| Abstract|| |
Although bone allografts are being widely used in dentistry, many of clinicians appear to be unfamiliar with their preparation and processing as well as their use as safe and effective graft materials. The major concerns associated with these materials are antigenicity and risk of disease transmission from donor to recipient. To minimize this risk, the production of an allograft worthy of distribution and implantation requires strict attention to detail through a comprehensive process. With an increasing clinical requirement for bone grafting procedures, there is a commensurate increase in patients' demands for assurance that bank bone will not be infected with pathogens. To ensure the patients, dental surgeons should be able to cite factual information and recommendations by responsible organizations regarding safety of allografts. Knowledge of human bone allograft procurement, processing, and tracking may allow dentists to better educate patients and address concerns about this valuable treatment option. The purpose of this review is to furnish and update the current knowledge on processing, safety, and efficacy of allograft materials.
Keywords: Bone allograft, efficacy, safety
|How to cite this article:|
Grover V, Kapoor A, Malhotra R, Sachdeva S. Bone allografts: A review of safety and efficacy. Indian J Dent Res 2011;22:496
Bone grafting is one of the therapeutic modalities employed to fulfill the ideal goal of periodontal therapy-the reconstruction of periodontal tissues that had been previously destroyed by the disease process. It has shown to produce a successful clinical result that lasts for longer than 20 years when patients effectively control plaque through good oral hygiene and regular periodontal maintenance visits.  The results of published studies indicate that following the use of bone grafts a significant bone filling can be expected against treatment by debridement, obtaining an average filling of the defect of between 60% and 65%. 
|How to cite this URL:|
Grover V, Kapoor A, Malhotra R, Sachdeva S. Bone allografts: A review of safety and efficacy. Indian J Dent Res [serial online] 2011 [cited 2013 May 24];22:496. Available from: http://www.ijdr.in/text.asp?2011/22/3/496/87084
Several types of bone grafts have been studied over the years and search is still continued for an ideal bone replacement material. Bone allograft material has been used in dentistry for the past four decades. Allografts are bone grafts taken from one individual for transplantation to another. Bone allografts are being widely used in the field of dentistry, ,,, orthopedics, ,, and craniofacial surgery. ,, They are generally used in two forms-freeze dried bone allograft (FDBA) and demineralized freeze dried bone allograft (DFDBA). In reconstructive craniofacial surgery, autogenous bone was the material of optimal choice despite serious shortcomings, before the emergence of demineralized allogenic bone which was accepted as the most promising alternative to autogenous bone in 1900s. 
FDBA was first used in periodontal therapy in early 1970s although it has been used clinically in orthopedic therapy since 1950s.  FDBA provides an osteoconductive scaffold for bone growth and elicits resorption when implanted in mesenchymal tissues.  DFDBA was first used in dentistry and medicine in 1965 but for the treatment of periodontal defects in humans it was utilized in 1975 for the first time.  DFDBA also provides osteoconductive surface, and in addition, it also acts as a source of osteoinductive factors. So, it elicits mesenchymal cell migration, attachment, and osteogenesis when implanted in well-vascularized bone; it induces endochondral bone formation when implanted in tissues that would otherwise not form bone. DFDBA contains bone morphogenic proteins (BMPs) such as BMP 2, 4, and 7, which help stimulate osteoinduction.  Thus, commercially prepared, allograft-retained proteins have the capacity to influence cell behavior in vivo. BMPs produce multiple effects on bone by: (1) acting as mitogens on undifferentiated mesenchymal cells and osteoblast precursors; (2) inducing the expression of the osteoblast phenotype (e.g., increasing alkaline phosphatase activity in bone cells; and (3) acting as chemoattractants for mesenchymal cells and monocytes as well as binding to extracellular matrix type IV collagen.  Studies have determined that the minimal effective amount of BMP necessary to affect bone growth is about 2 μg/40 mg wet weight of explants. The optimal amount is about 10 μg. 
