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

Articular cartilage is the load bearing, low friction tissue covering the ends of bones in synovial joints and can withstand great forces by distributing stress through compressive displacement [1,2]. This highly complex tissue is composed mainly of water, collagen fibers, and proteoglycans made of negatively charged GAG molecules attached to a protein core. The collagen fibers in articular cartilage are anisotropically organized into three zones along its thickness in a depth-dependent manner. From the articular surface (AS) to the cartilage–bone interface, or tidemark (TM), the zones include a superficial zone (SZ), transitional zone (TZ), and radial zone (RZ) in which collagen fibers are oriented parallel, randomly, and perpendicular to the AS, respectively. In healthy articular cartilage, molecular properties such as collagen content and GAG concentration increase through tissue depth [3,4] and give rise to increasing depth-dependent elastic modulus [5–8] through hydraulic and osmotic pressure, and electronegative repulsion of GAG molecules [9–11]. Over time, cartilage function can diminish due to disease such as osteoarthritis (OA), which affects most of the adult human population [12,13]. Patients experiencing advanced OA symptoms, such as chronic joint pain and stiffness, but who do not require full joint replacement, may instead select an osteochondral grafting procedure. A major shortcoming for a procedure of this nature is cryopreservation of osteochondral plugs prior to implantation. The depth-dependence and concentration of GAG are significantly altered due to physical or biochemical damage when stored without a cryoprotectant such as dimethyl sulfoxide, a chemical that protects against cryodamage but has high cellular toxicity [4]. Since the static load bearing capability of cartilage is highly dependent on GAG concentration [7], a reduction in GAG concentration could affect osteochondral graft function. Improper load distribution in cryopreserved cartilage may also have effects on chondrocyte survival. For example, Muldrew et al. found 1 yr after transplanting cryopreserved osteochondral grafts, chondrocytes were nonexistent in the intermediate zone (approximately 50% relative tissue depth) [14,15]. Reduced cartilage stiffness may cause overloading of chondrocytes, eventually causing annihilation. The understanding of intratissue load distribution is of great importance in cryobiology. Techniques used in cartilage repair need to promote long-term health benefits, and an understanding of depth-dependent compressive changes due to cryopreservation of cartilage is vital. By using a technique developed by Szarko and Xia [8], it is possible to visually track intratissue chondrocyte displacement using minimally altered tissue samples to obtain quantitative depth-dependent stiffness measurements. We hypothesize the reduction in depth-dependent GAG concentration will significantly reduce the depth-dependent elastic modulus, an incident that may lead to abnormal cartilage mechanics and decreased osteochondral graft viability. This investigation aims to measure the depth-dependent modulus alteration using one osteochondral cryopreservation method and to verify our ability to detect those changes. Gaining an understanding of cryopreservation effects on the depth-dependent modulus will aid in determining the most effective way to preserve osteochondral grafts.