Biomechanical study on the edge shapes for penetrating keratoplasty

A parametric study to investigate the compressive and the shear stress distributions for various edge shapes created during penetrating keratoplasty (PK) using femtosecond laser is reported. The finite element analysis has been implemented using ABAQUS to study the cornea with various edge shapes, namely the standard edge shape, the zigzag edge shape, the top hat edge shape and the mushroom edge shape for PK. The ratio of maximum compressive stress to maximum shear stress is used as the main factor to assess the relative merits of wound healing rate for different edge shapes. For the typical values of tissue mechanical properties, the zigzag edge shape has the highest ratio of maximum compressive stress to maximum shear stress (11.1 in the xy-direction and 3.7 in the yz-direction), followed by the mushroom edge shape (7.7 in the xy-direction and 3.2 in the yz-direction). The ratios for the top hat and the standard edge shapes are even lower in both directions. A sensitivity analysis of the model has been done to demonstrate that the zigzag edge shape always results in the highest ratios of stresses regardless of the difference in the tissue mechanical properties. The zigzag edge shape also gives the lowest dioptric power D = 45.4. The present results imply that the zigzag edge shape provides the best wound healing rate and optical outcome among the four edge shapes models for PK.     

Vascular endothelial wound closure under shear stress: role of membrane fluidity and flow-sensitive ion channels.

Sufficiently rapid healing of vascular endothelium following injury is essential for preventing further pathological complications. Recent work suggests that fluid dynamic shear stress regulates endothelial cell (EC) wound closure. Changes in membrane fluidity and activation of flow-sensitive ion channels are among the most rapid endothelial responses to flow and are thought to play an important role in EC responsiveness to shear stress. The goal of the present study was to probe the role of these responses in bovine aortic EC (BAEC) wound closure under shear stress. BAEC monolayers were mechanically wounded and subsequently subjected to either "high" (19 dyn/cm(2)) or "low" (3 dyn/cm(2)) levels of steady shear stress. Image analysis was used to quantify cell migration and spreading under both flow and static control conditions. Our results demonstrate that, under static conditions, BAECs along both wound edges migrate at similar velocities to cover the wounded area. Low shear stress leads to significantly lower BAEC migration velocities, whereas high shear stress results in cells along the upstream edge of the wound migrating significantly more rapidly than those downstream. The data also show that reducing BAEC membrane fluidity by enriching the cell membrane with exogenous cholesterol significantly slows down both cell spreading and migration under flow and hence retards wound closure. Blocking flow-sensitive K and Cl channels reduces cell spreading under flow but has no impact on cell migration. These findings provide evidence that membrane fluidity and flow-sensitive ion channels play distinct roles in regulating EC wound closure under flow.     

Macrophage cell morphology-imprinted substrates can modulate mesenchymal stem cell behaviors and macrophage M1/M2 polarization for wound healing applications

Mesenchymal stem cells and macrophages (MQ) are two very important cells involved in the normal wound healing process. It is well understood that topological cues and mechanical factors can lead to different responses in stem cells and MQ by influencing their shape, cytoskeleton proliferation, migration, and differentiation, which play an essential role in the success or failure of biomaterial implantation and more importantly wound healing. On the other hand, the polarization of MQ from proinflammatory (M1) to prohealing (M2) phenotypes has a critical role in the acceleration of wound healing. In this study, the morphology of different MQ subtypes (M0, M1, and M2) was imprinted on a silicon surface (polydimethylsiloxane [PDMS]) to prepare a nano-topography cell-imprinted substrate with the ability to induce anti-inflammatory effects on the mouse adipose-derived stem cells (ADSCs) and RAW264.7 monocyte cell line (MO). The gene expression profiles and flow cytometry of MQ revealed that the cell shape microstructure promoted the MQ phenotypes according to the specific shape of each pattern. The ELISA results were in agreement with the gene expression profiles. The ADSCs on the patterned PDMS exhibited remarkably different shapes from no-patterned PDMS. The MOs grown on M2 morphological patterns showed a significant increase in expression and section of anti-inflammatory cytokine compared with M0 and M1 patterns. The ADSCs homing in niches heavily deformed the cytoskeletal, which is probably why the gene expression and phenotype unexpectedly changed. In conclusion, wound dressings with M2 cell morphology-induced surfaces are suggested as excellent anti-inflammatory and antiscarring dressings.     