The advantages of allogeneic grafts include availability in adequate quantities, predictable results and the elimination of an additional donor site surgery.  The disadvantages of allografts include host incompatibility, potentially contaminated specimens resulting in recipient site infections and potential transmission of disease from donor to recipient of the allograft and impractical or biologically ineffective usefulness.  It is worth mentioning here about a media report of 2005  that has shaken the public's confidence in this treatment option. In late 2005, a human tissue recovery firm BTS (Biomedical Tissue Services) had been found responsible for harvesting high value body parts in unsanitary conditions and marketing them without testing for diseases according to FDA regulations. Although the matter had been solved and BTS was ordered to cease all manufacturing operations, the damage caused by this scandal may affect medical as well as dental professions for years to come. It is in the hands of clinicians using allografts to provide assurance to the patients regarding this treatment option and for this purpose they should be aware of tissue banking methodology currently in general use. This article reviews the bone tissue banking caveats and provides an insight into the concerns regarding safety and efficacy of bone allografts.
| Procurement of Allografts|| |
Use of any substitute for autogenous tissue requires consideration of its biological and biomechanical potential as a graft material and the possibility of transfer of disease from donor to recipient as well as the presence and significance of immune responses to foreign antigens.  Thus, bone banks accredited by responsible organizations exist for the purpose of supplying the surgeon with safe and effective bone tissue that is suitable for intended clinical application and are available whenever the need arises. The goals of bone banking are to preserve the physical integrity of the graft and the inductive protein, to reduce its immunogenicity, and to ensure sterility.  Bone banking has greatly increased the options for the periodontal therapist in the management of severe osseous defects. Bone graft procedures are no longer limited by available autogenous bone. The possibility of disease transfer with bone allografts is very unlikely if the material is procured and processed according to tissue-banking protocols.
There are certain organizations regulating allograft acquisition, processing, and use:
| Fda|| |
FDA Centre for Biologics Evaluation and Research (CBER) regulates human cells, tissues, and cellular-based products under federal law, title 21 of U.S. Code of Federal Regulations (CFR), parts 1270 and 1271. CFR Title 21 part 1271 requires HCT/P (Human Cellular and Tissue Based Products) manufacturers to register their companies and products with FDA CBER and comply with applicable FDA regulations.
| Aatb|| |
American Association of Tissue Banks is an independent non-profitable organization dedicated to ensuring and maintaining the safety, consistency, and availability of allografts in the United States. To fulfill this mission, the AATB publishes tissue-banking industry standards and offers rigorous accreditation for institutional members as well as a certification program for people working in the field. By accepting AATB accreditation, tissue banks agree to comply with on-site inspections of processing facilities, annual audits, and other various AATB-prescribed safety regulations. Additionally, by satisfying AATB accreditation, tissue banks help ensure their compliance with FDA HTC/P regulations.
Producing an allograft worthy of distribution and implantation requires strict attention to detail throughout a comprehensive process. This process begins with donor screening.
| Donor Screening and Testing|| |
The donor's medical/social history is screened for medical conditions or disease processes that would contraindicate the donation of tissues in accordance with current policies and procedures approved by bone banks meeting the standards established by FDA. A donor should be in good systemic health and free of infectious diseases having potential risk of transmission. The contraindications for bone tissue donation include:
Various steps in the preprocurement of human bone allografts are as follows: 
- Donor from high risk groups, as determined by medical testing and/or behavioral risk assessments.
- Donors testing positive for HIV antibody by ELISA
- Autopsy of donor reveals occult disease.
- Donor bone tests positive for bacterial contamination.
- Donor and bone test positive for Hepatitis B surface antigen (HBsAg) or Hepatitis C virus (HCV).
Procedural steps in manufacturing and processing of allografts ,,
- Notification of prospective donor's death - Hospitals or morgues notify tissue recovery agencies of human deaths.
- Determination of initial donor eligibility - The tissue recovery agency determines donor eligibility on the basis of readily available information (for example, age, cause of death, evidence of infection, history of systemic disease, evidence of drug use).
- Consent- If a potential donor is deemed acceptable, the tissue recovery agency obtains and documents consent from relatives or caretaker of the donor according to U.S. Food and Drug Administration regulations and state anatomical gift laws.
- Dispatch of recovery team - Most tissue recovery agencies use their own recovery teams to evaluate and procure potential donor tissues.
- Assignment of tracking number to prospective donor-The dispatched tissue recovery team assigns a unique tracking number to the potential donor.
- Determination of additional donor eligibility - The tissue recovery team confirms donor identity, reviews medical records, performs a full-body physical assessment, reviews critical time limits and verifies the temperature of the cadaver's storage.
- Tissue procurement - The tissue recovery team must procure the tissue within 12 hours of death for non refrigerated cadavers or within 24 hours for refrigerated cadavers.
- Autopsy - Some tissue procurement agencies perform autopsies on potential donors as anadditional screening procedure.
- Transport - The tissue recovery team transports harvested donor tissue, blood samples and relevant medical records to the tissue processing center.
