The use of scaffolds for tissue engineering involves aspects of mechanotransduction that are controlled by scaffold properties and structure at the local, cellular scale. For fibrous, electrospun scaffolds, such features include the local fiber stress-strain behavior, fiber density, and undulations in fiber orientation. These serve to provide variations in local stiffness and anisotropy that cannot be quantified through macroscopic testing alone. A combined computational-experimental approach is adopted whereby finite element simulations of electrospun scaffolds are used to link the macroscopic stress-strain response to underlying fiber geometry and fiber stress-strain response. These simulations capture the discrete fiber-straightening, reorientation, and fiber-fiber contact that occurs during scaffold deformation. They can also provide scaffold "Green's functions" to quantify local response to concentrated forces exerted by cells, enabling extraction of "cellular force footprints" in principle. The present simulations are informed by actual fiber geometries from high-resolution confocal microscopy images and macroscopic stress-strain data. An output is the local fiber stress-strain response, which is notoriously difficult to obtain by direct experimental measurement. The calibrated simulations underscore the highly non-uniform (non-affine) and anisotropic nature of the deformation. They also reveal the scale-dependent nature of mechanical response. The talk concludes with challenges to simulation "scale-up" and other pertinent issues. This work is supported by a Multidisciplinary Team Grant, Institute for Materials Research, The Ohio State University.
With the renewed excitement in the inducible stem cell field, regenerative medicine is poised at our ability to efficiently direct differentiation of stem cells into functional tissues and organ systems. Besides the vast amount of work currently addressing the mechanistic underpinnings of the directed differentiation process, practical tools to harness this into a clinical utility are lacking. In this talk, I present our work with low power lasers as an innovative tool for clinical regenerative applications. Our current work has uncovered the physical, chemical and molecular events mediating the molecular mechanism mediating these effects using a wide range of in vitro analytical techniques. These observations were validated in vivo assessing directed differentiation of adult dental stem cells in animal models. In summary, low power laser can directs differentiation of resident stem cells via activation of endogenous morphogen.
While the impact of single extracellular matrix (ECM) proteins and mechanical stiffness on cell function have been thoroughly probed individually, little work has been put into to understanding their interactions in the context of cell function. This is particularly important as the ECM is a complex mixture of proteins that change throughout normal development both in composition and stiffness. Recent work by others has demonstrated that cells respond differently to both static substrate stiffness and mechanical stretch when plated on substrates of different compositions. In addition, the effects of growth factor treatment can also be modulated by substrate composition and stiffness. In this talk I will cover our own recent work investigating the effects of alterations in stiffness and composition of the substrate on cardiac differentiation of stem cells and cardiomyocyte proliferation. The system we use is a polyacrylamide gel system with binding sites generated from solubilized decellularized cardiac ECM. This setup effectively allows us to decouple stiffness and composition to investigate their individual roles and any synergistic/ antagonistic effects. Preliminary data indicate that ECM composition and stiffness interact in a complex manner to effect cardiac differentiation of mesenchymal stem cells. Moreover, fetal cardiac ECM composition enhances neonatal cardiomyocyte proliferation over adult cardiac ECM or normal tissue culture plastic.
In reconstructive surgery, there is a tremendous clinical need for adequate implants to repair soft tissue defects resulting from tumor resection (e.g., breast reconstruction after mastectomy), traumatic injury, or congenital anomalies. Adipose tissue engineering holds the promise to provide answers to this still increasing demand. While many different biomaterials have been explored, a recent review of the field shows that cell carriers which are more adipose-specific are desirable. Current developments will be presented that focus on biomaterials based on structures that are derived from the extracellular matrix of adipose tissue. This includes decellularized native ECM, but also synthetic materials exhibiting characteristics of the adipose ECM, such as adhesion peptides and peptides that are substrates for matrix-degrading enzymes. Besides aiming at the generation of tissue constructs for clinical use, 3D engineered adipose tissue constructs can also be utilized as models for basic research investigating the influence of tissue-inherent factors on adipogenesis. A new model based on 3D spheroids made from human adipose-derived stem cells (ASC) will be presented that provides, without any exogenous material, cell-cell and cell-ECM interactions in a more in vivo-like context, as compared to conventional 2D cell culture. Different aspects with regard to the ECM that are investigated utilizing the ASC spheroids will be discussed.
