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Abstract
A composite that utilizes the osteoinductivity of demineralized bone matrix (DBM) and the attractive characteristics of polylactide (PL) may be useful as a tissue-engineered bone substitute. The objective of this study was to investigate the potential of a composite system consisting of DBM fragments and PL beads to support the attachment and proliferation of multipotent mouse marrow stromal cells and to provide a structure for the cells' differentiation into the osteoblast lineage. Furthermore, the overarching goal was to provide a preliminary assessment of the DBM/PL cultures in order to facilitate the development of injectable composite DBM/PL systems in the long term. Demineralized bone matrix fragments were produced from bovine femurs and polylactide beads were produced by a single emulsion process. Differential scanning calorimetry and gel-permeation chromatography were used to characterize the PL samples. Multipotent mouse marrow stromal cells were cultured on several different substrate mixtures including 100% DBM, 70% DBM:30% PL, 50% DBM:50% PL and 100% PL. Cells were analyzed using a LIVE/DEAD® Viability/Cytotoxicity kit as well as scanning electron microscopy. Lactic acid and glucose levels were measured throughout the study. Osteogenic differentiation of the MSCs was assessed with an alkaline phosphatase activity (ALP) assay and RT-PCR for expression of bone sialoprotein, osteocalcin and runt-related transcription factor 2. All cell types attached more readily to DBM fragments than PL beads resulting in more lactic acid production in the samples containing mostly DBM. The ALP activity and gene expression results indicate that the optimal mixture for the D1 line of multipotent mouse marrow stromal cells differentiation into osteoblasts is 100% PL. However, it is likely that the decreased pH in the DBM containing samples resulted in an environment that was not very conducive for osteogenic differentiation.
