Gut mucosal injury ranges from the subcellular membrane defects and microscopic erosions engendered by interaction with luminal contents and which normally heal within seconds or minutes to large deep ulcerations that may never heal. The biology of healing depends upon the nature of the injury as well as the mucosal milieu. Mucosal healing is itself known to be regulated by a variety of growth factors and cytokines. However, new evidence will be reviewed that suggests that the healing process may also be influenced by physical forces such as repetitive deformation and increased extracellular pressure in a complex frequency- and amplitude-dependent manner.
Bell's introduction of the fibroblast populated collagen lattice (FPCL) has facilitated the study of collagen-cell interactions. As a result of the numerous modifications of the casting of FPCL's, the in vivo applications of these in vitro findings has been confusing. The experimental FPCL contraction findings will be viewed with regard to three proposed mechanisms responsible for lattice contraction. The cellular mechanisms responsible for generating FPCL contraction are: cell contraction, cell tractional forces related to cell locomotion, and initial cell elongation and spreading. I will introduce a mathematical model of FPCL and some preliminary results.
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.
The appropriate function of inflammatory cells has generally been considered indispensable for successful wound healing. Multiple types of leukocytes migrate into healing wounds, and both the number and functions of these cells represent quantifiable components of the repair process. There is little argument that proper leukocyte activity assists in microbial decontamination of wounds. In addition, there are several logical arguments in support of a role for leukocytes in healing, even within sterile wounds. However, a number of recent studies challenge the established paradigm, and suggest that leukocytes are primarily detrimental to the healing process. Many questions remain, including the relative importance of the many interactions among inflammatory cells and other cell types in the wound, and the utility of modulating the inflammatory response at the site of an injury to improve the quality of healing.
Scarring resulting from any wound can cause considerable patient morbidity e.g. adverse aesthetic, functional or psychological sequelae. We discovered that wounds made on early embryos heal without scarring. Analysis of the cellular and molecular mechanisms underlying scar-free compared to scar-forming healing revealed a number of important mechanistic differences. Embryonic wounds have elevated levels of morphogenetic growth factors involved in skin development e.g. TGFß3, but lower levels of fibrotic growth factors such as TGFß1 and TGFß2 and a quantitatively and qualitatively different inflammatory response to injury compared to adult wounds, which heal with a scar. Elevation of the levels of TGFß3, reduction of the levels of TGFß1 and TGFß2 or modulation of the inflammatory response experimentally in animal models results in an improvement in scarring. We have investigated how alterations in gene and protein expression at the cellular level can result in altered tissue architecture. The speed and direction of migration of fibroblasts re-populating the wounded area is a critical component. Equally the timing of any therapeutic intervention is key: early application is essential in order to re-route in a self-reinforcing and autocatalytic fashion the healing response: clear example of systems biology.
We have translated these findings into potential new human pharmaceuticals. Human Recombinant Transforming Growth Factor Beta 3 (Avotermin, Juvista) has now been investigated in extensive Phase I and Phase II programmes involving 16 double blind, placebo controlled, within subject clinical trials in approximately 1500 human subjects. This has allowed the elucidation of the optimal dose, dosing regimes, clinical trial designs, endpoints for measuring scarring etc. Juvista treated wounds heal with less noticeable scars, which blend in better with the surrounding skin and which histologically have a dermal architecture that more closely resembles normal skin. Juvista treated wounds are also less red and the redness fades faster. These beneficial effects are first evident approximately 3 months after wounding and are stable at approximately 7-12 months post surgery. Juvista is now in Phase III clinical development in the European Union. If the drug is successful in these Phase III trials it will represent the first human pharmaceutical for the prophylactic improvement of scarring.
Neovascularization is essential for normal tissue repair. Bone marrow (BM)-derived vascular progenitor cells capable of contributing to new vessel formation have been postulated to play a critical role in ischemic neovascularization and are thought to have therapeutic potential as cell-based vectors to augment neovascularization following injury. However, the specific lineage of these cells remains unclear. Moreover early clinical trials using whole BM-derived cells to enhance neovascularization have yielded disappointing results, possibly due to heterogeneity in this population1, 2 More recently, mesenchymal stem cells (MSCs) from bone marrow or fat have been proposed as promising agents to improve the response to injury and promote tissue regeneration. However, once again the lineage and mechanism of action of these cells remains unknown3-5. It seems likely that a more precise characterization of these cells will be required to develop cell based therapeutics for regenerative medicine.
