Past Bone Seminars

2002-2003

___September 2002 ___
Speaker: Aaron S. Posner PhD Scientist Emeritus, Hospital For Special Surgery; Professor Emeritus, Cornell University Medical College
Topic: Bisphosphonates in Federal Patent Court

Dr. Posner’s Research Interests: Nature of hard tissue at the molecular level and the mechanisms of normal and pathological tissue mineralization

Abstract
This lecture recounts the adventures of a retired professor in a patent case: Merck & Co., Inc. (plaintiff) v. Teva Pharmaceuticals USA, Inc. and Zenith Goldline Pharmaceuticals, Inc. (defendants). The defendants claimed to the FDA that Merck's patent for the widely described bisphosphonate, Fosamax, was invalid and they intended to make and sell a generic of this drug; Merck sued to prevent this action. Dr. Posner was hired as an expert witness for Zenith because of his research and teaching in the hard tissue field. Background will be given in the biochemistry and clinical application of bisphosphonates, particularly in both bone loss and pathological calcification. It is their Ca-binding property that makes these compounds useful as both chemical detergents and clinical bone seekers. As an example of the latter, Technecium-labelled Etidronate (1, hydroxyethylidene bisphosphonate) has long had an important application in the radiation imaging of bone dyscrasias (skeletal scintigraphy). In the trial, the defendants' case rested upon two major points: (1) there is an earlier patent for alendronic acid which supersedes Merck's patent for this material; (2) Merck is making and selling a sodium salt of alendronic acid although they only hold the (disputed) patent for the acid form. The court arguments and the decision will be related in detail.

___October 2002 ___
Speaker: Karl J. Jepsen PhD, Assistant Professor, Department of Orthopaedics, Mount Sinai School of Medicine
Topic: Bones, Genes, and Everything in Between

Dr. Jepsen’s Research Interests: Mechanical testing of bone and the effects of heritability on the mechanical properties of bone

Abstract
Osteoporotic fracture incidence and underlying risk factors like low peak bone mass are heritable, but the genetic basis of osteoporosis remains poorly understood. Based on beam theory, stating that mechanical properties depend on both the amount and quality of a structure's constituent materials, we investigated the relationship between whole bone mechanical properties and a set of morphological and compositional traits in femurs of eight inbred mouse strains. K-means cluster analysis revealed that individual femora could be classified reliably according to genotype based on the combination of bone area (tissue amount), moment of inertia (tissue distribution) and ash content (tissue quality). This trait combination explained 66-88% of the inter-strain variability in four whole bone mechanical properties that describe all aspects of the failure process, including measures of brittleness. Stiffness and maximum load were functionally linked to cortical area, while measures of brittleness were linked to ash content.
In contrast, work-to-failure was not directly linked to a single trait but depended on a combination of trait magnitudes. Based on these findings, which were entirely consistent with established mechanical theory, we developed a hierarchical paradigm relating the mechanical properties that define bone fragility with readily measurable phenotypic traits that exhibit clear heritability. This paradigm may help guide the search for genes that underlie fracture susceptibility and osteoporosis; moreover, because the traits we examined appear to be measurable by non-invasive means, this approach may also prove directly applicable to osteoporosis risk assessment.

___November 2002 ___
Speaker: Clinton Rubin PhD, Professor and Chair, Department of Biomedical Engineering, Director, Center for Biotechnology, State University of New York, Stony Brook
Topic: Searching for Wolff's Law: The Osteogenic Potential of Low-Level Mechanical Signals

Dr. Rubin’s Research Interests: Understanding the cellular mechanisms responsible for the growth, healing, and homeostasis of bone. More specifically, how biophysical stimuli (i.e., mechanical, electrical, temperature, magnetic, pressure) mediate these responses. The clinical significance of this work is applicable to the inhibition of osteopenia, the promotion of bony ingrowth into prostheses or skeletal defects, and the acceleration of fracture healing. These goals are approached via interdisciplinary studies at the biochemical, molecular, cellular, tissue, organ, computational (e.g., FEM) and clinical levels.

