Prem S. Ramakrishnan

Oxidant conditioning protects cartilage from mechanically induced damage.

Articular cartilage degeneration in osteoarthritis has been linked to abnormal mechanical stresses that are known to cause chondrocyte apoptosis and metabolic derangement in in vitro models. Evidence implicating oxidative damage as the immediate cause of these harmful effects suggests that the antioxidant defenses of chondrocytes might influence their tolerance for mechanical injury. Based on evidence that antioxidant defenses in many cell types are stimulated by moderate oxidant exposure, we hypothesized that oxidant preconditioning would reduce acute chondrocyte death and proteoglycan depletion in cartilage explants after exposure to abnormal mechanical stresses. Porcine cartilage explants were treated every 48 h with tert‐butyl hydrogen peroxide (tBHP) at nonlethal concentrations (25, 100, 250, and 500 µM) for a varying number of times (one, two, or four) prior to a bout of unconfined axial compression (5 MPa, 1 Hz, 1800 cycles). When compared with untreated controls, tBHP had significant positive effects on post‐compression viability, lactate production, and proteoglycan losses. Overall, the most effective regime was 100 µM tBHP applied four times. RNA analysis revealed significant effects of 100 µM tBHP on gene expression. Catalase, hypoxia‐inducible factor‐1alpha (HIF‐1α), and glyceraldehyde 6‐phosphate dehydrogenase (GAPDH) were significantly increased relative to untreated controls in explants treated four times with 100 µM tBHP, a regime that also resulted in a significant decrease in matrix metalloproteinase‐3 (MMP‐3) expression. These findings demonstrate that repeated exposure of cartilage to sublethal concentrations of peroxide can moderate the acute effects of mechanical stress, a conclusion supported by evidence of peroxide‐induced changes in gene expression that could render chondrocytes more resistant to oxidative damage.

Selection of reference genes for normalization of quantitative real-time PCR in organ culture of the rat and rabbit intervertebral disc

BackgroundThe accuracy of quantitative real-time RT-PCR (qRT-PCR) is often influenced by experimental artifacts, resulting in erroneous expression profiles of target genes. The practice of employing normalization using a reference gene significantly improves reliability and its applicability to molecular biology. However, selection of an ideal reference gene(s) is of critical importance to discern meaningful results. The aim of this study was to evaluate the stability of seven potential reference genes (Actb, GAPDH, 18S rRNA, CycA, Hprt1, Ywhaz, and Pgk1) and identify most stable gene(s) for application in tissue culture research using the rat and rabbit intervertebral disc (IVD).FindingsIn vitro, four genes (Hprt1, CycA, GAPDH, and 18S rRNA) in rat IVD tissue and five genes (CycA, Hprt1, Actb, Pgk1, and Ywhaz) in rabbit IVD tissue were determined as most stable for up to 14 days in culture. Pair-wise variation analysis indicated that combination of Hprt1 and CycA in rat and the combination of Hprt1, CycA, and Actb in rabbit may most stable reference gene candidates for IVD tissue culture.ConclusionsOur results indicate that Hprt1 and CycA are the most stable reference gene candidates for rat and rabbit IVD culture studies. In rabbit IVD, Actb could be an additional gene employed in conjunction with Hprt1 and CycA. Selection of optimal reference gene candidate(s) should be a pertinent exercise before employment of PCR outcome measures for biomedical research.

Mechanical stress and ATP synthesis are coupled by mitochondrial oxidants in articular cartilage

Metabolic adaptation of articular cartilage under joint loading is evident and matrix synthesis seems to be critically tied to ATP. Chondrocytes utilize the glycolytic pathway for energy requirements but seem to require mitochondrial reactive oxygen species (ROS) to sustain ATP synthesis. The role of ROS in regulating ATP reserves under a mechanically active environment is not clear. It is believed that physiological strains cause deformation of the mitochondria, potentially releasing ROS for energy production. We hypothesized that mechanical loading stimulates ATP synthesis via mitochondrial release of ROS. Bovine osteochondral explants were dynamically loaded at 0.5 Hz with amplitude of 0.25 MPa for 1 h. Cartilage response to mechanical loading was assessed by imaging with dihydroethidium (ROS indicator) and a Luciferase‐based ATP assay. Electron transport inhibitor rotenone and mitochondrial ROS scavenger MitoQ significantly suppressed mechanically induced ROS production and ATP synthesis. Our findings indicate that mitochondrial ROS are produced as a result of physiological mechanical strains. Taken together with our previous findings of ROS involvement in blunt impact injuries, mitochondrial ROS are important contributors to cartilage metabolic adaptation and their precise role in the pathogenesis of osteoarthritis warrants further investigation.