Control of the shape of a thrombus-neointima-like structure by blood shear stress

Fluid mechanical factors are thought to influence vascular morphogenesis. Here we show how blood shear stress regulates the shape of a thrombus-neointima-like tissue on a polymer micro-cylinder implanted in the center of the rat vena cava with the micro-cylinder perpendicular to blood flow. In this model, the micro-cylinder is exposed to a laminarflow with a known shear stress field in the leading region and a vortexflow in the trailing region. At 1, 5, 10, 20, and 30 days after implantation, it was found that the micro-cylinder was encapsulated by a thrombus-neointima-like tissue with a streamlined body profile. The highest growth rate of the thrombus-neointima-like tissue was found along the trailing and leading stagnation edges of the micro-cylinder. Blood shear stress in the laminar flow region was inversely correlated with the rate of thrombus formation and cell proliferation, and the percentage of smooth muscle a actin-positive cells. These biological changes were also found in the trailing vortex flow region, which was associated with lowered shear stress. These results suggest that blood shear stress regulates the rate of thrombus and neointimal formation and, thus, influences the shape of the thrombus-neointima-like structure in the present model.     

Localized elasticity measured in epithelial cells migrating at a wound edge using atomic force microscopy

Restoration of lung homeostasis following injury requires efficient wound healing by the epithelium. The mechanisms of lung epithelial wound healing include cell spreading and migration into the wounded area and later cell proliferation. We hypothesized that mechanical properties of cells vary near the wound edge, and this may provide cues to direct cell migration. To investigate this hypothesis, we measured variations in the stiffness of migrating human bronchial epithelial cells (16HBE cells) approximately 2 h after applying a scratch wound. We used atomic force microscopy (AFM) in contact mode to measure the cell stiffness in 1.5-microm square regions at different locations relative to the wound edge. In regions far from the wound edge (>2.75 mm), there was substantial variation in the elastic modulus in specific cellular regions, but the median values measured from multiple fields were consistently lower than 5 kPa. At the wound edge, cell stiffness was significantly lower within the first 5 microm but increased significantly between 10 and 15 microm before decreasing again below the median values away from the wound edge. When cells were infected with an adenovirus expressing a dominant negative form of RhoA, cell stiffness was significantly decreased compared with cells infected with a control adenovirus. In addition, expression of dominant negative RhoA abrogated the peak increase in stiffness near the wound edge. These results suggest that cells near the wound edge undergo localized changes in cellular stiffness that may provide signals for cell spreading and migration.     

Cells Actively Stiffen Fibrin Networks by Generating Contractile Stress

During wound healing and angiogenesis, fibrin serves as a provisional extracellular matrix. We use a model system of fibroblasts embedded in fibrin gels to study how cell-mediated contraction may influence the macroscopic mechanical properties of their extracellular matrix during such processes. We demonstrate by macroscopic shear rheology that the cells increase the elastic modulus of the fibrin gels. Microscopy observations show that this stiffening sets in when the cells spread and apply traction forces on the fibrin fibers. We further show that the stiffening response mimics the effect of an external stress applied by mechanical shear. We propose that stiffening is a consequence of active myosin-driven cell contraction, which provokes a nonlinear elastic response of the fibrin matrix. Cell-induced stiffening is limited to a factor 3 even though fibrin gels can in principle stiffen much more before breaking. We discuss this observation in light of recent models of fibrin gel elasticity, and conclude that the fibroblasts pull out floppy modes, such as thermal bending undulations, from the fibrin network, but do not axially stretch the fibers. Our findings are relevant for understanding the role of matrix contraction by cells during wound healing and cancer development, and may provide design parameters for materials to guide morphogenesis in tissue engineering.     

