title: Dose-injury relationships for cryoprotective agent injury to human chondrocytes.
abstract: Vitrification of articular cartilage (AC) could enhance tissue availability but requires high concentrations of cyroprotective agents (CPAs). This study investigated relative injuries caused by commonly used CPAs. We hypothesized that the in situ chondrocyte dose-injury relationships of five commonly used CPAs are nonlinear and that relative injuries could be determined by comparing cell death after exposure at increasing concentrations. Human AC samples were used from four patients undergoing total knee arthroplasty surgery. Seventy μm slices were exposed in a stepwise protocol to increasing concentrations of 5 CPAs (max = 8 M); dimethyl sulfoxide (Me(2)SO), glycerol (Gly), propylene glycol (PG), ethylene glycol (EG), and formamide (FM). Chondrocyte viability was determined by membrane integrity stains. Statistical analysis included t-tests and nonlinear least squares estimation methods. The dose-injury to chondrocytes relationships for all CPAs were found to be nonlinear (sigmoidal best fit). For the particular loading protocol in this study, the data identified the following CPA concentrations at which chondrocyte recoveries statistically deviated significantly from the control recovery; 1 M for Gly, 4 M for FM and PG, 6 M for Me(2)SO, and 7 M for EG. Comparison of individual means demonstrated that Gly exposure resulted in the lowest recovery, followed by PG, and then Me(2)SO, FM and EG in no specific order. The information from this study provides an order of damage to human chondrocytes in situ of commonly used CPAs for vitrification of AC and identifies threshold CPA concentrations for a stepwise loading protocol at which chondrocyte recovery is significantly decreased. In general, Gly and PG were the most damaging while DMSO and EG were among the least damaging.
authors.affiliation: Division of Orthopaedic Surgery, Department of Surgery, University of Alberta, Edmonton, AB, Canada.
authors.forename: M D
authors.initials: MD
authors.lastname: Fahmy
authors.affiliation: Division of Orthopaedic Surgery, Department of Surgery, University of Alberta, Edmonton, AB, Canada.
authors.forename: K A
authors.initials: KA
authors.lastname: Almansoori
authors.affiliation: Division of Orthopaedic Surgery, Department of Surgery, University of Alberta, Edmonton, AB, Canada.
authors.forename: L
authors.initials: L
authors.lastname: Laouar
authors.affiliation: Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB, Canada.
authors.forename: V
authors.initials: V
authors.lastname: Prasad
authors.affiliation: Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB, Canada.
authors.forename: L E
authors.initials: LE
authors.lastname: McGann
authors.affiliation: Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB, Canada; Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB, Canada.
authors.forename: J A W
authors.initials: JA
authors.lastname: Elliott
authors.affiliation: Division of Orthopaedic Surgery, Department of Surgery, University of Alberta, Edmonton, AB, Canada. Electronic address: njomha@ualberta.ca.
authors.forename: N M
authors.initials: NM
authors.lastname: Jomha
mesh_terms: D002358:Cartilage, Articular / Q000166:cytology / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects*; D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D019855:Ethylene Glycol / Q000494:pharmacology; D005559:Formamides / Q000494:pharmacology; D005990:Glycerol / Q000494:pharmacology; D006801:Humans; D061251:Primary Cell Culture; D019946:Propylene Glycol / Q000494:pharmacology; D058989:Vitrification
chemical_list: D003451:Cryoprotective Agents; D005559:Formamides; C031066:formamide; D019946:Propylene Glycol; D019855:Ethylene Glycol; D005990:Glycerol; D004121:Dimethyl Sulfoxide
keywords: Chondrocytes; Cryopreservation; Cryoprotective agents; Dose; Human; Injury; Vitrification