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AAOS Now

Published 6/1/2016
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Adam I. Edelstein, MD; Ivan Cheng, MD

Questions to Ask About Demineralized Bone Matrix

Demineralized bone matrix (DBM) is composed of collagenous and noncollagenous extracellular matrix components that have been extracted from allogeneic cadaver bone. The presence of growth factors such as bone morphogenetic proteins (BMP) in the graft gives rise to osteoinductive properties; in some DBM products, graft particularity and three-dimensional structure provide osteoconductivity.

Differences in graft source, harvest techniques, sterilization measures, storage, and use of carriers result in significant variation in the molecular composition of DBM products. Although the exact processing measures used in preparation of off-the-shelf DBM products are often proprietary, a general knowledge of the relevant steps can be useful in understanding graft efficacy.

The following list of questions may be of particular value in selecting an appropriate DBM.

What is the screening process for potential bone tissue donors?
The allogeneic nature of DBM gives rise to a potential risk of disease transmission from donor to recipient. Donor tissue is tested according to serologic and microbiologic standards regarding suitability for use as DBM. Some manufacturers also screen donors based on medical records and social history.

Fortunately, the mineral extraction process drastically reduces the infectivity of viruses in the donor bone. Estimates of viral transmission related to use of DBM are well below 1 per 1,000,000 uses.

Although the American Association of Tissue Banks (AATB) provides standards for safe selection and handling of donor tissues, not every manufacturer of DBM uses AATB-certified tissue banks. Most DBM products achieve regulatory compliance under the U.S. Food and Drug Administration's (FDA) 510(k) pathway by establishing substantial equivalence to a product already on the market. However, this regulatory pathway—which is for devices and therefore demonstrates safety from a mechanical, rather than biologic, standpoint—can lack a rigorous demonstration of safety.

How was the graft demineralized?
To create DBM, the mineral phase of allogeneic bone must be removed. This typically entails exposing the bone to an organic solvent. Manufacturers may use different protocols with respect to type and concentration of demineralizing solvent, duration of exposure, and temperature.

Following extraction, the demineralized matrix must be washed to remove the solvent and subsequently dried. This preparation can affect the concentration and potency of each available growth factor within the final product.

What sterilization steps were used?
Creation of DBM starts with screening tissue-banked human bone. Although the graft is most often harvested in a sterile fashion, many manufacturers undertake additional sterilization. These extra steps may include treating the tissue with antibiotics, electron-beam sterilization, ethylene oxide exposure, or gamma irradiation. Exposure to radiation and ethylene oxide has been shown to degrade growth factors and has a dose-dependent negative impact on osteoinductivity of the final product. Temperature and the presence of an aqueous carrier can also affect the radiation-related activity loss.

How much growth factor is present?
The total concentration of growth factors in DBM is typically on the order of several micrograms per gram of graft. The amount of growth factors in specific formulations may be highly variable. In fact, studies have found greater variability between different lots of the same DBM product than between different DBM formulations.

The type and amount of growth factors in DBM have varied across published studies; in general, common DBMs will contain BMP-2 and BMP-7 in greatest abundance, followed by transforming growth factor (TGF)-β1, fibroblast growth factor (FGF)α, insulin-like growth factor (IGF)-1, and platelet-derived growth factor (PDGF). The source of the allograft bone may also determine growth factor content. For example, DBM derived from female donors contains significantly more BMP than that from male donors, whereas the age of the donor does not significantly affect growth factor content.

DBM products can be tested by protein extraction and quantification assays to determine the concentration of growth factors. Such data are not typically included in the package insert.

What carriers and adjuvants have been added?
DBM is often combined with a biologically compatible carrier material for enhanced handling properties and facilitation of delivery to the target site. Carriers may be hydrous or anhydrous. The addition of the carrier may drastically reduce the concentration of DBM in the final product. Commonly used carrier materials include the following:

  • glycerin
  • synthetic polymer
  • collagen
  • hyaluronate
  • lecithin
  • carboxymethylcellulose

DBM also may be used in particulate form without a carrier, which may be useful if it will be admixed to bone marrow aspirate or autograft. Manufacturers may add materials such as allogenic bone chips or calcium sulfate granules to DBM. The resulting final product may be powder, gel, putty, chips, or solid. The variability of formulations can subject the proteins to chemical and physical degradation.

Has the osteoinductive potential been assayed?
DBM grafts may undergo a variety of assays to verify their osteoinductive potential. These tests include chemical or immunologic assays, such as ELISA (enzyme-linked immunosorbent assay) testing, to verify the presence of specific growth factors in the DBM.

Alternatively, DBM may be tested in vitro to demonstrate an osteoinductive effect on cultured osteoblasts. Bioactivity also may be assayed by in vivo evaluation of de novo bone formation and alkaline phosphatase levels. All available in vitro assays evaluate DBMs without the added carrier, and few can be used to evaluate DBM that has undergone the full sterilization process. Only a few tissue banks use an in vivo model to measure the bioactivity of a final DBM product with its carrier.

How has the product been stored?
Various storage techniques are employed by DBM manufacturers. Some package the DBM in a dry (lyophilized) state and do not mix it with the carrier until immediately prior to graft implantation. Others premix the graft with the carrier so that it is ready for use as packaged.

The hydration status of the graft may have an impact on its shelf life and response to temperature extremes during storage. Studies have shown that DBM starts to lose osteoinductive potential when stored at temperatures of 65˚C or greater and that hydrated grafts are more susceptible to heat-related damage. Conversely, grafts that are stored in a hydrated state may have more rapid elution of growth factors following use. Some grafts are frozen, while others are stored at room temperature.

What evidence supports use of this product?
In general, high-quality evidence related to the use of DBM is lacking. Most of the literature surrounding DBM consists of preclinical data, retrospective case series, or retrospective comparative studies. Within the prospective literature, one randomized trial demonstrated that the use of DBM resulted in the formation of new bone, compared to no graft, in a critical sized fibular defect.

Several prospective studies from the spine literature have demonstrated noninferiority of DBM compared to autograft when used as a bone graft extender in posterolateral spinal fusion.

Conclusion
A wide array of DBM products are available to today's orthopaedic surgeons. These products vary in their form, content, and biologic activity. Consideration of the questions outlined in this article can serve as a starting point in selecting the most appropriate graft for a given clinical application.

Ivan Cheng, MD, is a member of the Biological Implants Committee. He can be reached at ivan.cheng@stanford.edu

Adam I. Edelstein, MD, is the resident member of the Biological Implants Committee. He can be reached at adam.edelstein@fsm.northwestern.edu

References:

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