Virginia L. Ferguson - US grants

1055989
Ferguson

The osteochondral (OC) interface is a region that presents a significant engineering challenge in that it experiences rigorous shear and compressive stresses, yet it joins together two highly dissimilar tissues: stiff bone and soft cartilage. Surprisingly, the OC interface is robust and rarely fails in vivo. Cartilage replacements for joint repair have had better success when integrated into the OC interface and underlying bone. However tissue-engineering approaches have not produced a viable replacement material, in part due to a limited understanding of how the biologic tissue is formed and structured to facilitate stress transfer across the interface. The transition between bone and cartilage necessitates sophisticated measures for functional grading of mineral and extracellular matrix content, composition, and organization. Only by mimicking both the properties and the underlying function of the biologic tissue can we successfully engineer solutions for joint repair that function like and integrate into surrounding healthy tissue. To realize the PI's long-term goal of reverse-engineering the osteochondral interface for successful joint repair, two aims are proposed:
Aim 1: Investigate mechanisms of load transfer across the osteochondral interface in synovial and cartilaginous joints by characterizing: (1) gradation of mechanical properties at multiple scales, (2) content, composition, organization and spatial distribution of mineral and extracellular matrix, and (3) the 3-D structure of the mineralized interface.
Aim 2: Engineer functionally-graded materials that recapitulate and enable study of specific microstructural characteristics of the osteochondral interface. We seek to engineer materials to investigate the mechanical contribution of single functional grading mechanisms at relevant length scales such as step-wise decreases in mineral content, mineralized particles that are graded in density and connectivity, or aligned collagen fibers that extend from the cartilage into the adjacent mineralized region. Finite element models will enable assessment of the biologic tissue and engineered materials. Finally, we aim to engineer a complex OC interface that includes several of the most important functional grading mechanisms; where material design will be informed by characterization in Aim 1.
The educational goals of this CAREER proposal are to: 1) continue inclusion of underrepresented minority and female students in the PI's research program and 2) improve recruitment and retention of 1st and 2nd year underrepresented minority and women students through graduate-student mentored research experiences that largely focus on rehabilitation or enabling technologies for those with disabilities. In pursuit of this goal, the PI is piloting a new program: "Your Own Undergraduate Research Experience at CU", YOU'RE @ CU, in 2010-11 that targets freshman and sophomores to work in research labs, improves retention in engineering, encourages vertical integration of learning, and engages undergraduates to seek graduate degrees. Graduate student mentors will gain mentoring experience and benefit from a work-life-career seminar series. The end goal is to generate a pipeline of engineers who consider research careers and especially to excite students about using engineering for applications in rehabilitation and enabling assistive technology development.
Intellectual Merit: This proposed research will enable advancements in engineering solutions for common, debilitating orthopedic problems, such as osteoarthritis and spinal disc degeneration, and an improved understanding of how nature anchors soft and hard materials to facilitate load transmission. Overall, this CAREER award will enable the PI to expand her current investigation of the osteochondral interface, further extend her research program into the areas of tissue engineering, and enable her to later study a range of clinically-relevant orthopedic research questions.
Broad Impact: Joint disease is one of the most frequent causes of disability in the United States, where osteoarthritis and degenerative disc disease in the spine affect 27 and 65 million Americans per year, respectively. Current efforts in osteochondral tissue-engineering are limited by the lack of understanding of how the native tissue transmits loads and resists failure. Further, engineering solutions, including surgical insertion of orthopedic devices, require improved understanding of the OC interface to ensure matching of functional behavior with the surrounding tissue. In addition, a multidisciplinary approach to study such problems will be used to develop student's critical thinking skills by using engineering concepts and tools to study biological and medical problems to develop solutions for rehabilitation and disabilities through the PI's laboratory and throughout the CU College of Engineering via a new program, YOU'RE@CU, where lowerclassmen will engage in a graduate student-mentored research experience

