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1.1 This guide covers general guidelines to evaluate the effectiveness of DBM-containing products intended to cause and/or promote bone formation when implanted or injected in vivo. This guide is applicable to products that may be composed of one or more of the following components: natural biomaterials (such as demineralized bone), and synthetic biomaterials (such as calcium sulfate, glycerol, and reverse phase polymeric compounds) that act as additives, fillers, and/or excipients (radioprotective agents, preservatives, and/or handling agents) to make the demineralized bone easier to manipulate. It should not be assumed that products evaluated favorably using this guidance will form bone when used in a clinical setting. The primary purpose of this guide is to facilitate the equitable comparison of unique bone-forming products in in vivo heterotopic models of osteoinductivity. The purpose of this guide is not to exclude other established methods.

1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.

1.3 This standard does not purport to address all of the safety concerns, if any, associated with the use of DBM-containing bone-forming/promoting products. It is the responsibility of the user of this standard to establish appropriate safety and health practices involved in the development of said products in accordance with applicable regulatory guidance documents and in implementing this guide to evaluate the bone-forming/promoting capabilities of the product.

4.1 This guide covers animal implantation methods and analysis of the explanted DBM-containing material to determine whether a material or substance possesses osteoinductive potential, as defined by its ability to cause bone to form in vivo at a site that would otherwise not support bone formation, that is, heterotopically in a skeletal muscle implant site. For in vitro evaluation see Test Method F2131 for in vitro assessment of rhBMP 2.

4.2 The test methods described here may be suitable for defining product specifications, cGMP lot release testing, research evaluation, regulatory submission, and so forth, but a positive outcome should not be presumed to indicate that the product will be osteoinductive in a human clinical application. At present, the only direct assays to assess new bone formation are in vivo, since the property of bone conduction or induction can only be assessed in a heterotopic or orthotopic site in a living animal. When these products are implanted in an orthotopic site, osteogenic factors already present at the implantation site may contribute to and enhance bone formation in conjunction with the osteoconductive nature of the product. Thus, orthotopic implantation of products may result in bone formation by acting on existing bone forming cells and not by causing mesenchymal stem cells to become osteochondroprogenitor cells. In contrast, when these products are implanted in a heterotopic site, no native osteogenic factors are present to contribute to or enhance bone formation. Thus, heterotopic implantation of products will only result in new bone formation by causing mesenchymal stem cells to become osteochondroprogenitor cells. In vitro assays have been described and some believe they may correlate to the results obtained from in vivo assays. However such in vitro assays measure only some of the biochemical marker(s) associated with in vivo bone formation and are therefore only indirect assays for osteoinductive activity or the capacity to promote new bone formation. Many factors or combination of factors contribute to osteoblast progenitor cells differentiating and/or proliferating into bone forming cells in vitro that are both osteoinductive and osteoconductive when they are implanted in vivo. Thus, only an in vivo assay method currently directly considers the many potential factors involved in new bone formation induced by DBM containing biomaterials. The qualification of a DBM or DBM-containing material should also encompass product characterization such as that described in Appendix X1.