R. Suderman (Sharon, CA)
Lunenfeld Tanenbaum Research InstitutePresenter Of 1 Presentation
P013 - Viability and Metabolic Changes in Chondrocytes During Prolonged Storage Evaluated Using a Fluorescent Tri-Stain Method
Abstract
Purpose
The Missouri Osteochondral Allograft Preservation System (MOPS) has been shown to extend the period of acceptable cell viability compared to the standard of care (SOC). Metabolic changes and mechanisms by which cells remain viable cannot be evaluated using common viability assessments. This study aimed to compare metabolic changes in chondrocytes after storage in MOPS or SOC conditions using a tri-stain method.
Methods and Materials
Ovine femoral condyles were stored using MOPS (proprietary media, 22-25°C storage) or SOC [Lactated Ringer’s solution, cefazolin (1 g/L), bacitracin (50,000 U/L), 4°C storage]. Cartilage was stained concurrently with Calcein-AM, Ethidium Homodimer-1, and Mitotracker and imaged using a confocal microscope following dissection (fresh control) or after 56 days of storage. Morphological features, including presence of a Mitotracker boundary (Fig. 1B-E) was used to develop a grading system (Fig. 1) ranging from Grade-1, representing highly metabolic and viable cells, to Grade 5, representing dead cells with no metabolic activity. Results were reported as percentage of total cells per grade, with % live calculated by summation of Grades 1 and 2 (no Ethidium Homodimer present). T-tests were performed.
Results
1513 cells were graded in 9 samples (Table 1). When compared to SOC, MOPS samples displayed higher percentage of living cells and Grade-2 cells, as well as less Grade-3 and Grade-4 cells. When compared to controls, both 56-day storage groups had significantly lower Grade-1 cells and significantly higher Grade-5 cells. The mechanism causing the Mitotracker boundary (Fig. 1B-E) is unknown with a possible explanation of intracellular organelle transfer due to cell stress signaling.
Conclusion
MOPS maintained more viable chondrocytes than SOC after 56 days of storage. The grading system revealed changes in metabolic activity and viability resulting from storage. The three-stain approach and novel grading system allowed categorization beyond the live/dead binary and may contribute to understanding how chondrocytes respond to prolonged storage.
Presenter Of 1 Presentation
P013 - Viability and Metabolic Changes in Chondrocytes During Prolonged Storage Evaluated Using a Fluorescent Tri-Stain Method
Abstract
Purpose
The Missouri Osteochondral Allograft Preservation System (MOPS) has been shown to extend the period of acceptable cell viability compared to the standard of care (SOC). Metabolic changes and mechanisms by which cells remain viable cannot be evaluated using common viability assessments. This study aimed to compare metabolic changes in chondrocytes after storage in MOPS or SOC conditions using a tri-stain method.
Methods and Materials
Ovine femoral condyles were stored using MOPS (proprietary media, 22-25°C storage) or SOC [Lactated Ringer’s solution, cefazolin (1 g/L), bacitracin (50,000 U/L), 4°C storage]. Cartilage was stained concurrently with Calcein-AM, Ethidium Homodimer-1, and Mitotracker and imaged using a confocal microscope following dissection (fresh control) or after 56 days of storage. Morphological features, including presence of a Mitotracker boundary (Fig. 1B-E) was used to develop a grading system (Fig. 1) ranging from Grade-1, representing highly metabolic and viable cells, to Grade 5, representing dead cells with no metabolic activity. Results were reported as percentage of total cells per grade, with % live calculated by summation of Grades 1 and 2 (no Ethidium Homodimer present). T-tests were performed.
Results
1513 cells were graded in 9 samples (Table 1). When compared to SOC, MOPS samples displayed higher percentage of living cells and Grade-2 cells, as well as less Grade-3 and Grade-4 cells. When compared to controls, both 56-day storage groups had significantly lower Grade-1 cells and significantly higher Grade-5 cells. The mechanism causing the Mitotracker boundary (Fig. 1B-E) is unknown with a possible explanation of intracellular organelle transfer due to cell stress signaling.
Conclusion
MOPS maintained more viable chondrocytes than SOC after 56 days of storage. The grading system revealed changes in metabolic activity and viability resulting from storage. The three-stain approach and novel grading system allowed categorization beyond the live/dead binary and may contribute to understanding how chondrocytes respond to prolonged storage.