Stephanie J. Bryant, Ph.D. - US grants

DESCRIPTION (provided by applicant): Heart disease is the leading cause of death in the U.S. During acute myocardial infarction (one of the most common diagnoses of early heart disease) a part of the heart muscle tissue is damaged beyond repair, which can lead to congestive heart failure, an incurable condition. Therefore, there is a strong need to develop treatments for patients with damaged heart tissue. The proposed research aims to develop novel 3D cell-scaffolds that guide tissue development. Since heart muscle tissue consists of highly aligned myofibers, a scaffold that promotes tissue formation and tissue alignment is essential. To accomplish these design requirements, a highly porous scaffold will be designed where channels are introduced through photo-patterning. To promote and maintain cell differentiation, skeletal myoblasts or primary cardiac myocytes will be suspended in a pre-hydrogel solution and seeded onto the scaffold. Upon gelation, the cells will be embedded in a 3D tissue-like microenvironment while the macroenvironment promotes unidirectional growth of myofibers. The micro-(gel) and macro-(porous scaffold) environments can be fine tuned independently to match tissue growth.

DESCRIPTION (provided by applicant): This career transition award will provide the candidate with the opportunity to investigate new areas of research: mechanotransduction and mechanical stimulation for regenerating condylar cartilage of the temporomandibular joint (TMJ). The environment in the Dept. of Chemical and Biological Eng. at the University of Colorado and the School of Dentistry at the Health Science Center will provide the candidate with fruitful collaborations, mentorship and a plethora of shared equipment. The long-term goals of the candidate are (i) to develop a successful research career in tissue engineering, (ii) to make a substantial advancement in developing tissue engineering strategies for treating patients with TMJ disorders, and (iii) to provide a positive learning environment for the next generation of researchers and tissue engineers. The overall objective of this proposal is to stimulate the regeneration of cartilage through mechanical conditioning of tissue engineering scaffolds for the replacement of condylar cartilage of the TMJ. The global hypothesis of this research is photopolymerized gels can be designed with high fidelity to provide insight into the mechanotransduction pathways of chondrocytes, and this knowledge can then be used to engineer gel environments that when subjected to mechanical conditioning will promote functional tissue development. Specifically, the aims of this research are to: Aim 1: Elucidate the chondrocyte's response (cell proliferation and ECM synthesis) to a range of loading conditions as a function of gel material properties and chemistries; Aim 2: Isolate and study the mechanotransduction pathways in chondrocytes that involve nitric oxide and intracellular calcium and their potential role in the cell's response to changes in cell deformation and streaming potentials and to the presence of cell-ECM interactions. Aim 3: Incorporate degradable crosslinks into the hydrogels and examine the macroscopic tissue composition and mechanical properties as a function of loading regimes and degradation profiles.

This award by the Biomaterials program in the Division of Materials Research to University of Colorado Boulder is to provide partial support for the attendance of graduate students, postdocs and young faculty members at a symposium on ?Engineering Biomaterials for Regenerative Medicine?. This symposium will be held as part of the Materials Research Society Fall Meeting in Boston, Massachusetts from November 30 to December 4, 2009. Scientific themes of the conference include: a) bio-inspired scaffold design, synthesis and modification; 2) interactions between stem cell and materials; 3) nanotechnology including nanoparticles and fibers; and (5) development of new technology for biomedical applications and microfabricated devises. The
objectives of the proposed conference are: a) to highlight recent advances in the design, synthesis and application of materials for fundamental understanding of cell biology and practical applications in tissue engineering; b) to provide a platform for material scientists, bioengineers, and biologists from academia and industry to establish connections, collaborations and exchange of ideas; and c) to facilitate opportunities for women, minority and junior faculty, postdoctoral fellows, and graduate students to present their research results, and establish collaborations and interactions for future research.

Broader impact of the conference is to bring together students, junior scientists and leading researchers from USA and abroad to present their recent research. The conference is expected to advance the knowledge base for design and development of new materials and technologies in the field of tissue engineering. In addition, this conference is expected to create interactions and collaborations among current and future researchers from academia and industry in this field.

ID: MPS/DMR/BMAT(7623) 0847390 PI: Bryant, Stephanie ORG: Colorado

Title: CAREER: Multi-structured Hydrogels to Control Biochemical and Biomechanical Cues to MSCs: An Integrative Plan to Promote Diversity

