Quantitative microcomputed tomography analysis of mineralization within three-dimensional scaffolds in vitro

Synthetic and naturally derived scaffold biomaterials in combination with osteogenic cells or bioactive factors have the potential to serve as bone graft substitutes. Porous poly(l-lactide-co-dl-lactide) (PLDL) scaffolds with mechanical properties comparable to trabecular bone and an oriented, interconnected porosity designed to enhance internal mass transport were recently developed. In this study, PLDL scaffolds were seeded with rat calvarial or rat stromal cells and cultured up to 8 weeks in media containing osteogenic supplements. Cell-seeded human demineralized trabecular bone matrix (DTBM) scaffolds were included for comparison. All constructs were imaged weekly from 4 to 8 weeks using microcomputed tomography (micro-CT) to nondestructively quantify the amount and distribution of mineralized matrix formation. The total mineralized matrix volume increased with time in culture for all construct groups. DTBM constructs contained significantly more mineralized matrix than PLDL constructs. However, an analysis of the acellular DTBM scaffolds exposed to osteogenic media revealed partial remineralization of the demineralized matrix whereas no mineralization was detected in acellular PLDL scaffolds. Differences in mineral distribution were also evident with cell-mediated mineralization found throughout the PLDL constructs but localized to the periphery of the DTBM constructs for both cell types. Expression of bone marker genes indicating osteoblast differentiation was demonstrated in all groups at 8 weeks using a quantitative reverse transcription polymerase chain reaction. Osteocalcin expression was significantly higher for calvarial cell constructs compared to stromal cell constructs, regardless of the type of scaffold. This study demonstrated that micro-CT imaging may be used to nondestructively and quantitatively monitor mineralization within three-dimensional scaffolds in vitro. PLDL scaffolds with an oriented microarchitecture were shown to support cell attachment, differentiation, and cell-mediated mineralization comparable to natural DTBM scaffolds.