Improved Preservation of Fresh Osteochondral Allografts for Clinical Use
Fresh osteochondral allografts (OCAs) have been used clinically to treat cartilage defects in the knee for over 30 years.1 The major advantages of OCAs over other currently available “biologic” treatment options for cartilage defects of the knee include implantation of hyaline cartilage and bone in a graft that is site- and size-matched with tissue architecture and material properties that can withstand the loads normally transmitted to the joint. Fresh OCA grafting has a reported 5- to 10-year functional survival rate of 75 to 85% for treatment of focal defects of the femoral condyle (FC).2–4 Additionally, the longevity of the fresh OCA tissue after implantation has been documented to be as long as 25 years,3 indicating that this procedure can provide a long-term solution for treatment of osteochondral defects. The factor most consistently reported to influence the long-term success of OCAs is the viability of chondrocytes in the transplanted tissue.3,5,6 However, in the United States, fresh OCA tissue has only been available commercially since 1998, so much of the long-term data are from centers that could harvest tissue from cadaveric donors, process and store the tissue, and perform the transplant procedure at a single site. In these centers, tissues were harvested from the donor and used for OCA transplant within 24 to 72 hours,1,3 which allowed for optimal maintenance of chondrocyte viability and tissue biochemical and biomechanical properties. However, concerns regarding disease transmission stimulated the implementation of a mandatory disease and contamination testing period of 14 days once tissue banks in the United States made OCAs commercially available.
With the 14-day testing period requirement, tissue banks had to develop protocols for storing OCAs in a way that would maintain sterility and chondrocyte viability until they could be delivered and implanted.
Initially, OCAs were stored in lactated Ringer solution (LRS) at 4°C based on standard protocol.1 However, chondrocyte viability in OCAs stored in LRS at 4°C rapidly declines to ∼ 60% of at-harvest levels by day 7,7,8 and ∼ 20% by day 147 after harvest. This essentially renders the grafts unusable for clinical patients. To maintain chondrocyte viability in OCAs for clinically relevant time periods, researchers have applied tissue preservation, rather than storage, methods to this problem by using cell culture protocols for OCAs.7–18
Use of various culture media preparations to preserve OCAs at 4°C has been reported to significantly improve chondrocyte viability compared with LRS.7,14,17,19 These media preparations can be separated into those that include fetal bovine serum (FBS)12,20,21 and those that do not.22 When OCA tissues were preserved in culture media without FBS, chondrocyte viability was maintained at 54 to 97% of at-harvest levels through 14 days of storage.7,8 However, chondrocyte viability dropped significantly to only 15 to 70% of at-harvest levels by day 28 of storage.7,12,14,23 When FBS was added to media, chondrocyte viability was maintained at 80 to 86% and 45 to 80% of at-harvest levels at days 14 and 28 postharvest, respectively. Chondrocyte viability levels were significantly higher with FBS when directly compared with FBS-free media at all time points.7,12,13,20 As such, the addition of FBS to media used to preserve OCAs at 4°C is the current standard of care for most tissue banks. However, even with the use of FBS, there is still a significant loss of cell viability by day 28 postharvest, especially in the superficial zone of the cartilage.13,19,20,22 With the mandatory disease testing period, this leaves a “clinical window for use” of only 14 days, which can be problematic when considering sizing, shipping, and scheduling requirements for tissue banks, surgeons, and patients. In addition, concerns regarding batch-to-batch variability and potential for zoonotic disease transmission and contamination associated with FBS make its use less desirable. Therefore, recent research efforts have focused on methods for improving chondrocyte viability of OCAs to extend their clinical window for use.
A significant increase in apoptotic gene expression has been observed in OCA tissues stored at 4°C,24 indicating that loss of chondrocyte viability observed during storage is at least partially due to apoptosis. There is evidence that indirectly blocking apoptosis using etanercept to block TNF-α signaling, a proapoptotic pathway, significantly improves chondrocyte viability in the superficial zone.13 However, total chondrocyte viability in the tissue was not significantly different from untreated controls at 28 days. Similarly, another study found that storing OCA tissue with ZVAD-fmk, a potent apoptosis inhibitor, did not improve chondrocyte viability through 28 days of storage.7 These data indicate that blocking the apoptosis pathway alone does not effectively increase the clinical window for use of OCAs.
As with the transition from LRS to cell culture medium and the addition of FBS, researchers continue to look to cell culturing procedures to improve maintenance of cell viability of OCA tissue during storage. Several groups have looked at cryopreservation of the tissue at subzero temperatures in various cryoprotectant formulations,8,25–34 commonly used for long-term storage of cells for culture. However, no study has shown consistent maintenance of chondrocyte viability in OCA tissue, especially in OCAs of sizes that are typically used clinically. Further, cryopre-served tissues have performed poorly when transplanted into in vivo animal models.32,34 Therefore, cryopreservation of OCA tissue is not currently considered a viable option for clinical use.