Work done in collaboration with Jürgen Groll.
Cardiac and skeletal muscle development and regeneration involve complex interactions among different cell types, biochemical and biophysical stimuli, and extracellular matrix molecules. Studies of how each of these components influences the tissue morphogenesis, function, and disease are often precluded by the complex structure of the native tissue and the inability to directly control or monitor cellular processes and fates in vivo. The use of tissue engineering technologies, on the other hand, allows for the generation and manipulation of 3-dimensional cell culture systems to both improve our understanding of the myogenic processes in vitro and enable the development of new therapies for heart and muscle disease. Recently, our efforts have been focused on the use of stem cells and natural hydrogels to construct cardio- and myo-mimetic tissue culture systems with enhanced functional output. We find that specific combinations of hydrogel composition, dynamic biochemical culture environment, boundary conditions imposed on cells, and supporting non-myogenic cell types can uniquely advance structural and electromechanical properties of engineered muscle to resemble those found in age-matched tissues in vivo. These studies provide insights into the principles of functional myogenesis that could lead to the development of efficient tissue engineering therapies for human disease.
Modeling cell-cell and cell-extracellular interactions is fundamental to many systems. In order to alter, predict, and ultimately control wound healing, developmental processes, and pathological conditions such as cancer a greater understanding of cell-cell and cell-extracellular interactions is needed. In wound healing various cell types are recruited to the wound region to repair the wound. More specifically the collective motion of epithelial cells is required to repair a defect in the tissue layer as cells migrate into the wound region restoring the integrity of the epidermis.
These cell-cell and cell-extracellular interactions are both force and biochemically based and are mediated by random processes. Thus when a cell contacts another cell the contact must persist for a meaningful interaction to take place. The duration of the contact can be described by a probability distribution. We propose to model the force interactions as a system of differential equations representing the location of the cell centers and their surrounding interaction sites. The terms linking the different equations represent the interactions between the sites and are random in nature. In this talk I will discuss some the insights gained by some simple simulations.
The normal healing response begins the moment the tissue is injured. As the blood components spill into the site of injury, the platelets come into contact with exposed collagen and other elements of the extracellular matrix. This contact triggers the platelets to release clotting factors as well as essential growth factors and cytokines such as platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-ß). Following hemostasis, the neutrophils then enter the wound site and begin the critical task of phagocytosis to remove foreign materials, bacteria and damaged tissue. As part of this inflammatory phase, the macrophages appear and continue the process of phagocytosis as well as releasing more PDGF and TGFß. Once the wound site is cleaned out, fibroblasts migrate in to begin the proliferative phase and deposit new extracellular matrix. The new collagen matrix then becomes cross-linked and organized during the final remodeling phase. In order for this efficient and highly controlled repair process to take place, there are numerous cell-signaling events that are required. In pathologic conditions such as non-healing pressure ulcers, this efficient and orderly process is lost and the ulcers are locked into a state of chronic inflammation characterized by abundant neutrophil infiltration with associated reactive oxygen species and destructive enzymes. Healing proceeds only after the inflammation is controlled. On the opposite end of the spectrum, fibrosis is characterized by excessive matrix deposition, contraction and reduced remodeling. New technologies utilizing PTFE tube implantation have been developed to analyze inflammation and tissue repair in humans. On days 3, 5, 7 and 14 the tubes are removed and the newly deposited cells and matrix components are characterized using histologic, immuno-staining and proteomic analysis. These ongoing studies will be discussed.
