The effect of methylprednisolone intravenous infusion on the expression of ciliary neurotrophic factor in a rat spinal cord injury model.

BACKGROUND CONTEXT: Methylprednisolone (MP) infusion after acute spinal cord injury (SCI) remains controversial despite large randomized studies, including the National Acute Spinal Cord Injury Studies (NASCIS). PURPOSE: To determine the effect of NASCIS protocol MP infusion on the expression of ciliary neurotrophic factor (CNTF), a neuroprotective cytokine, in a rat model after SCI. STUDY DESIGN: Animal laboratory study. METHODS: Thirty rats were randomized into an MP infusion group (intravenous [IV]-MP) versus normal saline (NS) control group (IV-NS) after a standardized SCI. Ciliary neurotrophic factor expression was measured by reverse transcription-polymerase chain reaction at 6, 12, 24, 48, and 72 hours post-SCI. RESULTS: Mean CNTF expression was diminished in the MP group at 12 (p=.006) and 24 (p=.008) hours postinjury compared with the control group. Expression of CNTF was not significantly different between the groups at 6, 48, and 72 hours post-SCI. CONCLUSIONS: Standardized MP infusion post-SCI reduces CNTF activation in a rat SCI model. Further study is needed to determine if this effect is seen in human SCIs.

An evaluation of carrier agents for desferoxamine, an up-regulator of vascular endothelial growth factor.

Avascularity and hypoxia result in avascular necrosis and play a negative role in fracture healing. The FDA-approved iron chelating agent, desferoxamine (DFO) in a liquid form, has been shown to induce angiogenesis and improve fracture healing through upregulation of the vascular endothelial growth factor. We were concerned that local injection of DFO would either fail to adequately deliver sufficient drug to the desired site or lead to undesired delivery to adjacent sites. Therefore, a sustained release delivery system was desirable to direct DFO to the intended site. Calcium sulfate pellets, collagen sponges, and demineralized cortical bone matrix were all evaluated as potentially controlled release systems for DFO using a fetal mouse metatarsal angiogenesis assay. Angiogenesis was analyzed using a vascularity grading scale, by measuring the mean vessel length of the 5 longest vessels, and by counting the mean number of vessels per metatarsal. Although there was some evidence of angiogenesis with all three carriers, DFO loaded CaSO4 pellets increased vascularity grading, the mean length of the five longest vessels, and the mean number of vessels, all by statistically significant margins versus the control. These results suggest that CaSO4 pellets could be used as a viable, nontoxic, controlled release system for DFO in clinical situations where increased angiogenesis and bone growth are desirable.

Annulus cells release ATP in response to vibratory loading in vitro.

Mechanical forces regulate the developmental path and phenotype of a variety of tissues and cultured cells. Vibratory loading as a mechanical stimulus occurs in connective tissues due to energy returned from ground reaction forces, as well as a mechanical input from use of motorized tools and vehicles. Structures in the spine may be particularly at risk when exposed to destructive vibratory stimuli. Cells from many tissues respond to mechanical stimuli, such as fluid flow, by increasing intracellular calcium concentration ([Ca(2+)](ic)) and releasing adenosine 5'-triphosphate (ATP), extracellularly, as a mediator to activate signaling pathways. Therefore, we examined whether ATP is released from rabbit (rAN) and human (hAN) intervertebral disc annulus cells in response to vibratory loading. ATP release from annulus cells by vibratory stimulation as well as in control cells was quantitated using a firefly luciferin-luciferase assay. Cultured hAN and rAN cells had a basal level of extracellular ATP ([ATP](ec)) in the range of 1-1.5 nM. Vibratory loading of hAN cells stimulated ATP release, reaching a net maximum [ATP] within 10 min of continuous vibration, and shortly thereafter, [ATP] declined and returned to below baseline level. [ATP] in the supernatant fluid of hAN cells was significantly reduced compared to the control level when the cells received vibration for longer than 15 min. In rAN cells, [ATP] was increased in response to vibratory loading, attaining a level significantly greater than that of the control after 30 min of continuous vibration. Results of the current study show that resting annulus cells secrete ATP and maintain a basal [ATP](ec). Annulus cells may use this nucleotide as a signaling messenger in an autocrine/paracrine fashion in response to vibratory loading. Rapid degradation of ATP to ADP may alternatively modulate cellular responses. It is hypothesized that exposure to repetitive, complex vibration regimens may activate signaling pathways that regulate matrix destruction in the disc. As in tendon cells, ATP may block subsequent responses to load and modulate the vibration response. Rabbit annulus cells were used as a readily obtainable source of cells in development of an animal model for testing effects of vibration on the disc. Human cells obtained from discarded surgical specimens were used to correlate responses of animal to human cells.