1338154
Ferguson

The proposed system facilitates multimodal analysis of a single ROI on one modular platform by integrating an Atomic Force Microscope, an Ultra Nanoindentation Tester (max depth = 100 ìm; load = 50 mN), a Nanoindentation Tester (1000 ìm; 500 mN), and an optical microscope through which 2-D Raman microscopy (514 or 785 nm lasers) is performed. A high precision translation stage moves samples between modules. This proposed instrument will facilitate combined mechanical analysis, across multiple small length-scales, with determination of chemical composition at a single ROI. AFM and nanoindentation are well-established techniques for assessing stiff, conventional engineering materials. Yet most commercially available nanoindenters fail to accurately test materials that are hydrated, soft, or subjected to thermal changes. The CSM Instruments platform adeptly overcomes limitations of existing indenter systems by using one independent probe to find a sample's surface and continuously monitor thermal drift while a second probe indents the sample - critical for soft or fluid-like surfaces. Integration with a Raman microscope will facilitate combined analysis of mechanical behavior and materials chemistry in one exact ROI. Acquisition of this system at CU Boulder will inform design of cutting-edge materials in an unparalleled fashion and enhance our activities in world-class materials research.

The osteochondral (OC) tissues of hyaline articular cartilage, calcified cartilage and subchondral bone operate collectively, thus the degeneration of any one inevitably influences the others. Pressurization of interstitial fluid in healthy cartilage protects cells from excessive tissue strain under large joint contact forces. In osteoarthritis (OA), extracellular matrix degradation results in vastly increased cartilage tissue permeability and diminished mechanical properties and function. Concomitantly, degenerative processes (e.g., vascular invasion and microfractures) create new routes for fluid efflux through calcified cartilage and into the underlying subchondral bone. Thus mechanical and biochemical signaling between OC tissues are perturbed. Moreover an interplay exists between tissue strain and interstitial fluid flows (iFFs) that stimulates bone cells to alter bone structure and, consequently, their mechanical environment (e.g., subchondral bone thickens substantially in late-stage OA). In particular, osteocytes, which are resident cells within bone, sense and respond to iFF changes, to disrupt homeostatic regulation of bone mass through several established mechanosensory pathways (e.g., PGE2 and Wnt/?-catenin signaling). Thus our overarching hypothesis for this research is that the changes to cartilage at the onset of OA, which lead to higher permeability, affect the iFF in subchondral bone and alter osteocyte signaling by releasing PGE2 among other molecules and activating Wnt/?-catenin signaling, which signals osteoblasts and leads to the observed pathophysiological phenotype of increased subchondral bone mass. To test this hypothesis, we will establish a novel 3D tri-layered hydrogel that emulates the complex flow behavior of OC tissues under dynamic compressive forces. Tri-layered hydrogels will be designed with a soft layer that experiences large strains and induces iFF into the bony layer, an intermediate layer whose crosslink density is tuned for permeability to control fluid velocity, and finally a stiff bony layer comprised of an engineered lacunocanalicular network of osteocytes that experience highly dynamic fluid flow with minimal strains. We have outlined two specific aims. In specific aim #1, we will develop tri-layer hydrogels that possess moduli and permeability characteristics to emulate iFF behavior of healthy and osteoarthritic bone tissue using a combined experimental and computational approach. In specific aim #2, we will perform a series of experiments to study the mechanisms by which osteocytes, when embedded in an engineered lacunocanalicular network in the stiff bony layer of the tri-layer hydrogels, respond to different levels of iFF. Upon completion of this project, we expect to have developed a tri-layered hydrogel that captures the unique iFF behavior in healthy and osteoarthritic OC tissues and determined the role of iFF in mediating osteocyte signaling. Long-term, this platform will enable us to study the dynamic conversation between chondrocytes, osteoblasts, and osteocytes under healthy and osteoarthritic mechanical environments and provide novel insights into the role of mechanical cues in the progression of whole joint OA. !

This Major Research Instrumentation award to acquire a high-resolution X-ray microtomography (XRM) imaging system will advance a broad spectrum of fundamental research, potentially leading to novel materials that enhance infrastructure resilience, next-generation medicine, and energy production. The instrumentation, which is not currently available to researchers in the Rocky Mountain region, uniquely combines an X-ray source with an objective turret to attain exceptional spatial resolution and unprecedented image quality. The instrumentation will advance critical research areas, including next-generation civil infrastructure materials, biological tissues and materials for tissue repair and regeneration, natural and archival materials, smart polymers, and energy collection and storage. As a publicly available resource, the XRM will be leveraged to advance the scientific missions of industry, individual researchers, and research institutions throughout the Rocky Mountain region. Annual working group meetings and a biannual materials imaging symposium will facilitate dissemination of state-of-the-art imaging science, enable continuous recruitment of new users, and catalyze new local and regional collaborations. The project will also support the education, training, and mentorship of a new generation of advanced instrumentalists, who will establish a regional expertise in high-resolution imaging of both hard and soft materials.

