Extractable bone morphogenetic protein and correlation with induced new bone formation in an in vivo assay in the athymic mouse model

Introduction: DBM contains BMPs that are essential factors for endochondral bone formation. Among proteins in the BMP family, BMP-4 is one of the most osteoinductive factors. At present, the most commonly used test method for demonstrating that a material or substance (DBM) is 'osteoinductive', as defined by its ability to cause bone to form in vivo at a site that would otherwise not support bone formation, for example heterotopic, is the athymic mouse or rat model. Although this heterotopic implant model is generally deemed to be suitable for specification acceptance, design purposes, service evaluation, regulatory statutes, etc., it should not be presumed to indicate suitability for forming bone in a clinical application. At present, the only reliable assays are in vivo, since the property of bone induction or conduction can only be assessed in a heterotopic orthotopic site in a living animal. In vitro assays, mostly cell culture based, have been described and correlations made to the results obtained in in vivo assays. However such in vitro assays measure only some biochemical marker presumed to be associated with in vivo bone formation and are therefore generally less well accepted as an assay for new bone formation. Thus, only the in vivo assay method is currently considered relevant as a standard for assessing new bone formation by biomaterials. The objective of this study was to investigate a possible relationship between extractable BMP-4 in DBM and the ability of that DBM to induce new bone formation in an athymic (nude) mouse assessment model. Materials and methods: DBM samples prepared from cortical bone derived from 40 donors were extracted by collagenase digestion and implanted heterotopically in an athymic (nude) mouse. BMP-4 in the protein extracts from these DBM samples were measured and quantitated using and ELISA assay method. The osteoinductivities of DBM from these donors were assessed using the in vivo athymic mouse assay. Correlation between BMP-4 level and osteoinductivity of DBM was analyzed by linear regression analysis (SPSS 10.00). Results: The extractable BMP-4 levels were variable and ranged between 1.82 and 7.94 ng/g of DBM. We revealed that the less residual calcium left in DBMincreased the extractability of BMP-4. The extractability of BMP-4 from DBM varied relative to the particle size such that DBM particles ranging from 550 to 710 lm resulted in the highest level of extractable BMP-4, whereas DBM particles less than 250 lm provided for the lowest level of extractable BMP-4. In the donor age and gender study, the extractable BMP-4 content appears to be age-dependent, with DBM from younger donors being most likely to have greater BMP-4 quantity. In contrast, no difference in the quantity of extractable BMP-4 was observed between male and female donors. Conclusions: DBM exhibiting high osteoinductivity in the nude mouse bioassay also contained greater BMP-4 levels in the protein extracts from this DBM than did DBM samples possessing low levels of osteoinductivity. There was a positive correlation between quantitative BMP-4 in vitro assay and osteoinduction (percentage of new bone formation) determined by the implantation of DBM in the athymic mouse assay with a correlation coefficient of 0.74 (p<0.001), providing that this quantitative BMP-4 in vitro assay could be an alternative means of assessing the osteoinductive potential of DBM. © Springer 2005.