Abstract
Background aims. Combining autologous bone precursor cells with cancellous bone allograft (CBA) offers an appealing strategy for skeletal regeneration. In this context, multipotent mesenchymal stromal cells (MSC) provide an excellent cell source because they are readily harvested from donors, expanded and differentiated in vitro. The aim of this study was to evaluate the proliferation, morphology, osteogenic differentiation and stem cell-related gene expression during static long-term ex vivo cultivation using human MSC and CBA under good manufacturing practice (GMP)-conforming conditions. Methods. MSC were isolated from healthy donors (n = 5) and cultivated on peracetic acid-sterilized CBA in the presence of 10% human platelet-rich plasma without osteogenic supplements. Total protein content, cell-specific alkaline phosphatase (ALP) activity and osteogenic marker gene expression levels were assessed. Stem cell-related gene expression was compared with MSC monolayer cultivation using microarray analysis. Furthermore, cellular distribution and morphology within the porous CBA were visualized by histology and scanning electron microscopy. Results. Effective adhesion, spreading, proliferation and intercellular contact of human MSC within the pores of CBA were observed during the study (≤42 days). Cell-specific ALP activity peaked after 3 weeks of cultivation. Gene expression of early, intermediate and late osteogenic marker genes was detectable during long-term cultivation. Microarray-based annotation and biologic interaction network data analysis indicated that expression levels of genes encoding crucial differentiation-regulating proteins and extracellular matrix components involved in the process of osteogenesis were induced in CBA-cultivated MSC. Conclusions. MSC-vitalized CBA offers an attractive GMP-grade bone-filling material. Further research is warranted to evaluate its bone-healing potential in vivo.
Acknowledgments
The authors wish to express their gratitude to Dr Angela Jacobi, Dr Ute Schröter-Bobsin and Dr Ute Hempel for their help during the study design and data collection. Dr Denis Corbeil is thanked for providing laboratory infrastructure. In addition, we thank Cornelia Liebers, Ines Kleiber, Anette Baatrup, Katja Richter and Nadine Münch for excellent technical assistance. Funding for this study was kindly provided (initials of author grant recipient in parentheses) by the Center for Regenerative Therapies, Dresden, Germany (seed grant number 09-08, MS and MB); Technical University, Dresden, Germany (MeDDrive38 grant number 60–130, MS); German Academic Exchange Service/German Federal Ministry of Education and Research (grant number 0315187, MS); Simon Fougner Hartmanns Family Foundation, Denmark (MS); Aase og Ejnar Danielsens Foundation, Denmark (MS); Augustinus Foundation, Denmark (MS); Erik Hørslev og hustru Birgit Hørslevs Foundation, Denmark (MS); Deutsche Forschungsgemeinschaft (SFB655, project B2, PS, CW and MB).
Declaration of interest: The authors indicate no potential conflicts of interest.