Articular cartilage is recalcitrant to endogenous repair and regeneration and thus a focus of tissue engineering and regenerative medicine strategies. A pre-requisite for articular cartilage tissue engineering is an understanding of the signal transduction pathways involved in mechanical compression during trauma or disease. We sought to explore the role of the extracellular signal-regulated kinase 1/2 (ERK 1/2) pathway in chondrocyte proliferation and proteoglycan synthesis following acute mechanical compression. Bovine articular cartilage explants were cultured with and without the ERK 1/2 pathway inhibitor PD98059. Cartilage explants were statically loaded to 40% strain at a strain rate of 1−sec for 5 seconds. Control explants were cultured under similar conditions but were not loaded. There were four experimental groups: 1) no load without inhibitor 2) no load with the inhibitor PD98059, 3) loaded without the inhibitor, and 4) loaded with the inhibitor PD98059. Explants were cultured for varying durations, from 5 minutes to 5 days. Explants were then analyzed by biochemical and immunohistochemical methods. Mechanical compression induced phosphorylation of ERK 1/2, and this was attenuated with the ERK 1/2 pathway inhibitor PD98059 in a dose-dependent manner. Chondrocyte proliferation was increased by mechanical compression. This effect was blocked by the inhibitor of the ERK 1/2 pathway. Mechanical compression also led to a decrease in proteoglycan synthesis that was reversed with inhibitor PD98059. In conclusion, the ERK 1/2 pathway is involved in the proliferative and biosynthetic response of chondrocytes following acute static mechanical compression.

Objective. To demonstrate posttraumatic chondrocyte apoptosis in the murine xiphoid after a crush-type injury and to ultimately determine the pathway (i.e., intrinsic or extrinsic) by which chondrocytes undergo apoptosis in response to mechanical injury. Design. The xiphoids of adult female wild-type mice were injured with the use of a modified Kelly clamp. Postinjury xiphoid cartilage was analyzed via 3 well-described independent means of assessing apoptosis in chondrocytes: hematoxylin and eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and activated caspase-3 staining. Results. Injured specimens contained many chondrocytes with evidence of apoptosis, which is characterized by cell shrinkage, chromatin condensation, nuclear fragmentation, and the liberation of apoptotic bodies. There was a statistically significant increase in the number of chondrocytes undergoing apoptosis in the injured specimens as compared with the uninjured specimens. Conclusions. Chondrocytes can be stimulated to undergo apoptosis as a result of mechanical injury. These experiments involving predominantly cartilaginous murine xiphoid in vivo establish a baseline for future investigations that employ the genetic and therapeutic modulation of chondrocyte apoptosis in response to mechanical injury.

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Traumatic joint injury is a significant cause of osteoarthritis. At the cellular level, injurious mechanical compression to articular cartilage leads to decreased proteoglycan synthesis and increased chondrocyte proliferation. The molecular pathways leading to these biochemical and cellular changes remain poorly understood. We explored the role of the ERK 1/2 pathway following injurious mechanical compression to bovine articular cartilage explants. Specifically, we investigated the involvement of the ERK 1/2 pathway in chondrocyte proliferation and proteoglycan synthesis.MethodsFollowing explantation and processing, bovine articular cartilage explants were cultured with and without the ERK 1/2 pathway inhibitor PD98059. Explants were then statically loaded to 40% strain at a strain rate of 1-sec to produce peak stresses of ˜35 MPa. Loading was maintained for 5 seconds to simulate an acute injurious event. Control explants were cultured under similar conditions but were not loaded. Thus, four experimental groups were created; 1) no load without inhibitor, 2) loaded without inhibitor, 3) no load with the inhibitor PD98059, and 4) loaded with the inhibitor PD98059. Explants were cultured for 0-120 hours after loading. Explants were then analyzed both biochemically and immunohistochemicaly. Western blot was used to determine the presence of phospho-ERK 1/2 at selected time points. 35SO4 incorporation was utilized as a measure of proteoglycan biosynthesis. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) was used as a marker of cellular proliferationResultsInjurious mechanical compression induced activation of the ERK 1/2 pathway via phosphorylation, with activation beginning at 30 minutes and returning to baseline at 48 hours. This activation was attenuated with the ERK 1/2 pathway inhibitor PD98059. Loading of explants resulted in increased chondrocyte proliferation and decreased proteoglycan biosynthesis. Addition of PD98059 to the culture medium of loaded explants led to significantly less chondrocyte proliferation following loading. This was observed at all time points. Furthermore, with application of inhibitor, the decrease in proteoglycan synthesis seen following loading was reversed.ConclusionsThe ERK 1/2 pathway is an integral component of the chondrocyte proliferative response and proteoglycan biosynthetic response following acute mechanical injury to articular cartilage.

Traumatic joint injury is a significant cause of osteoarthritis. At the cellular level, injurious mechanical compression to articular cartilage leads to decreased proteoglycan synthesis and increased chondrocyte proliferation. The molecular pathways leading to these biochemical and cellular changes remain poorly understood. We explored the role of the ERK 1/2 pathway following injurious mechanical compression to bovine articular cartilage explants. Specifically, we investigated the involvement of the ERK 1/2 pathway in chondrocyte proliferation and proteoglycan synthesis. Methods: Following explantation and processing, bovine articular cartilage explants were cultured with and without the ERK 1/2 pathway inhibitor PD98059. Explants were then statically loaded to 40% strain at a strain rate of 1 -sec to produce peak stresses of ~35 MPa. Loading was maintained for 5 seconds to simulate an acute injurious event. Control explants were cultured under similar conditions but were not loaded. Thus, four experimental groups were created; 1) no load without inhibitor, 2) loaded without inhibitor, 3) no load with the inhibitor PD98059, and 4) loaded with the inhibitor PD98059. Explants were cultured for 0-120 hours after loading. Explants were then analyzed both biochemically and immunohistochemicaly. Western blot was used to determine the presence of phospho-ERK 1/2 at selected time points. 35 SO4 incorporation was utilized as a measure of proteoglycan biosynthesis. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) was used as a marker of cellular proliferation Results: Injurious mechanical compression induced activation of the ERK 1/2 pathway via phosphorylation, with activation beginning at 30 minutes and returning to baseline at 48 hours. This activation was attenuated with the ERK 1/2 pathway inhibitor PD98059. Loading of explants resulted in increased chondrocyte proliferation and decreased proteoglycan biosynthesis. Addition of PD98059 to the culture medium of loaded explants led to significantly less chondrocyte proliferation following loading. This was observed at all time points. Furthermore, with application of inhibitor, the decrease in proteoglycan synthesis seen following loading was reversed. Conclusions: The ERK 1/2 pathway is an integral component of the chondrocyte proliferative response and proteoglycan biosynthetic response following acute mechanical injury to articular cartilage.