Programmed cell death is required for palate shelf fusion and is regulated by retinoic acid

Efficient wound healing including clotting and subsequent reepithelization is essential for animals ranging from insects to mammals to recover from epithelial injury. It is likely that genes involved in wound healing are conserved through the phylogeny and therefore, Drosophila may be an useful in vivo model system to identify genes necessary during this process. Furthermore, epithelial movement during specific developmental processes, such as dorsal closure, ressembles of those seen in mammalian wound healing. As puckered (puc) gene is a target of the JUN N-terminal kinase signaling pathway during dorsal closure, we investigated puc gene expression during wound healing in Drosophila. We showed that puc gene expression is induced at the edge of the wound in epithelial cells and Jun kinase is phosphorylated in wounded epidermal tissues, suggesting that the JUN N-terminal kinase signaling pathway is activated by a signal produced by an epidermal wound. In the absence of the Drosophila c-Fos homologue, puc gene expression is no longer induced. Finally, impaired epithelial repair in JUN N-terminal kinase deficient flies demonstrates that the JUN N-terminal kinase signaling is required to initiate the cell shape change at the onset of the epithelial wound healing. We conclude that the embryonic JUN N-terminal kinase gene cassette is induced at the edge of the wound. In addition, Drosophila appears as a good in vivo model to study morphogenetic processes requiring epithelial regeneration such as wound healing in vertebrates.     

Multiple forces contribute to cell sheet morphogenesis for dorsal closure in Drosophila

The molecular and cellular bases of cell shape change and movement during morphogenesis and wound healing are of intense interest and are only beginning to be understood. Here, we investigate the forces responsible for morphogenesis during dorsal closure with three approaches. First, we use real-time and time-lapsed laser confocal microscopy to follow actin dynamics and document cell shape changes and tissue movements in living, unperturbed embryos. We label cells with a ubiquitously expressed transgene that encodes GFP fused to an autonomously folding actin binding fragment from fly moesin. Second, we use a biomechanical approach to examine the distribution of stiffness/tension during dorsal closure by following the response of the various tissues to cutting by an ultraviolet laser. We tested our previous model (Young, P.E., A.M. Richman, A.S. Ketchum, and D.P. Kiehart. 1993. Genes Dev. 7:29-41) that the leading edge of the lateral epidermis is a contractile purse-string that provides force for dorsal closure. We show that this structure is under tension and behaves as a supracellular purse-string, however, we provide evidence that it alone cannot account for the forces responsible for dorsal closure. In addition, we show that there is isotropic stiffness/tension in the amnioserosa and anisotropic stiffness/tension in the lateral epidermis. Tension in the amnioserosa may contribute force for dorsal closure, but tension in the lateral epidermis opposes it. Third, we examine the role of various tissues in dorsal closure by repeated ablation of cells in the amnioserosa and the leading edge of the lateral epidermis. Our data provide strong evidence that both tissues appear to contribute to normal dorsal closure in living embryos, but surprisingly, neither is absolutely required for dorsal closure. Finally, we establish that the Drosophila epidermis rapidly and reproducibly heals from both mechanical and ultraviolet laser wounds, even those delivered repeatedly. During healing, actin is rapidly recruited to the margins of the wound and a newly formed, supracellular purse-string contracts during wound healing. This result establishes the Drosophila embryo as an excellent system for the investigation of wound healing. Moreover, our observations demonstrate that wound healing in this insect epidermal system parallel wound healing in vertebrate tissues in situ and vertebrate cells in culture (for review see Kiehart, D.P. 1999. Curr. Biol. 9:R602-R605).     

Cells Actively Stiffen Fibrin Networks by Generating Contractile Stress

During wound healing and angiogenesis, fibrin serves as a provisional extracellular matrix. We use a model system of fibroblasts embedded in fibrin gels to study how cell-mediated contraction may influence the macroscopic mechanical properties of their extracellular matrix during such processes. We demonstrate by macroscopic shear rheology that the cells increase the elastic modulus of the fibrin gels. Microscopy observations show that this stiffening sets in when the cells spread and apply traction forces on the fibrin fibers. We further show that the stiffening response mimics the effect of an external stress applied by mechanical shear. We propose that stiffening is a consequence of active myosin-driven cell contraction, which provokes a nonlinear elastic response of the fibrin matrix. Cell-induced stiffening is limited to a factor 3 even though fibrin gels can in principle stiffen much more before breaking. We discuss this observation in light of recent models of fibrin gel elasticity, and conclude that the fibroblasts pull out floppy modes, such as thermal bending undulations, from the fibrin network, but do not axially stretch the fibers. Our findings are relevant for understanding the role of matrix contraction by cells during wound healing and cancer development, and may provide design parameters for materials to guide morphogenesis in tissue engineering.     

