Survival of chondrocytes in cold-stored osteochondral grafts is believed to enhance the long-term performance of these tissues in reconstructive procedures. However, declining chondrocyte viability during the storage process compromises the potential advantage of transferring viable cells during the transplantation procedure. We therefore tested two hypotheses: (1) the composition of the media used during cold storage of osteochondral grafts would alter the viability of articular chondrocytes over time; and (2) the addition of recombinant insulin growth factor-1 (IGF-1) and/or the apoptosis inhibitor ZVAD-fmk to cell culture media would increase the survival of chondrocytes during cold storage of osteochondral grafts.
Several limitations of this study should be considered in interpreting our results. First, we used bovine osteochondral grafts rather than human grafts. Second, our data were limited to chondrocyte viability without any measures of metabolic function of those viable cells. Third, the exact composition of the commercial storage media provided by the Musculoskeletal Transplant Foundation is proprietary; therefore, the contribution of each component is difficult to assess. Nevertheless, our experimental data yielded a substantial number of important findings.
Lactated Ringer’s is an isotonic solution consisting of electrolytes and lactate but lacking any nutrients to sustain cellular growth. Historically, LR has been used for short-term graft storage, but its use in cartilage allograft banking has been largely discontinued in favor of other forms of nutrient-containing storage media. Our data confirm that the rapid decline in viability precludes the use of LR for longer-term storage.
One previous study of human osteochondral grafts demonstrated grafts stored in LR have a lower percent of viable chondrocytes in comparison to grafts stored in standard cell culture media [2
]. DMEM is a commonly used “minimal” cell culture media that contains basic nutrients capable of supporting cellular viability. Typical “minimal” cell culture media contain amino acids, salts, and glucose [19
]. Our data demonstrate the decline in chondrocyte viability of bovine osteochondral grafts stored in DMEM occurred at a more gradual rate in comparison to grafts stored in LR. However, mean viability still declined by approximately half after 2 weeks in cold storage and by greater than 85% after 4 weeks. In the United States, 2 weeks is typically the minimum amount of time required to complete testing of osteochondral grafts for microbial contamination. Therefore, these findings raise the question of whether “minimal” cell culture media such as DMEM is optimal for prolonged cold storage of osteochondral grafts. The extremely low chondrocyte viability observed in our study at later time points contrasts somewhat with data from similar studies using human osteochondral tissue that reported a chondrocyte viability from 27% to 83% after 28 days of cold storage [2
]. Bovine articular cartilage differs from human articular cartilage in terms of mechanical properties, cartilage thickness, and cell density [1
]. Therefore, some differences in its response to cold storage are not unexpected. Another contributing factor could be the studies with human specimens included storage media changes every 2 to 7 days, whereas no media changes were made in the present study in accordance with typical commercial storage protocols.
The storage media provided by a commercial tissue bank (Musculoskeletal Transplant Foundation) is based on DMEM/F-12 and contains FBS, HEPES buffer, antibiotics, and supplemental amino acids, vitamins, sugars, and salts. As expected, chondrocyte survival during cold storage in this media was higher than in either LR or DMEM. However, because of the complexity of the media and the unavailability of detailed information regarding the specific components, it is unclear which components are most responsible for the observed increase in chondrocyte survival. One component that likely contributed to improved chondrocyte viability was FBS. In a recent study on human osteochondral grafts, the addition of FBS augmented cell viability [13
]. However, the inclusion of FBS is not without disadvantages that include lack of consistency among different lots of serum, potential immunologic reactions, and potentially serious or even lethal infectious disease transmission [13
Theoretically, single well-characterized drugs such as recombinant growth factors could substitute for FBS. IGF-1 is an attractive candidate based on its ability to decrease chondrocyte cell death in other models of cartilage viability. IGF-1 is a key anabolic growth factor in normal cartilage tissue [9
]. We found chondrocyte viability in bovine osteochondral grafts declined at a slower rate in storage media supplemented with IGF-1 over the first 3 weeks of cold storage. However, no difference was observed after 4 weeks of storage. Because the media was not changed in these experiments, one possible explanation is the IGF-1 was degraded or otherwise inactivated during the storage period. This problem potentially could be overcome by repeated addition of IGF-1 throughout the storage period. However, any benefits of IGF-1 must be weighed against its primary disadvantage, cost. It is also likely additional, and potentially costly, agents would be necessary to fully substitute for FBS; therefore, the economic feasibility of developing a “defined” storage media that is equivalent or superior to media used commercially remains an unanswered question.
A recent study reported multiple key mediators of apoptosis are upregulated during cold storage of human osteochondral tissues [14
]. Theoretically, blockade of these mediators of apoptosis could decrease cell loss during cold storage. IGF-1 inhibits chondrocyte programmed cell death, which may contribute to its beneficial effects noted in our experiments [5
]. The prototypical apoptosis inhibitor ZVAD-fmk is a potent irreversible inhibitor of multiple caspases and has been used in previous studies of apoptosis inhibition in cartilage [3
]. The addition of ZVAD-fmk to DMEM increased chondrocyte viability compared with DMEM alone; however, we did not formally demonstrate the mechanism was prevention of apoptosis.
Our experiments using bovine osteochondral tissue provided data comparable to previous studies using human tissues; therefore, we believe bovine osteochondral grafts are a reasonable substitute for valuable human grafts in experiments testing parameters of graft storage. This study supports others that generally show an association between increased nutritional content of storage media with enhanced chondrocyte survival. The addition of recombinant IGF-1 and ZVAD-fmk to DMEM improved chondrocyte survival providing proof of concept for the development of a “defined” storage media that could exclude FBS with its associated problems. Extensive research will be needed to define the optimal components of a “defined” storage media because the combinatorial possibilities are essentially limitless. Nevertheless, we believe the end objective appears attainable.