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Endothelial progenitor cells (EPCs) contribute tode novoangiogenesis, tissue regeneration, and remodeling. Interleukin 10 (IL-10), an anti-inflammatory cytokine that primarily signals via STAT3, has been shown to drive EPC recruitment to injured tissues. Our previous work demonstrated that overexpression of IL-10 in dermal wounds promotes regenerative tissue repair via STAT3-dependent regulation of fibroblast-specific hyaluronan synthesis. However, IL-10's role and specific mode of action on EPC recruitment, particularly in dermal wound healing and neovascularization in both normal and diabetic wounds, remain to be defined.Therefore, inducible skin-specific STAT3knockdown mice were studied to determine IL-10's impact on EPCs, dermal wound neovascularization and healing, and whether it is STAT3-dependent. We show that IL-10 overexpression significantly elevated EPC counts in the granulating wound bed, which was associated with robust capillary lumen density and enhanced re-epithelialization of both control and diabetic (db/db) wounds at day 7. We noted increased VEGF and high C-X-C motif chemokine 12 (CXCL12) levels in wounds and a favorable CXCL12gradient at day 3 that may support EPC mobilization and infiltration from bone marrow to wounds, an effect that was abrogated in STAT3knockdown wounds. These findings were supportedin vitro. IL-10 promoted VEGF and CXCL12synthesis in primary murine dermal fibroblasts, with blunted VEGF expression upon blocking CXCL12 in the media by antibody binding. IL-10-conditioned fibroblast media also significantly promoted endothelial sprouting and network formation. In conclusion, these studies demonstrate that overexpression of IL-10 in dermal wounds recruits EPCs and leads to increased vascular structures and faster re-epithelialization.

Cancer is among the leading causes of death worldwide. In recent years, many cancer-associated biomarkers have been identified that are used for cancer diagnosis, prognosis, screening, and early detection, as well as for predicting and monitoring carcinogenesis and therapeutic effectiveness. Phosphatidylserine (PS) is a negatively charged phospholipid which is predominantly located in the inner leaflet of the cell membrane. In many cancer cells, PS externalizes to the outer cell membrane, a process regulated by calcium-dependent flippases and scramblases. Saposin C coupled with dioleoylphosphatidylserine (SapC-DOPS) nanovesicle (BXQ-350) and bavituximab, (Tarvacin, human–mouse chimeric monoclonal antibodies) are cell surface PS-targeting drugs being tested in clinical trial for treating a variety of cancers. Additionally, a number of other PS-selective agents have been used to trigger cytotoxicity in tumor-associated endothelial cells or cancer cells in pre-clinical studies. Recent studies have demonstrated that upregulation of surface PS exposure by chemodrugs, radiation, and external electric fields can be used as a novel approach to sensitize cancer cells to PS-targeting anticancer drugs. The objectives of this review are to provide an overview of a unique dual-role of PS as a biomarker/target for cancer imaging and therapy, and to discuss PS-based anticancer strategies that are currently under active development.

Diabetes mellitus is a chronic metabolic disorder that negatively impacts patient's health and results in significant economic burden healthcare around the globe. Chronic diabetic ulcers represent a major complication of diabetes and are characterized by dysregulated molecular and cellular wound microenvironment and persistent inflammation. Biomaterials such as films, foams, hydrogels, antimicrobials, and different types of dressings have been used in a variety of therapeutic applications for wound treatment. However, despite tremendous amount of research efforts, there is still no “ideal” treatment that is able to directly address the complexity of nonhealing diabetic ulcers. This chapter focuses on the recent advances and existing challenges in the development of biomaterials for the management and treatment of diabetic wounds, including major classes of biomaterials used in wound dressings and skin substitutes, their physiochemical properties, critical aspects of biomaterial processing, as well as emerging therapies for diabetic wound treatment that incorporate biomaterials as an key component. The discussion includes synthetic, natural and combined polymers and composites, self-assembling peptides, hydrogels and nanoparticles, microRNA-based materials, alternative biophysical therapies, cell- and drug-delivery therapies. Given the multitude of approaches being investigated, along with the current explosive pace of the biomaterial technology development, the analysis warrants cautious optimism for a breakthrough in the near future that would enable the regenerative healing of a diabetic wound and provide much-needed cure for chronic diabetic ulcers.

Aortic valve (AV) disease involves stiffening of the AV cusp with progression characterized by inflammation, fibrosis, and calcification. Here, we examine the relationship between biomechanical valve function and proteomic changes before and after the development of AV pathology in the Emilin1−/− mouse model of latent AV disease. Biomechanical studies were performed to quantify tissue stiffness at the macro (micropipette) and micro (atomic force microscopy (AFM)) levels. Micropipette studies showed that the Emilin1−/− AV annulus and cusp regions demonstrated increased stiffness only after the onset of AV disease. AFM studies showed that the Emilin1−/− cusp stiffens before the onset of AV disease and worsens with the onset of disease. Proteomes from AV cusps were investigated to identify protein functions, pathways, and interaction network alterations that occur with age- and genotype-related valve stiffening. Protein alterations due to Emilin1 deficiency, including changes in pathways and functions, preceded biomechanical aberrations, resulting in marked depletion of extracellular matrix (ECM) proteins interacting with TGFB1, including latent transforming growth factor beta 3 (LTBP3), fibulin 5 (FBLN5), and cartilage intermediate layer protein 1 (CILP1). This study identifies proteomic dysregulation is associated with biomechanical dysfunction as early pathogenic processes in the Emilin1−/− model of AV disease.

Evidence from prior studies of cutaneous trauma, burns, and chronic diabetic wound repair demonstrates that endothelial progenitor cells (EPCs) contribute tode novoangiogenesis, anti-inflammatory reactions, tissue regeneration, and remodeling. We have shown that IL-10, a potent anti-inflammatory cytokine, promotes regenerative tissue repair in an adult model of dermal scar formation via the regulation of fibroblast-specific hyaluronan synthesis in a STAT3 dependent manner. While it is known that IL-10 drives EPC recruitment and neovascularization after myocardial infarction, its specific mode of action, particularly in dermal wound healing and neovascularization in both control and diabetic wounds remains to be defined. Here we show that IL-10 promotes EPC recruitment into the dermal wound microenvironment to facilitate neovascularization and wound healing of control and diabetic (db/db) wounds via vascular endothelial growth factor (VEGF) and stromal-cell derived factor 1 (SDF-1α) signaling. Inducible skin-specific STAT3 knockout (KO) mice were studied to determine whether the impact of IL-10 on the neovascularization and wound healing is STAT3 dependent. We found that IL-10 treatment significantly promotes dermal wound healing with enhanced wound closure, robust granulation tissue formation and neovascularization. This was associated with elevated wound EPC counts as well as increased VEGF and high SDF-1α levels in control mice, an effect that was abrogated in STAT3 KO transgenic mice. These findings were supportedin vitro, wherein IL-10-enhanced VEGF and SDF-1α synthesis in primary murine dermal fibroblasts. IL-10-conditioned fibroblast media was shown to promote sprouting and network formation in aortic ring assays. We conclude that overexpression of IL-10 in the wound-specific milieu recruits EPCs and promote neovascularization, which occurs in a STAT3-dependent manner via regulation of VEGF and SDF-1α levels. Collectively, our studies demonstrate that IL-10 increases EPC recruitment leading to enhanced neovascularization and healing of dermal wounds.