Long bones are the source for periodontal bone allografts. Cortical bone is the material of choice because it has been found to be less antigenic than cancellous bone. BMP is located in the bone matrix and since the mass of bone matrix is greater in cortical than cancellous bone, the increased amount of BMP is present in cortical bone.  BMP concentration is greater in cortical than cancellous bone in quantities 1 mg/kg of wet weight of fresh bone. 
As a result of allograft processing, there is an exponential reduction in the potential for graft contamination, disease transfer, or both. With proper processing, allografts for dental purposes routinely achieve sterility assurance level (SAL) of 10 -6 . SAL is probability that an item will not be sterile after it has been subjected to a validated sterilization process.  With a SAL of 10 -6 , the odds of an organism's surviving after allograft processing are less than one in 1 million.  There is no need of secondary sterilization after procuring the bone as usually most bone banks procure the bone under sterile conditions. But if bone allograft is contaminated at the time of procurement, it has to be sterilized using ionizing radiation or ethylene oxide. 
- First of all soft tissue stripping is done to remove residual muscle, tendon, ligament, and so forth.
- The cortical bone is rough cut to particle size ranging from 500 μm to 5 mm. This fragmentation increases the efficiency of defatting of bone and subsequent decalcification.
- The graft material is then immersed in 100% ethyl alcohol for 1 h to remove fat that may inhibit osteogenesis and to inactivate viruses. Viral infectivity is undetectable within 1 min of treatment with 70% ethyl alcohol.
- The bone is frozen at -80°C for 1 to 2 weeks to interrupt the degradation process and the tissue water is removed by the process of lyophilization. This process is commonly referred to as freeze drying. During this time, the results from bacterial cultures, serologic tests, and antibody and direct antigen assays are analyzed. If contamination is found, the bone is discarded or sterilized by additional means.
- Freeze drying removes more than 95% of water content from the bone. Although freeze drying kills all cells, it has the advantage of facilitating long term storage and reducing antigenicity.
- The cortical bone is ground and sieved to a particle size of approximately 250 to 750 μm. Particle sizes within this range have been shown to promote osteogenesis, whereas a particle size below 125 μm can induce a significant foreign body giant cell response.
- The graft material is again immersed in 100% ethyl alcohol and washed repeatedly to remove chemicals used in processing.
- Decalcification with 0.6 N hydrochloric acid removes the calcium from the bone matrix and exposes the bone inductive proteins. This step is not needed if undemineralized freeze dried bone is the desired end product, such as in orthopedic and oral surgery procedures in which structural stability is necessary.
- The bone is washed in a sodium phosphate buffer to remove residual acid.
- If the bone is demineralized, it is refreeze dried.
- Vacuum sealing in glass containers protects against contamination and degradation of the material while permitting storage at room temperatures for an indefinite period of time.
After processing bone allograft has to undergo certain tests which include:
Puros (Zimmer Dental, Carlsbad, California) is a new allograft of cancellous bone in the market.  It is human bone that undergoes a patented tutoplast process. The patented Tutoplast process gently removes unwanted material such as fats, cells, antigens, and inactivates pathogens, while preserving the valuable minerals and collagen matrix, leading to complete and rapid bone regeneration.  This process involves delipidization with acetone and ultrasound, osmotic treatment, oxidation with hydrogen peroxide to destroy unwanted proteins, solvent dehydration with acetone to preserve the collagenous fiber structure, and low-dose gamma irradiation. Manufacturers believe that this new solvent preservation method preserves the trabecular pattern and mineral structure better than the freeze-drying process, thus providing a more osteoconductive material. Grafton DBM 21 (BioHorizons, Birmingham, Alabama) is another allograft processed from cadaver long bones by aseptically processing the bone to remove lipid, blood, and cellular components before it is frozen. Cortical bone is milled into elongated fibers of 0.5 mm in diameter or pulverized into particles of 100 to 500 mm. It is combined with a glycerol carrier to stabilize the proteins and improve the graft handling.
- Visual inspection test - Visual detection is done for problems such as gross graft contamination, packaging defects and product mislabeling.
- Residual moisture test - Testing of freeze-dried allografts is done to ensure residual moisture is 6 percent or less.
