ABSTRACT
The combination of supportive biomaterials and bioactive factors to stimulate endogenous progenitor cells is of key interest for the treatment of conditions in which intrinsic bone healing capacities are compromised. To address this need a "scaffold-decoration platform" was developed in which a biocompatible, biotin-functionalised 3D structural polymer network was generated through a solvent blending process, and used to recruit avidin modified nanoparticles within its 3D structure through biotin-avidin conjugation. This was enabled via the generation of a suite of poly(lactic-co-glycolic acid) (PLGA) nanoparticles, encapsulating two bioactive factors, vascular endothelial growth factor (VEGF) and l-ascorbic acid 2-phosphate (AA2P) and conjugated to streptavidin to allow attachment to the bone generating scaffold. The levels of encapsulated and released VEGF and AA2P were tailored to fall within the desired range to promote biological activity as confirmed by an increase in endothelial cell tubule formation and collagen production by osteoblast cells in response to nanoparticle release of VEGF and AA2P, respectively. The release of VEGF from the scaffolds produced a significant effect on vasculature development within the chick chorioallantoic membrane (CAM) angiogenic assay. Similarly, the scaffolds showed strong biological effects in ex vivo assays indicating the potential of this platform for localised delivery of bioactive molecules with applications in both hard and soft tissue engineering.
ABSTRACT
Inflammation is one of the key regulators of the repair process in bone tissues. Current data about the effect of interleukin-1ß (IL-1ß) on MSCs and osteoblasts are conflicting. We investigated the long-term effect of IL-1ß on direct osteogenic differentiation of hMSCs in vitro. IL-1ß-stimulated cells showed enhanced proliferation and entered maturation prior to non-stimulated ones, as monitored by ALP activity. The process of calcification was accelerated during long-term stimulation of hMSCs with IL-1ß. Since donor variability is a well-known issue, we suggest a new method to illustrate global changes of a random chosen donor population through collative analysis. We further demonstrate an absorbance assay to evaluate the degree of calcification during in vitro culture of monolayer expanded hMSCs. Our findings support the importance of IL-1ß in osteogenic differentiation of hMSCs in an in vitro monolayer culture model. A new online absorbance assay is a useful method to evaluate the osteogenic differentiation of hMSCs at early stages. These findings will be helpful in optimizing predifferentiation of hMSCs in vitro for bone tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.
Subject(s)
Calcification, Physiologic , Interleukin-1beta/pharmacology , Mesenchymal Stem Cells/cytology , Osteogenesis/drug effects , Adult , Aged , Bone and Bones/cytology , Calcinosis , Cell Differentiation , Cell Proliferation , Cells, Cultured , DNA/analysis , Female , Humans , Male , Middle Aged , Osteoblasts/cytology , Tissue Engineering/methods , Young AdultABSTRACT
INTRODUCTION: Runx2 is one of the most studied transcription factors expressed in mesenchymal stem cells (MSCs) upon their commitment toward an osteogenic differentiation. During endochondral bone formation in vivo, Sox9 directly interacts with Runx2 and represses its activity; however, the role of Sox9 in direct osteogenesis in vitro has been largely overlooked. METHODS: Bone marrow-derived human MSCs (hMSCs) were cultured in vitro either in the control or osteogenic medium supplemented with dexamethasone (DEX). To further investigate the role of Sox9 in direct osteogenesis in vitro, hMSCs were treated with Sox9 siRNA. RESULTS: We show here that Sox9 is the key early indicator during in vitro osteogenic differentiation of hMSCs. Osteogenic induction leads to a significant decrease of Sox9 gene and protein expression by day 7. Treatment of hMSCs with Sox9 siRNA enhanced mineralization in vitro, suggesting that downregulation of Sox9 is involved in direct osteogenesis. siRNA knockdown of Sox9 did not in itself induce osteogenesis in the absence of DEX, indicating that other factors are still required. CONCLUSION: Screening of not preselected donors of different ages and gender (n=12) has shown that the Runx2/Sox9 ratio on day 7 is correlated to the (45)Ca incorporation on day 28. The impact of Sox9 downregulation in the mineralization of human MSCs in vitro indicates a so far unprecedented role of Sox9 as a major regulator of direct osteogenesis. We propose that the Runx2/Sox9 ratio is a promising, early, in vitro screening method for osteogenicity of human MSCs.
Subject(s)
Core Binding Factor Alpha 1 Subunit/metabolism , Mesenchymal Stem Cells/metabolism , Osteogenesis , SOX9 Transcription Factor/metabolism , Adult , Aged , Core Binding Factor Alpha 1 Subunit/genetics , Dexamethasone/pharmacology , Down-Regulation/drug effects , Electroporation , Female , Humans , Male , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Middle Aged , Osteogenesis/drug effects , Osteogenesis/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , SOX9 Transcription Factor/genetics , Tissue Donors , Young AdultABSTRACT
The Alamar Blue assay is based on enzymatic reduction of indicator dye by viable cells and serves as an effective tool for assessing cell proliferation and as a screening technique. It can be applied in studies concentrating on animal, plant, yeast, and bacteria cells. Among the various methods for cell viability and cytotoxicity, it utilises all features of ideal and reliable test; it is one-step, sensitive, safe, non-toxic for cells, and cost-effective.