Reduced mobilization of endothelial progenitor cells (EPCs) from the bone marrow (BM) and impaired EPC recruitment into the wound represent a fundamental deficiency in the chronic ulcers. However, mechanistic understanding of the role of BM-derived EPCs in cutaneous wound neovascularization and healing remains incomplete, which impedes development of EPC-based wound healing therapies. The objective of this study was to determine the role of EPCs in wound neovascularization and healing both under normal conditions and using single deficiency (EPC) or double-deficiency (EPC + diabetes) models of wound healing. MMP9 knockout (MMP9 KO) mouse model was utilized, where impaired EPC mobilization can be rescued by stem cell factor (SCF). The hypotheses were: (1) MMP9 KO mice exhibit impaired wound neovascularization and healing, which are further exacerbated with diabetes; (2) these impairments can be rescued by SCF administration. Full-thickness excisional wounds with silicone splints to minimize contraction were created on MMP9 KO mice with/without streptozotocin-induced diabetes in the presence or absence of tail-vein injected SCF. Wound morphology, vascularization, inflammation, and EPC mobilization and recruitment were quantified at day 7 postwounding. Results demonstrate no difference in wound closure and granulation tissue area between any groups. MMP9 deficiency significantly impairs wound neovascularization, increases inflammation, decreases collagen deposition, and decreases peripheral blood EPC (pb-EPC) counts when compared with wild-type (WT). Diabetes further increases inflammation, but does not cause further impairment in vascularization, as compared with MMP9 KO group. SCF improves neovascularization and increases EPCs to WT levels (both nondiabetic and diabetic MMP9 KO groups), while exacerbating inflammation in all groups. SCF rescues EPC-deficiency and impaired wound neovascularization in both diabetic and nondiabetic MMP9 KO mice. Overall, the results demonstrate that BM-derived EPCs play a significant role during wound neovascularization and that the SCF-based therapy with controlled inflammation could be a viable approach to enhance healing in chronic diabetic wounds.

Background Prosthetic valves currently used in children lack the ability to grow with the patient and often require multiple reoperations. Small intestinal submucosa-derived extracellular matrix (SIS-ECM) has been used successfully as a patch for repair in various tissues, including vessels, valves, and myocardium. Objectives This study sought to assess the remodeling potential of a tubular tricuspid valve (TV) bioprosthesis made of SIS-ECM by evaluating its growth, structure, and function in a growing ovine model. Methods A total of 12 3-month-old lambs were studied for a period of 3 or 8 months. SIS-ECM TVs were placed in 8 lambs; conventional bioprosthetic valves and native valves (NV) were studied as controls. All lambs underwent serial echocardiography, measuring annulus diameter and valve and right ventricular function. Results The SIS-ECM valves demonstrated an incremental increase in annular diameter similar to NV. SIS-ECM valve function was normal in 7 of 8; 1 valve had severe regurgitation due to a flail leaflet. Explanted SIS-ECM valves approximated native tissue in gross appearance. Histopathology demonstrated migration of resident mesenchymal cells into the scaffold and trilaminar ECM organization similar to an NV, without inflammation or calcification at 8 months. Ex vivo mechanical testing of SIS-ECM valve tissue showed normalization of the elastic modulus by 8 months. Conclusions In an ovine model, tubular SIS-ECM TV bioprostheses demonstrate “growth” and a cell-matrix structure similar to mature NVs while maintaining normal valve function. The SIS-ECM valve may provide a novel solution for TV replacement in children and adults.

Aortic valve disease is a burgeoning public health problem associated with significant mortality. Loss of function mutations in NOTCH1 cause bicuspid aortic valve (BAV) and calcific aortic valve disease. Because calcific nodules manifest on the fibrosa side of the cusp in low fluidic oscillatory shear stress (OSS), elucidating pathogenesis requires approaches that consider both molecular and mechanical factors. Therefore, we examined the relationship between NOTCH loss of function (LOF) and biomechanical indices in healthy and diseased human aortic valve interstitial cells (AVICs). An orbital shaker system was used to apply cyclic OSS, which mimics the cardiac cycle and hemodynamics experienced by AVICs in vivo. NOTCH LOF blocked OSS-induced cell alignment in human umbilical vein endothelial cells (HUVECs), whereas AVICs did not align when subjected to OSS under any conditions. In healthy AVICs, OSS resulted in decreased elastin (ELN) and α-SMA (ACTA2). NOTCH LOF was associated with similar changes, but in diseased AVICs, NOTCH LOF combined with OSS was associated with increased α-SMA expression. Interestingly, AVICs showed relatively higher expression of NOTCH2 compared to NOTCH1. Biomechanical interactions between endothelial and interstitial cells involve complex NOTCH signaling that contributes to matrix homeostasis in health and disorganization in disease.

Aortic valve disease (AVD) and aortopathy are associated with substantial morbidity and mortality, representing a significant cardiovascular healthcare burden worldwide. These mechanobiological structures are morphogenetically related and function in unison from embryonic development through mature adult tissue homeostasis, serving both coordinated and distinct roles. In addition to sharing common developmental origins, diseases of the aortic valve and proximal thoracic aorta often present together clinically. Current research efforts are focused on identifying etiologic factors and elucidating pathogenesis, including genetic predisposition, maladaptive cell-matrix remodeling processes, and hemodynamic and biomechanical perturbations. Here, we review the impact of these processes as they pertain to translational research efforts, emphasizing the overlapping relationship of these two disease processes. The successful application of new therapeutic strategies and novel tissue bioprostheses for AVD and/or aortopathy will require an understanding and integration of molecular and biomechanical processes for both diseases.

Aortopathy is characterized by vascular smooth muscle cell (VSMC) abnormalities and elastic fiber fragmentation. Elastin insufficient (Eln +/− ) mice demonstrate latent aortopathy similar to human disease. We hypothesized that aortopathy manifests primarily in the aorto-pulmonary septal (APS) side of the thoracic aorta due to asymmetric cardiac neural crest (CNC) distribution. Anatomic (aortic root vs. ascending aorta) and molecular (APS vs. non-APS) regions of proximal aorta tissue were examined in adult and aged wild type (WT) and mutant (Eln +/− ) mice. CNC, VSMCs, elastic fiber architecture, proteoglycan expression, morphometrics and biomechanical properties were examined using histology, 3D reconstruction, micropipette aspiration and in vivo magnetic resonance imaging (MRI). In the APS side of Eln +/− aorta, Sonic Hedgehog (SHH) is decreased while SM22 is increased. Elastic fiber architecture abnormalities are present in the Eln +/− aortic root and APS ascending aorta, and biglycan is increased in the aortic root while aggrecan is increased in the APS aorta. The Eln +/− ascending aorta is stiffer than the aortic root, the APS side is thicker and stiffer than the non-APS side, and significant differences in the individual aortic root sinuses are observed. Asymmetric structure–function abnormalities implicate regional CNC dysregulation in the development and progression of aortopathy.