Rat spinal motion segment in organ culture: a cell viability study.

Study Design. This study investigated tissue integrity and viability of cells in an organ culture system of intervertebral disc (IVD) with adjoining vertebral bodies. Objective. The goal of this study was to design a methodology to maintain an IVD motion segment in organ culture, thereby preserving viability and tissue architecture. Summary of Background Data. Study of IVD mechanobiology in vitro necessitates availability of vertebral bodies for controlled application of complex loads. Methods. IVD motion segments were dissected from rat lumbar segments and maintained in organ culture and cell viability was evaluated histochemically using NitroBlue Tetrazolium. Tissue integrity and morphology were evaluated using conventional histologic techniques. Results. The in vitro organ culture of motion segments maintained the viability and tissue integrity for 14 days. More than 95% viability in all three regions of interest (anulus fibrosus, nucleus pulposus, end plates) was maintained for 14 days in culture. Conclusion. Our initial results suggest that long-term motion segment culture is practical, and the inclusion of vertebral bodies will facilitate anchoring during biomechanical stimulation. Thus, we expect the culture system to provide us with an excellent model for studying the pathomechanics of IVD degeneration and the effects of mechanical stimulation on the biology of IVD cells.

Injurious Loading of Articular Cartilage Compromises Chondrocyte Respiratory Function.

To determine whether repeatedly overloading healthy cartilage disrupts mitochondrial function in a manner similar to that associated with osteoarthritis (OA) pathogenesis.

Biocompatibility and preclinical feasibility tests of a temperature-sensitive hydrogel for the purpose of surgical wound pain control and cartilage repair

We recently introduced a novel pluronic F127 and hyaluronic acid-based hydrogel (HG) designed to deliver a broad range of therapeutics. The reverse-thermal responsive HG exhibits physical properties that seem to be ideal for the local delivery of drug- and cell-based therapies to specific anatomic sites through percutaneous injection. However, questions related to the HGs safety and efficacy must first be addressed. To address these issues, we performed standard in vitro cytotoxicity and drug release tests and in vivo biocompatibility tests in a rat model. In addition, we determined whether the HG was an effective stem cell carrier in a rat cartilage defect model. We found that the HG showed viability and biocompatibility levels similar to those reported for F127 or hyaluronic acid alone. In vitro drug release studies with bupivacaine, a drug used clinically for local pain relief, revealed that after an initial burst bupivacaine was released continuously for 10 days. Stem cells loaded in the HG were retained in situ and stimulated cartilage regeneration in experimental defects. Taken as a whole, these findings support further efforts to develop the HG as a versatile system for the delivery of a wide range of therapeutic agents in humans.

Organ culture stability of the intervertebral disc: Rat versus rabbit

There is a need to develop mechanically active culture systems to better understand the role of mechanical stresses in intervertebral disc (IVD) degeneration. Motion segment cultures that preserve the native IVD structure and adjacent vertebral bodies are preferred as model systems, but rapid ex vivo tissue degeneration limits their usefulness. The stability of rat and rabbit IVDs is of particular interest, as their small size makes them otherwise suitable for motion segment culture. The goal of this study was to determine if there are substantial differences in the susceptibility of rat and rabbit IVDs to culture‐induced degeneration. Lumbar IVD motion segments were harvested from young adult male Sprague–Dawley rats and New Zealand White rabbits and cultured under standard conditions for 14 days. Biochemical assays and safranin‐O histology showed that while glycosaminoglycan (GAG) loss was minimal in rabbit IVDs, it was progressive and severe in rat IVDs. In the rat IVD, GAG loss was concomitant with the loss of notochordal cells and the migration of endplate (EP) cells into the nucleus pulposus (NP). None of these changes were evident in the rabbit IVDs. Compared to rabbit IVDs, rat IVDs also showed increased matrix metalloproteinase‐3 (MMP‐3) and sharply decreased collagen type I and II collagen expression. Together these data indicated that the rabbit IVD was dramatically more stable than the rat IVD, which showed culture‐related degenerative changes. Based on these findings we conclude that the rabbit motion segments are a superior model for mechanobiologic studies.

