title: Viability and volume of in situ bovine articular chondrocytes-changes following a single impact and effects of medium osmolarity.
abstract: OBJECTIVE
  Mechanical stress above the physiological range can profoundly influence articular cartilage causing matrix damage, changes to chondrocyte metabolism and cell injury/death. It has also been implicated as a risk factor in the development of osteoarthritis (OA). The mechanism of cell damage is not understood, but chondrocyte volume could be a determinant of the sensitivity and subsequent response to load. For example, in OA, it is possible that the chondrocyte swelling that occurs renders the cells more sensitive to the damaging effects of mechanical stress. This study had two aims: (1) to investigate the changes to the volume and viability of in situ chondrocytes near an injury to cartilage resulting from a single blunt impact, and (2) to determine if alterations to chondrocyte volume at the time of impact influenced cell viability.
  
  
  METHODS
  Explants of bovine articular cartilage were incubated with the fluorescent indicators calcein-AM and propidium iodide permitting the measurement of cell volume and viability, respectively, using confocal laser scanning microscopy (CLSM). Cartilage was then subjected to a single impact (optimally 100g from 10 cm) delivered from a drop tower which caused areas of chondrocyte injury/death within the superficial zone (SZ). The presence of lactate dehydrogenase (LDH; an enzyme released following cell injury) was used to determine the effects of medium osmolarity on the response of chondrocytes to a single impact.
  
  
  RESULTS
  A single impact caused discrete areas of chondrocyte injury/death which were almost exclusively within the SZ of cartilage. There appeared to be two phases of cell death, a rapid phase lasting approximately 3 min, followed by a slower progressive 'wave of cell death' away from the initial area lasting for approximately 20 min. The volume of the majority (88.1+/-5.99% (n=7) of the viable chondrocytes in this region decreased significantly (P<0.006). By monitoring LDH release, a single impact 5 min after changing the culture medium to hyper-, or hypo-osmolarity, reduced or stimulated chondrocyte injury, respectively.
  
  
  CONCLUSIONS
  A single impact caused temporal and spatial changes to in situ chondrocyte viability with cell shrinkage occurring in the majority of cells. However, chondrocyte shrinkage by raising medium osmolarity at the time of impact protected the cells from injury, whereas swollen chondrocytes were markedly more sensitive. These data showed that chondrocyte volume could be an important determinant of the sensitivity and response of in situ chondrocytes to mechanical stress.
authors.affiliation: School of Biomedical and Clinical Laboratory Sciences, University Medical School, Hugh Robson Building, George Square, University of Edinburgh, Edinburgh EH8 9XD, Scotland, UK.
authors.forename: Peter G
authors.initials: PG
authors.lastname: Bush
authors.forename: Peter D
authors.initials: PD
authors.lastname: Hodkinson
authors.forename: Georgina L
authors.initials: GL
authors.lastname: Hamilton
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Hall
mesh_terms: D000818:Animals; D002358:Cartilage, Articular / Q000201:enzymology / Q000293:injuries / Q000473:pathology* / Q000503:physiopathology; D002417:Cattle; D048429:Cell Size; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000201:enzymology / Q000473:pathology* / Q000502:physiology; D007091:Image Processing, Computer-Assisted; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D018613:Microscopy, Confocal; D009994:Osmolar Concentration; D013314:Stress, Mechanical; D016474:Weight-Bearing / Q000502:physiology*
chemical_list: D007770:L-Lactate Dehydrogenase