INTELLECTUAL MERIT: This proposal describes an integrated research and educational plan based on the PI's expertise in biomaterials design for tissue engineering and together will provide unique and exciting opportunities for promoting diversity in STEM disciplines at the University of Colorado. The theme of the PI's overall research plan is to design innovative and intelligent hydrogels for applications in tissue engineering, which mimic nature's unique way of guiding complex tissue development and maintaining homeostasis. The proposed research builds upon recent developments in stem cell research, which suggest that stem cell interactions with the extracellular matrix (ECM) provide both biochemical and biomechanical cues to guide differentiation. Towards this end, the PI will build a fundamental connection between the hydrogel environment (via its chemistry and structure) and the translation of mechanical forces (in the form of dynamic compressive strains) through the hydrogel to the cells to guide stem cell differentiation in 3D. This fundamental knowledge will be used to develop innovative multi-structured hydrogels where the structure and chemistry are spatially controlled to regulate both biochemical and biomechanical cues in 3D and differentially guide stem cell differentiation into cartilage and bone in a single hydrogel for osteochondral tissue engineering. The 3D hydrogel stem cell niches developed by this work will enable certain biochemical (the ECM) and biomechanical cues to be isolated and their roles elucidated in MSC differentiation. By systematically controlling the hydrogel niche the PI will be able to elucidate fundamental mechanisms involved in mesenchymal stem cell (MSC) differentiation that involve their ECM and biomechanical environment. From this fundamental knowledge this research will build innovative and complex 3D hydrogels for tissue engineering which (1) recapitulate these cues in 3D to mimic, to a certain degree, the translation of biomechanical signals within a normal joint and (2) are designed to manipulate stem cell fate to drive MSCs down differentiated lineages within a single hydrogel. The proposed work will develop a new class of scaffolds for regenerating osteochondral tissues. Overall, this research will impact that the training of 4 graduate students and at least 15 undergraduates

BROADER IMPACTS: The proposed educational activities are closely intertwined with the PI?s overall research theme, which together will build a strong and diverse research program. The PI will work closely with Adams State College, a Hispanic Serving Institution, in rural Colorado to develop a nurturing path for students, particularly those who are from Hispanic or Latino origin, from low-income families and/or first generation college students, to pursue engineering careers. The proposed plan is rooted in creating strategies to increase participation in the pre-engineering program at Adams State College (ASC) by closely integrating the PI?s research in tissue engineering with educational programs. Through close collaboration with ASC, the PI will create opportunities for ASC students to explore the University of Colorado at Boulder (CU) campus and enhance their academic skills through a 10-week summer research program at CU-Boulder. Together, these objectives will prepare ASC students to transfer successfully to CU-Boulder into Engineering and obtain a B.S. degree in Engineering. The proposed educational activities will begin a life-long partnership with a rural Colorado college, ASC, and create a pilot summer program that ultimately can be expanded to broadly impact other rural colleges.

DESCRIPTION (provided by applicant): Our long-term research goal is to develop a surface modification strategy for synthetic-based cell-laden hydrogel scaffolds, which when placed in vivo, facilitates host tissue remodeling at the scaffold-host interface and promotes functional integration of the engineered tissue into the host tissue. Our motivation stems from the fact that when most non-biological materials are implanted into higher organisms, they elicit a foreign body reaction (FBR) resulting in the formation of a thick avascular fibrous capsule. From a tissue engineering perspective, the FBR has received little attention. Our global hypothesis is that the FBR to tissue engineering scaffolds will have a negative impact on cell function and tissue integration - ultimately limiting the utility of synthetic scaffolds in vivo. Specifically, this research tests two hypotheses: i) activation of host inflammatory cells (i.e., macrophages) negatively impacts the function of cells which are encapsulated in PEG-based hydrogels and ii) a thin coating applied to PEG-based hydrogels containing biological signals will inhibit macrophage activation permitting the encapsulated cells to function normally. To test this hypothesis, this research aims to: * evaluate and identify key factors involved in the in vivo host response to poly(ethylene glycol) (PEG)-based hydrogels, which are currently being explored in craniofacial regenerative medicine, by assessing the timing of macrophage recruitment, macrophage activation, and fusion into foreign body giant cells; * develop an in vitro model system that mimics the in vivo environment with respect to macrophage attachment, activation and fusion at the surface of cell-laden PEG-based hydrogels. This model will enable us to study macrophage response when cells are encapsulated in the hydrogel and to study the effect macrophages have on function of the cells encapsulated in the PEG-based hydrogels;and * utilize this model system to develop new strategies aimed at minimizing the FBR. At the completion of these studies, we expect to have demonstrated that macrophage activation and fusion negatively impact encapsulated cells, but that a surface modification strategy can be employed to mitigate this negative effect permitting normal function of the encapsulated cells. In future work, this model system will enable us to study more relevant tissue engineering strategies (e.g., involving stem cells) and to study a wide range of biological signaling molecules, which are currently being explored by Dr. Kyriakides and which have potential for inhibiting macrophage activation. This research will lay the groundwork for designing new tissue engineering strategies that modulate the host response, promote functional tissue development, and enhance integration. This award will position the PI and her collaborator to seek competitively a NIH R01 grant. Public Health Relevance: The clinical success of tissue engineering has been limited, in part, due to a lack of understanding of the host cell response to the material. This research aims to address this deficit by elucidating the host response to hydrogel scaffolds and to develop new methods to modulate this response to promote functional tissue integration.