Towards a new spatial representation of bone remodeling.

Irregular bone remodeling is associated with a number of bone diseases such as osteoporosis and multiple myeloma. Computational and mathematical modeling can aid in therapy and treatment as well as understanding fundamental biology. Different approaches to modeling give insight into different aspects of a phenomena so it is useful to have an arsenal of various computational and mathematical models. Here we develop a mathematical representation of bone remodeling that can effectively describe many aspects of the complicated geometries and spatial behavior observed. There is a sharp interface between bone and marrow regions. Also the surface of bone moves in and out, i.e. in the normal direction, due to remodeling. Based on these observations we employ the use of a level-set function to represent the spatial behavior of remodeling. We elaborate on a temporal model for osteoclast and osteoblast population dynamics to determine the change in bone mass which influences how the interface between bone and marrow changes. We exhibit simulations based on our computational model that show the motion of the interface between bone and marrow as a consequence of bone remodeling. The simulations show that it is possible to capture spatial behavior of bone remodeling in complicated geometries as they occur in vitro and in vivo. By employing the level set approach it is possible to develop computational and mathematical representations of the spatial behavior of bone remodeling. By including in this formalism further details, such as more complex cytokine interactions and accurate parameter values, it is possible to obtain simulations of phenomena related to bone remodeling with spatial behavior much as in vitro and in vivo. This makes it possible to perform in silica experiments more closely resembling experimental observations.

Oxidative Conditioning and Treatment for Osteoarthritis

Oxidative stress is associated with aging and is also implicated as a contributing factor in osteoarthritis, a degenerative joint disease resulting mainly due to progressive degradation of the articular cartilage. Avascular and aneural in nature, articular cartilage is a unique tissue thriving in a mechanically active environment that requires physical stimuli to maintain tissue health. Cartilage adaptation is achieved by modulating matrix synthesis and other protective biological features in an effort to tolerate increased mechanical demands. One potential mechano-transductive pathway that regulates functional adaptation is believed to be driven by oxidative stress, but the exact mechanisms of this phenomenon are not clear. As an important rate-limiting factor for cartilage metabolism, sublethal levels of oxidants play a protective role against injurious mechanical loads, probably by modulating multiple biochemical pathways that increase stress tolerance thresholds of cartilage. Antioxidant status, nuclear factor (NF-κB), and hypoxia-inducible factor (HIF-1α) are potential factors that may play a role in oxidant conditioning and cartilage adaptation.

Biomechanical disc culture system: feasibility study using rat intervertebral discs.

A small-scale biomechanical disc culture system was designed to stimulate intervertebral disc (IVD) ‘motion segment’ in culture environment with load-controlled compression and combined load (compression + shear). After 7 days of diurnal mechanical loading, cell viability of discs stimulated with static compression load (0.25 MPa) and static combined load (compression (0.25 MPa) + shear (1.5 N)) were similar (> 90 per cent) to unloaded controls. Mechanically stimulated discs showed decrease in static/dynamic moduli, early stress relaxation, and loss of disc height after 7 days of diurnal loading. Histological data of discs indicated load-induced transformations that were not apparent in controls. The feasibility of studying the mechanobiology of intact IVD as a motion segment was demonstrated. Media conditioning (improve tissue stability in long-term culture) and application of biochemical gene expression assays (differential tissue response to types of mechanical stimulation) are proposed as future improvements. The study suggests that the limitations in studying mechanobiology of IVD pathology in vitro can be overcome and it is possible to understand the physiologically relevant mechanism of IVD pathology.