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Hyzy Sharon L 201208 Mast.Pdf (762.5Kb) ADVERSE EFFECTS OF BONE MORPHOGENIC PROTEIN-2 DURING OSSEOINTEGRATION A Thesis Presented to The Academic Faculty by Sharon Leigh Hyzy In Partial Fulfillment of the Requirements for the Degree Masters of Science in the School of Biology Georgia Institute of Technology August 2012 ADVERSE EFFECTS OF BONE MORPHOGENIC PROTEIN-2 DURING OSSEOINTEGRATION Approved by: Dr. Barbara D. Boyan, Advisor Dr. Zvi Schwartz, Co-advisor School of Biomedical Engineering School of Biomedical Engineering Georgia Institute of Technology Georgia Institute of Technology Dr. Kirill Lobachev Dr. J. Todd Streelman School of Biology School of Biology Georgia Institute of Technology Georgia Institute of Technology Date Approved: May 03, 2012 ACKNOWLEDGEMENTS I would express my profound gratitude to my advisors, Dr. Barbara D. Boyan and Dr. Zvi Schwartz, for supporting my decision to begin my degree and for guidance of my research. I would like to thank the members of my committee, Dr. Kirill Lobachev and Dr. J. Todd Streelman, for their encouragement, difficult questions, and insightful comments during the completion of this thesis. I would like to acknowledge the members of the Boyan/Schwartz laboratory for their support during this process, particularly the staff members. My sincere thanks go to Dr. Rene Olivares-Navarrete for encouraging my scientific development, constant guidance, and patience with this thesis. I am grateful to the undergraduates who have worked with me, particularly Christian Tan and Rahul Basu. My parents, sister, grandparents, aunts, and uncles have been supportive of my postgraduate studies and unendingly understanding when my work consumes my time, for which I am eternally thankful. iii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS iii LIST OF TABLES v LIST OF FIGURES vi LIST OF SYMBOLS AND ABBREVIATIONS vii SUMMARY viii CHAPTER 1 INTRODUCTION 1 Specific Aims 5 2 BONE MORPHOGENIC PROTEIN 2 AND INTERLEUKINS 6 Introduction 6 Methods 8 Results 12 Discussion 20 Conclusion 23 3 BONE MORPOGENIC PROTEIN 2 AND OSTEOBLAST APOPTOSIS 24 Introduction 24 Methods 25 Results 29 Discussion 37 Conclusion 40 4 CONCLUSIONS 41 REFERENCES 43 iv LIST OF TABLES Page Table 2.1: Primer sequences used in real-time qPCR analysis 10 Table 3.1: Primer sequences used in real-time qPCR analysis 27 v LIST OF FIGURES Page Figure 2.1: Effect of surface microstructure and energy on anti-inflammatory gene expression 12 Figure 2.2: Effect of surface microstructure and energy on pro-inflammatory interleukin expression of MG63 cells 13 Figure 2.3: Effect of surface microstructure and energy on osteogenic differentiation of MG63 cells 14 Figure 2.4: Effect of surface microstructure and energy on interleukin production by MG63 cells 15 Figure 2.5: Effect of BMP2 on MG63 differentiation on microstructured Ti surfaces 16 Figure 2.6: Effect of BMP2 on inflammatory interleukin expression in MG63 cells cultured on microstructured Ti surfaces 17 Figure 2.7: BMP pathway modulation of inflammatory microenvironment produced by MG63 cells on microstructured Ti surfaces 19 Figure 3.1: Effect of BMP2 treatment on apoptotic proteins in NHOst cells 30 Figure 3.2: Effect of BMP2 on mesenchymal stem cell apoptosis 31 Figure 3.3: Effect of BMP2 on MG63 cell apoptosis 32 Figure 3.4: Effect of BMP2 on normal human osteoblast apoptosis 33 Figure 3.5: Effect of BMP2 treatment on NOG mRNA levels 34 Figure 3.6: Effect of NOG silencing on MG63 cell apoptosis 35 Figure 3.7: Effect of NOG silencing on BMP2-induced MG63 cell apoptosis 36 Figure 3.8: Effect of BMP pathway inhibition on BMP2-induced NHOst apoptosis 37 vi LIST OF SYMBOLS AND ABBREVIATIONS BMP2 Bone morphogenic protein 2 IL Interleukin MSC Mesenchymal stem cells NHOst Normal human osteoblasts Ti Titanium TCPS Tissue culture polystyrene GAPDH Glyceraldehyde 3-phosphate dehydrogenase TAK1 Transforming growth factor β–activated kinase 1 TAB TAK1-binding protein shNOG-MG63 Silenced Noggin MG63 cell line TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling vii SUMMARY Modifications of biomaterial surface properties are employed to increase osteoblast differentiation and bone formation. Microtextured metallic surfaces promote osteoblast differentiation and high surface energy- achieved by controlling surface hydrocarbon contamination- increases osteoblast differentiation and peri-implant bone formation. Recombinant human bone morphogenic protein 2 (BMP2) is approved to induce bone formation in a number of applications. It is used clinically in combination with biomaterials to improve peri-implant bone formation and osseointegration. The amount of BMP2 that is required is large and inflammatory (swelling/seroma) and bone- related (ectopic bone/bone resorption) complications have been reported after BMP2 treatment. The aim of this study was to examine potential deleterious effects of BMP2 on the inflammatory environment and apoptosis of osteoblasts. Surface roughness and energy decreased pro-inflammatory interleukins and increased anti-inflammatory interleukins. In contrast, BMP2 abolished the surface effect, increasing pro-inflammatory interleukin (IL) 6, IL8, and IL17 in a surface roughness- dependent fashion and decreasing anti-inflammatory IL10 on rough surfaces. 