DESCRIPTION (provided by applicant): Our long-term goal is to develop biodegradable synthetic hydrogels for regenerating articular cartilage, which are capable of supporting the normal forces in vivo while simultaneously permitting matrix deposition and new tissue growth. Current limitations in the development of such hydrogels can be summarized as follows: (a) highly cross-linked hydrogel can resist loads but restrict matrix diffusion, which prevents growth of new tissue (b) reversely, low cross-link density permits matrix diffusion but results in unacceptably weak bulk properties that cannot sustain normal forces. The objective of this work is thus to introduce a hydrogel system for which spatial and temporal degradation can be controlled to better match tissue development. Our global hypothesis is that a bimodal degrading hydrogels, incorporating localized and cell-mediated (enzymatic) and bulk (hydrolytic) degradation, maintains mechanical integrity while simultaneously allowing matrix development and that there exists an optimized design space to achieve the outcomes. To test our hypothesis, mathematical models will be developed in tandem with experiments in order to accurately describe the combined effects of gel degradation and matrix deposition. In particular, the specific aims of the project are to: 1. Develop, validate, and calibrate a mathematical model for bimodal degrading hydrogels. This aim will be decomposed in two parts. First, our existing model for matrix degradation will be validated against experimental measurement based on enzyme-loaded microparticles. Second, a model for ECM production and deposition, combined with hydrolytic degradation will be developed and validated against preliminary data. 2. Characterize degradation behavior and matrix evolution in single and dual mode degrading hydrogel. This aim will extend the mathematical model to the general case of a combination of bimodal degradation and ECM deposition in order to assess the effect of hydrogel parameters on the competition between gel degradation and ECM deposition. Two experimental strategies, testing both enzymatic and bimodal degradable gels, are then proposed to validate and calibrate the model. At the completion of this exploratory research, we expect to have developed a new class of bimodal degrading hydrogels based on crosslinked poly(ethyelene glycol) where the crosslinks can be degraded either through cell-mediated enzymatic degradation (i.e., aggrecanses secreted by entrapped chondrocytes) or hydrolytically (i.e., poly(lactic acid) segments). By merging experiments with modeling, we expect to clearly understand how a bimodal degradable gel can be used to maintain mechanical integrity while permitting macroscopic tissue evolution. In future work, this model system will enable us to develop superior degradable hydrogels, which will lay the foundation for seeking competitively a NIH R01 and to pursue their (pre)clinical utility.

DESCRIPTION (provided by applicant): Advancements in treatments for tendinopathy have been hampered, in part, because our basic understanding of the pathophysiology of tendinopathy and in general tendon mechanobiology is poor. Therefore, our long-term research goal is to develop a fundamental understanding of the mechanotransduction pathways in tenocytes that contribute to tendinopathy and the mechanotransduction pathways that contribute to tendon regeneration, towards identifying therapeutic strategies for treating tendinopathy and promoting functional healing. New evidence has given us key insights into how tendon functions under physiological loads. These micro-mechanical studies suggest that tendon sustains its loading environment by functioning as a typical fiber composite, where extension occurs through a combination of fiber sliding between adjacent collagen units and fiber extension. Thus, we can postulate that cells, situated along the fibers, are subjected to a complex loading environment encompassing varying levels of shear and tension during physiological loading. These observations have prompted us to form the central hypothesis for this research, which is local shear strains in combination with local tensile strains are key regulators of tenocyte metabolism, ultimately impacting the balance between anabolic and catabolic activity. Specific to the central hypothesis, we also hypothesize that these mechanotransduction events involve cellular Ca2+ signals, which represents one central pathway by which cells may detect and respond to their mechanical environment. Recently, we developed a synthetic fiber composite hydrogel material that captures aspects of the micromechanical behavior unique to tendons, encompassing local shear and tension. Our preliminary findings indeed point towards the importance of the local strain environment in regulating cell function. Therefore, the primary objective of this exploratory grant is to test our central hypothesis in the following three specific aims: Aim 1) Develop and characterize our new fiber composite material, optimizing methods for controlled manipulation of the micromechanics and cellular strains, which capture the micromechanics in healthy and damaged tendon. Aim 2) Define and characterize calcium signals in tenocytes in response to changes in their local mechanical environment using genetically encoded calcium sensors. Aim 3) Elucidate calcium-mediated events that direct tenocyte anabolic and catabolic activity in response to changes in their local environment. The proposed research is innovative because our new synthetic fiber composite exhibits well-controlled shear/tension ratios, the use of genetically encoded calcium sensors enables the nature of the signal to be defined in space and time, and the use of specially designed straining rigs enables in situ and real time assessment during the application of gross strains and when combined provide a unique platform for studying tenocyte mechanotransduction. Completion of these studies is expected to demonstrate that a microenvironment comprised of shear and tension regulates tenocyte metabolism and that the levels of shear/tension are critical to maintaining a healthy response. We also expect to have established a viable platform for in situ and real time tenocyte mechanotransduction research having provided new insights into Ca2+-mediated mechanotransduction events. PUBLIC HEALTH RELEVANCE: Tendon injuries represent a significant fraction of the more than thirty million musculoskeletal injuries reported each year in the US alone. However, treatment options offer imperfect solutions and this observation is partly attributed to our poor understanding of tendon biology. The proposed research aims to develop a new platform to better understand tenocyte, or tendon cell, mechanotransduction, a process thought to be key to regulating the balance between anabolic and catabolic metabolisms. By capturing the complex local strain behavior of healthy and damaged tendon in this new platform, this research aims to provide new insights into tendon mechanobiology which may help to identify better therapies for treating damaged tendons.