Conversely, numerous studies have reported promising results with respect to chondrocyte viability when OCAs are preserved at 37°C, the standard temperature for in vitro cell and tissue culture, when compared with 4°C.11,16,19,35–38 OCAs preserved at 37°C have consistently had significantly higher chondrocyte viability at day 28 compared with those preserved at 4°C.19,35,36
Importantly, these studies have also indicated that supplementation with FBS may not be required to maintain high levels of chondrocyte viability of the OCA preserved at 37°C tissue.35–37 In one study, there was no significant difference in chondrocyte viability between OCAs preserved with or without FBS at 37°C through 28 days after harvest.36 Another group used insulin, transferrin, and selenious acid to supplement the media in place of FBS, and reported no reduction in chondrocyte viability through 28 days in storage.37 Our group has reported maintenance of viable chondrocyte density at harvest levels through 56 days of preservation of OCAs at 37°C using a proprietary medium that does not include FBS (U.S. Patent pending).35 Therefore, it is possible to preserve osteochondral tissues with clinically applicable chondrocyte viability levels at 37°C without FBS supplementation, removing the possibility for zoonotic transfer of disease and the potential variability in storage media composition that can arise from differences in the variable composition of FBS39 while effectively tripling the clinical window of use for OCAs.
While the data for 37°C preservation of OCAs have been promising, it has not been adopted as the standard of care protocol for tissue banks. This is at least in part due to the concern that preservation of tissues at 37°C will increase the risk of bacterial and fungal contamination. While higher contamination rates have not been reported to date, there has not been a study specifically designed to determine if OCA preservation at 37°C results in higher rates of contamination, to the authors’ knowledge. The other significant hurdle for 37°C storage of OCAs is the associated costs of equipment, supplies, personnel, and training. The transition from preservation in refrigerators to CO2 incubators would be substantial. Therefore, the associated gain in OCA quality would have to be significant for tissue banks to make this investment and transition. If a safe and effective methodology for OCA preservation could be developed that did not require these large investments, its adoption might be more appealing to tissue banks.
Despite advances in OCA preservation methodology, a remaining problem affecting clinical efficacy of this procedure is related to the significant interspecimen variability in chondrocyte viability of OCAs at the time of implantation.22 For example, chondrocyte viability levels at 28 days postharvest in OCAs preserved using the same protocol in the same laboratory have ranged from 27 ± 13% to 70 ± 11%.12–14 Similarly, the highest levels of chondrocyte viability at 28 days posthar-vest, 60 to 70% have been reported using methods with and without FBS and at both 4 and 37°C.12–14,36 While this variability may be related to differences in harvest timing or technique, animal model used, or viability assessment technique, the inter- and intra-study variability in chondrocyte viability of OCAs is indicative of true variability in OCA tissues retrieved from a spectrum of organ donors by different harvest teams in different locations, which can have significant impact on graft quality and patient outcomes. Currently, OCA chondrocyte viability at the time of implantation is based on published reference ranges based on postharvest time point. This means that for the typical OCA sent from the tissue bank at 21 to 28 days postharvest, chondrocyte viability could be less than 25% to greater than 90% depending on all associated variables. Unfortunately, the tissue bank, surgeon, and patient currently have no idea what the viability of each graft is at the time of implantation. Because chondrocyte viability is known to influence outcomes for OCA transplantation, it is critical that we develop methods for determining viability in each graft. If the viability of OCAs can be accurately determined nondestructively before transplantation into the patient, samples with unacceptable viability can be discarded, improving success rates and decreasing costs associated with OCA surgery.
In an attempt to address the current limitations in consistently providing tissues of appropriate quality and quantity for use in osteochondral allografting procedures, our goal was to develop a preservation methodology that would maximize chondrocyte viability, minimize disease transmission and contamination potential, allow for nondestructive viability testing, and avoid large financial costs associated with preservation technique. Our hypothesis was that OCA tissues could be preserved at room temperature (∼ 25°C) without CO2 supplementation for at least 56 days with chondrocyte viability maintained at > 70% of at-harvest levels. Further, we hypothesized that we could nondestructively determine chondrocyte viability in OCAs using a novel assay technique that would strongly correlate (r > 0.7) to the “gold standard” technique of fluorescent microscopy.
Materials and Methods
Tissue Harvest and Culture
All procedures were performed under Animal Care and Use Committee approval. During the course of two studies, medial and lateral FCs from both knees of 14 adult canine cadavers were aseptically harvested within 4 hours of euthanasia performed for reasons unrelated to this study. The FCs were either used as time 0 (at harvest) controls (n = 7) or separated into one of the five test groups based on proprietary media composition (M-1, M-2, and M-3) and container condition (C-1, C-2, C-3) (U.S. Patent pending) such that each FC from a single animal was placed into a distinct group. The following media and container condition groupings were assessed for study one: M-1/C-1 (n = 7), M-1/C-2 (n = 4), M-1/C-3 (n = 7), and M-2/C-1 (n = 5). For study two the media and container condition groupings were M-1/C-1 (n = 5), M-1/C-3 (n = 5), and M-3/C-3 (n = 8), resulting in the five different OCA storage groups. The M-1 medium was designed to provide basic tissue nutrition, the M-2 medium was designed to be antidegradative, and the M-3 medium was designed to be anti- inflammatory. Tissues were stored at 25°C without CO2 supplementation in 60 mL of media for 63 days. The media were changed every 7 days and saved for biomarker analyses. At the end of the storage, osteochondral plugs were evaluated for tissue viability.