This study investigated the temporal composition of an osteogenic extracellular matrix construct generated by culturing mesenchymal stem cells in an electrospun biodegradable poly(epsilon-caprolactone) fiber mesh scaffold within a flow perfusion bioreactor. Constructs of different extracellular matrix maturities were analyzed for their protein and mineral composition at several culture durations by liquid chromatography-tandem mass spectrometry, scanning electron microscopy, energy-dispersive X-ray diffraction, X-ray diffraction, and calcium and phosphate assays. The analysis revealed that at short culture durations the cells deposited cellular adhesion proteins as a prerequisite protein network for further bone formation. At the later culture durations, the extracellular matrix was composed of collagen 1, hydroxyapatite, matrix remodeling proteins, and regulatory proteins. These results suggest that the later culture duration constructs would allow for improved bone regeneration due to the ability to mineralize and the capabilities for future remodeling.