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Title: Pre-culture and BMP-2 stimulation under serum free conditions enhances osteochondrogenic differentiation and BMP signalling in human periosteum derived
Other Titles: Pre-culture and BMP-2 stimulation under serum free conditions enhances osteochondrogenic differentiation and BMP signalling in human periosteum derived
Authors: Bolander, Johanna ×
Bloemen, Veerle
Chai, Yoke Chin
Ji, Wei
Schrooten, Jan
Luyten, Frank #
Issue Date: 11-Jul-2013
Host Document: Pre-culture and BMP-2 stimulation under serum free conditions enhances osteochondrogenic differentiation and BMP signalling in human periosteum derived
Conference: Biomaterials and Regenerative Medicine location:Riva Del Garda, Italy date:8-12th July 2013
Abstract: Bone morphogenetic proteins (BMPs) are under investigation as a part of engineered constructs that combine osteoinductive growth factors, osteoconductive carriers and differentiating osteoprogenitor cells to induce and enhance in vivo bone formation. A promising cell source for this combined approach is the use of human periosteum-derived cells (hPDC) as it is the main cell population contributing to bone fracture repair in vivo. In addition, their osteochondrogenic potential has been proven in vitro and in vivo. Standard expansion protocols of these cells in vitro involve the use of culture media containing 10 % Fetal Bovine Serum (FBS). Although FBS is known to be directly correlated to cell proliferation, when engineering osteogenic constructs with hPDCs and growth factors such as BMPs, proliferation induced by higher serum levels may interfere with the effect of the growth factor. Also, towards clinical relevance, the use of FBS represents concerns related to introducing xenogeneic antigens to patients as well as the risk of transmitting zoonosis. In addition, previous 2D in vitro studies have shown that 10 % FBS in culture medium decreases the response to BMP ligands significantly compared to lower serum levels, reflected by a reduced ALP-activity. This effect is suggested to be due to proteins and cytokines present in the serum, which bind to BMPs and therefore interfere with the BMP-cell signalling.
The aim of this study was to identify the effect of media containing different serum levels (0-10 %) on BMP-2 induced osteochondrogenic differentiation in vitro. Initially, DNA content and metabolic activity was measured to determine optimal serum level in the culture media to maintain cell survival rather than to induce cell proliferation. Media that maintained high cell survival were further investigated by supplementing them with BMP-2 for osteochondrogenic differentiation of hPDCs. The effect of different serum levels during preculture and BMP-2 stimulation on cell proliferation and differentiation was analysed by DNA content, ALP-activity and gene expression. Standard culture media containing 10 % FBS was used as control.
Results showed that culture media containing 1 % FBS maintained cell survival and did not significantly induce cell proliferation. Therefore, were selected for further investigation of BMP-2 induced in vitro osteogenesis. DNA content showed that BMP-2 supplemented media significantly induced cell proliferation in all media conditions compared to non-supplemented. The ALP-activity was significantly higher in cells cultured in media supplemented with BMP-2. Gene expression analysis confirmed a robust osteochondrogenic profile in the same group, based on significantly upregulated expression of Sox9, Aggrecan, Runx2 and Osterix. In addition, elevated activation of downstream BMP-signalling was found by enhanced expression of Id1, Dlx5 and Msx2 in cells cultured in BMP-2 supplemented CDM.
Together, these data show that by lowering the serum level in culture media, an enhanced effect of BMP-2 on osteochondrogenic cell proliferation, differentiation and downstream BMP signalling can be achieved.
Publication status: published
KU Leuven publication type: IMa
Appears in Collections:Skeletal Biology and Engineering Research Center (+)
Materials Technology TC, Campus Group T Leuven
Technologiecluster Materialentechnologie
Department of Materials Engineering - miscellaneous
× corresponding author
# (joint) last author

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