Ana Chee

MicroRNA-146a reduces IL-1 dependent inflammatory responses in the intervertebral disc

Because miR-146a expression in articular chondrocytes is associated with osteoarthritis (OA), we assessed whether miR-146a is linked to cartilage degeneration in the spine. Monolayer cultures of nucleus pulposus (NP) cells from the intervertebral discs (IVD) of bovine tails were transfected with a miR-146a mimic. To provoke inflammatory responses and catabolic extracellular matrix (ECM) degradation, cells were co-treated with interleukin-1 (IL-1). Transfection of miR-146a decreases IL-1 induced mRNA levels of inflammatory genes and catabolic proteases in NP cells based on quantitative real-time reverse transcriptase PCR (qRT-PCR) analysis. Similarly, miR146a suppresses IL-1 induced protein levels of matrix metalloproteinases and aggrecanases as revealed by immunoblotting. Disc segments from wild type (WT) and miR-146a knockout (KO) mice were cultured ex vivo in the presence or absence of IL-1 for 3days. Histological and immuno-histochemical (IHC) analyses of disc organ cultures revealed that IL-1 mediates changes in proteoglycan (PG) content and in-situ levels of catabolic proteins (MMP-13 and ADAMTS-5) in the nucleus pulposus of the disc. However, these IL-1 effects are more pronounced in miR-146a KO discs compared to WT discs. For example, absence of miR-146a increases the percentage of MMP-13 and ADAMTS-5 positive cells after treatment with IL-1. Thus, miR-146a appears to protect against IL-1 induced IVD degeneration and inflammation. Stimulation of endogenous miR-146a expression or exogenous delivery of miRNA-146a are viable therapeutic strategies that may decelerate disc degeneration and regain a normal homeostatic balance in extracellular matrix production and turn-over.

Intervertebral disk repair by protein, gene, or cell injection: a framework for rehabilitation-focused biologics in the spine.

Low back pain carries an enormous socioeconomic burden. Current treatment modalities for symptomatic intervertebral disk (IVD) degeneration have limited and often inconsistent clinical benefits. Novel approaches with the potential to halt or even reverse disk degeneration and restore physiologic disk function, such as biological treatments, are therefore very attractive. The following barriers are impeding the development of successful therapeutic interventions: (1) the biology and pathophysiology of disk degeneration are not well understood, and (2) the precise relationship between IVD degeneration and low back pain remains unclear. This article reviews the structural changes that take place during IVD degeneration and their relationship to diskogenic back pain. It also presents treatment modalities that currently are under laboratory investigation and are being studied in clinical trials. The authors of recent studies have shown that the content of large proteoglycans, such as aggrecan and versican, decreases with aging and IVD degeneration, whereas the content of certain small proteoglycans, such as biglycan, increases. Proinflammatory cytokines such as interleukin‐1 and tumor necrosis factor‐α also are associated with IVD degeneration and are potential biomarkers of IVD degeneration and repair. Our group of investigators and others have developed in vitro models of IVD cell and explant culture in addition to in vivo animal models to study IVD degeneration and repair. With the use of these models, we have tested candidate therapeutic agents to assess their therapeutic potential for matrix restoration. When a rabbit annular puncture model of IVD degeneration was used, injections of either bone morphogenetic protein‐7 (also known as osteogenic protein‐1) or bone morphogenetic protein‐14 (also known as growth differentiation factor‐5) were shown to be effective in restoring IVD structures. On the basis of these data, the Food and Drug Administration has recently allowed the initiation of Investigational New Drug clinical trials on osteogenic protein‐1 and growth differentiation factor‐5 in the United States. Protein therapies such as other growth factors, inhibitors of degradation enzymes or cytokines, and cell therapies also are being investigated in laboratory settings with the goal of restoring disk function and alleviating back pain symptoms. These therapies may be used by physiatrists with the skills required to administer intradiskal injections and supervise a comprehensive rehabilitation program after the procedures. Ultimately, the clinical use of any biological treatment discussed in this article would require the collective efforts of clinicians and researchers.

Human umbilical cord blood-derived mesenchymal stem cells in the cultured rabbit intervertebral disc: a novel cell source for disc repair.