Abstract
There is increasing evidence that extremely low magnitude (<100 microstrain) mechanical signals can be strongly osteogenic if applied at a high frequency (15 to 60 Hz). Such high frequency low magnitude strains comprise a dominant component of a bone's strain history, indicating that these mechanical events represent a significant determinant of bone morphology. With this in mind, we have been examining if small perturbations in high frequency loading, induced non-invasively into the lower appendicular skeleton, will stimulate an increase in bone mass without sacrificing bone quality. Short term animal studies provide evidence that very low intensity (<10 microstrain) mechanical stimuli are strongly anabolic if applied above 20Hz. Extremely low-level strains, if induced at 20Hz, promote osseointegration. Ten minutes per day of these low level signals (0.25g), induced non-invasively using an oscillating platform, are able to retain bone mass despite 23 hours and 50 minutes of disuse, while ten minutes of normal weight bearing fails to do so. Longer term animal studies (one year), have shown that low level mechanical loading, inducing cortical strains on the order of 5 microstrain, can increase cancellous bone volume fraction, thicken trabeculae, increase trabecular number 6 and enhance bone stiffness and strength. Considering these strain levels are far below (<1/1000th) those which may cause damage to the tissue, we believe these signals hold great potential as a mechanical prophylaxis for osteoporosis.

___December 2002 ___
Speaker: Adele L. Boskey PhD. Starr Chair in Mineralized Tissue Research and Director of the Mineralized Tissue Laboratory, Hospital for Special Surgery; Professor of Biochemistry, Weill Medical College of Cornell University; Adjunct Professor of Biomedical Engineering, The City College of New York
Topic: Mechanisms of Action of Matrix Phosphoproteins in the Regulation of Biomineralization

Dr. Boskey’s Research Interests: Factors regulating mineral deposition, mineral growth, and remodeling in bones and teeth. These questions have a bearing on treatment of diseases in which mineralization is altered (osteoporosis, osteogenesis imperfecta, osteomalacia, osteopetrosis, etc.), in the prevention of dystrophic calcification in arteries, prosthetic valves, and other soft tissues, and in engineering bone replacement.

Abstract
The matrices of many species that deposit intracellular or extracellular minerals is rich in anionic proteins. These proteins are thought to regulate mineral deposition acting as ion reservoirs, nucleators, and regulators of growth. In bone, calcified cartilage, and dentin there are several proteins that are differentially phosphorylated by the action of kinases and phosphatases. The extent of phosphorylation varies with tissue site. It is hypothesized that the extent of phosphorylation determines the ways in which each of these proteins regulates the mineralization process. To validate this hypothesis for each protein, four pieces of evidence are required. First the protein must be changed (in content or phosphorylation state) at the site of mineralization. Second solution based studies should demonstrate distinct effects of the phosphorylated/ dephosphorylated proteins on apatite (bone-like mineral) formation and/or crystal growth. Third, the phosphorylated and dephosphorylated proteins should have distinct effects on cell mediated in vitro mineralization. Finally, animal models or examples of human diseases, in which the protein is ablated or over-expressed, should show alterations in mineral and hence mechanical properties. Verification of this hypothesis based on osteopontin and dentin matrix protein-1 (DMP-1) will be discussed.

___March 2003 ___
Speaker: Nicola C. Partridge PhD, Professor and Chairman, Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ
Topic: Parathyroid Hormone Regulation of Gene Expression in the Osteoblast

Dr. Partridge’s Research Interests: We have shown that parathyroid hormone (PTH) induces the transcription of collagenase-3 in osteoblastic cells. This involves the protein kinase A pathway and induction of activator protein-1 transcription factors as well as phosphorylation of another transcription factor, core binding factor a1. Thus, PTH regulates both transcription factors through this pathway, either by increasing their expression or altering their phosphorylation. Our hypothesis of the functions of these proteins is that they interact physically in a nucleosomal structure, recruiting other proteins such as co-activators, modifiers of the nucleosome and the general transcription factors. In another project, we are investigating the signal transduction pathways whereby PTH induces anabolic effects on bone and determining novel genes regulated by this hormone. In two other projects we have shown that extracellular concentrations of collagenase-3 are regulated by the existence of a specific receptor that binds the enzyme. Subsequent internalization and degradation of collagenase-3 require transfer to the endocytotic receptor, the low-density lipoprotein receptor-related protein. We have recently purified and identified the specific receptor as a 170-kDa protein. We are now further characterizing the collagenase-3 removal process in osteoblasts, fibroblasts as well as chondrocytes from patients with osteoarthritis. The latter work could lead to new treatments for this disease.