DESCRIPTION (provided by applicant): Our long-term goal is to engineer functionally competent cartilage for replacing damaged or diseased cartilage. While it is known that engineering a functionally competent cartilage depends on the mechanical environment, choosing the appropriate loading environment has proven challenging. This observation is largely in part due to the fact that the biomechanical cues sensed by the cells will be dictated by the mechanical structure and chemistry of the scaffold and will be dynamic in time as the scaffold degrades and neotissue develops. To overcome these challenges, the global hypothesis for this research is that a dynamic culture environment that detects and responds to changes in a tissue-engineered scaffold improves the quality of the engineered cartilage. Central to our hypothesis is a novel dynamic compressive bioreactor recently designed, constructed and validated with collaborators at NIST, which is equipped with online, nondestructive measurement capabilities comprised of individual load cells for assessing mechanical properties and an ultrasonic transducer coupled with a video microscope for assessing development and quality of the engineered tissue. To test the global hypothesis, the specific aims of the project are to: 1. Design a dual enzyme degrading polyethylene glycol (PEG) hydrogel with cell-mediated local degradation and 'on demand' bulk degradation capabilities. This aim tests the hypothesis that a hydrogel with crosslinks containing oligopeptides that are degraded by cells, leading to local degradation (critical for local matrix elaboration without sacrificing mechanical integrity) and polycaprolactone that is degraded 'on demand' by exogenous delivery of lipase, leading to bulk degradation (critical for macroscopic tissue development) yields improved engineered cartilage. 2. Develop and validate a dynamically responsive 'smart' bioreactor using a heuristic control loop to modify the biochemical and mechanical environment in real time. This aim tests the hypothesis that real time changes to bulk degradation and mechanical loading in response to the developing tissue will lead to improved engineered cartilage. We will achieve this aim by incorporating a fuzzy controller with a set of heuristic control actions into our current bioreactor where the output variables, the quality index estimated from ultrasound and mechanical properties, will be related to input variables that include strain amplitude, duty cycle, and enzyme addition. At the completion of this exploratory research, we expect to have developed i) a new class of dual enzyme degrading hydrogels based on cross-linked polyethylene glycol where degradation is more closely matched spatially and temporally to tissue growth and elaboration and ii) a dynamically responsive 'smart' bioreactor that is capable of detecting and responding in accord with tissue growth. We also expect to have answered the fundamental question; does a dynamically responsive culture environment lead to improved tissue elaboration and functional properties over a constant culture environment? It is anticipated that such a bioreactor would enable facile adaption to engineering cartilage from multiple cell sources (donor, age, species), which inherently have different dynamics and timescales for tissue development, and can readily be adapted to other scaffold types. Findings from this grant will lay the foundation for seeking competitively a NIH R01 and to pursue their (pre)clinical utility.

DESCRIPTION (provided by applicant): The overall goal of this proposal is to better understand the role of the foreign body reaction (FBR) in tissue engineering and in particular the dynamic interplay between interrogating macrophages and the cells residing within a scaffold. While the FBR has been investigated with respect to implantable biomedical materials including scaffolds for tissue engineering, the impact of the FBR on cells residing with the scaffold has not been addressed. Our preliminary studies have provided two important observations: 1) there exists a dynamic communication between interrogating macrophages (the primary orchestrators of the FBR) and the encapsulated cells which impacts the overall response (both FBR and neotissue formation) and 2) the severity of the FBR appears to depend on the differentiation stage of the encapsulated cell. Based on these observations we have formulated the following hypothesis to be tested in this proposal. Specifically, we will test the hypothesis that inflammatory macrophages hinder the biosynthetic ability of cells encapsulated in biodegradable hydrogels but mesenchymal stem cells (MSCs) and differentiating MSCs alter macrophage phenotype and improve the overall outcome of the engineered tissue. To test this hypothesis we have developed two specific aims: Aim 1) We will determine whether the stage of differentiation of encapsulated cells in biodegradable PEG-based hydrogels affects the activation of interrogating macrophages and in turn influences the encapsulated cells. Aim 2) We will evaluate the in vivo performance of cell-laden biodegradable PEG-based hydrogels when cells at different stages of differentiation are encapsulated. To accomplish our proposed aims, we will develop a tissue engineering model system for bone tissue engineering where MSCs at varying stages of osteogenic differentiation are encapsulated in a bone biomimetic hydrogel. In Aim 1, we will use our established in vitro co-culture model, which simulates macrophages interrogating a cell-laden hydrogel in an inflammatory environment to elucidate the dynamic interplay between encapsulated cells and macrophages. In Aim 2, we will use syngeneic cell-laden hydrogels implanted subcutaneously in immumocompetent animals to elucidate the dynamic interplay between encapsulated cells and the complex FBR. By understanding the dynamic interplay between interrogating macrophages and encapsulated cells at different stages of differentiation, we have the potential to identify a balance between differentiation (required for neotissue formation) and anti-inflammatory properties (to reduce the severity of the FBR), which together we hypothesize will lead to significantly improved neotissue growth long-term.