Several biological processes depend on interactions between cells as their extracellular matrix (ECM). Cells typically attach to the ECM via specific focal adhesions and exert traction stresses on the ECM by generating internal contractile or tensile forces. The generation of these contractile forces is essential to numerous biological and pathological processes including development, growth, wound healing and cancer cell migration/invasion. A common technique used to assess the contractile properties of cells is Traction Force Microscopy (TFM). In TFM, the displacements of fluorescently labeled beads embedded into the ECM or substrate are measured and converted into a traction stress field using inverse analytical or finite element methods. Recently, standard 2D TFM methods have been extended into 3D and the traction stress field generally used as a measure of cell contractility. However, we hypothesize that changes in several biomechanical and morphological properties (i.e. cell stiffness, cell-substrate adhesion and aspect ratio) may alter the traction stress field measured by TFM. The fact that multiple parameters other than contractility can influence TFM measurements may lead to inappropriate interpretation of experimental results. Therefore, we have developed an idealized finite element model of 2D and 3D TFM and have used this model to evaluate how cell mechanical and morphological properties influence TFM measurements. Results indicate that even for equivalent contractile stress, changes in cell stiffness (Young's Modulus) and adhesion energy can lead to dramatic changes in the contractility as determine by TFM. These results therefore indicate that in addition to TFM, several other biomechanical measurements of stiffness and adhesion are required to appropriate interpret TFM measurements and assess changes in cell contractility.
This work was supported by NSF CAREER 0852417 and NSF 1134201.
This talk will discuss mathematical and computational modeling of cell-matrix interactions at different length scales. Agent-based models were developed where cells and ECM fibers are each considered as systems of multiple interacting agents. In these models, the physical properties of cell and fibers are based on a set of conceptually reasonable mechanical processes and the dynamics of cell-matrix binding and unbinding are considered as simple position- and strain-dependent processes. From these relatively simple sets of rules, complex behaviors arise that are consistent with experimental data including cell-mediated compaction of ECM fiber networks, the alignment of ECM fibers between cells, and the directed migration of pairs of cells towards one another. In addition, there is evidence consistent with durotaxis and haptotaxis arising as emergent behaviors from these simple sets of rules. The implications of the peri-cellular distribution of ECM predicted in these agent-based models and observed experimentally are explored in a mathematical model of cell-mediated compaction of ECM.
Joint work with James Reinhardt, Mark Stevenson, Victor Barocas, and Alisha L. Sarang-Sieminski. This work was supported by NSF CMMI-0928739.
In nature, self-assembly is a common process for living systems to build large-scale architectures without pre-existing positional cues from scaffolds or templates. At molecular scales, scaffold-free self-assembly has also been used to create bio-mimetic supra-molecular architectures and sub-cellular components. However, the principles of cell-microenvironment self-assembly have not been widely used to construct large-scale functional cellular devices. For example, current engineering of un-branched epithelial tubules of lengths above hundred micrometers is achieved on premade scaffolds only. Here, we show that adult epithelial cells and type I collagen molecules can self-assemble into centimeter-long, hundred micrometer-wide and un-branched tubules under scaffold-free conditions. These tubules develop apicobasal polarity and lumens similar to those found in situ. The observed tubule self-assembly requires cell-collagen interactions in the liquid phase to form the initial linear templates. In contrast with conventional thoughts, such template formation is primarily mediated by long-range (~ 600 micrometers) mechanical interactions rather than gradients of soluble factors between cells. The stability of linear templates increases with their lengths. Moreover, the ability of the cells to form tubules depends on the initial and boundary conditions of the culturing systems, and could be prevented by cell-substrate interactions. Only large culture systems containing a sufficient density of cells cultured on low-adhesive substrates enables the formation of centimeter-long, un-branched tubules. Our findings illustrate the feasibility of building long tubules under scaffold-free conditions and provide insights into the principles of cell-microenvironment self-assembly that could be used for the engineering of functional organs.
In addition to chemical signals, cells are able to communicate via mechanical signaling. We are investigating the hypothesis that the fibers in the extracellular matrix (ECM) are the key factor for transmitting mechanical signals between fibroblast cells as an alternative to the hypothesis in the literature that the strain-hardening behavior of the ECM is responsible for long-range cell-cell mechanical signals. To compare the two hypotheses, confocal reflectance microscopy was used to develop image-based 2-D finite-element models of stress transmission within fibroblast-seeded collagen gels. Models that include the gel's fibers were compared with homogenous linear-elastic and strain-hardening models to investigate the mechanisms of stress propagation. Experimentally, cells were observed to contract collagen fibers centripetally and align collagen fibers between neighboring cells within 24 hours. Finite-element analysis revealed that stresses generated by a centripetally contracting cell are concentrated in the relatively stiff ECM fibers and are propagated farther in a fibrous matrix as compared to linear elastic or strain-hardening homogenous materials. These results support the hypothesis that ECM fibers play the key role in long-range stress transmission, and that strain-hardening effects are secondary.
