Timing is of the essence for allograft transplants

Theodore I. Malinin, MD, advises surgeons to obtain osteochondral allografts from tissue banks that perform all necessary laboratory tests.

Issue: June 2006

ByDouglas W. Jackson, MD

Dr. Theodore I. Malinin, director of the tissue bank at the University of Miami, is a major contributor in the field of allograft transplants in orthopedics. I have read Ted’s publications over his professional career and have had the pleasure of interacting with him professionally. We are fortunate to be able to publish his succinct responses to what I feel are pertinent questions on the current use of osteochondral allografts. As many more surgeons are turning to and/or considering osteochondral allografts in select patients, the information and insight he shares should be very helpful.

Douglas W. Jackson, MD: What does cold storage of an osteochondral allograft entail?

Theodore I. Malinin, MD [photo]
Theodore I. Malinin

Theodore I. Malinin, MD: Cold storage entails placement of an allograft in a balanced salt solution, tissue culture media, tissue culture media with serum, etc., and maintaining the same at refrigerator temperatures, usually 2°C to 6°C. This procedure is used to keep tissues in a viable state short-term so they can be transplanted in an optimal state. Freezing of cartilage produces irreversible damage. However, metabolic activity of tissues maintained at hypothermia is still present, although metabolism proceeds at a reduced rate. Therefore, storage time at hypothermia is finite.

An autograft transplanted into a canine knee after 20 days of storage in a tissue culture medium. The surface is irregular and the center of the graft is liquefied.

Images: Malinin TI.

Within a certain period, metabolites become exhausted and cartilage becomes first irreversibly injured, then necrotic. It is important to know when irreversible injury occurs and to transplant cartilage before this takes place. If an osteoarticular autograft or allograft is stored too long, it will disintegrate during transplantation, as has been repeatedly shown in a variety of animal models.

Storage of tissues at hypothermia is not a new concept. Surgeons have gained extensive experience with the technique over the years with skin, corneas and other tissues.

Jackson: What is the clinical significance of chondrocyte survival on the success of a transplant?

Malinin: Cartilage is a complex structure and not just an accumulation of chondrocytes. Obviously, to be transplantable, cartilage must contain a critical number of viable cells and an intact matrix. Precisely what percentage of viable living cartilage cells are required to sustain cartilage transplant is not known, however.

In vitro techniques that differentiate between viable and nonviable chondrocytes are poor predictors of transplant survival. Integrity of the cartilage as a whole, and revascularization of subchondral bone, are prerequisites for the “take” of osteochondral grafts. Revascularization of subchondral bone and, thus, the nutrient supply of the cartilage are needed for long-term survival of transplanted cartilage. (Malinin TI, Ouelette EA. Osteoarth & Cartilage. 2000;8:483).

Jackson: What are some of the unique cellular and matrix properties that change with time of cold storage?

Malinin: The changes are gradual and concomitant. There is a progressive loss of chondrocytes. After 20 days of storage, about one half of the chondrocytes disappear, but even if chondrocytes are present, the cartilage integrity might be distorted. There is also a loss in the ability of cartilage to grow in tissue culture.

Image A shows an allograft transplanted into a knee of a baboon after 21 days of cold storage. Image B is an osteochondral allograft transplanted after 28 days of cold storage. Images C and D show histological sections with the presence of chondrocytes in the degenerated cartilage; distribution of Safranin-positive material is uneven, while E shows how most chondrocytes have been lost.

Tinctorial properties of the matrix also undergo a change. By the 14th or 15th day, the intensity of Safranin O staining is diminished. By 20 days, the cartilage plate is frequently separated from the subchondral bone. Thereafter, the matrix is stained only weakly or not at all.

Jackson: What are your main concerns, and how does a surgeon obtain grafts within three weeks of harvesting?

Malinin: First of all, the surgeon must have confidence in the graft he or she is about to transplant. Several surgeons recently observed graft failures in cartilage grafts transplanted after more than 20 days of storage. These are manifested either as cartilage degeneration, pitting or fragmentation. These kinds of problems are avoidable.

Since there are no reliable laboratory tests to show whether or not the cartilage is transplantable, we must rely on storage time limitations. It is entirely possible and feasible to obtain osteochondral allografts for transplantation stored for less than three weeks. This in no way compromises the safety of the graft. Results of serologic tests are available within 72 hours, as are preliminary results from an autopsy.

The problem lies with microbiological testing. Allografts can be repeatedly sampled for microbiological studies during the first two days after the excision, with the first multiple samples obtained at the time of excision. Even if one uses thioglycolate culture medium, which is usually incubated for 14 days, there is still sufficient time to transplant the graft within three weeks.

On the other hand, if one uses Columbia broth/trypticase soy broth blood culture media, which require only seven days of incubation, the results become available much sooner than the 14-day limit, a time framework which appears to be a turning point in allograft becoming nontransplantable.

So, in summary, surgeons are advised to obtain osteochondral allografts from tissue banks that will perform all necessary laboratory testing designed to safeguard the recipient within the time framework of allograft transplantability.