DESCRIPTION (provided by applicant): Success of Autologous Chondrocyte Implantation (ACI) for treating damaged cartilage in the knee has been marginal and limited to young, healthy, and active patients. With the advent of second generation ACI referred to as Matrix-Assisted ACI (MACI), a new opportunity arises. We hypothesize that if the design of the matrix is patient-specific (i.e., specific to the tissue synthesis capabilities of the cell), it will be pssible to not only improve the effectiveness of ACI long-term, but expand its indication to a wider patient population regardless of age or health. Thus, the overarching goal of this research project is to personalize MACI. Our innovative approach to personalizing MACI combines the following two highly interconnected themes: (a) A new class of highly tunable hydrogels with spatiotemporal control over degradation (to enable patient-matched tissue synthesis capabilities), high moduli capabilities (to restore function), and matrix-retention capabilities (t minimize tissue loss). (b) The introduction of a universal computational tool based on a well-established theoretical framework, which will analyze data related to the response of a patient-specific cell and, based on this information, predict the corresponding hydrogel structure and degradation that enables tissue growth and sustained mechanical integrity in a dynamic loading environment (such as that in the knee). To accomplish our overall research goals, the specific aims are as follows. We aim to determine model constants that enable the design of personalized hydrogels, first in the absence of mechanical loading (Aim 1) then in the presence of mechanical loading (Aim 2). We will accomplish this through an integrated experimental and simulation campaign combined with the use of a self-learning algorithm. This will lead to the construction of the data- driven predictive computational model. Once developed, we will test the predictive capability of the mathematical model in personalized MACI using a large animal model, specifically to treat a chondral lesion in the knee of a swine (Aim 3). At the completion of this five year research project, we expect to have developed a predictive computational tool and established a novel and highly tunable hydrogel platform for personalizing MACI. The universal nature of the computational predictive tool enables it to be broadly applied in future research to other scaffolds and cells, including osteoarthritic chondrocytes and stem cells.

While hydrogels offer a facile method for in situ delivery of cells, they are not conducive to simultaneously withstanding the large forces found in joints (requiring high moduli) and promoting stem cell differentiation (requiring low moduli). Moreover, a mismatch in mechanical properties between scaffold and the adjacent tissue can lead to mechanical destabilization and eventually degeneration in the surrounding joint tissue. This points to the need for a mechanically robust scaffold that can withstand normal joint loads. In osteochondral tissues, cells reside in their own niche and are largely protected from large forces by the extracellular matrix. The proposed tissue engineering solution lies in mimicking nature's solution to this complex problem. Specifically, we will decouple the structural (i.e., load-bearing) component from the cellular niche within our hydrogel design. A stiff and functionally graded, load-bearing structural hydrogel component will withstand large forces and transfer appropriate strains (i.e., mechanical signals) to each cellular niche. Independently, three cellular niches will capture chemistries and degradation appropriate to hyaline cartilage, calcified cartilage and bone. When combined with dynamic loading that transfers mechanical cues from the structural component to each cellular niche, stem cell mediated OC tissue regeneration will be achieved. Our approach is possible by the enabling technologies of digital projection photolithography and highly tunable photoclickable hydrogels. Thus the overarching hypothesis for this research is: a structurally stiff and functionally graded material embedded within a soft material containing stem cells supports normal joint loads, minimizes damage to the surrounding tissue, and promotes OC tissue regeneration. To test this hypothesis, we have outlined three specific aims. In specific aim #1, we will design architecturally-controlled 3D OC mimetic hydrogel materials to support stresses similar to native OC tissue in vivo and transfer appropriate strains to each layer of the OC mimetic hydrogel. We will test the ability of an acellular and mechanically stable OC mimetic hydrogel to minimize damage to tissue surrounding an OC defect in swine knees. In specific aim #2, we will investigate MSC differentiation and OC tissue regeneration when MSCs are incorporated in the soft cellular component that is designed with biochemical and mechanical cues appropriate to each OC niche and cultured in custom bioreactors that mimic aspects of the in vivo loading environment. In specific aim #3, degradable and mechanically stiff OC mimetic hydrogels with autologous MSCs will be implanted in a swine OC knee defect for 12 weeks and evaluated for engineered OC tissue and damage to tissues surrounding the defect. Upon completion of this project, we expect to have demonstrated a mechanically stiff hydrogel with encapsulated MSCs is capable of (a) withstanding large forces, (b) promoting stem cell mediated OC tissue regeneration and (c) maintaining the health of the tissue surrounding the defect. Long-term, we are developing a miniaturized and portable printing technology that will be easily accessible to surgeons via an arthroscopic platform.  

