Vitrification of particulated articular cartilage via calculated protocols

Abstract

Preserving viable articular cartilage is a promising approach to address the shortage of graft tissue and enable the clinical repair of articular cartilage defects in articulating joints, such as the knee, ankle, and hip. In this study, we developed two 2-step, dual-temperature, multicryoprotectant loading protocols to cryopreserve particulated articular cartilage (cubes ~1 mm3 in size) using a mathematical approach, and we experimentally measured chondrocyte viability, metabolic activity, cell migration, and matrix productivity after implementing the designed loading protocols, vitrification, and warming. We demonstrated that porcine and human articular cartilage cubes can be successfully vitrified and rewarmed, maintaining high cell viability and excellent cellular function. The vitrified particulated articular cartilage was stored for a period of 6 months with no significant deterioration in chondrocyte viability and functionality. Our approach enables high-quality long-term storage of viable articular cartilage that can alleviate the shortage of grafts for use in clinically repairing articular cartilage defects.

A Cryoprotectant-Gel Composite Designed to Preserve Articular Cartilage during Frozen Osteoarticular Autograft Reconstruction for Malignant Bone Tumors: An Animal-Based Study

“…For CGC, the total concentration of cryoprotectants would be around 11.2 M, which is higher than VS55 and other proposed protocols. 14 , 15 , 18 , 42 According to experiments performed by Jomha and his colleagues, at 22 °C, around 34.6% and 66.8% of EG and around 39.5% and 75.4% of DMSO would permeate into porcine cartilage tissue at 5 minutes and 15 minutes, respectively. 35 The predicted permeated concentration of total cryoprotectants after applying CGC for 15 minutes would be more than 6.5 M in our porcine model, which met the concentration required to protect porcine articular cartilage mentioned in previous studies.…”

“… 35 The predicted permeated concentration of total cryoprotectants after applying CGC for 15 minutes would be more than 6.5 M in our porcine model, which met the concentration required to protect porcine articular cartilage mentioned in previous studies. 10 , 11 , 42 In the rat experiment, because the bone is smaller and the cartilage thinner than in the porcine model, the exposure time was shortened to 5 minutes.…”

Biomolecular Pathways of Cryoinjuries in Low-Temperature Storage for Mammalian Specimens

“…They can be preserved in two states: either at deep cryogenic temperatures in frozen/vitrified states with totally suspended animation for months or even years (cryopreservation usually refers to temperatures from −80 to −196 °C) or at hypothermic temperatures without phase transition with partially decreased animation for hours or days (hypothermic storage around 4 °C) [ 6 , 7 ]. Due to its indispensable role in the supply chain of biological specimens, biopreservation is ubiquitously used in biomedical and clinical research and applications, such as assisted reproduction [ 8 , 9 , 10 ], stem cell therapy [ 11 , 12 ], regenerative medicine [ 13 ], tissue engineering [ 14 ], and mRNA medicine [ 15 ]. However, mammalian specimens under low-temperature preservation are far from their normal physiological conditions, which inevitably induces biophysical and biochemical changes and injuries to the preserved cells.…”

Vitrified Particulated Articular Cartilage for Joint Resurfacing: A Swine Model

“… 7 , 9 , 19 , 25 Multiple attempts have been made to cryopreserve AC; however, it was not until recently that successful cryopreservation protocols were developed. 7 , 9 , 18 , 19 , 37 , 39 Optimized protocols for the long-term storage of osteochondral dowels 18 and more recently particulated AC cubes (~1-mm 3 cubes) have been developed. 37 Based on extensive previous research on the vitrification of AC, # mathematical modeling has been used to identify and screen potential loading parameters that included cryoprotectant concentration, exposure time, toxicity, and temperature.…”

“… 37 Based on extensive previous research on the vitrification of AC, # mathematical modeling has been used to identify and screen potential loading parameters that included cryoprotectant concentration, exposure time, toxicity, and temperature. 39 Protocols were tested on swine and human particulated cartilage to ensure that chondrocyte viability was maintained (>80% viable) after vitrification and rewarming. The optimized vitrification protocol for particulated AC is a 2-step dual-temperature method that uses multiple cryoprotectants.…”