Abstract
Parathyroid hormone (PTH) plays a central role in regulation of calcium metabolism but also appears to have a role as an anabolic hormone for bone. The hormone has multiple actions, including many direct changes in the functions of the osteoblast. To date, all of its skeletal effects appear to be mediated by binding to a single receptor on osteoblasts. In this process, PTH causes a change in osteoblastic gene expression and function. Apart from producing osteoclast-activating factors such as RANKL and interleukin -6 (IL-6) in response to PTH, the osteoblast appears to also have a direct role in matrix degradation in response to this hormone. For instance, PTH induces collagenase-3 gene transcription in osteoblastic cells through a cAMP-dependent pathway requiring de novo protein synthesis. Thus, this is a secondary effect that involves the induction and activation of specific transcription factors acting on this gene. We identified the PTH-response elements as being the activator protein-1 (AP-1) and the core binding factor a1 (Cbfa1) binding sites in the collagenase-3 promoter. We have demonstrated a PTH-dependent cooperative interaction between the sites and the proteins binding to them. By gel shift analysis, we have shown enhanced binding of c-Fos and c-Jun proteins at the AP-1 site upon treatment with PTH but no significant change in the level of Cbfa1 binding to its site. Supporting our earlier work, the PKA pathway was shown to be the only pathway regulating the collagenase-3 promoter as a mediator of PTH action. The importance of this pathway was demonstrated by the fact that PTH stimulates the transactivation of activation domain-3 in Cbfa1 through its PKA site. PTH regulates both transcription factors through this pathway, either by increasing their expression or altering their phosphorylation. Our hypothesis of the functions of these proteins is that they interact physically in a nucleosomal structure, recruiting other proteins such as co-activators, modifiers of the nucleosome and the general transcription factors. If there is time, I will talk about some of our new work on PTH regulation of novel genes in osteoblasts.

___April 2003 ___
Speaker: Helen H. Lu PhD, Department of Biomedical Engineering, Columbia University
Topic: Design Considerations in Orthopedic Tissue Engineering of Bone, Soft Tissue, and Interfaces

Dr. Lu’s Research Interests: The regeneration of a functional interface between bone and ligaments/tendons, as well as the interface connecting bone and cartilage. Providing a mechanically functional interface between the biomaterial and bone tissue, and between bone and soft tissue will significantly improve the long-term stability of the implant. Dr. Lu's research group at the Biomaterials and Interface Tissue Engineering Laboratory at Columbia University are developing in vitro culturing systems to mimic the formation of the interface between bone and soft tissue (cartilage and ligament) in vivo. These systems are used to examine the effect of co-culturing on the growth and differentiation of osteoblasts, chondrocytes and ligament fibroblasts. Results from these studies are being utilized to design 3-D, tissue engineered scaffold systems that can be applied in the treatment of osteoarthritis and anterior cruciate ligament injuries.

Abstract
Optimal treatment modalities in orthopedics are needed to meet the demands of an aging yet still active population. Due to limitations associated with existing biological and synthetic grafts, tissue engineering has emerged as an alternative approach in orthopedic repair and regeneration. An area of recent interest is the design of interfaces to facilitate the integration of bone with tissues such as muscle, cartilage, ligaments, and tendon. The nature of the tissue-tissue interface is important in the fixation of existing implants to bone, and in the integration of a variety of tissues formed in vitro using tissue engineering approaches. Development of a bone-soft tissue interface is a highly complex problem, involving the engineering of both soft and hard tissue, as well as the interfacial region. This talk will first describe our research efforts in bone tissue engineering utilizing a composite scaffold of biodegradable polymers and bioactive ceramics seeded with bone-forming cells, as well as the use of bone morphogenetic proteins in promoting mineralization by varied cell sources. Next, results from our work in tissue engineering of the anterior cruciate ligament using a three-dimensional, porous, biodegradable, and braided construct will be presented. The design, in vitro and in vivo characterizations, and optimization of both soft and hard tissue engineering constructs will be discussed. Finally, current efforts in the integration of soft and hard tissues will be described, and new research directions will be proposed.

