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Artificial extracellular matrices for bone tissue engineering
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ABSTRACTS / Bone 42 (2008) S17–S110
Mesenchymal stem cells (MSC) are the source for bone and cartilage regeneration. In osteoporosis anabolic and antiresorptive drugs are applied which respectively stimulate the differentiation of MSC to osteoblasts (e.g. PTH) or inhibit osteoclasts (e.g. bisphosphonates (BP)). The aminobisphosphonate zoledronic acid (ZA) inhibits the farnesylsynthase of osteoclasts which leads to inhibition of posttranslational prenylation of proteins like Ras and Rho resulting in osteoclast apoptosis. It was shown that a once-yearly infusion of ZA 5 mg during a 3year period significantly reduced the risk of osteoporotic fractures. Osteoblasts and precursor cells are also targets of BP but little is known about short-time and chronic effects of ZA on MSC cultures and their osteogenic offspring. To clarify the influence of ZA on MSC, cells were treated with 5, 20 and 50 μM ZA for 6 and 16 h and the gene expression pattern was determined by RT-PCR. Additionally, MSC were treated with 5– 50 μM ZA for 3 h and the osteogenic differentiation potential of MSC was analyzed after 4 weeks in osteogenic medium. Cells were harvested and osteogenic marker genes were amplified by RT-PCR. Mineralization of the cell monolayer was analyzed by alizarin red staining. For chronic stimulation with BP MSC were treated with 5, 20 and 50 uM ZA for 24, 48 and 72 h and apoptosis and proliferation capacity of the cells were determined. Stimulation of MSC with 50 uM ZA for 6 to 16 h increased the expression of Dkk1 and runx3 and decreased the expression of L1CAM. Short-time treatment of MSC with 20 and 50 μM ZA for 3 h stimulated osteogenic differentiation capacity after 4 weeks compared to untreated controls which was shown by alizarin red staining. Osteogenic markers as runx3 and osteopontin were upregulated dose-dependently while osteocalcin was downregulated. In contrast, long-time treatment of MSC with 20 and 50 μM for 48 and 72 h led to increased apoptosis and decreased proliferation capacity. After 24 h no effect was detected. We show here that a 3 h exposure to μM concentrations of ZA is already sufficient to enhance osteogenic differentiation of MSC in vitro while long-time exposure to ZA impairs MSC proliferation and induces MSC apoptosis. In osteoporosis treatment this might be of clinical relevance to better determine the dosing and the upper threshold of BP accumulation in the bone microenvironment when constantly high local BP concentrations start to impair osteogenic effects in bone. doi:10.1016/j.bone.2007.12.130
121 Artificial extracellular matrices for bone tissue engineering Martin Ehrbar a, Matthias P. Lütolf b, Simone C. Rizzi a, Jeffrey A. Hubbell b, Franz E. Weber a a Department of Cranio-Maxillofacial Surgery, University Hospital Zurich, Zurich, Switzerland b Institute of Bioengineering, EPFL, Lausanne, Switzerland Materials from natural sources were successfully used as cell delivery or tissue regeneration devices in medical applications due to their innate biological properties. Artificial extracellular
matrices (aECMs) represent interesting alternatives for biopolymers as they can be specifically designed with respect to their mechanical properties, susceptibility to proteolytic activity and presentation of cell adhesion ligands or morphogens. Here we present modularly designed poly(ethylene glycol) (PEG)-based aECMs that are enzymatically formed under physiological conditions. They are a new class of biomaterials that enable the formation of matrices containing multiple tethered bioactive molecules and cell-responsive enzymatic substrates in a simple one step reaction. In the context of bone tissue engineering the aECM was designed to be degradable by cell secreted matrix metalloproteinase MMP-1 and to present bioactive molecules such as fibronectin derived cell adhesion peptide (RGD), vascular endothelial growth factor (VEGF) and bone morphogenic protein (BMP-2). Spreading and migration of MC3T3-E1 preosteoblastic cells in 2D and 3D cultures was strongly dependent on incorporated RGD-peptides. The cell migration behavior could be controlled by the responsiveness of the aECM to MMP-1 or the crosslinking density of the matrix. MC3T3-E1 cells and human mesenchymal stromal cells differentiated in response to BMP-2 which was entrapped within the gel or administered to the culture medium. This differentiation process was more pronounced in 3D cultures than in conventional 2D cultures. When implanted in rat cranial defects, aECMs were remodeled by proteolytically invading cells during 5 weeks. When BMP was entrapped within the matrix bone formation was induced on the hydrogel tissue interface resulting in the replacement of the provisional matrix by bony tissue. When VEGF was co-delivered with BMP a trend towards more robust bone formation could be observed. In contrast to natural materials our artificial ECMs allow the control of properties including matrix stiffness, protease susceptibility and presentation of biological cues. The tailoring of these properties enables us to rationally control cell behavior in both in vitro and in vivo contexts. These matrices could be useful tools for experimental cell biology as well as for in vivo applications such as bone tissue regeneration. doi:10.1016/j.bone.2007.12.131
122 New mechanisms for old targets: Novel approaches to steroid receptors in drug discovery Leonard P. Freedman Women’s Health and Musculosketal Biology, Wyeth Research, Collegeville, USA Steroid hormones such as estrogens and androgens play critical roles in normal and patho-physiology. Through their binding to nuclear hormone receptors, they also provide attractive targets for drug discovery. Considerable effort has been exerted over the past several years in designing so-called selective estrogen or androgen receptor modulators (SERMs and SARMs, respectively) that recapitulate the desired effects of these steroids on specific target tissues (i.e., the inhibition of bone resorption or stimulation of bone formation), without undesired