We have developed a novel technique for high-throughput single cell gene expression analysis (microfluidic large-scale integration) to characterize putative stem cells (ESCs, MSCs). Using a panel of 48 genes contained on a microfluidic chip, we are able to define the transcriptional activity of genes important for pluripotency, differentiation fates and cell cycle regulation in every cell individually in any given population. To analyze the data we employ fuzzy c-means clustering, optimized with Akaike Information Criterion (AIC), to detect discrete sub-populations and generate associated characteristic marker profiles. Using this approach we have developed a standardized metric for comparison of population heterogeneity based on transcriptional variation over relevant gene sets. In a murine model of diabetes, where alterations in progenitor cells have been suggested in human and animal systems, we are able to demonstrate reduced expression of several important stem cell genes within BM-derived MSCs and deletion of entire sub-populations of progenitors. These results suggest that derangements in specific progenitor sub-populations may underlie the impairments in neovascularization characteristic of diabetes.
Support: NIH: RO1 AG-25016, RO1 DK-074095, DoD #W81XWH-08-2-0032-3
Introduction. Our clear understanding of the mechanism of blunt trauma to the vascular wall would be of a significant clinical importance. Early diagnosis may result in better survival, justifying the search for potential risk factors and diagnostic tests. In addition, it may improve safety features of motor vehicles.
The aim of this research was to investigate the mechanism of blunt injury to the vascular wall.
Methods. Multi-phase equations have been used. The system of equations with certain boundary conditions was solved numerically by applying the finite-difference method with order of approximation equal to 0.0001.
Results. Trauma may result in transfer of laminar boundary layer into turbulent boundary layer and specific conditions such as compression of at least 25% and velocity is greater than 2.4 m/s may damage endothelium. Damage to the endothelium can occur if or if compression is at least 10% and velocity is greater than 2.9 m/s. In addition, trauma can lead to the appearance of zones with high (directly damaging the endothelium) or low (within the zone of boundary layer separation) shear stress. Finally, substantial variations in the geometric parameters of human vessels, and, therefore, the degree of curvature (the Dean number) may become highly informative about the shear stress on the external surface of the vessel.
Conclusion. Certain injury conditions can cause damage to the endothelium (for instance, the significant degree of compression resulting from injury). The mathematical model created in this study will improve our understanding of the complex mechanism of injury to the vascular wall.
We have constructed a mathematical model of the protein-protein interactions and protein modifications that comprise the G2 to M transition and the G2 DNA damage checkpoint. This model was constructed from interactions known to play a role in these processes. This construction allows us to determine if behaviors observed in this system can be accounted for by the known interactions or if additional mechanisms are required, giving us insight into how the G2 to M transition and G2 checkpoint operate. Additionally, this model provides a platform to rapidly simulate experiments to help determine what physical experiments might be significant such as the model prediction that depletion of the protein Wee1 will result in an accumulation of inactive MPF (a complex of Cyclin B and CDK1 which triggers mitosis) in the nucleus during a DNA damage arrest of the cell cycle. In addition, the model provides a means to investigate situations which are difficult or impossible to reproduce experimentally such as the depletion or overexpression of several proteins simultaneously.
Some mathematical models for the growth of biological tissue will be outlined, particular focus being given to the hole-closure problem that describes the behaviour as the tissue grows to fill in the entire domain.
The ability of a living cell to control its three-dimensional structure is critical to normal tissue physiology. An individual cell derives this morphological control from its cytoskeleton, the three-dimensional network of biopolymers whose collective dynamics and mechanics define cell shape and enable cells to sense, process, and respond to a variety of physical cues in the environment, including mechanical force and the geometry and stiffness of the extracellular matrix (ECM). I will describe several experimental approaches my colleagues and I have taken to understanding how cytoskeletal polymers contribute to cellular mechanics and biophysical crosstalk with the ECM, which include the use of various micro/nanoscale technologies to probe the biophysical properties of contractile and adhesive structures within living cells. I will also discuss our recent efforts to determine the role of cell-ECM mechanobiology in influencing the growth and invasion of tumors of the nervous system, as well as our attempts to leverage cell-ECM mechanobiology to engineer cell fate and assembly in bottom-up tissue engineering systems.