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.

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.

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. !

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.

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, cartilage tissue within the physis is often replaced by unwanted bony tissue, forming a ?bony bar? that can lead to angular deformities or even completely halt longitudinal bone growth. Children with a bony bar that spans less than half of the physis usually undergo bony bar resection and insertion of a permanent interpositional material, such as a fat graft, to prevent bony bar recurrence and allow the surrounding uninjured physeal tissue to restore longitudinal bone growth. Clinical success for this procedure is <35% and often the bony bar and associated growth impairments return. Children who are not candidates for bony bar resection due to a physeal bar that spans greater than half of their physis, undergo corrective osteotomy or bone lengthening procedures. These approaches are complex and result in multiple hospitalizations. There is a critical need to develop effective treatments that prevent bony bar formation and regenerate physeal cartilage in order to restore normal bone elongation. Mesenchymal stem cells (MSCs) from the underlying subchondral bone have been shown to migrate into the injured physis, undergo osteogenesis, and form the bony bar. This suggests that MSCs play a central role in bony bar formation, and are a potential target for treatment strategies directed towards physeal injuries. Our hypothesis is that a cartilage- biomimetic hydrogel that provides cartilage-specific physiochemical cues to MSCs prevents bony bar formation by directing MSCs towards chondrogenesis and not osteogenesis, restores the physis by promoting cartilage-specific repair tissue, and allows normal bone elongation after physeal injury. This hypothesis will be tested in two aims. In Aim 1, we will evaluate a temporary biomimetic hydrogel, which has chondrogenic physiochemical cues, for its ability to direct endogenous MSCs towards chondrogenesis, thus preventing bony bar formation in a rat model of physeal injury when compared to a hydrogel without chondrogenic cues and to an untreated injured model. In Aim 2, we will deliver exogenous syngeneic MSCs within the cartilage-biomimetic hydrogel to facilitate rapid and robust cartilage repair tissue formation and evaluate its ability to serve as a template for bone elongation after physeal injury in a rat model. Findings from each aim have clinical significance. Children who are candidates for bony bar resection would benefit from the implantation of a temporary interpositional material that prevents bony bar formation and augments the surrounding healthy physis to continue longitudinal bone growth. On the other hand, children that are not candidates for resection would benefit from the delivery of a cartilage-biomimetic hydrogel with MSCs to form robust cartilage formation within the injured site to restore longitudinal bone growth. Ultimately this work will have a high impact on the clinical management of physeal injuries and the quality of life of affected children.

The foreign body response (FBR) represents a major challenge in the application and clinical success of current and future biomaterial-based treatments of musculoskeletal injuries and diseases. The FBR is orchestrated by macrophages and occurs ubiquitously to all implanted non-biological materials. Although the mechanisms driving the FBR remain to be elucidated, it is generally understood the FBR is initiated by inflammatory cells that recognize the material as foreign through surface-adsorbed proteins, which can unfold, exposing epitopes known as damage-associated molecular patterns (DAMPs). These DAMPs are directly influenced by the highly dynamic and heterogeneous behavior of proteins in near-surface environments, including transient unfolding and refolding, rapid exchange of folded and unfolded protein molecules between the surface and bulk solution, and intermittent diffusion on the surface. While such interfacial processes are likely involved in the FBR, their roles have been all but ignored due to the lack of experimental techniques to directly observe these processes. The overarching aim of this research is to investigate the extent to which interfacial protein dynamics influences the presence of DAMPs using novel single-molecule (SM) biophysical methods, which are uniquely sensitive to interfacial protein dynamics. Such methods will be combined with poly(ethylene glycol)/poly(sulfobetaine) copolymer brushes that are tuned to control interfacial dynamic behavior of proteins, in vitro macrophage activation assays, and in vivo mouse studies to elucidate the role of surface-induce protein unfolding in the FBR. We will test the hypothesis that the presentation of unfolded proteins (i.e., DAMPs) as a result of the complex and heterogeneous behavior of proteins in near-surface environments triggers macrophage activation via toll like receptor (TLR) signaling and contributes to the FBR. In particular, toll-like receptors (TLRs) are a class of membrane proteins that are involved in innate immune signaling; and specifically, TLR2 and 4 have been shown to recognize host proteins acting as DAMPs. Thus, in Aim 1, we will confirm the role of TLR2/4 signaling in the activation of macrophages to known DAMPs (Aim 1.1) and identify the dynamic behaviors that lead to the presentation of unfolded proteins and in turn to macrophage activation (Aim 1.2). In Aim 2, the amount of transient unfolded protein will be further correlated to the FBR in vivo via TLR2/4 signaling using knockout mouse models. Combined, these aims will 1) identify that transient unfolded proteins contribute to macrophage activation and the FBR via TLR2/4 signaling and 2) elucidate the mechanisms by which near surface environments influence transient protein unfolding. These new insights will provide the foundation for new research aimed at designing novel chemistries that control complex protein dynamics and thus we may, for the first time, be able to prevent protein unfolding at surfaces and potentially eliminate the FBR.

