Marc J. Brouillette

Use of Recombinant Human Stromal Cell–Derived Factor 1α–Loaded Fibrin/Hyaluronic Acid Hydrogel Networks to Achieve Functional Repair of Full-Thickness Bovine Articular Cartilage Via Homing of Chondrogenic Progenitor Cells

Articular cartilage damage after joint trauma seldom heals and often leads to osteoarthritis. We previously identified a migratory chondrogenic progenitor cell (CPC) population that responds chemotactically to cell death and rapidly repopulates the injured cartilage matrix, which suggests a potential approach for articular cartilage repair. This study was undertaken to determine whether recombinant human stromal cell–derived factor 1α (rhSDF‐1α), a potent CPC chemoattractant, would improve the quality of cartilage regeneration, hypothesizing that increased recruitment of CPCs by rhSDF‐1α would promote the formation of cartilage matrix upon chondrogenic induction.

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.

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.

A validated model of the pro- and anti-inflammatory cytokine balancing act in articular cartilage lesion formation.

Traumatic injuries of articular cartilage result in the formation of a cartilage lesion and contribute to cartilage degeneration and the risk of osteoarthritis (OA). A better understanding of the framework for the formation of a cartilage lesion formation would be helpful in therapy development. Toward this end, we present an age and space-structured model of articular cartilage lesion formation after a single blunt impact. This model modifies the reaction-diffusion-delay models in Graham et al. (2012) (single impact) and Wang et al. (2014) (cyclic loading), focusing on the “balancing act” between pro- and anti-inflammatory cytokines. Age structure is introduced to replace the delay terms for cell transitions used in these earlier models; we find age structured models to be more flexible in representing the underlying biological system and more tractable computationally. Numerical results show a successful capture of chondrocyte behavior and chemical activities associated with the cartilage lesion after the initial injury; experimental validation of our computational results is presented. We anticipate that our in silico model of cartilage damage from a single blunt impact can be used to provide information that may not be easily obtained through in in vivo or in vitro studies.

Characteristics of meniscus progenitor cells migrated from injured meniscus.

Serious meniscus injuries seldom heal and increase the risk for knee osteoarthritis; thus, there is a need to develop new reparative therapies. In that regard, stimulating tissue regeneration by autologous stem/progenitor cells has emerged as a promising new strategy. We showed previously that migratory chondrogenic progenitor cells (CPCs) were recruited to injured cartilage, where they showed a capability in situ tissue repair. Here, we tested the hypothesis that the meniscus contains a similar population of regenerative cells. Explant studies revealed that migrating cells were mainly confined to the red zone in normal menisci: However, these cells were capable of repopulating defects made in the white zone. In vivo, migrating cell numbers increased dramatically in damaged meniscus. Relative to non‐migrating meniscus cells, migrating cells were more clonogenic, overexpressed progenitor cell markers, and included a larger side population. Gene expression profiling showed that the migrating population was more similar to CPCs than other meniscus cells. Finally, migrating cells equaled CPCs in chondrogenic potential, indicating a capacity for repair of the cartilaginous white zone of the meniscus. These findings demonstrate that, much as in articular cartilage, injuries to the meniscus mobilize an intrinsic progenitor cell population with strong reparative potential.

Targeting mitochondrial responses to intra-articular fracture to prevent posttraumatic osteoarthritis

Inhibiting mitochondrial oxidant production after surgical fixation of an intra-articular fracture prevents osteoarthritis in a porcine model. Osteoarthritis—A mitochondrial malady Articular cartilage—the smooth, avascular tissue that covers the bones in joints—can be damaged by traumatic injury, which can lead to osteoarthritis. In response to injury, chondrocytes ramp up mitochondrial activity, producing reactive oxygen species that can cause further tissue damage and cell death. Coleman and colleagues treated intra-articular fractures in a porcine model with an antioxidant or an inhibitor of the mitochondrial electron transport chain. Regulating mitochondrial metabolism prevented osteoarthritis. This work suggests that the mighty mitochondrion is a therapeutic target for posttraumatic osteoarthritis. We tested whether inhibiting mechanically responsive articular chondrocyte mitochondria after severe traumatic injury and preventing oxidative damage represent a viable paradigm for posttraumatic osteoarthritis (PTOA) prevention. We used a porcine hock intra-articular fracture (IAF) model well suited to human-like surgical techniques and with excellent anatomic similarities to human ankles. After IAF, amobarbital or N-acetylcysteine (NAC) was injected to inhibit chondrocyte electron transport or downstream oxidative stress, respectively. Effects were confirmed via spectrophotometric enzyme assays or glutathione/glutathione disulfide assays and immunohistochemical measures of oxidative stress. Amobarbital or NAC delivered after IAF provided substantial protection against PTOA at 6 months, including maintenance of proteoglycan content, decreased histological disease scores, and normalized chondrocyte metabolic function. These data support the therapeutic potential of targeting chondrocyte metabolism after injury and suggest a strong role for mitochondria in mediating PTOA.

