Effects of Cryopreservation on the Depth Dependent Elastic Modulus in Articular Cartilage and Implications for Osteochondral Grafting

Abstract

Cryopreservation of articular cartilage is often used in storage of experimental samples and osteochondral grafts, but the depthdependence and concentration of glycosaminoglycan (GAG) are significantly altered when cryogenically stored without a cryoprotectant, which will reduce cartilage stiffness and affect osteochondral graft function and longterm viability. This study investigates our ability to detect changes due to cryopreservation in the depthdependent elastic modulus of osteochondral samples. Using a directvisualization method requiring minimal histological alterations, unconfined stepwise stress relaxation tests were performed on four fresh (never frozen) and three cryopreserved (−20 آ°C) canine humeral head osteochondral slices 125 آ±â€‰5 خ¼m thick. Applied force was measured and tissue images were taken at the end of each relaxation phase using a 4أ— objective. Intratissue displacements were calculated by tracking chondrocytes through consecutive images for various intratissue depths. The depthdependent elastic modulus was compared between fresh and cryopreserved tissue for samedepth ranges using analysis of variance (ANOVA) with Tukey posttest with a 95% confidence interval. Cryopreservation was found to significantly alter the force–displacement profile and reduce the depthdependent modulus of articular cartilage. Excessive collagen fiber folding occurred at 40–60% relative depth, producing a “black lineâ€‌ in cryopreserved tissue. Force–displacement curves exhibited elongated toeregion in cryopreserved tissue while fresh tissue had nonmeasurable toeregion. Statistical analysis showed significant reduction in the elastic modulus and GAG concentration throughout the tissue between samedepth ranges. This method of cryopreservation significantly reduces the depthdependent modulus of canine humeral osteochondral samples.