Joint work with Xiaoyue Ma, Maureen Weber, Mark D. Stevenson, Alisha L. Sarang-Sieminski, Keith J. Gooch, and Samir N. Ghadiali. This work was supported by NSF CMMI-0928739.
The mechanics of healing myocardial infarcts are a critical determinant of left ventricular function. We recently showed that infarcts healing in different mechanical environments develop different collagen fiber structures and mechanical properties. We developed an agent-based model to evaluate the mechanisms by which mechanical environment directs collagen deposition and remodeling by cardiac fibroblasts and to better understand the effects of therapeutic interventions on the evolving scar structure. Parameters for this model were derived from a combination of published literature and new experiments in engineered tissue-equivalents, focusing particularly on features of cell-matrix interaction such as contact guidance and collagen remodeling. Our results suggest that different environmental cues regulate scar formation in different tissues. In contrast to previous models of skin wound healing showing that chemokine gradients are a dominant regulator of scar formation, we find that mechanical environment is the dominant regulator of evolving scar structure following myocardial infarction. Our results suggest that a number of regenerative and device therapies that alter infarct mechanics, including stem cell injection, polymer injection, surgical reinforcement, and peri-infarct pacing, will also alter scar structure. Our computational model of infarct healing should enable in silico screening of novel therapies for potential adverse effects on scar formation.
The periosteum is a composite tissue that provides a niche for stem cells and exhibits a remarkable regenerative capacity to generate bone de novo within critical sized defects, even in the absence of the medullary cavity (e.g. when it is filled by an intramedullary nail for mechanical stabilization after tumor resection). Clinical reports and recent experiments indicate that the regenerative capacity of the periosteum is enhanced by mechanical loading. Furthermore, implementation of directional delivery implants designed as periosteum substitutes show that periosteum-derived cells as well as other biologic factors intrinsic to periosteum play a key role for infilling of critical sized defects. In this talk we will review the mechanobiological factors shown to promote emergence of anisotropic structure and function by stem cells, to facilitate de novo tissue building by periosteum derived cells, as well as surgical and engineering approaches to unleash the power of the periosteum for tissue engineering as well as trauma and reconstruction surgery.
The importance of growth regulation cannot be overstated. It exists in simple organisms that can only change size, as well as in complex organisms with complex morphology and differentiated tissues. It is vital in maintaining stem cell niches. It marks the difference between homeostasis and neoplasm. Growth regulation is so fundamental that we would like to understand it from a fundamental level. In this talk, we focus on the relationship between the chemical and mechanical growth regulatory mechanisms in a simple system which is important in tissue engineering: solid spheroids. We develop a model of transport and growth in epithelio-mesenchymal interactions. Analysis shows that sustained volumetric growth requires four generic mechanisms. The model reveals a bifurcation delineating two distinct morphogenetic regimes: (A) steady growth, (B) growth arrested by capsule formation in the mesenchyme. The bifurcation is determined by just two ratios representing the relative strengths of growth and proteolytic activity. The model provides a theoretical basis for explaining observations of growth arrest despite proteolysis of surrounding tissue, and gives a quantitative framework for the design and interpretation of experiments involving spheroids and tissues which are locally equivalent to spheroids. The results also provide guidance for optimizing the production of replacement tissue from spheroids.
Recent advancements in computational fluid dynamics have enabled researchers to efficiently explore problems that involve moving elastic boundaries immersed in fluids for problems such as cardiac fluid dynamics, fish swimming, and the movement of bacteria. These advances have also made modeling the interaction between a fluid and a neuromechanical model of an elastic organ feasible. The tubular hearts of some ascidians and vertebrate embryos offers a relatively simple model organ for such a study. Blood is driven through the heart by either peristaltic contractions or valveless suction pumping through localized periodic contractions. Models considering only the fluid-structure interaction aspects of these hearts are insufficient to resolve the actual pumping mechanism. The electromechanical model presented here will integrate feedback between the conduction of action potentials, the contraction of muscles, the movement of tissues, and the resulting fluid motion.