ObjectiveBack pain associated with symptomatic disc degeneration is a common clinical condition. Intervertebral disc (IVD) cell apoptosis and senescence increase with aging and degeneration. Repopulating the IVD with cells that could produce and maintain extracellular matrix would be an alternative therapy to surgery. The objective of this study was to determine the potential of human umbilical cord blood–derived mesenchymal stem cells (hUCB-MSCs) as a novel cell source for disc repair. In this study, we intended to confirm the potential for hUCB-MSCs to differentiate and display a chondrocyte-like phenotype after culturing in micromass and after injection into the rabbit IVD explant culture. We also wanted to confirm hUCB-MSC survival after transplantation into the IVD explant culture. DesignThis study consisted of micromass cultures and in vitro rabbit IVD explant cultures to assess hUCB-MSC survival and differentiation to display chondrocyte-like phenotype. First, hUCB-MSCs were cultured in micromass and stained with Alcian blue dye. Second, to confirm cell survival, hUCB-MSCs were labeled with an infrared dye and a fluorescent dye before injection into whole rabbit IVD explants (host). IVD explants were then cultured for 4 wks. Cell survival was confirmed by two independent techniques: an imaging system detecting the infrared dye at the organ level and fluorescence microscopy detecting fluorescent dye at the cellular level. Cell viability was assessed by staining the explant with CellTracker green, a membrane-permeant tracer specific for live cells. Human type II collagen gene expression (from the graft) was assessed by polymerase chain reaction. ResultsWe have shown that hUCB-MSCs cultured in micromass are stained blue with Alcian blue dye, which suggests that proteoglycan-rich extracellular matrix is produced. In the cultured rabbit IVD explants, hUCB-MSCs survived for at least 4 wks and expressed the human type II collagen gene, suggesting that the injected hUCB-MSCs are differentiating into a chondrocyte-like lineage. ConclusionsThis study demonstrates the abiity of hUBC-MSCs to survive and assume a chondrocyte-like phenotype when injected into the rabbit IVD. These data support the potential for hUBC-MSCs as a cell source for disc repair. Further measures of the host response to the injection and studies in animal models are needed before trials in humans.

Intervertebral Disc Cells Produce Interleukins Found in Patients with Back Pain.

ObjectiveTo examine the link between cytokines in intervertebral disc (IVD) tissues and axial back pain. DesignIn vitro study with human IVD cells cultured from cadaveric donors and annulus fibrosus (AF) tissues from patients. ResultsCultured nucleus pulposus (NP) and AF cells were stimulated with interleukin (IL)-1&bgr;. IL-8 and IL-7 gene expression was analyzed using real-time polymerase chain reaction. IL-8 protein was quantified by enzyme-linked immunosorbent assay. After IL-1&bgr; stimulation, IL-8 gene expression increased 26,541 fold in NP cells and 22,429 fold in AF cells, whereas protein released by the NP and AF cells increased 2,389- and 1,784-fold, respectively. IL-7 gene expression increased 3.3-fold in NP cells (P < 0.05).Cytokine profiles in AF tissues collected from patients undergoing surgery for back pain (painful group) or scoliosis (controls) were compared by cytokine array. IL-8 protein in the AF tissues from patients with back pain was 1.81-fold of that in controls. IL-7 and IL-10 in AF tissues from the painful group were 6.87 and 4.63 times greater than the corresponding values in controls, respectively (P < 0.05). ConclusionInflammatory mediators found in AF tissues from patients with discogenic back pain are likely produced by IVD cells and may play a key role in back pain.

Allogeneic Articular Chondrocyte Transplantation Downregulates Interleukin 8 Gene Expression in the Degenerating Rabbit Intervertebral Disk In Vivo.

