Allograft Tissue Safety and Technology

Disclosure Statement

Dr. Moore, Mr. Samsell, and Dr. McLean are employees of LifeNet Health, a nonprofit organization.

Introduction

Biologic tissues are among the many clinical options available to orthopedic surgeons, with over 1 million annual implants of human allografts alone. Such biologic tissues may be considered structural, for example, tendons or cortical bone struts, or nonstructural, such as ground demineralized bone matrices (DBMs) or amniotic membranes. Their intended use may mimic their original anatomical function, such as using a patellar ligament for anterior cruciate ligament (ACL) reconstruction or a cortical bone segment to repair a long-bone fracture. In other circumstances, their use may differ from original anatomy, such as using a dermal layer for rotator cuff repair or a ground amniotic membrane combined with demineralized cortical bone for a spine fusion procedure. This chapter focuses on the intent and science of various means of processing human allograft tissues to ensure safety and clinical use.

Although xenografts, derived from nonhuman sources, have found clinical use in a variety of surgical disciplines, including the use of porcine heart valves for heart valve replacement surgery and ground bovine bone for dental procedures, their use in orthopedic surgery has met with more limited use and success. In particular, porcine-derived small intestinal submucosa did not perform well as a tissue augmentation material, and bovine tendons are not commonly used ; thus, xenografts will not be discussed in this chapter.

Although autografts, taken directly from the patient, are widely used for procedures such as ACL reconstruction or spinal implant of an iliac crest segment, these grafts are not subject to any significant processing other than some cleaning, shaping, or suture attachment before intrasurgery transplantation and therefore are also not covered in this chapter. Other autograft materials, such as those used for autologous cartilage implantation or blood-derived preparations such as platelet-rich plasma, are described elsewhere in this book.

Before being used by the orthopedic surgeon, allografts are commonly processed through physical, chemical, or biochemical means. Such processing steps are typically performed to accomplish one or more objectives such as:

to reduce risk of disease transmission (e.g., through various disinfection or sterilization steps);
to reduce immunogenic response (e.g., through decellularization);
to reduce barriers to optimal physiological activity (e.g., by demineralizing cortical bone to enhance bioavailability of growth factors);
to physically convert grafts into more usable forms (e.g., shaping a bone graft for placement as an intervertebral body spacer);
to combine grafts with synthetics to enhance ease of use (e.g., by combining ground demineralized bone with a carrier to produce a putty-like material);
to preserve tissue to increase shelf life or simplify storage (e.g., lyophilization of ground bone that enables retention at ambient temperatures).

With the focus here on human allografts, a brief historical and regulatory perspective is provided as background, followed by sections on reduction of disease transmission, enhancing fusion potential of bone void fillers, lowering immunogenic response by decellularization, preservation methods, and future directions.

Background

Human allografts have been used in orthopedic surgery for many decades. In the 19th century William Macewen described the successful use of allogeneic cortical bone fragments to graft a replacement for a missing humeral mid-shaft. Over 100 years ago, Fred Albee published a long list of surgical uses of bone allografts including applications and stated, “ [I have] been able during the past 2 years to avoid entirely the use of metal … for internal bone fixation purposes … made possible, largely, by utilizing the best of well known mechanical devices hitherto rarely, if at all, used in surgery, such as bone inlays, wedges, dowels, tongue and groove joints, mortised and dove-tailed joints. ”

Through the 20th century bone grafts continued to be used, and tendon allografts started gaining widespread acceptance by the 1980s. During the mid- to late-20th century, most “bone banks” for allografts were hospital-based with tissue derived from deceased or amputated patients. These tissues often underwent only minimal chemical processing, such as antibiotic or disinfectant soaks. In addition, any physical alteration of these tissues was typically performed by the surgical team at the time of implantation and included bone shaping or grinding or tendon trimming or suturing. In answer to the need for better defined methods for ensuring graft safety and consistency, as well as appropriate respect for the tissue donors and their families, more formal systems began to be established by the Navy Tissue Bank in Bethesda, MD.

Tissue Bank Standards and Regulation

In the latter half of the 20th century, the methodologies and practices developed by the Navy Tissue Bank were increasingly adopted by other organizations. In 1976 the American Association of Tissue Banks (AATB) was formed, and in 1984 it issued the first set of tissue-processing standards, which established guidelines such as acceptable time from death to recovery, storage conditions for tissues, microbial testing requirements, definitions of demineralization, freeze-drying, and so forth. A certification program was also established to assure the surgical community that qualified tissue banks met AATB standards.

Similarly, around the turn of the 21st century, the US Food and Drug Administration (FDA) developed the classification of Human Cell and Tissue Products (HCT/Ps) as a separate regulatory classification into which most human tissue transplants fell. This classification applies to most human tissue transplants. It differs from a medical device classification, in which, to qualify as an HCT/P, the tissue needs to meet standards of not exceeding “minimal manipulation” such that the “original relevant characteristics” are not altered, and also “homologous use” meaning the tissue is used clinically in a similar manner to that intended in the body of origin. A clear example of a tissue meeting these requirements would be a hamstring tendon that is recovered intact, disinfected, sterilized, and then used for tendon replacement. Conversely, although bone void filler putty containing DBM would be considered for homologous use, it may be considered more than minimally manipulated because of the addition of a synthetic carrier and thus be classified as a medical device requiring FDA clearance before distribution. In further example, a disinfected and freeze-dried cortical bone segment used as an intervertebral body spacer is considered both minimally manipulated and intended for homologous use. As processors continue to become more innovative in the use and treatment of human tissues, these definitions will undoubtedly be tested and clarified.

