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Demineralized bone matrix (DBM) is used in humans to induce bone formation Spinal fusion surgery, osteoporotic fracture

Bone Matrix Production Using Canine Osteosarcoma Cells Embedded in Calcium Alginate Beads Cyndi H. T. Edwards, Aaron M. Fifer, Andrew K. Nickerson School of Chemical, Biological, and Environmental Engineering Oregon State University, Corvallis, OR. Results. Modeling. Introduction.

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Demineralized bone matrix (DBM) is used in humans to induce bone formation Spinal fusion surgery, osteoporotic fracture

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  1. Bone Matrix Production Using Canine Osteosarcoma Cells Embedded in Calcium Alginate Beads Cyndi H. T. Edwards, Aaron M. Fifer, Andrew K. Nickerson School of Chemical, Biological, and Environmental EngineeringOregon State University, Corvallis, OR Results Modeling Introduction Experiment 1 • Demineralized bone matrix (DBM) is used in humans to induce bone formation • Spinal fusion surgery, osteoporotic fracture healing, and dental procedures • Harvested from human cadaver bones • High variability in quality due to factors such as age, gender, and diet • Major drawbacks can be overcome by producing osteoinductive proteins in vitro • The goal of the project is to determine if gel encapsulation of bone-forming osteoblasts is a feasible method of cell stabilization for commercial DBM production. • Osteoblasts prefer to grow attached to a surface – the alginate matrix of a bead provides sites for adhesion • Bone cells respond to physical stress by producing more protein, so agitation may increase yield • Alginate weight percent is involved in shear stress transduction, and effects pore size Cells The percentage of viable cells digested from 20 beads from each of the four treatments at Day 9. A cross-sectional slice of a gel bead stained with trypan blue shows the distribution of cells within a bead taken at 4x magnification. Cells can be seen as small dark specks. Experiment 2 Materials and Methods Gel Beads Incubator – 37°C, 5% CO2 Potential Improvements Beakers • Cell viabilities in all experiments were lower than anticipated. In the first experiment, it was due to low inoculation density resulting in poor cell-to-cell signaling. In the second experiment, the viability was higher at about 60% on Day 2, but still low. In the future if work is continued on this project, the following improvements will be made: • Purchase Type-1 collagenase for bead digestion • Use ELISA (Enzyme-Linked Immunosorbent Assay to determine trace protein concentrations • Additional controls for the protein assay • Do not use expired materials Orbital Shaker Protein concentration as measured by Bradford assay in samples being prepared for SDS-PAGE (Sodium Dodecyl Sulfate – PolyAcrylamide Gel Electrophoresis). T-flasks Day 2 After the medium is removed, the gel beads remain pink because they have taken up medium. Here a bead is being taken for imaging. WaterPan Acknowledgements We would like to thank Dr. Russell Turner and the Skeletal Research Lab for providing lab space and equipment, Dr. Philip Harding for funding and support, Dr. Kevin Marley for providing cells, supplies, and wisdom, Dr. Adam Higgins for advice on freezing cells, and Dr. Christine Kelly for guidance, reference materials, and assistance with assays. Day 6 References Dead Cell SDS-PAGE results: other than ladder, which is composed of a mixture of proteins of known molecular weight, most of the lanes resemble the media control. Flask 2 and flask 6 are T-flask controls. Exp. 2-A, 2-B, and 2-C are beads that could not be digested that were soaked in protein extraction solution. Healthy Cell Bae et al. “Intervariability and Intravariability of Bone Morphogenetic Proteins in Commercially Available Demineralized Bone Matrix Products.” Spine 2006; 31(12): 1229-1306. Chen, Huang-Chi and Yu-Chen Hu. “Bioreactors for tissue engineering.” Biotechnology Letters 2006; 28: 1415-1423. Freshney, R. Ian. Culture of Animal Cells. New York: Wiley-Liss, 2000 Phelan, Mary C. “Basic Techniques for Mammalian Cell Tissue Culture.” Current Protocols in Cell Biology (1998) 1.1.1-1.1.10 Sandhu et al. “Demineralized bone matrix, bone morphogenetic proteins, and animal models of spinal fusion: an overview.” European Spine Journal 2001; 10:S122-131. Unpublished data, Oregon State University Skeletal Research Lab. Unhealthy Cell Viable Cell A hemacytometer is used to determine cell viability. Healthy osteosarcoma cells appear stretched out, while unhealthy cells appear spherical in a T-flask (10x magnification). Day 9

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