- Residual calcium test - Testing of demineralized freeze-dried bone allograft is done to ensure residual calcium content is 8% or less.
| Debate over DFDBA Effectiveness|| |
The effectiveness of demineralized bone matrices might differ depending on the age and gender of the donor, the residual mineral, the particle size, or the preparation method. ,, According to Sayler et al. the success and safety of demineralized bone implants as well as different characteristics of the product, including its osteoinductive potential, depend on the technological process used to produce them. , Studies have examined the ability of commercial DFDBA to induce new bone formation in vivo in order to assess if the broad variation in clinical response was due to differences in the preparations or to variations in host response. It was found that wide variations in commercial bone bank preparations of DFDBA do exist, including the ability to induce new bone formation, even within the same bank. Commercial bone banks do not verify the specific amount of BMPs or any level of inductive capacity in any graft material they sell. Therefore, graft quality cannot be considered standardized. Delaying the procurement of donor bone after death, improper storage conditions or other processing factors may play a significant role in the bioactivity of allograft that makes the way to the clinician's office. In addition, age, gender and medical status of deceased donors may also affect osteogenic activity in grafts taken from them.
Another concern is what happens to DFDBA when placed in periodontal defect over time. If DFDBA particles remain in the site for longer than a year acting as bone matrix, they may weaken the host bone and delay normal bone formation possibly by interfering with the osteoclasts' ability to resorb the DFDBA particles. When DFDBA is used in particulate form, particle size also appears to be an important variable in the success of DFDBA as a bone inductive material. Particles in the range of 125 to 1000 μm possess a higher osteogenic potential than do particles below 125 microns.  Optimal particle size appears to be between 100 to 300 μm.  This may be due to a combined effect of surface area and packing density. Very small DFDBA particles elicit a macrophage response and are rapidly resorbed with little or no new bone formation. Tissue banks providing DFDBA for dental use will usually have this graft material in various particle sizes, and the range from 250 to 750 μm is the most frequently available. Glowacki and Mulliken developed the technology of preparing demineralized bone implants in powder form. Powder provides the maximum surface area necessary for interaction with recipient target cells, which stimulates endochondral proliferation. Glowacki and colleagues demonstrated that the extent of bone induction is a function of the surface area of the implanted bone. 
Gendler introduced microperforations in the demineralized bone, which, according to his long-term experience, are centers of new bone formation. He assumed that the mechanism of osteoinduction of demineralized perforated bone is similar to that described for other forms of demineralized bone matrix although the microperforations, in his opinion, enhance osteoinduction. , So the presence of microperforations in demineralized bone allograft also affects the osteogenic potential of the graft.
With the increased risk of disease transmission, gamma irradiation or ethylene oxide are used to secondarily sterilize the allografts. Residuals due to inadequate evacuation following ETO or radiation sterilization may also contribute to the variability in response. Residual levels of ethylene oxide in the graft have been shown to be toxic to fibroblasts and appear to cause morphologic changes in fibroblasts that may or may not be reversible. Ethylene oxide may produce inflammation and impair healing, unless it or its by-products are removed from the graft. 
Some researchers suggest that irradiation interferes with osteoinduction. Others suggest that dose of radiation (2.5 Mrad) used by most tissue banks for the sterilization of bone tissue doesn't destroy bone induction properties of allografts.  Thus, sterilization processing may be an important contributor to variability in DFDBA's osteoinductive properties. Thus more predictable results might be achieved with allografts if bone banks standardize graft material by instituting strict standards on the sources and time frames associated with procuring grafts and by developing a way to test quickly the inductive capacity of any graft material they supply.
| Safety of Bone Allografts|| |
There are two major concerns regarding the use of bone allografts, antigenicity, and the risk of disease transmission.
| Antigenicity|| |
A concern about the antigenicity of the donor material arises with any dental/medical procedure using tissues derived from human donors. The Proceedings from the State of Art Workshop 1 held in 1982 stated "a principal concern with allografts is the problem of graft rejection." In humans, chromosome 6 contains the major histocompatibility complex (MHC), which codes for the human lymphocyte antigens (HLA). These antigens are expressed on the cell surface of nearly every nucleated cell in the body and represent the primary stimulus for transplant tissue rejection when HLA mismatches occur between donor and recipient. Detection of donor-specific anti-HLA antibody formation in a patient receiving allografts is an important measure of the clinical immunogenicity of the respective graft material.  A comparison between bone grafts and whole organ transplant yields fundamental differences in the desired clinical outcomes. During healing of allograft, revascularization and osteoclastic activity would ideally result in eventual replacement of the allograft with host bone, eliminating the original defect. If this revascularization and replacement were observed in another tissue organ transplant, it would be analogous to classic graft rejection. Therefore, the popular concept of "graft rejection" may not apply to periodontal allografts.