___May 2003 ___
Speaker: Stephen C. Cowin PhD, The City College of New York
Topic: Bones Have Ears

Dr. Cowin’s Research Interests: Mechanics of materials, particularly in determining the influence of microstructure on the gross mechanical behavior of granular, composite, and biological materials. Dr. Cowin concentrated on bone mechanics for many years and has been interested in tissue building process in skeletal tissues in recent years.

Abstract
The structural adaptations of living require a cell-based mechanosensing system with a sensor cell that perceives the mechanical deformation of the mineralized matrix in which the cell resides, a cell-based mechanosensing system not unlike that in the ear. One of the most perplexing features of this mechanosensory system in bone is the very low strain level that a whole bone experiences in vivo compared to that needed to produce a response in cells. The amplitudes of the in vivo strains generally fall in the range 0.04 to 0.3 percent for animal locomotion and seldom exceed 0.1 percent. These strains are nearly two orders of magnitude less than those needed (1% to 10%) to elicit biochemical signals necessary for communication of the sensing cells with the cells that deposit and resorb bone tissue. There is a paradox in the bone mechanosensing system in that the strains that activate the bone cells are at least an order of magnitude larger than the strains to which the whole bone organ is subjected. A hierarchical model ranging over length scales that differ by 9 orders of magnitude, from the subcellular level to the whole bone level, is used to resolve this paradox. Using this extended model, it is possible to explain how the fluid flow around a bone cell process can lead to strains on the cell process structure that are two orders of magnitude greater than the ambient strains in the mineralized matrix in which the cell resides. This bone mechanosensory system has many features in common with the auditory system.

___June 2003 ___
Speaker: Mitchell B. Schaffler PhD, Professor of Orthopaedics, Cell Biology, and Anatomy, Director of Orthopaedic Research, Department of Orthopaedics, Mount Sinai School of Medicine; Co-Director, New York Center for Biomedical Engineering, The City College of New York
Topic: Mechanical Factors and Remodeling of Compact Bone Phosphoproteins in the Regulation of Biomineralization

Dr. Schaffler’s Research Interests: Bone biomechanics and tissue physiology, with emphasis on understanding mechanical wear and tear (fatigue) processes in skeletal tissues, and the cellular/molecular mechanisms used in the detection and repair of connective tissue matrix injury. Related areas of interest extend to aging and skeletal fragility, including osteoporosis, and the healing and regeneration of bone.

Abstract
Skeletal tissues maintain a balance between mechanical wear and tear (i.e. fatigue) damage and intrinsic, matrix-level repair. Imbalance in this damage-repair homeostasis, either because of excessively rapid damage accumulation or because of ineffective, inadequate or inappropriate biological responses to chronic injury, leads to pathology and, ultimately, mechanical failure of skeletal elements. These processes are implicated in a wide range of conditions, including overuse injuries, tissue fragility in aging, tendon and ligament failures and degenerative joint disease.
A major function of Haversian (osteonal) remodeling is to remove and replace regions of compact bone that accumulate microdamage due to fatigue. However, little is known about the damage or remodeling responses that occur at the levels of fatigue expected to result from normal wear and tear. In particular, how bone-remodeling units "target" microscopically damaged areas of bone is unknown. Our recent studies of remodeling-repair of microdamage find that intracortical resorption effectively removes both linear-type microcracks and diffuse matrix damage. Alterations of osteocyte and canalicular integrity are observed in microdamaged areas. Resorption spaces were also seen within areas of cortex in which no bone matrix damage occurred, but alterations of osteocyte and canalicular integrity were evident. Recent studies indicate that these alterations of osteocyte integrity correspond to osteocyte apoptosis, or programmed cell death. Thus, osteocyte death or damage may provide a key stimulus for this signaling or targeting the remodeling process in bone.