal TLRs may lead to development of therapeutic interventions aimed at controlling corneal inflammation. This ex vivo model can be used to clarify the molecular events of bacterial‐corneal tissue interactions and their inflammatory consequences.