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.

Linking Cellular and Mechanical Processes in Articular Cartilage Lesion Formation: A Mathematical Model

Post-traumatic osteoarthritis affects almost 20% of the adult US population. An injurious impact applies a significant amount of physical stress on articular cartilage and can initiate a cascade of biochemical reactions that can lead to the development of osteoarthritis. In our effort to understand the underlying biochemical mechanisms of this debilitating disease, we have constructed a multiscale mathematical model of the process with three components: cellular, chemical, and mechanical. The cellular component describes the different chondrocyte states according to the chemicals these cells release. The chemical component models the change in concentrations of those chemicals. The mechanical component contains a simulation of a blunt impact applied onto a cartilage explant and the resulting strains that initiate the biochemical processes. The scales are modeled through a system of partial-differential equations and solved numerically. The results of the model qualitatively capture the results of laboratory experiments of drop-tower impacts on cartilage explants. The model creates a framework for incorporating explicit mechanics, simulated by finite element analysis, into a theoretical biology framework. The effort is a step toward a complete virtual platform for modeling the development of post-traumatic osteoarthritis, which will be used to inform biomedical researchers on possible non-invasive strategies for mitigating the disease.

Differential Effects of Superoxide Dismutase Mimetics after Mechanical Overload of Articular Cartilage

Post-traumatic osteoarthritis can develop as a result of the initial mechanical impact causing the injury and also as a result of chronic changes in mechanical loading of the joint. Aberrant mechanical loading initiates excessive production of reactive oxygen species, oxidative damage, and stress that appears to damage mitochondria in the surviving chondrocytes. To probe the benefits of increasing superoxide removal with small molecular weight superoxide dismutase mimetics under severe loads, we applied both impact and overload injury scenarios to bovine osteochondral explants using characterized mechanical platforms with and without GC4403, MnTE-2-PyP, and MnTnBuOE-2-PyP. In impact scenarios, each of these mimetics provides some dose-dependent protection from cell death and loss of mitochondrial content while in repeated overloading scenarios only MnTnBuOE-2-PyP provided a clear benefit to chondrocytes. These results support the hypothesis that superoxide is generated in excess after impact injuries and suggest that superoxide production within the lipid compartment may be a critical mediator of responses to chronic overload. This is an important nuance distinguishing roles of superoxide, and thus superoxide dismutases, in mediating damage to cellular machinery in hyper-acute impact scenarios compared to chronic scenarios.

A Validated Model of the Pro- and Anti-in ammatory Cytokine Balancing Act in Articular Cartilage Lesion Formation under Mechanical Strain

Joint (articular) cartilage is designed to sustain certain level of impact. However, when a large enough force is applied, the cells in the cartilage release pro- and anti-inflammatory chemicals that can affect the cell population, the chemical feedback loop, as well as the cartilage itself, causing lesions. Modeling all these processes requires several components: cellular, chemical, and mechanical. The cellular component deals with the population densities of different cells and their interactions and transitions from one type into another. These transitions are induced by, and in turn affect, the chemicals which are released as a result of the impact. The first two components have already been modeled in published papers, using a nonlinear system of age-structured, parabolic PDEs with a radial spacial dimension. The next step is modeling the mechanics of the impact as more than a constant force term, but as a time-dependent effect on the physical and chemical processes in the cartilage. This requires an additional spacial dimension for the depth of the cartilage. Increasing the dimensions, besides adjusting the original equations accordingly, requires the addition of a system of hyperbolic PDEs, to model the effect of the impact using linear elasticity. The current model develops and solves the system numerically, and is validated by experimental results.