R. Suderman (Sharon, CA)

Lunenfeld Tanenbaum Research Institute

Presenter Of 1 Presentation

Poster Cartilage Imaging and Functional Testing

P013 - Viability and Metabolic Changes in Chondrocytes During Prolonged Storage Evaluated Using a Fluorescent Tri-Stain Method

Presentation Topic
Cartilage Imaging and Functional Testing
Date
13.04.2022
Lecture Time
09:30 - 09:30
Room
Exhibition Foyer
Session Name
7.3 - Poster Viewing / Coffee Break / Exhibition
Session Type
Poster Session
Disclosure
No Significant Commercial Relationship

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.icrs_fig1.jpg

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.

icrs_table1.jpg

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.

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Presenter Of 1 Presentation

Cartilage Imaging and Functional Testing

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.icrs_fig1.jpg

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.

icrs_table1.jpg

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.

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