As the gold standard in materials imaging, high-resolution XRM with in situ mechanical testing, temperature-controlled capabilities, and dynamic, time-resolved imaging provides a non-destructive means to image and differentiate internal micro- and nanostructures of materials with 700 nm spatial resolution at large working distances, <70 nm voxel resolution, and exceptional phase contrast for both small and large sample sizes (up to 300 mm). Advanced capabilities permit in situ augmentation of standard tests to image material behavior in 3D/4D under controlled temperature, compression, tension, and flexure, enabling previously unobservable damage and failure mechanisms at the sub-micron scale. Beyond quantifying microstructural features and empirically analyzing physical and mechanical properties in situ, image data can be directly imported into numerical simulations and manipulated with stress, strain, temperature, pressure, and fluid flow to computationally model, predict, and observe microscale material behaviors, ultimately enabling more sophisticated design of highly complex synthetic and biomimetic materials.

Physeal injuries account for 30% of all pediatric fractures and can result in impaired bone growth. The physis (or, ?growth plate?) is a cartilage region at the end of children's long bones that is responsible for longitudinal bone growth. Once damaged, mesenchymal stem cells from the underlying subchondral bone migrate into the injured physis, undergo osteogenesis, and form unwanted bony tissue, referred to as a ?bony bar?. This can lead to angular deformities or completely halt longitudinal bone growth, which is devastating for children that are still growing. Current surgical treatments involve the removal of the bony bar. The site is often filled either with a soft fat graft or a hard, non-degradable plastic, both of which offer imperfect solutions leading to collapse of the resection site or the dislodgement of the biomaterial, respectively. Thus, the overall goal of this project is to develop an improved treatment option that utilizes 3D printing technology to engineer a biomimetic of growth plate cartilage containing mechanically-graded 3D stiff structures in-filled with a soft cartilage biomimetic hydrogel. Our hypothesis is that a 3D printed biomimetic of growth plate cartilage prevents collapse at the resection site through its structure and simultaneously recruits MSCs to direct them through zonally appropriate physiochemical cues to a chondrogenic, not osteogenic, lineage and prevents bony bar formation by replacing it with a cartilaginous repair tissue. Thus, long-term the 3D printed biomimetic will allow normal bone elongation after physeal injury. To test this hypothesis, we have developed two aims for the R21 phase and two aims for the R33 phase. In the R21 phase, we will (1) print a 3D construct that mimics the morphology and mechanical properties of growth plate cartilage (Aim 1) and (2) evaluate the ability of a 3D printed biomimetic of growth plate cartilage to prevent bony bar formation in a rabbit model of physeal injury (Aim 2). At the conclusion of the 2-year exploratory phase, we expect to have established a novel biomimetic of growth plate cartilage designed through 3D printing technology and confirmed that a 3D printed stiff structure mimicking that of the growth plate and infilled with a soft hydrogel prevents bony bar reformation. In the R33 phase, we will (1) assess cartilage formation in the implanted 3D printed biomimetic construct in a rabbit model of physeal injury through the recruitment of endogenous stem cells (Aim 3), and (2) evaluate the ability of a 3D printed biomimetic of growth plate cartilage to enable longitudinal bone growth in a rabbit model of physeal injury, which is followed for 1 year after implantation. At the conclusion of the 3-year R33 phase, we expect to have demonstrated that filling the site after bony bar resection with a 3D printed biomimetic of growth plate cartilage prevents bony bar reformation and supports cartilage formation that is eventually converted into new bone following growth to skeletal maturity. By providing a solution to restore normal bone growth, this 3D printed biomimetic of growth plate cartilage has the potential to be translated into the clinic to improve the quality of life of affected children.