Tissue engineering constructs comprising cells and extracellular matrix-like scaffolds in fiber form are increasingly used for regenerative medicine applications. While cell types, fibers and modes of attachment vary, a simple classification can be made regarding the relative cell size versus fiber diameter. We became interested in the situation when the two dimensions are comparable, during a project aiming at the colonization with endothelial progenitor cells (EPC) of scaffolds made of polymeric fibers obtained by electrospinning. The goal is to use these cell-seeded scaffolds as carriers for various purposes, such as manipulating neovascularization at the site of implantation. In the process, we found that EPC engage with the fibers by a novel filamentous (F) actin-rich, highly dynamic cytoskeletal structure, that we named 'actin grip'. These F-actin bundles are finely regulated by the intracellular levels of reactive oxygen species. Therefore, they could be instrumental for controlled attachment to and detachment of the cells from the fibers, independent of their adhesiveness. To quantify the number and geometry of these 'grips', their co-localization with signaling molecules, and their impact on cell density and shape within the scaffolds, we are developing an automatic analysis program of three dimensional confocal microcopy images. I will discuss the methods and the main results obtained in this project so far.
Articular cartilage is a connective tissue lining bony surfaces in diarthrodial joints (knees, hips, and shoulders). When degradation exceeds the synthesis of extracellular matrix constituents, cartilage will start eroding and fragmenting, eventually leading to defects. Since cartilage has a limited capacity for growth and repair, defects due to osteoarthritis and injury may rapidly progress to complete tissue degradation, necessitating a joint replacement. Prior to complete tissue degradation, defects could be filled with cell-seeded biocompatible porous scaffolds, with the hopes of repairing and regenerating new tissue. In order to achieve biological and mechanical functionality of these constructs, many factors need to be considered. This talk will highlight key aspects such as nutrient transport, cell proliferation and migration, matrix synthesis, and mechanical interactions in the context of cartilage regeneration. Previous computational work will be described that ranges from the use of neural networks, reaction diffusion models, level set methods, and continuum modeling frameworks to investigate and understand aspects of cartilage tissue engineering.
Engineered tissues must reproduce the biological and mechanical function of their native counterparts if they are to provide health benefits to society. However, the current generation of engineered skin (ES) fails to match the mechanical properties of native skin, limiting its use in vivo. This is due, in part, to the static, non-physiological conditions used during synthesis. Using mechanical stimuli during tissue culture is known to improve function and strength in engineered tissues. However, this advance has not been fully realized in skin or other complex, hierarchical tissues with multiple cell lineages and extracellular environments. This presentation will focus on the use of novel mechanical-bioreactor technology in conjunction with materials processing techniques, computational modeling and biological tools to understand the role of scaffold mechanics, stress gradients and cell communication during mechanical stimulation of engineered skin.
Multiscale mechanical interactions are scale spanning physical interactions between the tissue and the extracellular matrix (ECM). They are involved in a variety of biological phenomena, including tissue growth, remodeling, disease, and damage. These interactions are important to characterize because they control both the mechanical behavior of the tissue and the manner in which mechanical signals are propagated to the cellular level. In this talk I will discuss recent work where we incorporate (1) fiber-level rules that govern enzymatic degradation and growth and (2) contractile elements that simulate cell compaction and a redistribution of forces within the surrounding fiber networks into our multiscale modeling framework. Understanding the role of these processes is crucial to comprehending and controlling the integrated response of the mechanical environment in a number of biological contexts.
In non-linear, strain stiffening biopolymers, localized sources of strain can give rise to local alignment and large strain out to a certain radius, and very small strain outside. We give evidence for this effect and estimate the 'horizon' for strain propagation.
Understanding how matrix properties influence cell behavior has implications for our understanding of many processes and applications, including fundamentals of cell and developmental biology, wound healing, tumor formation, and design of biomaterials for tissue engineering. While it is widely appreciated that the mechanical properties of the matrix, as well as cell force generation, are important aspects of the differentiated functions of numerous cell types, the interplay between these passive and active mechanical forces is less well understood. We have used an in vitro model of microvascular network (MVN) formation to investigate the dynamic and reciprocal relationship between cells and biomaterial supports. Macroscopic matrix remodeling due to endothelial cell force generation, as indicated by biomaterial gel compaction, is a key feature of MVN formation. To understand this phenomenon at the cellular length scale, we have employed confocal microscopy to image endothelial cells and their surrounding collagen matrix during various stages of MVN formation. Gathering of matrix around cells precedes cell elongation and MVN formation and decreases as a function of increasing cell-cell distance and collagen stiffness. Additionally, the patterns of matrix gathering are consistent with the hypothesis that nearby cells form lines of matrix tension or alignment that they elongate along in order to form multi-cellular structures. This work fits into the larger picture of increasing our understanding of the role of cell-derived forces and matrix remodeling in differentiated cell functions with implications for mechanobiology and design of new tissue engineering scaffolds.
