Partial-thickness cartilage injuries do not heal effectively, potentially leading to degeneration as occurs in post-traumatic osteoarthritis (PTOA). The role of chondrocytes could be crucial in determining the nature of the repair; however, their response to this injury is poorly understood. We have utilised an in vitro bovine osteochondral partial-thickness scalpel injury model and determined chondrocyte properties at and distant from the injury in the presence/absence of (a) serum-free DMEM (340 mOsm), (b) synovial fluid DMEM (SF-DMEM), (c) foetal calf serum DMEM (FCS-DMEM), (d) hyperosmolar serum-free DMEM (600 mOsm), or (e) hyperosmolar FCS-DMEM for up to two weeks. Chondrocytes were fluorescently-labelled with 5-chloromethylfluorescein-diacetate (CMFDA)/propidium iodide (PI) for live/dead cells and imaged using confocal microscopy. Quantitative data were obtained on chondrocyte properties (cell volume, clusters, morphology) at and distant from the injury. In serum-free DMEM, chondrocyte morphology at the injury remained unaffected throughout culture. However, with SF-DMEM or FCS-DMEM the chondrocytes displayed an increase in volume (p < 0.0001), cluster formation (FCS; p < 0.01) and abnormal morphology (p < 0.001) compared to serum-free DMEM. Cluster formation and shape changes during FCS-DMEM culture were more pronounced than with SF-DMEM. SF-DMEM or FCS-DMEM stimulated these changes to chondrocytes at the injury with only small effects on distant cells. Hyperosmolarity inhibited the morphological and volume changes to chondrocytes induced by FCS-DMEM (p < 0.001) and the injured cartilage had the appearance of that in serum-free DMEM. Raised osmolarity may therefore have benefit in preserving the morphological phenotype of chondrocytes at the site of injury, and thus promote more effective integrative repair in partial-thickness cartilage injury.