Of the myriad of growth factors implicated in wound healing, TGF-beta has the broadest spectrum of effects, promoting re-epithelialization, granulation tissue formation and tissue remodeling, as have been demonstrated in animal models. However, clinical results in humans have been modest, possibly due to inappropriate timing of administration or unavailability of the delivered factor. An alternative approach would be to manipulate endogenous TGF-beta action locally using regulatory molecules. Our group has recently identified a novel TGF-beta co-receptor, CD109, which negatively regulates TGF-beta signaling and inhibits extracellular matrix synthesis in skin cells. Our results indicate that the mechanism by which CD109 exerts this effect involves targeting TGF-beta signaling receptors for degradation. To examine CD109 function in vivo, we generated transgenic mice overexpressing CD109 in the epidermis. Using a bleomycin-induced skin fibrosis model, we show that the transgenic mice display diminished TGF-beta signaling, more organized collagen deposition and decreased dermal thickness, as compared to their wild-type littermates. Together these results demonstrate that CD109 is an important regulator of TGF-beta signaling, and may represent a potential molecular target for the treatment of skin disorders such as hypertrophic scarring.
The broad goal of tissue engineers is to grow functional tissues and organs in the laboratory to replace those which have become defective through age, trauma, and disease and which can be used in drug screening applications. To achieve this goal, tissue engineers aim to control accurately the biomechanical and biochemical environment of the growing tissue construct, in order to engineer tissues with the desired composition, biomechanical and biochemical properties (in the sense that they mimic the in vivo tissue). The growth of biological tissue is a complex process, resulting from the interaction of numerous processes on disparate spatio-temporal scales. Advances in the understanding of tissue growth processes promise to improve the viability and suitability of the resulting tissue constructs. In this talk, I highlight some of our recent mathematical modelling work that aims to provide insights into tissue engineering applications.
Normal wound healing process involves the reparative phases of inflammation, proliferation, and remodeling. Interruption of any phase during the wound healing process may result in chronically unhealed wounds, amputation, or even patient death. Accurate characterization of structural, functional, and molecular changes at each phase of the wound healing process will help to quantitatively guide the therapeutic process and objectively assess the clinical outcome. However, many existing techniques and clinical procedures for wound assessment are qualitative and subjective. Limited tools are available for clinicians to systemically evaluate and document wound healing progression or regression.
We developed a portable multimodal imaging system for quantitative imaging of wound. The imaging system can be used for multiple clinical applications such as wound margin detection, hypoxia imaging, infection detection, perfusion assessment, and therapeutic guidance. We also developed a biodegradable and biocompatible carrier for targeted delivery of multiple contrast enhancement agents and drugs. In this talk, we will show our preliminary results and discuss about potential clinical applications.
A long term goal of regenerative medicine is to restore normal size, shape and function of organs following injury or disease. During embryonic development, these same parameters are determined by groups of cells called "organizing centers," whose primary functions are to secrete growth factors and support patterned growth. Organizing centers demonstrate a remarkable level of self-regulation, preventing insufficient or overgrowth of tissue. Surprisingly, many organizing centers are even capable of reforming after experimental ablation. Understanding the robust developmental mechanisms that regulate organizing centers may be critical to achieving the goals of wound repair. The role and regulation of organizing centers in the skin is not completely known. Here we discuss the organizing centers of the skin and show that a key homeostatic mechanism in maintaining organizing centers in the skin is mediated through the RAS/MAPK pathway. Using gain and loss-of-function genetic models, we find that RAS regulates skin surface area, hair follicle size, and other ectodermal organs. RAS/MAPK signals are interpreted through a second organizing center in the hair follicle, which translates increased or decreased RAS signals into reciprocal changes in Sonic Hedgehog expression levels. We discuss these results in the context of a family of human congenital diseases collectively called RAS/MAPK syndromes, which support the model that RAS signal strength plays a role in regulating organizing centers and pattern in human skin. Lastly, we propose that manipulating organizing centers through RAS/MAPK signaling could be used to re-create normal amounts of tissue during wound repair.
Endogenous wound electric fields were measured at wounds centuries ago. Recent experiments provide compelling evidence that the wound electric fields may play a far more important role than generally perceived. Electric fields of the strength that can be measured in vivo override many well accepted directional cues (such as contact inhibition release, population pressure and chemical gradients) and guide the migration of epithelial cells in wound healing. Genetic study demonstrates that PI3 kinase/Akt and Pten are essential molecules in the response and are activated asymmetrically by the electric fields. Continuous medium perfusion and genetic decoupling experiments argue that the electric field-directed cell migration is not at least exclusively mediated by chemotaxis. The endogenous DC electric fields thus may represent a fundamental signaling mechanism to give cells and tissues a direction to heal and to regenerate in wound healing.