Diabetes-induced cardiomyopathy is characterized by cardiac remodeling, fibrosis, and endothelial dysfunction, with no treatment options currently available. Hyperglycemic memory by endothelial cells may play the key role in microvascular complications in diabetes, providing a potential target for therapeutic approaches. This study tested the hypothesis that a proangiogenic environment can augment diabetes-induced deficiencies in endothelial cell angiogenic and biomechanical responses. Endothelial responses were quantified for two models of diabetic conditions: 1) an in vitro acute and chronic hyperglycemia where normal cardiac endothelial cells were exposed to high-glucose media, and 2) an in vivo chronic diabetes model where the cells were isolated from rats with type I streptozotocin-induced diabetes. Capillary morphogenesis, VEGF and nitric oxide expression, cell morphology, orientation, proliferation, and apoptosis were determined for cells cultured on Matrigel or proangiogenic nanofiber hydrogel. The effects of biomechanical stimulation were assessed following cell exposure to uniaxial strain. The results demonstrate that diabetes alters cardiac endothelium angiogenic response, with differential effects of acute and chronic exposure to high-glucose conditions, consistent with the concept that endothelial cells may have a long-term “hyperglycemic memory” of the physiological environment in the body. Furthermore, endothelial cell exposure to strain significantly diminishes their angiogenic potential following strain application. Both diabetes and strain-associated deficiencies can be augmented in the proangiogenic nanofiber microenvironment. These findings may contribute to the development of novel approaches to reverse hyperglycemic memory of endothelium and enhance vascularization of the diabetic heart, where improved angiogenic and biomechanical responses can be the key factor to successful therapy.

Cellular mechanotransduction is an integral part of many crucial physiological processes, but non-invasive tools for quantifying intracellular strain in vivo are not available for complex tissues such as bone. As a first step to address this gap, we have utilized a novel, non-invasive approach to quantify cellular strain in vitro by employing a transfected alpha-actinin Forster Resonance Energy Transfer (FRET) sensor. Following validation experiments, mouse fibroblasts transfected to express FRET sensors were seeded to a silicone membrane and subjected to up to 10% tensile strain mounted on a multi-photon microscope. During tensile strain, fluorescent emission of acceptor (YFP) and donor (CFP) proteins was quantified. YFP/CFP ratio was normalized to the initial baseline (unstretched) ratio for each cell which demonstrates a negative linear correlation between the relative proximity ratio of emission spectra and cell strain, with a mean decrease of 1.017% normalized ratio for every percent strain experienced by the cell. The exciting implications of our findings are that the discovery of the stable correlation between loss of FRET and experimentally applied strain opens intriguing possibilities for future use of this technology with in vivo research, leading to discoveries improving disease treatments in mechanically sensitive tissues such as bone.

This paper presents a novel numerical method for simulating the fluid---structure interaction (FSI) problems when blood flows over aortic valves. The method uses the immersed boundary/element method and the smoothed finite element method and hence it is termed as IS-FEM. The IS-FEM is a partitioned approach and does not need a body-fitted mesh for FSI simulations. It consists of three main modules: the fluid solver, the solid solver and the FSI force solver. In this work, the blood is modeled as incompressible viscous flow and solved using the characteristic-based-split scheme with FEM for spacial discretization. The leaflets of the aortic valve are modeled as Mooney-Rivlin hyperelastic materials and solved using smoothed finite element method (or S-FEM). The FSI force is calculated on the Lagrangian fictitious fluid mesh that is identical to the moving solid mesh. The octree search and neighbor-to-neighbor schemes are used to detect efficiently the FSI pairs of fluid and solid cells. As an example, a 3D idealized model of aortic valve is modeled, and the opening process of the valve is simulated using the proposed IS-FEM. Numerical results indicate that the IS-FEM can serve as an efficient tool in the study of aortic valve dynamics to reveal the details of stresses in the aortic valves, the flow velocities in the blood, and the shear forces on the interfaces. This tool can also be applied to animal models studying disease processes and may ultimately translate to a new adaptive methods working with magnetic resonance images, leading to improvements on diagnostic and prognostic paradigms, as well as surgical planning, in the care of patients.

Aortic valve disease (AVD) occurs in 2.5% of the general population and often requires surgical intervention. Aortic valve malformation (AVM) underlies the majority of cases, suggesting a developmental etiology. Elastin haploinsufficiency results in complex cardiovascular problems, and 20-45% of patients have AVM and/or AVD. Elastin insufficient (Eln+/-) mice demonstrate AVM and latent AVD due to abnormalities in the valve annulus region. The objective of this study was to examine extracellular matrix (ECM) remodeling and biomechanical properties in regional aortic valve tissue and determine the impact of early AVM on late AVD in the Eln+/- mouse model. Aortic valve ECM composition and remodeling from juvenile, adult, and aged stages were evaluated in Eln+/- mice using histology, ELISA, immunohistochemistry and gelatin zymography. Aortic valve tissue biomechanical properties were determined using micropipette aspiration. Cartilage-like nodules were demonstrated within the valve annulus region at all stages identifying a developmental abnormality preceding AVD. Interestingly, maladaptive ECM remodeling was observed in early AVM without AVD and worsened with late AVD, as evidenced by increased MMP-2 and MMP-9 expression and activity, as well as abnormalities in ADAMTS-mediated versican processing. Cleaved versican was increased in the valve annulus region of aged Eln+/- mice, and this abnormality correlated temporally with adverse alterations in valve tissue biomechanical properties and the manifestation of AVD. These findings identify maladaptive ECM remodeling in functional AVM as an early disease process with a progressive natural history, similar to that seen in human AVD, emphasizing the importance of the annulus region in pathogenesis. Combining molecular and engineering approaches provides complementary mechanistic insights that may be informative in the search for new therapeutic targets and durable valve bioprostheses.

Overcoming the effects of hyperglycemic conditions on matrix remodeling by cardiac fibroblasts represents a novel therapeutic approach to stimulate regeneration of cardiac tissue in the context of diabetes. The purpose of this study was to investigate the effects of diabetic conditions on the extracellular matrix (ECM) protein expression and biomechanical response of cardiac fibroblasts. Two types of diabetic conditions were used: in vivo using rat model of diabetic cardiomyopathy (DCM), or in vitro using high glucose culture medium. A commercially available Flexcell system was used to apply uniaxial stretch to rat cardiac fibroblasts, and cell response was assessed, including MMP-2 (ECM remodeling), collagen I (ECM deposition), and vascular endothelial growth factor (cell migration stimulus) expression as well as fibroblast morphology, orientation, proliferation and apoptosis. The results demonstrate differential cell responses to the two models of diabetic condition. Cardiac fibroblasts from the in vivo rat model of DCM exhibited an overall reduced synthetic activity and proliferation, while high glucose conditions lead to apparent increases in cell responses. In the in vivo rat model, exposure to cyclic strain appeared to act toward restoration of cell proliferation and collagen I expression that were decreased with diabetes.