Tendons are an important tissue for musculoskeletal function -- they connect muscles to bones. They are also a very slow healing tissue, due in part to the limited blood supply and small concentration of cells within the highly fibrous structure. If tendon cells, tenocytes, are going to be stimulated to enhance tendon growth and regeneration, it is vitally important that the understanding of how these cells respond to their mechanical environment is substantially increased. This project will use a combination of computer modeling and experimental studies to investigate how tenocytes respond to complex loading normally seen in a tendon. The work will focus on the micro-level -- in order to understand how variations in the mechanical environment just around the cell relate to the cellular response. In addition to advancing knowledge that will support future work in tissue engineering of tendons and other fibrous connective tissue, this project includes several educational and outreach activities. First, the research team will integrate students from high school through graduate school in order to help them understand the importance of research in this area and of integrating experimental and computational techniques. Next, there will be an opportunity for graduate students to travel to an international collaborator's laboratory to enhance their scientific preparation. Finally, hands-on workshops will be developed to provide participants with a broader understanding of connective tissue biomechanics.

The overall objectives for this project are to: (1) determine the micromechanics and the type and magnitude of the local cellular strains as a function of fiber composite properties under physiologically relevant tensile strains; (2) determine tenocyte anabolic and catabolic metabolism as a function of the type and magnitude of the local cellular strains and the typed of integrin-ligand interaction; and (3) elucidate intracellular signaling pathways based on MAP kinase signaling that are involved in integratin-mediated mechanotransduction within tenocytes. This will be done through the development of a computational model of tenocytes within a fiber-reinforced structure and the validation of this model with experimental studies using a novel hydrogel-fiber composite mimetic that captures aspects of the tendon's fiber composite mechanics.

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.

The foreign body response (FBR) represents a major challenge in the application and clinical success of current and future biomaterial-based treatments of musculoskeletal injuries and diseases. While macrophages are known to orchestrate the FBR, a therapeutic synthetic-based biomaterial that effectively targets macrophages and evades fibrous encapsulation has yet to be developed. This exploratory project examines prostaglandin-E2 (PGE2) signaling as a potential target to prevent the FBR. PGE2 with its four receptors (EP1-EP4) is an important modulator of inflammation that also affects fibrosis in a range of diseases. Studies have shown that macrophages synthesize PGE2 in response to an inflammatory stimulus and, through EP2 and EP4, PGE2 limits the severity of fibrosis. However, PGE2 signaling is complex. While EP2 and EP4 have been shown to attenuate inflammation EP1 and EP3 potentiate inflammation. An alternative strategy to using PGE2 as a therapeutic is to target the receptors, EP2 or EP4. In the context of the FBR, the role of EP2 and EP4 has yet to be identified. To this end, this research tests the hypotheses that a) macrophages limit the severity of the FBR through autocrine and paracrine signaling via activation of EP2 and EP4 and b) if EP2 is targeted in a biomaterial strategy at the onset of the FBR, it is possible to abrogate the FBR and prevent fibrous encapsulation. For the latter, we chose to target EP2 over EP4 for several reasons. EP4 requires internalization to induce signaling, while EP2 does not. EP4 signaling is more complex, leading to crosstalk that affects pathways not involved in inflammation. Two specific aims were developed for the proposed project. Specific aim #1 will identify the roles of EP2 and EP4 on macrophages in limiting the FBR. This aim tests the hypothesis that inhibiting either EP2 or EP4 signaling in macrophages enhances the FBR by sustaining inflammation, which leads to an increase in the number and size of foreign body giant cells and a thicker fibrous capsule. In this aim, we will develop mouse conditional knockout models for EP2 and EP4 in macrophages and perform in vitro and in vivo studies to examine the role of each receptor in inflammatory macrophage activation, macrophage fusion, and fibrous encapsulation. Specific aim #2 will develop a biomaterial strategy that targets EP2 and assess its ability to suppress the FBR. This aim tests the hypothesis that activating the EP2 receptor via a selective agonist that is immobilized on the surface of biomaterial implants inhibits fibrous encapsulation. In this aim, we propose to covalently attach an EP2 agonist to the biomaterial surface and assess inflammatory macrophage activation, macrophage fusion and fibrous encapsulation in vitro and in vivo in wild-type mice. At the conclusion of this exploratory project, we expect to have a better understanding of how macrophages control and limit the FBR. From a translational perspective, we expect that targeting EP2 will induce immunosuppressive actions in macrophages and in turn reduce FBGC formation and prevent fibrous encapsulation. This will lay the foundation to investigate new medical devices and their function when fibrous encapsulation can be evaded.