5Z-7- Oxozeaenol and Dorsomorphin, but not H-8, blocked the effect of BMP2 on IL1A expression. There was an increase in expression of IL6 when treated with BMP2 for the control and H-8 groups, but both 5Z-7-Oxozeaenol and Dorsomorphin blocked the effect. Both 5Z-7-Oxozeaenol and H-8 blocked the effect of BMP2 on IL10 expression. BMP2 treatment had little effect on apoptosis in human mesenchymal stem cells (MSCs). Exogenous BMP2 had no effect on TUNEL. Caspase-3 activity was viii increased only at 200ng/ml BMP2. BAX/BCL2 decreased in MSCs treated with 50 and 100ng/ml BMP2. In contrast, BMP2 increased caspase-3 activity and TUNEL at all doses in normal human osteoblasts (NHOst). BAX/BCL2 increased in NHOst treated with BMP2 in a dose-dependent manner. Cells treated with 200 ng/ml BMP2 had an 8-fold increase in BAX/BCL2 expression in comparison with untreated cells. Similarly, BMP2 increased DNA fragmentation in NHOst cells. The BMP2-induced increase in DNA fragmentation was eliminated by 5-Z7-Oxozeaenol and Dorsomorphin. The results suggest that while surface features modulate an initial controlled inflammatory response, the addition of BMP2 induces a pro-inflammatory response. The effect of BMP2 on apoptosis depends on cell maturation state, inducing apoptosis in committed osteoblasts. BMP2 together with microtextured orthopaedic and dental implants may increase inflammation and possibly delay bone formation. Dose, location, and delivery strategies are important considerations in BMP2 as a therapeutic and must be optimized to minimize complications. ix CHAPTER 1 INTRODUCTION Bone morphogenetic proteins Bone morphogenetic proteins (BMPs) were first described in 1965 by Marshall Urist as the active component of demineralized bone matrix able to induce ectopic bone formation. BMPs are members of the TGF-β growth factor superfamily (1). BMPs are generated by proteolytic cleavage of the transcribed preproproteins (2). After cleavage, mature BMPs are secreted from the cell and form homodimers or heterodimers to activate signaling (3, 4). BMPs transduce signals through membrane serine/threonine kinase receptors (5-7). These receptors, categorized as type I and type II receptors, have differential specificity for BMP ligands (8, 9). Dimers of type I receptors combine with dimers of type II receptors to transduce signaling. It has been demonstrated that the order of receptor recruitment and binding determines signaling (9). BMP binding to preformed complexes activates Smad signaling, while BMP-induced formation of heteromeric complexes activates non-canonical BMP signaling (9). Smads are intracellular signaling proteins that are categorized as either receptor-regulated Smads, common-partner Smad, or inhibitory Smads (5, 10). Type I BMP receptors phosphorylate Smad1/5/8. The activated Smad then complexes with common-partner Smad4, translocates to the nucleus, and initiates downstream signaling. In addition to this canonical Smad signaling, BMP signaling can also activate mitogen activated kinase signaling molecules (ERK1/2, p38, Jun N-terminal kinase) through activation of transforming growth factor β–activated kinase 1 (TAK1) and TAK1-binding protein (TAB) (6, 11, 12). BMP signaling is required for multiple processes in embryonic and adult tissue formation and homeostasis (1, 13-15). BMP signaling controls mesoderm patterning, chondrogenesis and osteogenesis in skeletogenesis, cartilage and bone formation, and limb development. In vitro, BMPs induce adipogenic, chondrogenic, and osteogenic differentiation of progenitor cells (16-18). BMP signaling also promotes patterning of craniofacial structures, and limb development. Programmed cell death, or apoptosis, is important in several of these processes. BMP2, BMP4, and BMP7 initiate apoptosis in the developing limb (19-21). While the effects of BMPs on apoptosis are clear, the controlling mechanisms in embryonic and adult processes remain to be elucidated. Besides roles in development, BMP2 is essential in bone regeneration. In a mouse model, limb-specific deletion of BMP2 expression delayed endochondral bone formation and led to micro-fractures in animals by post-natal day 14 (22, 23). Recombinant BMP2 and BMP7 (OP-1) are approved by the Food and Drug Administration for use in bone formation (24). BMP2 is used in fracture
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