Inherent pathologies associated with diabetic wound microenvironment including increased proteolysis, inflammatory dysregulation, and impaired neovascularization prevent timely resolution of chronic diabetic ulcers. It is hypothesized that augmentation of local wound microenvironment with a stable provisional matrix formed by proteolysis-resistant angiogenic peptide nanofibers (NFs) will create permissive environment for attenuated inflammation, enhanced neovascularization, and improved diabetic wound healing. Using murine excisional wound healing models, full-thickness dorsal skin wounds were treated with either NFs or control solutions (phosphate buffered saline; hyaluronic acid) and analyzed for morphology, inflammatory response, neovascularization, and biomechanical properties. NF treatment of diabetic wounds stimulated formation of a robust pro-angiogenic in situ tissue-engineered provisional matrix leading to a significant decrease in wound inflammatory cell infiltration and proinflammatory interleukin-6 levels, a significant increase in endothelial and endothelial progenitor cell infiltration, vascular endothelial growth factor levels, and neovascularization (day 7), as well as improved wound morphology, accelerated wound closure, and significantly stronger repair tissue (day 28). These results suggest that appropriate design of provisional matrix may compensate for some of the complex disruptions in diabetic wound microenvironment and provide missing cues to cells and direct in situ responses toward improved healing, which is promising for future development of new therapies for diabetic ulcers.

Objective This study aimed to use magnetoencephalography (MEG) to examine the question of whether Mandarin-English bilingual speakers recruit the same cortical areas or develop distinct language-specific networks without overlaps for word processing. Methods Eight healthy Mandarin-English bilingual adults and eight healthy English monolingual adults were scanned while single-word paradigms were audio-visually presented. Results Our results showed significantly stronger beta-band power suppression in the right inferior parietal lobe (IPL) covering the supramarginal gyrus (BA 40) and angular gyrus (BA 39) for bilinguals when processing Mandarin versus English. Moreover, there were no significant differences between bilinguals and monolinguals in the left inferior frontal cortex (LIFC, BA 44/45) when both were processing their first language. Conclusions These results support the view that Mandarin-English bilinguals have a shared neural system for word processing in both the first and second language, which is highly similar to monolinguals’, but with stronger right hemisphere involvement. Significance To our knowledge, this is the first MEG study to investigate the spatio-temporal and frequency characteristics between bilinguals and monolinguals, which provides us a new angle to better understand the language system in bilinguals’ and monolingual’s brain.

Reduced mobilization of endothelial progenitor cells (EPCs) from the bone marrow (BM) and impaired EPC recruitment into the wound represent a fundamental deficiency in the chronic ulcers. However, mechanistic understanding of the role of BM-derived EPCs in cutaneous wound neovascularization and healing remains incomplete, which impedes development of EPC-based wound healing therapies. The objective of this study was to determine the role of EPCs in wound neovascularization and healing both under normal conditions and using single deficiency (EPC) or double-deficiency (EPC + diabetes) models of wound healing. MMP9 knockout (MMP9 KO) mouse model was utilized, where impaired EPC mobilization can be rescued by stem cell factor (SCF). The hypotheses were: (1) MMP9 KO mice exhibit impaired wound neovascularization and healing, which are further exacerbated with diabetes; (2) these impairments can be rescued by SCF administration. Full-thickness excisional wounds with silicone splints to minimize contraction were created on MMP9 KO mice with/without streptozotocin-induced diabetes in the presence or absence of tail-vein injected SCF. Wound morphology, vascularization, inflammation, and EPC mobilization and recruitment were quantified at day 7 postwounding. Results demonstrate no difference in wound closure and granulation tissue area between any groups. MMP9 deficiency significantly impairs wound neovascularization, increases inflammation, decreases collagen deposition, and decreases peripheral blood EPC (pb-EPC) counts when compared with wild-type (WT). Diabetes further increases inflammation, but does not cause further impairment in vascularization, as compared with MMP9 KO group. SCF improves neovascularization and increases EPCs to WT levels (both nondiabetic and diabetic MMP9 KO groups), while exacerbating inflammation in all groups. SCF rescues EPC-deficiency and impaired wound neovascularization in both diabetic and nondiabetic MMP9 KO mice. Overall, the results demonstrate that BM-derived EPCs play a significant role during wound neovascularization and that the SCF-based therapy with controlled inflammation could be a viable approach to enhance healing in chronic diabetic wounds.

Regulation of cell function by a non-thermal, physiological-level electromagnetic field has potential for vascular tissue healing therapies and advancing hybrid bioelectronic technology. We have recently demonstrated that a physiological electric field (EF) applied wirelessly can regulate intracellular signalling and cell function in a frequency-dependent manner. However, the mechanism for such regulation is not well understood. Here, we present a systematic numerical study of a cell-field interaction following cell exposure to the external EF. We use a realistic experimental environment that also recapitulates the absence of a direct electric contact between the field-sourcing electrodes and the cells or the culture medium. We identify characteristic regimes and present their classification with respect to frequency, location, and the electrical properties of the model components. The results show a striking difference in the frequency dependence of EF penetration and cell response between cells suspended in an electrolyte and cells attached to a substrate. The EF structure in the cell is strongly inhomogeneous and is sensitive to the physical properties of the cell and its environment. These findings provide insight into the mechanisms for frequency-dependent cell responses to EF that regulate cell function, which may have important implications for EF-based therapies and biotechnology development.

Prosthetic valves currently used in children lack the ability to grow with the patient and often require multiple reoperations. Small intestinal submucosa-derived extracellular matrix (SIS-ECM) has been used successfully as a patch for repair in various tissues, including vessels, valves, and myocardium.This study sought to assess the remodeling potential of a tubular tricuspid valve (TV) bioprosthesis made of SIS-ECM by evaluating its growth, structure, and function in a growing ovine model.A total of 12 3-month-old lambs were studied for a period of 3 or 8 months. SIS-ECM TVs were placed in 8 lambs; conventional bioprosthetic valves and native valves (NV) were studied as controls. All lambs underwent serial echocardiography, measuring annulus diameter and valve and right ventricular function.The SIS-ECM valves demonstrated an incremental increase in annular diameter similar to NV. SIS-ECM valve function was normal in 7 of 8; 1 valve had severe regurgitation due to a flail leaflet. Explanted SIS-ECM valves approximated native tissue in gross appearance. Histopathology demonstrated migration of resident mesenchymal cells into the scaffold and trilaminar ECM organization similar to an NV, without inflammation or calcification at 8 months. Ex vivo mechanical testing of SIS-ECM valve tissue showed normalization of the elastic modulus by 8 months.In an ovine model, tubular SIS-ECM TV bioprostheses demonstrate "growth" and a cell-matrix structure similar to mature NVs while maintaining normal valve function. The SIS-ECM valve may provide a novel solution for TV replacement in children and adults.