Micropatterned structural control suppresses mechanotaxis of endothelial cells

Vascular endothelial cell migration is critical in many physiological processes including wound healing and stent endothelialization. To determine how preexisting cell morphology influences cell migration under fluid shear stress, endothelial cells were preset in an elongated morphology on micropatterned substrates, and unidirectional shear stress was applied either parallel or perpendicular to the cell elongation axis. On micropatterned 20-μm lines, cells exhibited an elongated morphology with stress fibers and focal adhesion sites aligned parallel to the lines. On 115-μm lines, cell morphology varied as a function of distance from the line edge. Unidirectional shear stress caused unpatterned cells in a confluent monolayer to exhibit triphasic mechanotaxis behavior. During the first 3 h, cell migration speed increased in a direction antiparallel to the shear stress direction. Migration speed then slowed and direction became spatially heterogeneous. Starting 11-12 h after the onset of shear stress, the unpatterned cells migrated primarily in the downstream direction, and migration speed increased significantly. In contrast, mechanotaxis was suppressed after the onset of shear stress in cells on micropatterned lines during the same time period, for the cases of both parallel and perpendicular flow. The directional persistence time was much longer for cells on the micropatterned lines, and it decreased significantly after flow onset. Migration trajectories were highly correlated among micropatterned cells within a three-cell neighborhood, and shear stress disrupted this spatially correlated migration behavior. Thus, presetting structural morphology may interfere with mechanisms of sensing local physical cues, which are critical for establishing mechanotaxis in response to hemodynamic shear stress.     

Continuum model of collective cell migration in wound healing and colony expansion

Collective cell migration plays an important role during wound healing and embryo development. Although the exact mechanisms that coordinate such migration are still unknown, experimental studies of moving cell layers have shown that the primary interactions governing the motion of the layer are the force of lamellipodia, the adhesion of cells to the substrate, and the adhesion of cells to each other. Here, we derive a two-dimensional continuum mechanical model of cell-layer migration that is based on a novel assumption of elastic deformation of the layer and incorporates basic mechanical interactions of cells as well as cell proliferation and apoptosis. The evolution equations are solved numerically using a level set method. The model successfully reproduces data from two types of experiments: 1), the contraction of an enterocyte cell layer during wound healing; and 2), the expansion of a radially symmetric colony of MDCK cells, both in the edge migration velocity and in cell-layer density. In accord with experimental observations, and in contrast to reaction-diffusion models, this model predicts a partial wound closure if lamellipod formation is inhibited at the wound edge and gives implications of the effect of spatially restricted proliferation.     

Scaffold-free cartilage by rotational culture for tissue engineering

Our objective was to investigate the hypothesis that tissue-engineered cartilage with promising biochemical, mechanical properties can be formed by loading mechanical stress under existing cell-cell interactions analogous to those that occur in condensation during embryonic development. By loading dedifferentiated chondrocytes with mechanical stress under existing cell-cell interactions, we could first form a scaffold-free cartilage tissue with arbitrary shapes and a large size with promising biological, mechanical properties. The cartilage tissue which constituted of chondrocytes and ECM produced by inoculated dedifferentiated chondrocytes to a high porous simple mold has arbitrary shapes, and did not need any biodegradable scaffold to control the shape. In contrast, scaffold-free cartilage tissue cultured under static conditions could not keep their shapes; it was fragile tissue. The possibility of scaffold-free organ design was suggested because the cartilage tissue increases steadily in size with culture time; indeed, the growth of cartilage tissue starting from an arbitrary shape might be predictable by mathematical expression. For tissue-engineered cartilage formation with arbitrary shapes, biochemical and mechanical properties, loading dedifferentiated chondrocytes with mechanical stress under existing cell-cell interactions has prominent effects. Therefore, our scaffold-free cartilage model loaded mechanical stress based on a simple mold system may be applicable for tissue-engineered cartilage.     