Properly-regulated inflammation is central to homeostasis, but in adequate or overly-robust inflammation can lead to disease. Like many biological processes, inflammation and its various manifestations in disease are multi-dimensional. The advent of multiplexed platforms for gathering biological data, while providing an unprecedented level of detailed information about the dynamics of complex biological systems such as the inflammatory response, has paradoxically also flooded investigators with data they are often unable to use. Systems approaches, including data-driven and mechanistic computational modeling, have been used to decipher aspects of the inflammatory responses that characterize trauma/hemorrhage and sepsis. Through combined data-driven and mechanistic modeling based on LuminexTM datasets, computational models of acute inflammation in mice, rats, swine, and humans were generated. These studies suggest that acute inflammation goes awry when the positive feedback loop of inflammation -> tissue damage/dysfunction -> inflammation, driven by damage-associated molecular pattern molecules, fails to resolve under the influence of anti-inflammatory/pro-healing mediators. This positive feedback loop also underlies agent-based computational models of inflammation and tissue injury in skin and liver, leading to tissue-realistic simulations and tissue-specific outcomes including chronic non-healing wounds and liver fibrosis, cirrhosis, and hepatocellular carcinoma, respectively. These systems-based insights have led to novel, rationally-designed therapies based on a biohybrid framework.
Chemotaxis is the active movement of cells or organisms in response to external chemical signals. The chemotactic movement of bacteria populations can lead to intricate spatial and temporal patterns. Traditionally, these patterns have been modeled using continuum approaches, which describe the evolution of the cell density by partial differential equations (PDEs). However, these models are phenomenological and their connection with mechanistic cellular processes such as signal transduction and cell movement is not well understood. In the first part of this talk, I will present cell-based models of bacterial chemotaxis, which integrate great details of fundamental cellular mechanisms, and address several bacterial patterns. For problems that involve large numbers of cells, cell-based models are computationally intensive and time-consuming. In the second part of this talk, I will show connections between the continuum/phenomenological models and cell-based/mechanistic models derived by asymptotic methods.
Objectives: Low power lasers (LPL) have been widely reported to enhance tissue regeneration in various settings, but the biological mechanism linking LPL to regeneration is unclear. This work addressed the hypothesis that LPL directs differentiation of stem cells via activation of endogenous morphogens.
Methods: Fluorescent probes were used to assess LPL-generated specific ROS. To evaluate LPL-activated complexes, a novel biochemical screen was developed. Activation of specific complexes were validated by immunoassays and reporter studies. Human dental stem cells were isolated and characterized from extracted tooth specimens. A rat molar pulp capping model was used to evaluate dentin induction by LPL using high resolution micro-computed tomographic (uCT) imaging, immunoassays and histology. Two approaches, a transgenic approach with a conditional knockout (DSPPCreTGF-βRIIFlox) mice and a biomaterials-based controlled delivery of inhibitor or neutralizing antibodies in the rat model were employed to validate in vivo efficacy of LPL-TGF-β1 in dentin induction.
Results: LPL irradiation induced specific ROS, namely superoxide, hydrogen peroxide and hydroxyl radicals. A screen demonstrated activation of several complexes, including TGF-β1, in biological fluids. TGF-β1 activation required a specific ROS-sensitive methionine in its latent complex for LPL-ROS mediated activation. LPL irradiation of human dental stem cells demonstrated decreased stem cell expression (CD44, CD90, CD106, CD117 and Stro-1) with concurrent up-regulation of dentin matrix expression (ALP, DSP, DMP1 and OPN). The rat pulp capping model demonstrated the ability of LPL to differentiate dental stem cells in vivo and induce a mineralized reparative dentin response. Neutralization of TGF-β in vivo with both strategies validated the role of LPL-activated endogenous TGF-β1 in LPL directed dentin induction.