Regulatory promise of electric field (EF) as a non-pharmacological, non-invasive tool to control cellular functions is of great therapeutic interest. However, biophysical mechanisms for the cell-EF interactions are not understood. We developed a theoretical-experimental approach to investigate EF effects on cells in electrode-free physiologically-relevant configuration, i.e. with cells attached to a substrate. Cell is modeled as a membrane-enclosed hemisphere with realistic parameters. Our numerical results demonstrated that EF frequency is the major parameter that controls the mechanism of EF interactions with cells in realistic environment. Non-oscillating or low-frequency EF leads to charge accumulation on the cell surface membrane and results in field screening from the cytoplasm, suggesting that in this regime, cell responses are regulated by EF interactions with the surface membrane receptors. In contrast, high-frequency EF penetrates the cell membrane and reaches cell cytoplasm, where it may directly activate intracellular responses. Theoretical simulation predicts the non-uniform distribution of the induced field within the cell membrane, which depends on the applied EF frequency. Importantly, substrate properties significantly affect both magnitude and distribution of the induced field on the cell membrane, underscoring the need for a comprehensive, physiologically-relevant modeling approach for EF-cell interactions. These theoretical predictions were confirmed in our experimental studies of the effects of applied EF on responses of vascular cells. Results show that non-oscillating EF increases vascular endothelial growth factor (VEGF) expression while field polarity controls cell adhesion rate. High-frequency, but not low-frequency, EF provides differential regulation of cytoplasmic focal adhesion kinase and VEGF expression depending on the substrate, with increased expression in cells cultured on RGD-rich synthetic hydrogels, and decreased expression for basement membrane (matrigel) culture. These results advance our understanding of complex mechanisms underlying cell-EF interactions and may contribute to future EF-based therapies.

Objective: The effect of chronic hyperglycemic exposure on endothelial cell (EC) phenotype, impaired wound neovascularization, and healing is not completely understood. The hypotheses are: 1) chronic exposure to diabetic conditions in vivo impairs the angiogenic potential of ECs and 2) this deficiency can be improved by an extracellular microenvironment of angiogenic peptide nanofibers. Approach: Angiogenic potential of microvascular ECs isolated from diabetic (db/db) and wild type (wt) mice was assessed by quantifying migration, proliferation, apoptosis, capillary morphogenesis, and vascular endothelial growth factor (VEGF) expression for cell cultures on Matrigel (Millipore, Billerica, MA) or nanofibers under normoglycemic conditions. The in vivo effects of nanofiber treatment on wound vascularization were determined using two mouse models of diabetic wound healing. Results: Diabetic ECs showed significant impairments in migration, VEGF expression, and capillary morphogenesis. The nanofiber microenviron...

Because an adequate blood supply to and within tissues is an essential factor for successful tissue regeneration, promoting a functional microvasculature is a crucial factor for biomaterials. In this study, we demonstrate that short self-assembling peptides form scaffolds that provide an angiogenic environment promoting long-term cell survival and capillary-like network formation in three-dimensional cultures of human microvascular endothelial cells. Our data show that, in contrast to collagen type I, the peptide scaffold inhibits endothelial cell apoptosis in the absence of added angiogenic factors, accompanied by enhanced gene expression of the angiogenic factor VEGF. In addition, our results suggest that the process of capillary-like network formation and the size and spatial organization of cell networks may be controlled through manipulation of the scaffold properties, with a more rigid scaffold promoting extended structures with a larger inter-structure distance, as compared with more dense structures of smaller size observed in a more compliant scaffold. These findings indicate that self-assembling peptide scaffolds have potential for engineering vascularized tissues with control over angiogenic processes. Since these peptides can be modified in many ways, they may be uniquely valuable in regeneration of vascularized tissues.

Periods of reduced joint loading have been shown to induce changes in the biochemical composition, metabolism and mechanics of articular cartilage. In this study, changes in cartilage swelling behavior were studied following a 4-week period of joint immobilization, using a recently developed osmotic loading technique [J. Biomech. 32 (1999) 401–408]. The magnitude and distribution of swelling strains were measured in cartilage–bone samples equilibrated in physiological and hypotonic saline, relative to a hypertonic reference NaCl solution. Physicochemical parameters (glycosaminoglycan fixed charge density and water volume fraction) were determined in site-matched cartilage samples. The experimental data for swelling strains, fixed charge density and water volume fraction were used with a triphasic mechano-chemical theory [J. Biomech. Eng. 113 (1991) 245–258] to determine the effect of joint immobilization on the tensile modulus of the cartilage solid matrix. Four weeks of immobilization resulted in a significant increase in the magnitude of swelling-induced strains, and a significant decrease in fixed charge density in cartilage, as compared with the contralateral controls. Joint immobilization also resulted in decreases in values for the modulus of cartilage, as compared with the contralateral controls. Our results suggest that 4 weeks of joint immobilization had a significant effect on cartilage mechanical function that may be linked to collagen changes in the cartilage extracellular matrix.

Articular cartilage is one of several biological tissues in which swelling effects are important in tissue mechanics and function, and may serve as an indicator of degenerative joint disease. This work presents a new approach to quantify swelling effects in articular cartilage, as well as to determine the material properties of cartilage from a simple free-swelling test. Samples of nondegenerate and degenerate human patellar cartilage were subjected to osmotic loading by equilibrating the tissue in solutions of varying osmolarity. The resulting swelling-induced strains were measured using a noncontacting optical method. A theoretical formulation of articular cartilage in a free-swelling configuration was developed based on an inhomogeneous, triphasic mechano-chemical model. Optimization of the model predictions to the experimental data was performed to determine two parameters descriptive of material stiffness at the surface and deeper cartilage layers, and a third parameter descriptive of thickness of the cartilage surface layer. These parameters were used to determine the thickness-averaged uniaxial modulus of cartilage, H A . The obtained values for H A were similar to those for the tensile modulus of human cartilage reported in the literature. Degeneration resulted in an increase in thickness of the region of "apparent cartilage softening," and a decrease in the value for uniaxial modulus at this layer. These findings provide important evidence that collagen matrix disruption starts at the articular surface and progresses into the deeper layers with continued degeneration. These results suggest that the method provides a means to quantify the severity and depth of degenerative changes in articular cartilage. This method may also be used to determine material properties of cartilage in small joints in which conventional testing methods are difficult to apply.