Optimal patterns for suturing wounds of complex shapes to foster healing.

Stress is one of the many biological factors that plays an important role in wound healing. It is therefore essential to analyze stresses around the wound closure theoretically, especially when no invasive/noninvasive technique to measure stress directly is available. The objective of this paper is to determine the regions of high stresses and the optimal pattern of suturing wounds of complex shapes. It is hypothesized that the optimal pattern of suturing wounds is that pattern which will produce minimum principal stresses. The finite element method (FEM) employing the basic equations of elasticity theory for orthotropic materials is utilized to compute the principal stresses and displacements resulting from suturing fusiform, elliptical and triangular wounds in human abdominal skin. The optimal suturing pattern for the triangular wound is determined. The average stress indices for varying suturing density are also determined which can provide useful clinical information for the surgeon. Since regions of high stresses in surgical closures produce adverse affects on healing and scar production, this work of predicting areas of high stresses is useful in indicating regions of slow healing in wounds.     

Strain tunes proteolytic degradation and diffusive transport in fibrin networks

Proteolytic degradation of fibrin, the major structural component in blood clots, is critical both during normal wound healing and in the treatment of ischemic stroke and myocardial infarction. Fibrin-containing clots experience substantial strain due to platelet contraction, fluid shear, and mechanical stress at the wound site. However, little is understood about how mechanical forces may influence fibrin dissolution. We used video microscopy to image strained fibrin clots as they were degraded by plasmin, a major fibrinolytic enzyme. Applied strain causes up to 10-fold reduction in the rate of fibrin degradation. Analysis of our data supports a quantitative model in which the decrease in fibrin proteolysis rates with strain stems from slower transport of plasmin into the clot. We performed fluorescence recovery after photobleaching (FRAP) measurements to further probe the effect of strain on diffusive transport. We find that diffusivity perpendicular to the strain axis decreases with increasing strain, while diffusivity along the strain axis remains unchanged. Our results suggest that the properties of the fibrin network have evolved to protect mechanically loaded fibrin from degradation, consistent with its function in wound healing. The pronounced effect of strain upon diffusivity and proteolytic susceptibility within fibrin networks offers a potentially useful means of guiding cell growth and morphology in fibrin-based biomaterials.     

Force-Induced Changes in Subnuclear Movement and Rheology

Extracellular mechanical forces result in changes in gene expression, but it is unclear how cells are able to permanently adapt to new mechanical environments because chemical signaling pathways are short-lived. We visualize force-induced changes in nuclear rheology to examine short- and long-time genome organization and movements. Punctate labels in the nuclear interior of HeLa, human umbilical vein endothelial, and osteosarcoma (Saos-2) cells allow tracking of nuclear movements in cells under varying levels of shear and compressive force. Under adequate shear stress two distinct regimes develop in cells under mechanical stimulation: an initial event of increased intranuclear movement followed by a regime of intranuclear movements that reflect the dose of applied force. At early times there is a nondirectionally oriented response with a small increase in nuclear translocations. After 30 min, there is a significant increase in nuclear movements, which scales with the amount of shear or compressive stress. The similarities in the nuclear response to shear and compressive stress suggest that the nucleus is a mechanosensitive element within the cell. Thus, applied extracellular forces stimulate intranuclear movements, resulting in repositioning of nuclear bodies and the associated chromatin within the nucleus.     

A morphoelastic model for dermal wound closure

We develop a model of wound healing in the framework of finite elasticity, focussing our attention on the processes of growth and contraction in the dermal layer of the skin. The dermal tissue is treated as a hyperelastic cylinder that surrounds the wound and is subject to symmetric deformations. By considering the initial recoil that is observed upon the application of a circular wound, we estimate the degree of residual tension in the skin and build an evolution law for mechanosensitive growth of the dermal tissue. Contraction of the wound is governed by a phenomenological law in which radial pressure is prescribed at the wound edge. The model reproduces three main phases of the healing process. Initially, the wound recoils due to residual stress in the surrounding tissue; the wound then heals as a result of contraction and growth; and finally, healing slows as contraction and growth decrease. Over a longer time period, the surrounding tissue remodels, returning to the residually stressed state. We identify the steady state growth profile associated with this remodelled state. The model is then used to predict the outcome of rewounding experiments designed to quantify the amount of stress in the tissue, and also to simulate the application of pressure treatments.     