Microplate live cell assay system for early events in mechanotransduction.

For studying mechanotransduction in cultured cells, we developed a microplate assay using a fluorescence/luminescence plate reader equipped with software-controlled injectors to deliver a reproducible mechanical stimulus (adjustable for both timing and force) and immediately measure adenosine 5(')-triphosphate (ATP) release and calcium mobilization. Suspension or adherent chondrocyte cultures in 96-well plates were incubated with firefly luciferase and luciferin for the ATP assay or loaded with Fluo-3-acetoxy methylester for intracellular calcium measurement. Steady state ATP release was measured in resting cells; then mechanical stimulation was delivered by injection of an equal volume of buffer into the wells. Serial integrations of 20 to 500ms allowed real-time analysis of the time course of ATP release. Luminescence increased within 500ms indicating the rapidity of ATP release in chondrocyte mechanotransduction. Subsequent injection of a cell lysis solution allowed quantitation of total cellular ATP as an internal control of cell viability and number. Intracellular calcium was also elevated within 500ms of fluid injection. This assay is easily adapted for changes in intracellular pH or other ions by use of different commercially available fluorescent indicators. The live-cell assay using fluid injection as a mechanical stimulus is a valuable tool for dissecting the role of signaling pathways in mechanotransduction.

Extracellular nucleotides, cartilage stress, and calcium crystal formation.

Nucleotides are released by chondrocytes at rest and in response to mechanical stimulation. Extracellular nucleotides are metabolized by a variety of ectoenzymes, producing free phosphate (Pi) or pyrophosphate (PPi) and promoting matrix mineralization. Ectoenzymes are differentially localized in cartilage and may be co-released with nucleotides during mechanical stimulation. Extracellular nucleotides can also serve as substrates and/or modulators of enzymes such as tissue transglutaminase and ecto-protein kinases that modify matrix proteins and regulate crystal deposition or growth. Understanding the evolution of osteoarthritis and calcium crystal deposition diseases will require clearer knowledge of the functions of nucleotides and ectoenzymes in the cartilage extracellular matrix.

Role of pericellular matrix in development of a mechanically functional neocartilage.

The role of the chondrocyte pericellular matrix (PCM) was examined in a three-dimensional chondrocyte culture system to determine whether retention of the native pericellular matrix could stimulate collagen and proteoglycan accumulation and also promote the formation of a mechanically functional hyaline-like neocartilage. Porcine chondrocytes and chondrons, consisting of the chondrocyte with its intact pericellular matrix, were maintained in pellet culture for up to 12 weeks. Sulfated glycosaminoclycans and type II collagen were measured biochemically. Immunocytochemistry was used to examine collagen localization as well as cell distribution within the pellets. In addition, the equilibrium compressive moduli of developing pellets were measured to determine whether matrix deposition contributed to the mechanical stiffness of the cartilage constructs. Pellets increased in size and weight over a 6-week period without apparent cell proliferation. Although chondrocytes quickly rebuilt a PCM rich in type VI collagen, chondron pellets accumulated significantly more proteoglycan and type II collagen than did chondrocyte pellets, indicating a greater positive effect of the native PCM. After 5 weeks in chondron pellets, matrix remodeling was evident by microscopy. Cells that had been uniformly distributed throughout the pellets began to cluster between large areas of interterritorial matrix rich in type II collagen. After 12 weeks, clusters were stacked in columns. A rapid increase in compressive strength was observed between 1 and 3 weeks in culture for both chondron and chondrocyte pellets and, by 6 weeks, both had achieved 25% of the equilibrium compressive stiffness of cartilage explants. Retention of the in vivo PCM during chondrocyte isolation promotes the formation of a mechanically functional neocartilage construct, suitable for modeling the responses of articular cartilage to chemical stimuli or mechanical compression.