Swelling effects in cartilage originate from an interstitial osmotic pressure generated by the presence of negatively charged proteoglycans in the tissue. This swelling pressure gives rise to a non-zero residual strain in the cartilage solid matrix in the absence of externally applied loads. Previous studies have quantified swelling effects in cartilage as volumetric or dimensional change of excised samples in varying osmotically active solutions. This study presents a new optical technique for measuring two-dimensional swelling-induced residual strain fields in planar samples of articular cartilage attached to the bone (i.e., in situ). Osmotic loading was applied to canine cartilage–bone samples by equilibration in external baths of varying NaCl concentration. Non-zero swellinginduced strains were measured in physiological saline, giving evidence of the existence of residual strains in articular cartilage. Only one component of planar strain (i.e., in thickness direction) was found to be non-zero. This strain was found to be highly non-uniform in the thickness direction, with evidence of compressive strain in the deep zone of cartilage and tensile strain in the middle and surface zones. The obtained results can be used to characterize the material properties of the articular cartilage solid matrix, with estimated values of 26 MPa for the tensile modulus for middle zone cartilage. The method provides the basis to obtain material properties of the cartilage solid matrix from a simple, free-swelling test and may be useful for quantifying changes in cartilage properties with injury, degeneration and repair.

Introduction Heart valve disease affects over 2% of the population, leading to at least 20,000 deaths and $1 billion in direct costs in the US each year. Calcific aortic valve disease (CAVD) is linked to loss of function mutations in NOTCH1 that result in osteogenic gene induction. In vitro animal studies have established that multiple osteogenic targets are mediated through induction of p-ERK 1/2 signaling; however, the relationship between NOTCH1 and p-ERK has not been examined in primary human aortic valvar interstitial cells (AVICs). Hypothesis We tested the hypothesis that chemically-induced NOTCH1 suppression results in mineralization due to p-ERK 1/2 activation in human AVICs. Methods Human AVICs were isolated from CAVD specimens at the time of valve replacement. AVICs were cultured for two weeks in complete media containing DMEM and 10% FBS, and treated with calcific media (10mM b-glycerol phosphate; 60μM ascorbic acid-2-phosphate; 1μM dexamethasone), which has been shown to suppress NOTCH1 signaling. Standard real-time RT-PCR, western blot, and immunocytochemistry were used to compare groups. Results Treatment with calcific media resulted in a 2.3 fold decrease in the level of NOTCH1 expression and a 3 fold increase in p-ERK 1/2 expression, assessed with both western blots and immunocytochemistry. Interestingly, calcific media also induced a 3.5 fold decrease in VEGF, consistent with early osteogenic differentiation. Additionally, the AVICs treated with calcific media demonstrated overt calcification by Alizarin Red staining. Conclusions Human AVICs demonstrated calcification associated with p-ERK 1/2 activation and reduced NOTCH1 levels, suggesting p-ERK inhibition may mitigate the effects of NOTCH loss of function, thereby representing a potential pharmacologic therapy for CAVD. Further investigation of the molecular mechanisms linking NOTCH1 and p-ERK are warranted, including studies that specifically inhibit NOTCH1 signaling and define the impact of mechanical forces on molecular pathways, thereby identifying mechanisms underlying CAVD progression.

Diabetic cardiomyopathy (DCM) is a diabetic complication, which results in myocardial dysfunction independent of other etiological factors. Abnormal intracellular calcium ([Ca(2+)](i)) homeostasis has been implicated in DCM and may precede clinical manifestation. Studies in cardiomyocytes have shown that diabetes results in impaired [Ca(2+)](i) homeostasis due to altered sarcoplasmic reticulum Ca(2+) ATPase (SERCA) and sodium-calcium exchanger (NCX) activity. Importantly, altered calcium homeostasis may also be involved in diabetes-associated endothelial dysfunction, including impaired endothelium-dependent relaxation and a diminished capacity to generate nitric oxide (NO), elevated cell adhesion molecules, and decreased angiogenic growth factors. However, the effect of diabetes on Ca(2+) regulatory mechanisms in cardiac endothelial cells (CECs) remains unknown. The objective of this study was to determine the effect of diabetes on [Ca(2+)](i) homeostasis in CECs in the rat model (streptozotocin-induced) of DCM. DCM-associated cardiac fibrosis was confirmed using picrosirius red staining of the myocardium. CECs isolated from the myocardium of diabetic and wild-type rats were loaded with Fura-2, and UTP-evoked [Ca(2+)](i) transients were compared under various combinations of SERCA, sarcoplasmic reticulum Ca(2+) ATPase (PMCA) and NCX inhibitors. Diabetes resulted in significant alterations in SERCA and NCX activities in CECs during [Ca(2+)](i) sequestration and efflux, respectively, while no difference in PMCA activity between diabetic and wild-type cells was observed. These results improve our understanding of how diabetes affects calcium regulation in CECs, and may contribute to the development of new therapies for DCM treatment.

VX2 rabbit liver cancer, treated in vivo using bulk ultrasound ablation by miniaturized image-ablate arrays, was histologically analyzed using TTC vital stain and DAPI nucleic acid stain. VX2 cells were implanted into rabbit liver lobes and allowed to grow for 11-21 days. Liver lobes containing solid VX2 tumors were then treated with 4.8 MHz, 22.5-38.5 W/cm2 in situ intensity, unfocused ultrasound for exposure times of 20-120 s. After animal sacrifice, thermal lesions were bisected along the imaging/treatment plane, one face stained with TTC, and the other with DAPI. Levels of TTC uptake (no uptake, partial uptake, and complete uptake) in liver parenchyma corresponded to three discrete regions of tan, pink and red color. By processing images of DAPI-stained parenchymal tissue from these three regions, cellular damage was quantified. A viability index parameter incorporating the size and shape of DAPI-stained nuclei correlated significantly with levels of TTC uptake, and thus with local tissue viability. F...

Diabetes is a serious problem in the United States, afflicting 7.8% of the population with annual medical costs estimated at $116B in 2007 (1). Diabetic cardiomyopathy (DCM) is a cardiovascular complication of diabetes resulting in pathological alterations to the myocardium including circulatory defects, impaired heart muscle contraction, and progressive fibrosis. Cardiac fibrosis is associated with an imbalance between the deposition of the extracellular matrix (ECM) proteins by cardiac fibroblasts and the ECM proteolytic degradation via matrix metalloproteinases (MMPs). Recent studies have demonstrated that in the diabetic heart, expression and activity of MMP-2 are reduced, resulting in increased collagen accumulation and cardiac dysfunction (2). These observations suggest that a MMP-related mechanism may contribute to cardiac fibrosis, and that it may be attenuated through stimulation of native MMP-2 expression or delivery of exogenous MMP-2. Therefore, reduced MMP-2 activity in DCM may represent a novel target for therapeutic treatment (3). To achieve this, a special proteolytically-stable delivery scaffold would be needed, because native ECM is rapidly degraded by MMPs. The goal of this study is to determine if self-assembling peptide nanofibers can be used for long-term (several weeks) MMP delivery and enhancement of cardiac remodeling. This study tests the hypothesis that increased MMP-2 concentration (native or exogenous) in the nanofiber environment will promote matrix remodeling in diabetic cardiac fibroblasts in vitro.Copyright © 2011 by ASME