In adding further regulatory safeguards especially with respect to the risk of disease transmission, FDA issued the “Interim Rule” in 1993 to “require certain infectious disease testing, donor screening, and recordkeeping facilities to help prevent the transmission of AIDS and hepatitis through human tissue used in transplantation.” In general, methods to reduce risk of disease transmission, such as antibiotic soaks, peroxide disinfection, and sterilization using radiation methods, are considered to be no more than minimal manipulation. Further on the regulatory front, in 2005, FDA established, in conjunction with tissue bank input, a standard of good tissue practices to “create a unified registration and listing system for establishments that manufacture HCT/Ps and to establish donor eligibility, current good tissue practice, and other procedures to prevent the introduction, transmission, and spread of communicable disease.”

In summary, the US system of AATB Standards and FDA regulations and inspections provide many safeguards for the provision of effective transplantable allograft tissues.

Reducing Risk of Disease Transmission in Transplanted Allografts

Human tissue carries an inherent, yet minimal, risk of disease transmission, which has been made essentially negligible through advancements in cleaning and processing methodologies. A 2005 survey from AATB estimated an overall allograft-associated infection rate of 0.014%. It is important to note that this survey preceded widespread implementation of more advanced methods aimed at reducing the risk of disease transmission, including FDA-mandated and sensitive nucleic acid testing (NAT) for certain viruses as well as routine terminal sterilization. Although well-documented cases of disease transmission have occurred, modern tissue bank practices and processes have successfully diminished incidences over the last few decades. In a case beginning in 1985, four organs and 54 allograft tissues were distributed from an HIV-infected donor who had a favorable screening history and negative serology test. All four organ recipients and the three recipients who received fresh frozen bone tissue tested positive for HIV. Investigators suspected that the transmissions occurred because the seronegative donor had very recently become infected and, being in the “window” period, had not yet developed an HIV-1 antibody detectable by the tests in use at the time. In 2002 another case surfaced where 40 recipients received organs and tissues from a donor infected with hepatitis C virus (HCV), and 8 consequently developed HCV infections. As with the earlier HIV transmission case, the donor was seronegative for HCV, and NAT had not been performed. Subsequent NAT of stored serum from the donor detected the virus, which highlighted the importance of using more sensitive testing before releasing donor tissue. NAT for HIV and HCV is now required by FDA after being added as a requirement by AATB in 2005. There have also been reported cases of transmission of tuberculosis, various Clostridium species , Group A Streptococci , and rabies. Overall, the relative risk of disease transmission is small considering the millions of allografts transplanted; however, the possibility of transmission makes avoidance, control, and reduction of microbial and viral bioburden integral to tissue-processing practices.

Reducing Risk of Disease Transmission: Approach

Allograft tissue providers reduce risk of disease transmission by three primary means:

1.
minimizing occurrence of processing donor tissue with unacceptable bioburden;
2.
controlling environment and tissue-handling practices to avoid contamination; and
3.
reducing any remaining bioburden through disinfection and sterilization techniques.

In the first category, the occurrence of recovering or processing tissue that is contaminated can be minimized through stringent bioburden tests, including anaerobic and aerobic culture tests for bacteria and fungi, as well as serological testing and NAT to detect specific viruses. Specific tests are required by FDA and also to meet AATB standards. Note that proper infectious disease tests must be validated for use specifically with cadaveric specimens. A detailed donor-screening process is also critical for minimizing bioburden; medical records and social history, such as travel, tattoos, high-risk sexual behavior, illicit drug use, and incarceration, as well as physical examination, help assess donor eligibility.

In the second category, bioburden loads can be controlled by using aseptic handling techniques during recovery and processing to prevent contamination of the tissue by pathogens. However, it is important to note that using aseptic conditions by themselves can only prevent additional contamination but will not reduce or eliminate any existing bioburden. For the third risk-reduction category, processing methods designed to reduce any remaining bioburden are addressed in the following sections.

Bioburden Reduction Methods

Bioburden loads can be reduced by cleaning and disinfecting the tissue. These steps vary by tissue type and may include:

debridement;
low doses of preirradiation (irradiation before other chemical processing steps);
physical methods such as lavage, pulsatile fluid flow, centrifuge, fluid bath rotation, and sonication;
enzymatic digestion of cellular material;
penetrating agents such as supercritical CO ₂ in combination with chemical activators;
milder chemicals including alcohol, detergents, and antibiotics;
more aggressive chemicals such as NaOH, acetone, and peroxide.

Cleaning processes can remove bone marrow elements, lipids, and low-molecular-weight proteins, thus reducing any graft immunogenic potential as well as bacterial, viral, and fungal contamination. More aggressive agents that may be commonly used to disinfect bone, such as hydrogen peroxide, are not typically used for soft tissue grafts. At least one process that includes the use of hydrogen peroxide on tendons was correlated with a significant increase in risk for revision ACL repair. In this case, however, the contribution of the hydrogen peroxide to apparent graft weakening is unclear because the process also includes pulsatile fluid flow. Cellular remnants and, presumably, associated infectious agents may also be removed through tissue decellularization methods, which will also be discussed in a later section. These methods include the use of chemicals such as nondenaturing anionic detergent, recombinant endonuclease, sodium dodecyl sulfate, sodium hydroxide, sodium peroxide, sodium chloride, and antibiotics.

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