Macrophages are key players in the foreign body response (FBR) to implanted biomaterials, in which an avascular fibrous capsule walls off the implant. However, the cellular mechanisms that contribute to the fibrous capsule have not yet been elucidated. As a result, synthetic-based biomaterials have been limited to those that the body tolerates and which function despite a FBR and does not lead to biomaterial-host integration. In this highly exploratory project, we put forth a novel hypothesis that is based on our team?s recent published findings in tissue fibrosis. We demonstrated that inhibition of cellular FLICE-like inhibitory protein (cFLIP), which is a major regulator of macrophage cell fate, can prevent tissue fibrosis. In this project, we test the hypothesis that that macrophage persistence in the FBR is mediated by intracellular cFLIP and inhibiting cFLIP resensitizes macrophages to apoptotic death signals to prevent or resolve fibrous encapsulation. We developed two specific aims to test the hypothesis and to translate this idea to a biomaterial strategy that targets cFLIP in macrophages to prevent fibrous encapsulation. In specific Aim #1, we will determine the kinetics of macrophage persistence in the FBR to distinct implants. This aim will use the hCD68-rtTA transgenic mouse that is coupled with a Tet-on Cre system and fluorescent tdTomato expression. Using this mouse model, a series of lineage tracing experiments will be performed that combine multiparameter flow cytometry to identify myeloid subsets, including recruited and tissue-resident macrophages, distinguish their temporal patterns in the FBR and determine changes in their expression profiles for fibrosis-relevant genes. In specific Aim #2, we will temporally inhibit c-FLIP in macrophages to promote their programmed cell death and attenuate formation and maintenance of the fibrous capsule in the FBR. The first part of Aim #2 will determine the temporal effects of cFLIP inhibition in macrophages using a similar transgenic mouse model as in Aim 1, but which is coupled with a tet-On Cre system that deletes cFLIP. This mouse model will enable the temporal effects of cFLIP deletion to be determined on both the formation of fibrous capsule and on its dissolution. The second part of Aim #2 will focus on designing a phototriggerable biomaterial to inhibit cFLIP temporally and locally in macrophages. This will be achieved through photo-labile microparticles that are embedded within a biomaterial, which when triggered by light lead to the slow release of YM155, a small molecule inhibitor of cFLIP. By tightly controlling when YM155 is released, the temporal effects of local cFLIP inhibition by a biomaterial-based strategy will be determined. At the conclusion of this project, we will have a) determined the temporal patterns of macrophage accumulation and when they begin to persist in the FBR, b) elucidated the role of cFLIP in mediating pro-survival programming in macrophages and its effect on fibrous encapsulation, c) identified the optimal timing for depleting cFLIP, and d) developed novel strategies that can be applied for preventing or resolving the FBR.

The growth plate is a cartilage tissue located at the end of children’s long bones that is responsible for bone growth. It begins as a cluster of stem cells that become specialized and organize themselves into columns to form a functioning growth plate. This process is driven by both chemical cues and mechanical forces, although it is unclear how they work together to form the structure and function of the growth plate. This Reproducible Cells and Organoids via Directed-Differentiation Encoding (RECODE) project will develop a reproducible growth plate organoid that will allow one to study how stem cells form a mature growth plate, which can lead to novel approaches for bone and cartilage regeneration particularly in children. This project will train a diverse group of graduate, undergraduate, and high school students in mathematical modeling, biomaterial development, and stem cell and developmental biology and will provide opportunities to the broader community through outreach activities and events open to the public.

The overarching goal for this RECODE project is to gain fundamental insight into the link between biophysical cues, cellular differentiation, and cellular organization that leads to the development of a functioning growth plate. This project combines experimental and computational approaches to gain insight into the local cues that govern directional cell division (Task 1), chondrogenic differentiation followed by columnar organization (Task 2), and hypertrophic differentiation, the characteristic phenotype of the growth plate (Task 3). This project will uncover how biophysical cues combined with spatially localized biochemical cues dovetail to drive the self-assembly of stem cells into a growth plate organ with the appropriate structure and function. By utilizing novel tools in biology, advanced biomaterials in 3D printing, and physics-based mathematical modeling, this project will create the first growth plate organoid to date. This organoid will provide a model system for deeper study of stem cell and chondrocyte differentiation, in normal and abnormal bone growth. Understanding the mechanisms that direct the differentiation of MSCs into a mature growth plate organoid will help guide the design of novel biomaterials for regenerative medicine approaches to treat growth plate injury, an area that currently lacks a viable and clinically accepted treatment.

This RECODE award is co-funded by the Physiological and Structural Systems Cluster in the Division of Integrative Organismal Systems, the Mechanics and Engineering Materials Cluster in the Division of Civil, Mechanical, and Manufacturing Innovation, and the Engineering Biology and Health Cluster in the Division of Chemical, Bioengineering, Environmental, and Transport Systems.

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.