Nadr M. Jomha

Background: The use of particulated articular cartilage for repairing cartilage defects has been well established, but its use is currently limited by the availability and short shelf life of donor cartilage. Vitrification is an ice-free cryopreservation technology at ultralow temperatures for tissue banking. An optimized vitrification protocol has been developed for particulated articular cartilage; however, the equivalency of the long-term clinical efficacy of vitrified particulated articular cartilage compared with fresh articular cartilage has not yet been determined. Hypothesis: The repair effect of vitrified particulated cartilage from pigs would be equivalent to or better than that of fresh particulated cartilage stored at 4°C for 21 days. Study Design: Controlled laboratory study. Methods: A total of 19 pigs were randomly divided into 3 experimental groups: fresh particulated cartilage group (n = 8), vitrified particulated cartilage group (n = 8), and negative control group (no particulated cartilage in the defect; n = 3). An additional pig was used as the initial cartilage donor for the first set of surgical procedures. Pigs were euthanized after 6 months to obtain femoral condyles, and the contralateral condyle was used as the positive (no defect) control. Samples were evaluated for gross morphology using the Outerbridge and Osteoarthritis Research Society International (OARSI) scoring systems, histology (safranin O, collagen type I/II, DAPI), and chondrocyte viability using live-dead membrane integrity staining. Results: There were no infections after surgery, and all 19 pigs were followed for the duration of the study. The OARSI grades for the fresh and vitrified particulated cartilage groups were 2.44 ± 1.35 and 2.00 ± 0.80, respectively, while the negative control group was graded significantly higher at 4.83 ± 0.29. Analysis of histological and fluorescent staining demonstrated that the fresh and vitrified particulated cartilage groups had equivalent regeneration within cartilage defects, with similar cell viability and densities and expression of proteoglycans and collagen type I/II. Conclusion: The implantation of fresh or vitrified particulated cartilage resulted in the equivalent repair of focal cartilage defects when evaluated at 6 months after surgery. Clinical Relevance: The vitrification of particulated cartilage is a viable option for long-term storage for cartilage tissue banking and could greatly increase the availability of donor tissue for transplantation.

Articular cartilage (AC) injuries do not heal primarily and large lesions progress to degenerative osteoarthritis. Osteochondral allograft transplantation is an effective surgical treatment but is limited by the lack of donor tissue availability. Fresh allografts can be stored hypothermically up to 28–45 days after which the tissue is no longer viable for transplantation. Vitrification is a method of cryopreservation with the potential to extend the storage time of AC. A specific protocol has been demonstrated to preserve high chondrocyte viability; however, its effect on various mechanical properties of the extracellular matrix (ECM) remains unknown and is the focus of this initial study. Porcine AC was subject to a defined vitrification protocol, using fresh and frozen samples as positive and negative controls, respectively; n = 20 for all three groups. Unconfined compression testing was used to assess mechanical properties of the tissue under rapid load, stress relaxation, and equilibrium conditions. The stress relaxation time constants (modeled with a 2-term Prony series) [Formula: see text] and [Formula: see text] were significantly lower for frozen ( p = 0.014, p < 0.001) and vitrified ( p = 0.009, p = 0.003) tissue compared to fresh, with no differences between frozen and vitrified samples ( p = 0.848 and 0.105 for [Formula: see text] and [Formula: see text], respectively). These values indicate that frozen and vitrified samples relaxed more rapidly than fresh, which may suggest altered matrix composition and permeability post-treatment. These results represent the initial study in our experimental path to evaluate differences in mechanical properties of vitrified tissues.