Development of selective tolerance to interleukin-1beta by human chondrocytes in vitro.

Interleukin-1 induces release of NO and PGE(2) and production of matrix degrading enzymes in chondrocytes. In osteoarthritis (OA), IL-1 continually, or episodically, acts on chondrocytes in a paracrine and autocrine manner. Human chondrocytes in chondron pellet culture were treated chronically (up to 14 days) with IL-1beta. Chondrons from OA articular cartilage were cultured for 3 weeks before treatment with IL-1beta (0.05-10 ng/ml) for an additional 2 weeks. Spontaneous release of NO and IL-1beta declined over the pretreatment period. In response to IL-1beta (0.1 ng/ml), NO and PGE(2) release was maximal on Day 2 or 3 and then declined to near basal level by Day 14. Synthesis was recovered by addition of 1 ng/ml IL-1beta on Day 11. Expression of inducible nitric oxide synthase (iNOS), detected by immunofluorescence, was elevated on Day 2 and declined through Day 14, which coordinated with the pattern of NO release. On the other hand, IL-1beta-induced MMP-13 synthesis was elevated on Day 3, declined on Day 5, and then increased again through Day 14. IL-1beta increased glucose consumption and lactate production throughout the treatment. IL-1beta stimulated proteoglycan degradation in the early days and inhibited proteoglycan synthesis through Day 14. Chondron pellet cultures from non-OA cartilage released the same amount of NO but produced less PGE(2) and MMP-13 in response to IL-1beta than OA cultures. Like the OA, IL-1beta-induced NO and PGE(2) release decreased over time. In conclusion, with prolonged exposure to IL-1beta, human chondrocytes develop selective tolerance involving NO and PGE(2) release but not MMP-13 production, metabolic activity, or matrix metabolism.

Tissue transglutaminase localization and activity regulation in the extracellular matrix of articular cartilage.

Tissue transglutaminase (tTG) catalyzes a Ca2+-dependent transglutaminase (TGase) activity which cross-links proteins and stabilizes many tissues [C.S. Greenberg et al. FASEB J. 5 (1991) 3071]. Because cartilage is subjected to great stress in vivo, an enzyme that strengthens and stabilizes tissue could play an integral role in maintaining cartilage integrity. The purpose of this study was to determine if active tTG is present in the extracellular matrix (ECM) of adult human osteoarthritic articular cartilage. Using a TGase activity assay along with immunolabeling for tTG of cartilage sections, TGase activity and tTG immunoreactivity were localized in the ECM in cartilage sections, predominantly in the superficial layer. Previous in vitro studies have demonstrated that the Mg-GTP complex inhibits the TGase activity of tTG [T.S. Lai et al. J. Biol. Chem. 273 (1998) 1776]. To investigate the in situ regulation of the TGase activity of tTG, a TGase activity assay was done with a dose response of GTP, measuring incorporation of fluorescein cadaverine. TGase activity was inhibited by GTP in a similar manner as in vitro. These results not only confirm tTG presence in the ECM. but also indicate tTG as the major TGase activity of the ECM. Secondly, the study provides a possible mechanism by which extracellular tTG is regulated in vivo.