Also, with tissue processing, cell death occurs, whether this is performed after aseptic procurement or during terminal sterilization, the magnitude of a possible immune reaction is considerably diminished.
| Risk of Disease Transmission Associated with use of Allografts|| |
The potential for disease transfer particularly viral transmission and even more particularly HIV is a crucial factor associated with use of bone allografts. The first case of HIV transmission through allogenic bone was reported in 1988.  A femoral head specimen from a 52-year-old man was resected as a part of hip arthroplasty and implanted in recipient 24 days after its procurement in November 1984. The recipient developed lymphadenopathy, diarrhea, nausea and vomiting, and night sweats within 21 days of surgery. In February 1988, she was tested and found positive for HIV antibody. This case of HIV transmission represents the violation of basic principles in handling allogeneic tissues. Donor's medical record revealed history of intravenous drug use, as well as history of lymph node biopsy, the results of which suggested HIV infectivity and thus represent significant donor exclusion criteria. The second element of concern is the hospital bone bank that processed the tissue. Indeed many of these "bone banks" exist, yet, unlike bonafide tissue banks, they may not be accredited by AATB and therefore are not subject to same quality control that exists in tissue banks accredited with responsible organizations.
Most frequently used methods of assuring graft sterility is irradiation because of the belief that irradiation will prevent HIV transmission, thus making it worthwhile to trade safety for the loss of osteoinduction and alteration of biomechanical properties of the bone. However, a study performed by Smith et al. showed that, even at doses at which tissue quality begins to be compromised (1.5-2.5 Mrads), irradiation failed to be virucidal for HIV type 1.  Although four cases of HIV have been reported till 1996 , following procedures using frozen bone allografts, it should be emphasized that frozen and fresh allografts typically are not being used in periodontal therapy. The delay required to process DFDBA and FDBA ensures that there is adequate time for testing for potential pathogens, helping to assure the safety of these materials. Moreover tissue banks have adopted rigorous exclusionary techniques, testing for HIV antigen, and HIV antibody and lymph node biopsy in order to reduce this potential risk. Additionally, mere freezing of bone allografts reduces the risk of disease transfer to 1 in 8 million.  Treatment of cadaveric bone spiked with viral particles and cortical bone procured from a donor who had died of AIDS, with a viricidal agent and demineralization in hydrochloric acid has been found to inactivate HIV in both cases. , The probability of HIV transfer following appropriate DFDBA preparation has been calculated to be 1 in 2.8 billion.  Thus, DFDBA offers a further margin of reliability over FDBA in a highly charged consumer environment of concern with possible viral contaminants. 
| Human Bone Allograft Tracking|| |
FDA regulations require human bone allografts must be tracked so that tissue banks and clinicians can notify recipients in the event of a product recall. CFR Title 21 part 1271.290 addresses tracking protocols for human bone allografts to facilitate the investigation of actual or suspected transmission of communicable diseases. According to this regulation, HCT/P processing facilities must label each manufactured HCT/P with a unique alphanumeric identification code that does not contain the donor's name or Social Security number as shown in [Figure 1]. This code allows each manufacturer to record and track the donor graft to its recipient and vice versa. Most tissue banks supply a self-addressed prepaid postage tracking form with each human bone allograft. These forms consist of triplicate copies: one for the patient's record, one for the practitioner's record and one for the tissue bank. In the event of an HCT/P recall, tissue banks refer to these records to notify practitioners who have used the products in question. Clinicians who have used recalled allografts should immediately notify patient recipients and test them for suspected pathogens for a minimum of 6 months after implantation of the product.
| Conclusion|| |
Ever increasing use of human bone allografts reflects positively on the usefulness and safety of these products. Progressive FDA policies and industry self-regulation through agencies such as the AATB have allowed reputable tissue-processing facilities to uphold their fiduciary responsibility to the public. As is recommended for medical surgeons, when using human bone allografts in the practice of dentistry, practitioners should investigate carefully and be familiar with the institutions that they are patronizing. Purchasing products from HCT/P manufacturers such as those accredited by the AATB may provide practitioners with peace of mind, knowing that these institutions accept and adhere to strict and reliable safety measures in the creation of their products. So, when purchasing allografts, we must choose the products accredited by responsible organizations to ensure the safety and quality of the product and better educate the patients regarding concerns about this valuable treatment option.
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Department of Periodontology and Oral Implantology, National Dental College and Hospital, Gulabgarh, Derabassi, Distt. SAS Nagar, Mohali, Punjab