ABSTRACTS / Bone 42 (2008) S17–S110
Mesenchymal stem cells (MSC) are the source for bone and cartilage regeneration. In osteoporosis anabolic and antiresorptive drugs are applied which respectively stimulate the differentiation of MSC to osteoblasts (e.g. PTH) or inhibit osteoclasts (e.g. bisphosphonates (BP)). The aminobisphosphonate zoledronic acid (ZA) inhibits the farnesylsynthase of osteoclasts which leads to inhibition of posttranslational prenylation of proteins like Ras and Rho resulting in osteoclast apoptosis. It was shown that a once-yearly infusion of ZA 5 mg during a 3year period significantly reduced the risk of osteoporotic fractures. Osteoblasts and precursor cells are also targets of BP but little is known about short-time and chronic effects of ZA on MSC cultures and their osteogenic offspring. To clarify the influence of ZA on MSC, cells were treated with 5, 20 and 50 μM ZA for 6 and 16 h and the gene expression pattern was determined by RT-PCR. Additionally, MSC were treated with 5– 50 μM ZA for 3 h and the osteogenic differentiation potential of MSC was analyzed after 4 weeks in osteogenic medium. Cells were harvested and osteogenic marker genes were amplified by RT-PCR. Mineralization of the cell monolayer was analyzed by alizarin red staining. For chronic stimulation with BP MSC were treated with 5, 20 and 50 uM ZA for 24, 48 and 72 h and apoptosis and proliferation capacity of the cells were determined. Stimulation of MSC with 50 uM ZA for 6 to 16 h increased the expression of Dkk1 and runx3 and decreased the expression of L1CAM. Short-time treatment of MSC with 20 and 50 μM ZA for 3 h stimulated osteogenic differentiation capacity after 4 weeks compared to untreated controls which was shown by alizarin red staining. Osteogenic markers as runx3 and osteopontin were upregulated dose-dependently while osteocalcin was downregulated. In contrast, long-time treatment of MSC with 20 and 50 μM for 48 and 72 h led to increased apoptosis and decreased proliferation capacity. After 24 h no effect was detected. We show here that a 3 h exposure to μM concentrations of ZA is already sufficient to enhance osteogenic differentiation of MSC in vitro while long-time exposure to ZA impairs MSC proliferation and induces MSC apoptosis. In osteoporosis treatment this might be of clinical relevance to better determine the dosing and the upper threshold of BP accumulation in the bone microenvironment when constantly high local BP concentrations start to impair osteogenic effects in bone. doi:10.1016/j.bone.2007.12.130
121 Artificial extracellular matrices for bone tissue engineering Martin Ehrbar a, Matthias P. Lütolf b, Simone C. Rizzi a, Jeffrey A. Hubbell b, Franz E. Weber a a Department of Cranio-Maxillofacial Surgery, University Hospital Zurich, Zurich, Switzerland b Institute of Bioengineering, EPFL, Lausanne, Switzerland Materials from natural sources were successfully used as cell delivery or tissue regeneration devices in medical applications due to their innate biological properties. Artificial extracellular
matrices (aECMs) represent interesting alternatives for biopolymers as they can be specifically designed with respect to their mechanical properties, susceptibility to proteolytic activity and presentation of cell adhesion ligands or morphogens. Here we present modularly designed poly(ethylene glycol) (PEG)-based aECMs that are enzymatically formed under physiological conditions. They are a new class of biomaterials that enable the formation of matrices containing multiple tethered bioactive molecules and cell-responsive enzymatic substrates in a simple one step reaction. In the context of bone tissue engineering the aECM was designed to be degradable by cell secreted matrix metalloproteinase MMP-1 and to present bioactive molecules such as fibronectin derived cell adhesion peptide (RGD), vascular endothelial growth factor (VEGF) and bone morphogenic protein (BMP-2). Spreading and migration of MC3T3-E1 preosteoblastic cells in 2D and 3D cultures was strongly dependent on incorporated RGD-peptides. The cell migration behavior could be controlled by the responsiveness of the aECM to MMP-1 or the crosslinking density of the matrix. MC3T3-E1 cells and human mesenchymal stromal cells differentiated in response to BMP-2 which was entrapped within the gel or administered to the culture medium. This differentiation process was more pronounced in 3D cultures than in conventional 2D cultures. When implanted in rat cranial defects, aECMs were remodeled by proteolytically invading cells during 5 weeks. When BMP was entrapped within the matrix bone formation was induced on the hydrogel tissue interface resulting in the replacement of the provisional matrix by bony tissue. When VEGF was co-delivered with BMP a trend towards more robust bone formation could be observed. In contrast to natural materials our artificial ECMs allow the control of properties including matrix stiffness, protease susceptibility and presentation of biological cues. The tailoring of these properties enables us to rationally control cell behavior in both in vitro and in vivo contexts. These matrices could be useful tools for experimental cell biology as well as for in vivo applications such as bone tissue regeneration. doi:10.1016/j.bone.2007.12.131
122 New mechanisms for old targets: Novel approaches to steroid receptors in drug discovery Leonard P. Freedman Women’s Health and Musculosketal Biology, Wyeth Research, Collegeville, USA Steroid hormones such as estrogens and androgens play critical roles in normal and patho-physiology. Through their binding to nuclear hormone receptors, they also provide attractive targets for drug discovery. Considerable effort has been exerted over the past several years in designing so-called selective estrogen or androgen receptor modulators (SERMs and SARMs, respectively) that recapitulate the desired effects of these steroids on specific target tissues (i.e., the inhibition of bone resorption or stimulation of bone formation), without undesired