Flow in t-bifurcations: Effect of the sharpness of the flow divider

The local geometry of a bifurcation has been hypothesized to be a potential geometrical risk factor for the development of atherosclerosis. While flow division and branch area ratios clearly affect the flow field, the importance of the flow divider shape is not as clear. A fast spectral element computational fluid mechanics (CFD) solver was used to simulate flow through 90 ° T-bifurcations with three different flow divider shapes. Other factors, such as flow partition, area ratio, and bifurcation angle, were kept constant. A Reynolds number range of 15 to 350 was studied to bracket experimental results in the literature. The variation in the sharpness of the corners had a dramatic effect on both the flow field and wall shear stress distribution in the side branch, but little effect on the flow in the main tube. The magnitude of reverse velocities and wall shear stress in the side branch increased linearly over a physiological range of Reynolds number and corner shape. This paper verifies the accuracy and usefulness of spectral element CFD in studying three-dimensional hemodynamics.     

Endocytosis-dependent coordination of multiple actin regulators is required for wound healing

The ability to heal wounds efficiently is essential for life. After wounding of an epithelium, the cells bordering the wound form dynamic actin protrusions and/or a contractile actomyosin cable, and these actin structures drive wound closure. Despite their importance in wound healing, the molecular mechanisms that regulate the assembly of these actin structures at wound edges are not well understood. In this paper, using Drosophila melanogaster embryos, we demonstrate that Diaphanous, SCAR, and WASp play distinct but overlapping roles in regulating actin assembly during wound healing. Moreover, we show that endocytosis is essential for wound edge actin assembly and wound closure. We identify adherens junctions (AJs) as a key target of endocytosis during wound healing and propose that endocytic remodeling of AJs is required to form “signaling centers” along the wound edge that control actin assembly. We conclude that coordination of actin assembly, AJ remodeling, and membrane traffic is required for the construction of a motile leading edge during wound healing.     

Laminar shear stress induces the expression of aquaporin 1 in endothelial cells involved in wound healing

Laminar shear stress (LSS) due to blood flow contributes to the maintenance of endothelial health by multiple mechanisms including promotion of wound healing. The present study examined the hypothesis that the induction of water channel aquaporin 1 (AQP1) expression by LSS might be functionally associated with endothelial wound healing. When human umbilical vein endothelial cells were exposed to LSS at 12 dyn cm^?2 for 24 h, significant increases in AQP1 expression were observed at the mRNA and protein levels as compared with static control. In the in vitro scratch wound healing assay, LSS treatments before and after wound creation enhanced endothelial wound healing and this effect was significantly attenuated by selective suppression of AQP1 expression using small interfering RNA. Ectopic expression of AQP1 enhanced wound healing in the absence of LSS. This study demonstrated that LSS stimulates the endothelial expression of AQP1 that plays a role in wound healing.     

Expression of synaptopodin in endothelial cells exposed to laminar shear stress and its role in endothelial wound healing

We examined the hypothesis that certain actin binding proteins might be upregulated by laminar shear stress (LSS) and could contribute to endothelial wound healing. Analysis of mRNA expression profiles of human umbilical vein endothelial cells under static and LSS-exposed conditions provided a list of LSS-induced actin binding proteins including synaptopodin (SYNPO) whose endothelial expression has not been previously reported. Additional studies demonstrated that SYNPO is a key mediator of endothelial wound healing because small interfering RNA-mediated suppression of SYNPO attenuated wound closure under LSS whereas overexpression of exogenous SYNPO enhanced endothelial wound closure in the absence of LSS. This study suggests that LSS-induced actin binding proteins including SYNPO may play a critical role in the endothelial wound healing stimulated by LSS.