Mechanical injury results in chondrocyte death in articular cartilage. The purpose of the present study was to determine whether medium osmolarity affects chondrocyte death in injured articular cartilage.
Osteochondral explants (n = 48) that had been harvested from the metacarpophalangeal joints of three-year-old cows were exposed to media with varying osmolarity (0 to 480 mOsm) for ninety seconds to allow in situ chondrocytes to respond to the altered osmotic environment. Explants were then wounded with a scalpel through the full thickness of articular cartilage, incubated in the same media for 2.5 hours, and transferred to 340-mOsm Dulbecco's Modified Eagle Medium (control medium) with further incubation for seven days. The spatial distribution of in situ chondrocyte death, percentage cell death, and marginal cell death at the wounded cartilage edge were compared as a function of osmolarity and time (2.5 hours compared with seven days) with use of confocal laser scanning microscopy.
In situ chondrocyte death was mainly localized to the superficial tangential zone of injured articular cartilage for the range of medium osmolarities (0 to 480 mOsm) at 2.5 hours and seven days. Therefore, a sample of articular cartilage from the superficial region (which included the scalpel-wounded cartilage edge) was studied with use of confocal laser scanning microscopy to compare the effects of osmolarity on percentage and marginal cell death in the superficial tangential zone. Compared with the control explants exposed to 340-mOsm Dulbecco's Modified Eagle Medium, percentage cell death in the superficial tangential zone was greatest for explants exposed to 0-mOsm (distilled water) and least for explants exposed to 480-mOsm Dulbecco's Modified Eagle Medium at 2.5 hours (13.0% at 340 mOsm [control], 35.5% at 0 mOsm, and 4.3% at 480 mOsm; p <or= 0.02 for paired comparisons) and seven days (9.9% at 340 mOsm [control], 37.7% at 0 mOsm, and 3.5% at 480 mOsm; p <or= 0.01 for paired comparisons). Marginal cell death in the superficial tangential zone decreased with increasing medium osmolarity at 2.5 hours (p = 0.001) and seven days (p = 0.002). There was no significant change in percentage cell death from 2.5 hours to seven days for explants initially exposed to any of the medium osmolarities.
Medium osmolarity significantly affects chondrocyte death in wounded articular cartilage. The greatest chondrocyte death occurs at 0 mOsm. Conversely, increased medium osmolarity (480 mOsm) is chondroprotective. The majority of cell death occurs within 2.5 hours, with no significant increase over seven days.
"CMFDA is permeable only to viable cells, and in the cytoplasm is metabolized by cellular esterases to produce a membrane-impermeant fluorescent product that stains the cytoplasm of viable cells green. PI however is only capable of crossing plasma membranes of dying/dead cells and stains the nuclei of the dead cells, red [Amin et al., 2008]. Bones were then fixed in 4% (v/v) paraformaldehyde overnight, and stored at 48C in PBS until visualized by confocal scanning laser microscopy (CLSM). "
"Only tibial plateau cartilage with intact SZ (grades 0/1) and underlying bone attached was used. Tissue blocks were trimmed to ∼2 mm × 5 mm × 5 mm,28 then incubated in DMEM with CTB (5 µM; 2 h; 37°C) to fluorescently label live in situ chondrocytes. Samples were snap-frozen in freezing hexane (−80°C), sections cut (25 µm; −25°C) using OCTC, mounted on silane-coated slides, and stored (−80°C) until required. "
[Show abstract][Hide abstract] ABSTRACT: Early osteoarthritis (OA) is poorly understood, but abnormal chondrocyte morphology might be important. We studied IL-1β and pericellular collagen type VI in morphologically normal and abnormal chondrocytes. In situ chondrocytes within explants from nondegenerate (grade 0/1) areas of human tibial plateaus (n = 21) were fluorescently labeled and visualized [2-photon laser scanning microscopy (2PLSM)]. Normal chondrocytes exhibited a "smooth" membrane surface, whereas abnormal cells were defined as demonstrating ≥1 cytoplasmic process. Abnormal chondrocytes were further classified by number and average length of cytoplasmic processes/cell. IL-1β or collagen type VI associated with single chondrocytes were visualized by fluorescence immuno-histochemistry and confocal laser scanning microscopy (CLSM). Fluorescence was quantified as the number of positive voxels (i.e., 3D pixels with fluorescence above baseline)/cell. IL-1β-associated fluorescence increased between normal and all abnormal cells in the superficial (99.7 ± 29.8 [11 (72)] vs. 784 ± 382 [15 (132)]; p = 0.04, positive voxels/cell) and deep zones (66.5 ± 29.4 [9 (64)] vs. 795 ± 224 [9 (56)]; p = 0.006). There was a correlation (r(2) = 0.988) between the number of processes/cell (0-5) and IL-1β, and an increase particularly with short processes (≤5 µm; p = 0.022). Collagen type VI coverage and thickness decreased (p < 0.001 and p = 0.005, respectively) with development of processes. Abnormal chondrocytes in macroscopically nondegenerate cartilage demonstrated a marked increase in IL-1β and loss of pericellular type VI collagen, changes that could lead to cartilage degeneration.
Journal of Orthopaedic Research 11/2010; 28(11):1507-14. DOI:10.1002/jor.21155 · 2.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This systematic review provides (1) a synthesis of existing clinical evidence that helps identify factors associated with the development of glenohumeral chondrolysis after arthroscopy (PAGCL), (2) a consolidated conceptualization of potential causal pathways that elucidate proposed mechanisms leading to PAGCL, and (3) a summary of implications for practice, policy, and future research.
A computerized literature review using an iterative search process identified 245 publications in English between January 1960 and April 2009. After we applied inclusion and exclusion criteria, 35 articles were stratified into 4 categories of factors related to PAGCL: (1) patient factors, (2) surgical factors (preoperative and intraoperative), (3) postoperative factors, and (4) causal pathways.
The majority of studies (61%) focused on surgical factors correlated with PAGCL, and most were laboratory based (n = 21). Publications involving human subjects were descriptive case reports (n = 15), not epidemiologic studies. A total of 88 patients (91 shoulder surgeries) with PAGCL were identified in case reports. The majority of patients (55%) was male, and the mean age was 27.9 years (range, 13.1 to 64 years). Among patients, 68% (n = 53) had implants/anchors, 67% (n = 59) received local anesthetics through a pain pump, and 45% (n = 41) had surgeries involving radiofrequency devices. The causal pathways to PAGCL likely involve initiating and secondary cartilage injury due to mechanical, thermal, or chemical events. The result is a cascade of interactive cellular responses that may include inflammation and chondrocyte apoptosis causing disturbance of cellular metabolism with subsequent loss of the gliding surface, congruity, and synovial fluid, leading to increased friction and accelerated wear that ultimately yield PAGCL.
The literature is limited to correlates, rather than true risk factors, for PAGCL. Well-designed epidemiologic studies that examine various exposures in relation to health outcomes, while controlling for potential confounders, are needed to determine relative risks that allow causal inference, thereby facilitating sound practice and policy decision making.
Level IV, systematic review.
Arthroscopy The Journal of Arthroscopic and Related Surgery 11/2009; 25(11):1329-42. DOI:10.1016/j.arthro.2009.06.001 · 3.21 Impact Factor
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