Diabetic cardiomyopathy (DCM) is a serious diabetes-associated complication that results in myocardial dysfunction independent of other etiological factors [1]. Pathological alterations to the myocardium associated with DCM include circulatory defects, impaired heart muscle contraction, and abnormal calcium (Ca2+) homeostasis in cardiac cells[2]. In myocardium, endothelial cells play an essential role in maintaining intracellular Ca2+ hemostasis in response to stimuli and regulating cardiac function [3]. External stimulus may cause abrupt changes in Ca2+ balance, including Ca2+ release from sarco-endoplasmic reticulum (ER) [4]. Subsequent return of the Ca2+ level to basal levels occurs due to Ca2+ decay mechanism, which is mainly regulated by sarco-endoplasmic reticulum Ca2+ ATPase pumps (SERCA) present at ER membrane which are responsible for Ca2+ sequestration [5]. Studies have shown that the mechanisms by which Ca2+ homeostasis alters cardiac function in diabetic cardiomyocytes include reduced activity of the SERCA pumps [6]. However, no information is available regarding the effects of diabetes on Ca2+ hemostasis and the underlying Ca2+ sequestration mechanism in diabetic cardiac endothelial cells[7]. This study tested the hypothesis that diabetic endothelial cells will exhibit disruptions in Ca2+ decay kinetics via alterations in the sequestration mechanism.Copyright © 2011 by ASME

RAD16-II peptide nanofibers are promising for vascular tissue engineering and were shown to enhance angiogenesis in vitro and in vivo, although the mechanism remains unknown. We hypothesized that the pro-angiogenic effect of RAD16-II results from low-affinity integrin-dependent interactions of microvascular endothelial cells (MVECs) with RAD motifs. Mouse MVECs were cultured on RAD16-II with or without integrin and MAPK/ERK pathway inhibitors, and angiogenic responses were quantified. The results were validated in vivo using a mouse diabetic wound healing model with impaired neovascularization. RAD16-II stimulated spontaneous capillary morphogenesis, and increased β(3) integrin phosphorylation and VEGF expression in MVECs. These responses were abrogated in the presence of β(3) and MAPK/ERK pathway inhibitors or on the control peptide without RAD motifs. Wide-spectrum integrin inhibitor echistatin completely abolished RAD16-II-mediated capillary morphogenesis in vitro and neovascularization and VEGF expression in the wound in vivo. The addition of the RGD motif to RAD16-II did not change nanofiber architecture or mechanical properties, but resulted in significant decrease in capillary morphogenesis. Overall, these results suggest that low-affinity non-specific interactions between cells and RAD motifs can trigger angiogenic responses via phosphorylation of β(3) integrin and MAPK/ERK pathway, indicating that low-affinity sequences can be used to functionalize biocompatible materials for the regulation of cell migration and angiogenesis, thus expanding the current pool of available motifs that can be used for such functionalization. Incorporation of RAD or similar motifs into protein engineered or hybrid peptide scaffolds may represent a novel strategy for vascular tissue engineering and will further enhance design opportunities for new scaffold materials.

Inherent pathologies associated with diabetic wound microenvironment including increased proteolysis, inflammatory dysregulation, and impaired neovascularization prevent timely resolution of chronic diabetic ulcers. It is hypothesized that augmentation of local wound microenvironment with a stable provisional matrix formed by proteolysis-resistant angiogenic peptide nanofibers (NFs) will create permissive environment for attenuated inflammation, enhanced neovascularization, and improved diabetic wound healing. Using murine excisional wound healing models, full-thickness dorsal skin wounds were treated with either NFs or control solutions (phosphate buffered saline; hyaluronic acid) and analyzed for morphology, inflammatory response, neovascularization, and biomechanical properties. NF treatment of diabetic wounds stimulated formation of a robust pro-angiogenic in situ tissue-engineered provisional matrix leading to a significant decrease in wound inflammatory cell infiltration and proinflammatory interleukin-6 levels, a significant increase in endothelial and endothelial progenitor cell infiltration, vascular endothelial growth factor levels, and neovascularization (day 7), as well as improved wound morphology, accelerated wound closure, and significantly stronger repair tissue (day 28). These results suggest that appropriate design of provisional matrix may compensate for some of the complex disruptions in diabetic wound microenvironment and provide missing cues to cells and direct in situ responses toward improved healing, which is promising for future development of new therapies for diabetic ulcers.

Rationale: Elastin is a ubiquitous extracellular matrix protein that is highly organized in heart valves and arteries. Because elastic fiber abnormalities are a central feature of degenerative valve disease, we hypothesized that elastin-insufficient mice would manifest viable heart valve disease. Objective: To analyze valve structure and function in elastin-insufficient mice ( Eln +/− ) at neonatal, juvenile, adult, and aged adult stages. Methods and Results: At birth, histochemical analysis demonstrated normal extracellular matrix organization in contrast to the aorta. However, at juvenile and adult stages, thin elongated valves with extracellular matrix disorganization, including elastin fragment infiltration of the annulus, were observed. The valve phenotype worsened by the aged adult stage with overgrowth and proteoglycan replacement of the valve annulus. The progressive nature of elastin insufficiency was also shown by aortic mechanical testing that demonstrated incrementally abnormal tensile stiffness from juvenile to adult stages. Eln +/− mice demonstrated increased valve interstitial cell proliferation at the neonatal stage and varied valve interstitial cell activation at early and late stages. Gene expression profile analysis identified decreased transforming growth factor-β–mediated fibrogenesis signaling in Eln +/− valve tissue. Juvenile Eln +/− mice demonstrated normal valve function, but progressive valve disease (predominantly aortic regurgitation) was identified in 17% of adult and 70% of aged adult Eln +/− mice by echocardiography. Conclusions: These results identify the Eln +/− mouse as a model of latent aortic valve disease and establish a role for elastin dysregulation in valve pathogenesis.

Interactions between endothelial and stromal cells are important for vascularization of regenerating tissue. Fibroblasts (FBs) are responsible for expression of angiogenic growth factors and matrix metalloproteinases, as well as collagen deposition and fibrotic myocardial remodeling. Recently, self-assembling peptide nanofibers were described as a promising environment for cardiac regeneration due to its synthetic nature and control over physiochemical properties. In this study, peptide nanofibers were used as a model system to quantify the dual role of fibroblasts in mediating angiogenesis chemically via expression of angiogenic factors and mechanically via cell-mediated scaffold disruption, extracellular matrix deposition, and remodeling. Human microvascular endothelial cells (ECs), FBs, or cocultures were cultured in three-dimensional nanofibers for up to 6 days. The peptide nanofiber microenvironment supported cell migration, capillary network formation, and cell survival in the absence of detectable scaffold contraction and proteolytic degradation. FBs enhanced early capillary network formation by “assisting” EC migration and increasing vascular endothelial growth factor and Angiopoietin-1 expression in a temporal manner. EC-FB interactions attenuated FB matrix metalloproteinase-2 expression while increasing collagen I deposition, resulting in greater construct stiffness and a more stable microenvironment in cocultures. Whereas FBs are critical for initial steps of angiogenesis in the absence of external angiogenic stimulation, coordinated efforts by ECs and FBs are required for a balance between cell-mediated scaffold disruption, extracellular matrix deposition, and remodeling at later time points. The findings of this study also emphasize the importance of developing a microenvironment that supports cell-cell interactions and cell migration, thus contributing toward an optimal environment for successful cardiac regeneration strategies.