ObjectiveThe aim of this study was to investigate whether repopulating the degenerating intervertebral disk (IVD) with articular chondrocytes will decrease inflammation in the degenerating rabbit IVD. DesignThis was a biologic study in a rabbit IVD-injury model in vivo. Dual cell tracking methods (infrared dye labeling and adenovirus transduction) were used to demonstrate the viability of allogeneic articular chondrocytes injected into degenerating rabbit IVDs. Interleukin 8 gene expression was determined via real-time polymerase chain reaction. Infiltrating inflammatory cells (macrophages, T cells, or neutrophils) were examined with immunohistochemistry. The IVDs were also examined by routine histology. ResultsArticular chondrocytes labeled with infrared dye were detected in the degenerating IVDs at both 2 and 8 wks after injection. At the 2-wk time point, interleukin 8 gene expression was comparable in IVDs injected with chondrocytes and in intact disks as control (P = 0.647), whereas its expression in IVDs injected with saline increased 50-fold (P = 0.028). Transgene expression of red fluorescent protein, &bgr;-galactosidase, and human bone morphogenetic protein 7 diminished at 8 wks after injection. IVDs injected with chondrocytes overexpressing human bone morphogenetic protein 7 did not show lower interleukin 8 gene expression or improved histology. Macrophages were consistently detected by immunohistochemistry in the cartilage formed around the needle insertion sites in both the saline and chondrocyte groups, whereas neither T cells nor neutrophils were detected. ConclusionsAllogeneic rabbit articular chondrocyte survived in the degenerating rabbit IVDs for at least 8 wks. Cell treatment resulted in reduced IVD inflammation but did not significantly improve IVD structure.

Rho-Associated Kinase Inhibitor Immortalizes Rat Nucleus Pulposus and Annulus Fibrosus Cells: Establishment of Intervertebral Disc Cell Lines With Novel Approaches.

Study Design. Establishment of immortalized cell lines derived from rat intervertebral disc cells by Rho-associated kinase (ROCK) inhibitor, Y-27632. Objective. To determine whether rat nucleus pulposus (NP) and annulus fibrosus (AF) cells could be immortalized, retain their phenotype, and used as cell lines for in vitro cell biology. Summary of Background Data. Intervertebral disc degeneration is a major factor for most low-back pain. However, the mechanism of the disease is not well understood by the limitation to obtain sufficient amounts of primary disc cells. Therefore, the establishment of disc cell lines will help in vitro molecular signaling studies to understand the mechanism of degenerative disc disease. Methods. Cells were isolated from the NP and AF tissues of lumbar discs of adult Sprague Dawley rat. Tissues were digested and cultured with DMEM/Hams F-12 (1:1) and 20% FBS and antibiotics. From day 3, cells were grown in the presence of 10 &mgr;M Y-27632, a well-characterized inhibitor of the ROCK, and subcultured by trypsinization, passaging them 1:3 onto 100 mm culture dishes. Morphologic and genetic analyses were performed on the different passaged cells. Results. ROCK inhibitor successfully immortalized rat NP and AF cells. They passaged for over 50 generations with sustained expression levels of several NP and AF cell markers. In addition, they retained phenotypic features similar to the primary parental NP and AF cells when the cells were challenged with different cytokines and growth factors. Conclusion. We established immortalized rat NP and AF cell lines using a method of treating cells with ROCK inhibitor Y-27632 and demonstrated that these immortalized cells retain the properties of primary cells and could serve as useful tools for in vitro signaling studies or drug screening studies to develop novel therapeutic strategies. Level of Evidence: N/A

Retroperitoneal approach to the intervertebral disc for the annular puncture model of intervertebral disc degeneration in the rabbit

BACKGROUND CONTEXT The rabbit annular puncture model of degeneration is among the most widely used models of intervertebral disc (IVD) degeneration. There are no published reports of the specific surgical technique used to produce this model. PURPOSE To describe the model in detail in an effort to reduce center-to-center variability and hopefully improve the reproducibility of future experimental results. STUDY DESIGN Technical report of surgical approach and experience. PATIENT SAMPLE New Zealand White Rabbits. METHODS A detailed report of the annular puncture technique in rabbits is provided including preparation of the animals, instrumentation, a description of retroperitoneal approach to the lumbar area, and the technique for IVD injury. Common pitfalls and complications of the procedure are described. CONCLUSIONS The methods described can serve to standardize the implementation of this important preclinical model of disc degeneration.

