Major drawbacks associated with autologous bone grafting are the risk of donor site morbidity and its limited availability. Sterilized bone allograft, however, lacking osteoinductive properties, carries the risk of graft failure resulting from insufficient osseointegration of the graft. The aim of this study was to vitalize bone allograft with human osteoprogenitor cells under GMP-conform conditions. For this purpose we investigated proliferation, osteogenic differentiation and large-scale gene expression of human MSCs cultured three-dimensionally on peracetic acid (PAA)-treated spongious bone chips. MSCs were isolated from healthy donors (N=5) and seeded onto PAA-treated spongious bone samples (~5×5×5 mm, DIZG, Germany) under GMP-conform conditions. Proliferation (total protein assay), osteogenic differentiation (cell-specific ALP activity assay, quantitative gene expression analysis of selected osteogenic marker genes), and morphology were assessed. RNA was isolated and microarray analysis was performed using the PIQORTM Stem Cell Microarray system (Miltenyi Biotec) including 942 target sequences. Increasing cellularity was observed during the 42 d observation period while cell-specific ALP activity peaked at day 21. Effective proliferation and adhesion of human MSCs on PAA-treated spongious bone was confirmed by histology, scanning electron and confocal laser scanning microscopy. Gene expression of early (Runx-2), intermediate (ALP), and late (osteocalcin) osteogenic marker genes was present during 42 days of cultivation. Microarray analysis of MSCs cultivated on bone allograft versus 2-D tissue culture demonstrated temporal upregulation of genes involved in extracellular matrix synthesis (e.g., matrix metalloproteases, collagens), osteogenesis (e.g., BMPR1b, Runx-2) and angiogenesis (angiopoietin, VEGF). PAA-treated spongious bone allograft is a biocompatible carrier matrix for long-term ex vivo cultivation of MSCs as observed by favorable proliferation, cell distribution, gene expression profile, and persisting osteogenic differentiation. GMP-grade vitalisation of bone allograft by cultivation with autologous MSCs represents a promising clinical application for the treatment of osseous defects.
