Bone regeneration with recombinant human bone morphogenetic protein-2 (rhBMP-2) using absorbable collagen sponges...

The effects of variability in three parameters (mass, cross-linking with CH2O, and EtO sterilization) of three surgically implantable absorbable collagen sponges (ACS) were studied. Sponges soaked with recombinant human bone morphogenetic protein-2 (rhBMP-2) solution were analyzed for pH, conductivity, and rhBMP-2 precipitation. A method using trinitrobenzenesulfonic acid was developed to quantify the free amino groups of the collagen sponge. With up to 240 min exposure to CH2O, the amount of free amino groups was reduced to 80%. In comparison, the denaturation temperature as determined by differential scanning calorimetry (DSC) after the sponges were soaked with phosphate-buffered saline, increased from 48 to 55 degrees C, indicating stronger interactions due to cross-linking. Subsequent sterilization with EtO caused a marked decrease in the amount of free amino groups (approximately 33% of nonsterilized controls) independent of previous CH2O treatment. However, the denaturation temperature was on average 5 degrees C lower in sterilized sponges than in nonsterilized material. In contrast to CH2O exposure, the strong reaction with EtO appeared to weaken the collagen structure. Resistance of the sponge to collagenase correlated with the degree of collagen cross-linking but was slightly reduced by sterilization. In addition, the pH of ACS soaked with water was substantially increased by sterilization. Protein precipitation was a function of pH and salt concentration but there was no effect due to collagen alone. Results indicated that ACS weight has to be limited to avoid rhBMP-2 precipitation.