Cell Therapy with Human Dermal Fibroblasts Enhances Intervertebral Disk Repair and Decreases Inflammation in the Rabbit Model

Study Design Pilot study using the rabbit model. Objective Low back pain is often associated with disk degeneration. Cell therapy for degenerating disks may promote tissue regeneration and repair. Human dermal fibroblasts, obtained from the patients skin tissue or donated tissue, may be a promising cell therapy option for degenerating disks. The objective of these studies is to determine the effects of intradiscal transplantation of neonatal human dermal fibroblasts (nHDFs) on intervertebral disk (IVD) degeneration by measuring disk height, magnetic resonance imaging (MRI) signal intensity, gene expression, and collagen immunostaining. Methods New Zealand white rabbits (n = 16) received an annular puncture to induce disk degeneration and were treated with nHDFs or saline 4 weeks later. At 2 and 8 weeks post-treatment, X-ray and MRI images were obtained. IVDs were isolated and examined for changes in collagen staining and gene expression. Results In the nHDF-treated group, there was a 10% increase in the disk height index after 8 weeks of treatment (p ≤ 0.05), and there was no significant difference in the saline-treated group. When compared with the saline-treated disks, disks treated with nHDFs showed reduced expression of inflammatory markers, a higher ratio of collagen type II over collagen type I gene expression, and more intense immunohistochemical staining for both collagen types I and II. Conclusions Human dermal fibroblast introduction into the disk reduced inflammation and promoted tissue rich in both type I and type II collagens. The results of this study suggest that nHDFs would be a feasible cell therapy option for disk degeneration.

Fibronectin splice variation in human knee cartilage, meniscus and synovial membrane: Observations in osteoarthritic knee

Fibronectin (FN) is a widely expressed molecule that can participate in development of osteoarthritis (OA) affecting cartilage, meniscus, and synovial membrane (SM). The alternatively spliced isoforms of FN in joint tissues other than cartilage have not been extensively studied previously. The present study compares FN splice variation in patients with varying degrees of osteoarthritic change. Joint tissues were collected from asymptomatic donors and patients undergoing arthroscopic procedures. Total RNA was amplified by PCR using primers flanking alternatively spliced Extra Domain A (EDA), Extra Domain B (EDB) and Variable (V) regions. EDB+, EDB− and EDA− and V+ variants were present in all joint tissues, while the EDA+ variant was rarely detected. Expression levels of EDB+ and EDV+ variants were similar in cartilage, synovium, and meniscal tissues. Synovial expression of V+ FN in arthroscopy patients varied with degree of cartilage degeneration. Two V− isoforms, previously identified in cartilage, were also present in SM and meniscus. Fibronectin splicing in meniscus and SM bears striking resemblance to that of cartilage. Expression levels of synovial V+ FN varied with degree of cartilage degeneration. V+ FN should be investigated as a potential biomarker of disease stage or progression in larger populations.

Biglycan Inhibits Capsaicin-Induced Substance P Release by Cultured Dorsal Root Ganglion Neurons.

ObjectiveThe purpose of this study was to examine the inhibitory effects of biglycan on substance P release from cultured sensory neurons in response to capsaicin. Study DesignIn vitro study of cultured primary sensory neurons from the rabbit dorsal root ganglion (DRG). We interrogated the culture system function with capsaicin. Biglycan is an important structural component of the intervertebral disc that may regulate growth factors and inflammatory mediators. We tested the hypothesis that biglycan inhibits substance P release in response to capsaicin. ResultsThe DRG cultures were shown to contain both neurons and astrocytes by immunostaining using antibodies recognizing neuron and glial cell markers. Cultured DRG cells respond to capsaicin in a dose- and time-dependent manner (capsaicin dose ranges from 5 to 500 &mgr;mol/L; stimulation time ranges from 0 to 60 minutes). The neurons preincubated with biglycan released 27% less substance P compared with neurons without biglycan (n = 4, P = 0.036). ConclusionWe have established a DRG cell culture system, which contains both sensory neurons and the supporting astrocytes. Biglycan, an inhibitor of substance P release by DRG cultures, may serve as an ingredient in intradiscal injectables to reduce back pain.