Polymer reinforced composites are materials that combine reinforcement materials such as carbon or glass fiber, or glass particles with a polymeric base material to produce a material with enhanced mechanical properties. Utilization of these materials has revolutionized industries involved in aerospace, automotive, and sporting goods manufacture. Increasingly, industry is turning to additive manufacturing, or 3D printing, to realize customized components with complex geometries. However, stereolithography, an additive manufacturing process that uses light to locally cure (harden) a liquid polymer resin in layers to build up a solid part, cannot successfully produce polymeric reinforced composites. Nor can the process easily incorporate material property gradients within a single build. This Grant Opportunities for Academic Liaison with Industry (GOALI) project seeks to overcome these limitations by understanding the material processing interactions occurring during a modified stereolithography printing process capable of combining polymers and nanoparticles to produce printed polymer composite materials. Success will advance the performance and range of polymeric materials that can be printed via stereolithography, and in doing so will realize the 3D printing of high performance, customizable, functionally graded components. This has the potential to advance the competitiveness of core US industries involved in the manufacture of aerospace, automotive, and medical components. As Align Technology, a manufacturer utilizing stereolithography in their custom-made orthodontics fabrication process, is a collaborator on this project the students involved in the project will not only be exposed to advanced material science and manufacturing technologies but will also gain an understanding of industrial challenges and drivers. Extended online courses will be made available to students and practicing engineers, providing flexible learning opportunities to keep informed of new developments in materials science and manufacturing.

The primary goal of this project is to elucidate the structure/property relationships of gradient composite polymers printed by gray scale stereolithography of a matrix polymer followed by swelling with a reactive filler containing nanoparticles. A secondary goal is to reduce, control or eliminate the large internal stresses caused by polymerization shrinkage and solvent swelling of stereolithographic parts. The latter will be achieved by employing covalent adaptable matrices, e.g. addition-fragmentation chain transfer backbones that rearrange to relax stress in the presence of radicals. To achieve these goals the following tasks will be conducted; 1) precise, macroscopic characterization of matrix monomer-to-polymer conversion as a function of processing conditions and how this partial conversion controls swelling of the filler, 2) validation of the macroscopic predictions on the micron scale via gray-scale stereolithography of the matrix followed by swelling and polymerization of the filler, 3) validation of the predicted viscoelastic behavior of inhomogeneous printed nanocomposites, and 4) demonstration that reversible addition-fragmentation chain transfer chemistry can be leveraged to provide local stress control in bulk composites. If successful the knowledge gained will be used to print and verify the predicted properties of a printed trinary nanocomposite with photo-induced plasticity.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

This project will study the mechanisms that lead to tissue degradation in microgravity. Exposure to spaceflight or reduced mechanical loading on Earth induces marked bone loss, muscle atrophy, and degradation of soft tissues. Despite advances in exercise and pharmacologic countermeasures designed to reduce musculoskeletal deterioration on Earth and in spaceflight, the protection is incomplete and response to these interventions are variable. Many studies have focused on the molecular and cellular mechanisms that underlie atrophy and subsequent recovery. However, little attention has been given to the extracellular matrix (ECM), the material in which cells reside. The ECM is a complex network that varies in composition between specific tissues depending on the mechanical and functional demands. Changes to the ECM can permanently alter tissue material properties, prevent resident cells from re-establishing homeostasis, and affect functionality. There is a need to better understand the fundamental changes that occur in ECM composition due to microgravity that compromise tissue functionality.

In this study, temporal changes in ECM proteins in response to, and after recovery from, microgravity will be investigated to test the following hypothesis: disuse of the musculoskeletal system irreversibly affects the ECM by reducing protein turnover and increasing enzymatic and non-enzymatic cross-links, leading to the incomplete restoration of functionality after a return to 1g. Unlike gene expression that significantly varies within minutes, a common measure to assess for the physiological effects of microgravity, the proteome changes over hours to days, and provides a more stable indicator of physiology that can withstand reentry, landing, and transport of mice from the ISS. Metabolic labeling and proteomic methods will be combined to track ECM turnover in vivo by providing mice with non-canonical amino acids (ncAAs) that are incorporated into proteins using endogenous cellular machinery. ncAAs possess bioorthogonal handles that enable the enrichment of newly synthesized proteins through click reactions with complementary chemical groups. The murine proteome will be labeled with ncAAs at different timepoints after the onset of microgravity to provide a robust “time-stamp” of proteins formed in all tissues in the body in response to unloading. The study will first identify the optimal parameters to label newly synthesized ECM proteins on Earth and the ISS, then test response of the musculoskeletal ECM to two different unloading paradigms: hindlimb suspension on Earth and spaceflight on the International Space Station. Concomitant study of three musculoskeletal tissues: bone, tendon, and skeletal muscle, and the use of mass spectrometry, will reveal biomarkers and pathways that are important to disuse atrophy and subsequently recovery across musculoskeletal tissues. In addition, the study will provide an unprecedented resource for CASIS to disseminate ncAA-labeled tissues through established tissue-sharing mechanisms that will benefit a wide range of secondary science investigators.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.