Scaffolds were fixed using 10% formalin, and mineralization was quantified by μCT imaging using Scanco 35 (Scanco Medical AG, Bruttisellen, Switzerland) in triplicate for each time point. Scans were performed at medium resolution with a source voltage E of 70 kVp and with a current I of 114 μA. The images had a final element size of 12.5 μm. Images were analyzed using software supplied from Scanco (Image Processing Language version 5.6) and reconstructed into 3D volumes of interest. Optimum arbitrary threshold values of 20 (containing scaffold and mineralization) and 80 (containing mineralization alone) were used uniformly for all specimens to quantify mineralized areas from surrounding unmineralized scaffold. Analysis of 3D reconstructions was performed using Scanco Evaluation script no. 2 (3D segmentation of two volumes of interest: solid dense in transparent low-density object) and script no. 6 (bone volume/density-only bone evaluation) for volume determinations.

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