Quantitative Micro-CT analysis of mineralized matrix formation on natural and synthetic 3D scaffolds in vitro

This study quantified mineralized matrix formation within novel poly(L-lactide-co-D, L-lactide 70:30) (PLDL) and human demineralized trabecular bone matrix (DTBM) porous scaffolds in vitro. Scaffolds were seeded with either rat calvarial or rat stromal cells and cultured for 8 weeks in osteogenic media. Microcomputed tomography (μCT) imaging was used to nondestructively monitor mineralized matrix formation within the constructs as a function of time. From weeks 4 to 8, the constructs were imaged weekly under sterile conditions to quantify mineralization and then returned to tissue culture. Analysis of μCT images showed that total mineralization within each of the constructs increased consistently with time. Mineralization was distributed throughout PLDL constructs but was localized to the periphery of DTBM constructs for both cell types. Total volume of mineralization was significantly higher within DTBM constructs compared to PLDL constructs. However, analysis of acellular control scaffolds exposed to osteogenic media indicated some remineralization of the DTBM scaffolds only. Histological analysis confirmed that DTBM constructs contained both cell-mediated matrix mineralization and regions of acellular remineralization. Expression of several bone marker genes indicating osteoblast differentiation was demonstrated in all groups at 8 weeks using quantitative RT-PCR. Alkaline phosphatase, osteonectin, osteocalcin, and osteopontin expression were significantly higher (p<0.05) in calvarial/PLDL constructs compared to calvarial/DTBM constructs.