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Bone and soft-tissue allografts processing and safety

By Steven Gitelis, MD, and Ross Wilkins, MD

Human bone and soft-tissue allografts are increasingly being used in surgery. The Musculoskeletal Transplant Foundation reports that more than 900,000 allografts are used each year in the United States. However, all allografts are not created equally. Surgeons must be familiar with their tissue processing bank and its procedures.

Although disease transmission by an allograft is rare, it can be devastating. Contaminated allografts have been associated with patient mortality, although this is rare; the last reported case was in 2001. Tissue bank processes have a significant impact on the risk of disease transmission. A general knowledge of how bone and soft-tissue allografts are recovered and processed is critical for the surgeons so they can provide orthopaedic patients with the tools to make an informed decision and be an active participant in the care plan.

The difference between an aseptic (free of pathogenic micro-organisms) and a sterile (free of all micro-organisms) graft may also be confusing. The AAOS strongly encourages members to use only tissue from banks that have been inspected by the U.S. Food and Drug Administration and that rigorously adhere to the standards of the American Association of Tissue Banks (AATB). This article provides an update on tissue procurement, screening, processing, and the effect of these techniques on the allograft performance for orthopaedic surgeons and other providers.

Donor screening
When a tissue recovery bank is notified of a potential donor, it first obtains the necessary information to determine donor suitability. A detailed medical/social history is obtained from the donor’s next of kin or from someone who can provide reliable donor historical information. This evaluation consists of more than 50 questions on medical history, high-risk social behavior, unusual environmental exposure, and medical conditions such as cancer. The decision to accept or reject the donor tissue is based on the responses to these questions.

If the donor is suitable, a tissue procurement team is sent to acquire the tissue from the operating room, morgue, or tissue bank facility. The tissue needs to be obtained aseptically within 24 hours of the donor’s death, using techniques similar to those used by orthopaedic surgeons in the operating room and under strict guidelines imposed by AATB accreditation.

The donor’s limbs are prepped and draped using standard surgical principles, and the tissue is recovered by an experienced surgical staff. Many types of tissues can be recovered, including stem cells, bone, soft tissues (tendon, ligament) and skin (Fig. 1). All grafts are cultured prior to processing to determine the level of bacterial contamination (Fig. 2). Fresh articular cartilage is initially refrigerated and then placed in a culture medium to maintain its viability for up to 28 days (Fig. 3).

If tissue culture results indicate the presence of “virulent organisms” such as Clostridia species, enterococci, or fungi, the tissues are discarded. Typically, the recovery/procurement and preprocessing cultures are taken using a swab-culturing technique. Although swab culture methodology is helpful in determining tissue contamination, it is not completely reliable and will give both false positive and false negative results. Thus, as part of the final sterility tests, newer techniques, such as bacterial extraction, are used to detect any low levels of bacterial growth.

Blood is also drawn when the tissues are procured, and a series of serologic tests—including screening for human immunodeficiency virus (HIV), hepatitis B and C, and syphilis are performed. Because a potential window exists when serologic tests may give a false negative for an infected donor, nucleic acid tests for the viral antigens are now being used to diminish the risk of disease transmission. The reported risk of HIV transmission is less than one in one million with current testing procedures. Similarly, the transmission risk for the other mentioned viruses is also extremely rare.

Tissue processing
Tissue can either be processed aseptically or be secondarily “sterilized.” This requirement for tissue sterility differs from that imposed for medical devices because typical sterilization processes (high-dose radiation and harsh chemical treatments) have deleterious effects on tissue properties. To be considered sterile, allografts should have fewer than 10-6 microorganisms; for medical devices, the requirement is fewer than 10-3 microorganisms.

Various methods may be used to achieve sterilization, including combinations of washing with or without pressurization, centrifuging with various chemicals such as alcohol or detergents, and combining antibiotics with low-dose radiation. Regardless of the technique used, the objective is to remove all infectious elements with minimal impact on the performance of the allograft.

After the grafts are cleansed and processed, they are packaged and stored. The designated shelf life depends on the packaging and storage methods used (such as deep frozen, freeze-dried, or fresh).

Grafts are stored at the tissue bank during the quarantine period until the required serologic and bacteriologic test results and autopsy reports (when required) are received. The tissue bank Medical Director then makes a determination of donor eligibility before releasing the graft.

At this time the allograft is safe for human implantation. Once it is received at the surgical facility, it must be logged in and tracked as required by Joint Commission regulations.

Impact of processing on allografts
Various methods of processing and fashioning are used in the production of tissue allografts. Grafts such as tendons or pieces of bone for skeletal reconstruction, which are used in structural situations, have to be processed in a manner that does not significantly alter their inherent mechanical properties. These grafts are cleaned and processed using low-dose irradiation, a chemical technique, or both to achieve “sterility.”

Allografts may also be processed aseptically without any secondary attempts at sterilization. These grafts must be rigorously controlled for bacterial contamination and are usually stored in a frozen state. Freezing an allograft has little impact on the mechanical properties of the tissue and will diminish its immunogenicity (Table 1). Freezing will affect the viability of articular cartilage unless some form of cryopreservation is employed.

Tissue can also be freeze dried. Freeze drying further diminishes the immunogenicity of the graft and may be helpful in eliminating the bacterial contamination (Table 2). It does however, have some negative effect on the mechanical strength of the grafts, especially if rehydration is not done properly in the operating room. Gamma radiation is effective in killing bacteria, fungi, spores, and, to a lesser degree, viruses. Depending on the dose, however, gamma radiation can weaken the graft. Doses below 1.5 mrad do not seem to adversely affect the tissue strength.

“Nonstructural” products, which do not require mechanical properties, include demineralized or “filler” products to encourage new bone growth. An assay of the bioactivity of demineralized bone matrix may be available and should be considered when choosing an inductive product. These products can be processed either sterile or aseptically. Gamma irradiation and excessive heat (> 60 degrees C) is known to damage bone proteins. Freezing or freeze drying does not appear to affect the healing properties of bone.

Conclusion
In considering the use of allograft tissue for orthopaedic reconstruction procedures, the surgeon must understand both the source and the processing techniques used to produce the graft. Only then can he or she take advantage of this valuable material to rebuild and replace skeletal structures biologically.

Steven Gitelis, MD, and Ross Wilkins, MD, are members of the AAOS Biological Implants Committee. Dr. Gitelis can be reached at sgitelis@rush.edu; Dr. Wilkins can be reached at DrRMW@aol.com

Disclosure information: Dr. Gitelis—Wright Medical Technology, Inc.; Dr. Wilkins—Wright Medical Technology, Inc., allosource, AATB

AAOS Now
May 2011 Issue
http://www.aaos.org/news/aaosnow/may11/research7.asp