Conclusions: A novel mechanism involving LPL generated ROS activation of TGF-β1 to direct differentiation of dental stem cells is presented. This process highlights the use of endogenous factors to direct differentiation of native stem cells. LPL is an innovative clinical tool with a broad potential for various applications in regenerative medicine.
Work done in collaboration with Tristan D Hunt, Andrew Cho, Gursimran Sidhu, Kyungsup Shin, Aaron Chiao-Chen, Eason Hahm, Bonnie L Padwa, Michael R Hamblin, Ashok Kulkarni and David J Mooney.
Hypertension, diabetes, and obesity are often associated with impaired microvascular function and structural adaptation. A.R. Pries and T.W. Secomb have developed a mathematical model of structural adaptation based on known physiological responses to shear stress, circumferential stress, and metabolic demand under healthy conditions. While this model captures key aspects of microvascular remodeling, it does not explicitly incorporate signaling pathways. As altered signaling pathways are a prominent feature of many disease states, in its current state this model cannot be used to predict the effects of diseases on vessel remodeling. Using the Pries and Secomb model as a framework, we have developed a model that incorporates relevant signaling pathways in the structural adaptation of microvessels. In our model, diameter changes with nitric oxide, a vasodilator, and endothelin-1, a vasoconstrictor, which are both functions of shear stress. Wall area changes with circumferential stress as well as the growth and death of vascular smooth muscle cells. Currently our model reflects the steady state trends in vessel geometry under normal conditions. Work is ongoing to further validate the model and examine vascular remodeling in disease states. Our long term goal is to improve the understanding of vascular adaptation in disease states and to create modeling tools which provide biologically testable hypotheses for experimentalists and clinicians.
Introduction: Fibrin-based tissue constructs show adaptation of collagen production in response to constant strain amplitude cyclic stretch, limiting the benefits of mechanical stimulation for tissue improvement. Precisely predicting the time course of adaptation is difficult, making it impossible to develop an ideal incremental regime. The hypothesis of this study was that monitoring collagen expression during bioreactor culture would predict the optimal timing for incrementing mechanical stimulation. To address this hypothesis, we assayed constructs during bioreactor conditioning, and incremented the strain amplitude when collagen expression decayed.
Materials and Methods: Neonatal human dermal fibroblasts (0.5 million/ml), fibrinogen (3.3 mg/ml), and thrombin (0.2 U/ml) were mixed and injected into molds to catalyze the formation of a tubular fibrin gel with embedded cells. All of the cells were stably transfected with a reporter plasmid consisting of the luciferase gene driven by the human type I collagen alpha 1 promoter. Constructs were cultured statically for ten days to allow radial compaction and ensuing circumferential alignment of cells and matrix fibers. Constructs were then maintained in static culture or mounted on a pulse-flow-stretch bioreactor that provided tunable pulsatile flow through the lumen of the vessel simultaneous with vessel distension. During culture, constructs were immersed in substrate (Luciferin-EF, Promega, 1 mM) and imaged for luminescence (IVIS, Caliper). In response to the luminescence data, increases in flow were implemented at 10 and 17 days of bioreactor culture. After three weeks of bioreactor culture, constructs were harvested for collagen content analysis by a modified hydroxyproline assay.
Results: Mechanical stimulation induced increased collagen transcription within 4 days, but this benefit decayed by 10 days of constant amplitude mechanical stimulation. Increasing the strain amplitude at 10 days failed to induce an increase in collagen transcription, but a second increase at 17 days resulted in an additional spike in collagen transcription. Collagen content was significantly increased (1.85?0.22 vs. 4.05?0.51 mg/ml) in the mechanically stimulated constructs.
Discussion: Collagen transcription was elevated only transiently by constant amplitude mechanical stimulation, in line with findings that an incremental regime of stimulation further increases the collagen content of engineered vessels. Real-time monitoring of collagen transcription in engineered tissues presents a novel strategy to optimize bioreactor regimes "on the fly" for a wide variety of cellular tissue engineering applications.
Acknowledgements: National Institutes of Health F32 HL104768 and R01 HL083880.