European Cells and Materials Vol. 14. Suppl. 1, 2007 (page 36) ISSN 1473-2262
Controlling osteogenesis by human mesenchymal stem cells
Ramakrishnaiah Siddappa, Ruud Licht, Jun Liu, Clemens van Blitterswijk and
Jan de Boer
Department of Tissue Regeneration
, Institute for Biomedical Technology, University of
Twente, Enschede, The Netherlands.
INTRODUCTION: We use human
mesenchymal stem cells (hMSCs) as a source for autologous bone tissue engineering and previously demonstrated bone formation upon implantation in mouse models and in a phase III clinical trial. However, bone deposition is not sufficient to fully reconstruct lost bone tissue and suboptimal hMSC differentiation protocols partly underlie this. In vitro differentiation recapitulates only a small segment of the complex signaling hierarchy of bone formation in vivo and as such, we are not yet able to fully unleash the osteogenic potential of hMSCs. We will describe a number of approaches taken to enhance the osteogenic potential of hMSCs and discuss the application in bone tissue engineering.
METHODS: hMSCs were isolated as the
adherent fraction of bone marrow from donors who had given written informed consent. Culturing and analysis were as described1. Bone formation in vivo was assessed by seeding onto porous ceramic materials and implantation in immune-deficient mice. Bone formation was visualised by histomorphometry.
RESULTS: Human mesenchymal stem cells
form bone in vivo but substantially less than cells from goats or rats (see Figure 1).
Fig. 1: Ectopic bone formation by equal number of rat and human. Arrows indicate basic fuchsine stained newly formed bone.
We hypothesised that bone formation could be improved by further enhancing osteogenic differentiation in vitro using a pharmaceutical approach. We cultured hMCS in the presence of chemicals affecting the activity of a number of proteins implicated in osteogenesis: histone deacetylases, Wnt signaling, protein kinase C and protein kinase A. Wnt2 and PKC activation inhibited hMSC osteogenesis (data not shown),
although the former induced proliferation. A number of HDAC inhibitors strongly induced osteogenesis and mineralisation but the compounds had a very strong effect on viability, with a negative net result in bone
tissue engineering3. dex 3 5 10 15 25 30 0 20 40 60 80 100 120 Ca lcium A c c u mula tion/2 5cm 2 f lask ( µg)
Figure 2. Short-term cAMP exposure (in days) significantly stimulates calcium deposition of hMSCs.
Activation of the PKA pathway using e.g. cAMP was very effective in inducing osteogenic differentiation (Figure 2). Gene expression profiling indicated that PKA does not only stimulate osteogenesis cell-autonomously through activation of BMP target genes, but also systemically through activation of a number of pro-osteogenic cytokines and growth factors. Implantation of cAMP-treated hMSCs resulted in bone formation approaching levels seen in goat and rat MSCs implantation.
DISCUSSION & CONCLUSIONS: hMSCs
are potentially useful in bone tissue engineering pending further optimisation of in vitro proliferation and differentiation. We are currently establishing high throughput screening protocols to find small molecules and genes that improve the osteogenic potential of hMSCs.
REFERENCES: 1 Siddappa et al, J Orth Res
(2007). 2 de Boer et al, Tissue Eng. 10: 393-401 2004). 3 de Boer et al, Tissue Eng. (2006)