Site-specific biomechanical properties of the aortic valve play an important role in native valve function, and alterations in these properties may reflect mechanisms of degeneration and disease. Small animals such as targeted mutagenesis mice provide a powerful approach to model human valve disease pathogenesis; however, physical mechanical testing in small animals is limited by valve tissue size. Aortic valves are comprised of highly organized extracellular matrix compartmentalized in cusp and annulus regions, which have different functions. The objective of this study was to measure regional mechanical properties of mouse aortic valve tissue using a modified micropipette aspiration technique. Aortic valves were isolated from juvenile, adult and aged adult C57BL/6 wild type mice. Tissue tensile stiffness was determined for annulus and cusp regions using a half-space punch model. Stiffness for the annulus region was significantly higher compared to the cusp region at all stages. Further, aged adult valve tissue had decreased stiffness in both the cusp and annulus. Quantitative histochemical analysis revealed a collagen-rich annulus and a proteoglycan-rich cusp at all stages. In aged adult valves, there was proteoglycan infiltration of the annulus hinge, consistent with the observed mechanical differences over time. These findings indicate that valve tissue biomechanical properties vary in wild type mice in a region-specific and age-related manner. The micropipette aspiration technique provides a promising approach for studies of valve structure and function in small animal models, such as transgenic mouse models of valve disease.

Abnormal angiogenesis (formation of capillaries) plays an important role in the impaired diabetic wound healing and has emerged as a new target area for therapeutic interventions. Pulsed magnetic field therapy, which was initially used for healing of bone fractures, has been recently introduced as a potential therapy to treat diabetic and chronic wounds [1], although the mechanisms responsible for improved healing are still unclear. Electromagnetic fields (EMF) have been shown to act as a directional cues in cellular responses such as migration and activations of several signal transduction cascades [2]. Recent literature delineates an important role of GHz EMF (i.e., with an oscillation period of a fraction of a nanosecond) in inducing rapid and sustained phosphorylation of mitrogen-activated kinase and extracellular-signal-regulated kinase (MAPK/ERK) [3]. Recent studies have also implicated MAP kinase in mediating the phosphorylation of Connexin-43 (Cx43) that accompanies regulation of cell-cell communication via connexin gap junctions [4]. Importantly, both MAPK/ERK pathway and Cx43 signaling are involved in the process of angiogenesis [5,6]. Therefore, the goal of this study was to test the hypothesis that high-frequency (7.5GHz) EMFs promote angiogenesis in vitro via MAPK/ERK and/or Cx43 signaling. We used a custom-built EMF exposure setup and a self-assembling peptide nanoscaffold as a controlled angiogenic microenvironment [7] to quantify the effect of EMF on capillary formation and underlying cellular responses.Copyright © 2009 by ASME

Chronic ulcers are a leading cause of morbidity in diabetic patients. Diabetes is associated with major changes in the wound microenvironment and disruption of normal wound healing process, characterized by a prolonged inflammatory phase with elevated levels of wound proteases and increased degradation of extracellular matrix (ECM) components [1]. This impedes wound healing due to a lack of provisional matrix, impaired recruitment and survival of endothelial (EC) and endothelial precursor (EPC) cells, and insufficient neovascularization, resulting in delayed healing. Therefore, strategies focused on restoring the diabetic wound microenvironment by decreasing ECM degradation and promoting neovascularization are promising for development of new therapies to treat chronic diabetic ulcers.Copyright © 2009 by ASME

Revascularization is critical for successful regeneration of ischemic cardiac tissue after injury. To achieve revascularization in engineered cardiac grafts, it is necessary to understand the interactions between major cardiac cell types. The importance of cardiomyocyte-endothelial interactions in angiogenesis is well documented [1]; however, less is known about interactions between endothelial and stromal cells, fibroblasts in particular. Studies indicate that during capillary assembly, fibroblasts (FBs) provide chemical signaling via growth factor expression and endothelial activation and proliferation [2]. In addition, fibroblasts deposit extracellular matrix (ECM) proteins [3] and also play a role in matrix remodeling. The objective of our study was to further investigate the role of endothelial-fibroblast interactions in angiogenesis with a focus on FB regulation of the extracellular mechanical environment. We and others have recently shown that self-assembling peptide nanoscaffold is a promising material for cardiac tissue regeneration, enhancing angiogenesis in vitro and promoting tissue neovascularization in vivo [1, 4–5]. An important advantage of this nanoscaffold is the ability to control its material properties, such as stiffness and rate of MMP degradation, through its sequence and/or concentration [6]. In this study, RAD16-II peptide nanoscaffold was used as a controlled system to test the hypothesis that fibroblasts regulate angiogenesis via modifying the extracellular mechanical environment through mechanisms including cell-mediated scaffold disruption and matrix remodeling.Copyright © 2009 by ASME

Supravalvular aortic stenosis (SVAS) [1] is a disease of the cardiovascular system that leads to narrowing of the large arteries in humans. Studies have shown [2] that SVAS is caused by mutations or deletions in the elastin gene resulting in elastin haploinsufficiency. Elastin haploinsufficiency results in systemic hypertension [3], thinner and more numerous elastic lamellae [4], and altered arterial mechanics [5]. Genetically modified elastin deficient mice (ELN+/-) recapitulates the human phenotype including obstructive arterial disease and decreased arterial compliance [1,3]. Elastin deficiency in these mice is associated with changes in the mechanical microenvironment in the vascular wall [6], including enhanced wall thickness, increased smooth muscle cell (SMC) proliferation [7] and stiffening of arteries [8]. However, the molecular mechanisms for these changes are not fully understood. Also from a developmental perspective, no information is available regarding initiation and progression of aortic pathology in ELN+/− mice with time. The objectives of this study were to determine the temporal effects of elastin haploinsufficiency on the functional properties of aortic tissue and the aortic cell phenotype, using the elastin deficient mouse model (ELN+/-). We hypothesized that elastin haploinsufficiency will result in progressive abnormalities in aortic stiffness and dynamic alterations in aortic smooth muscle cell phenotype.Copyright © 2008 by ASME

Cardiac tissue engineering studies have demonstrated the importance of revascularization in engineered grafts for successful implantation and regeneration [1]. Understanding the myocardium’s complex cellular organization and the interactions between the major cardiac cell types (cardiomyocytes, endothelial cells, and cardiac fibroblasts) is critical for revascularization. Our previous studies have shown the importance of cardiomyocyte-endothelial interactions [2]. However, there is limited information available on endothelial-fibroblast interactions. We and others have previously observed that during capillary assembly, fibroblasts provide chemical signaling via expression of growth factors [3, 4]. In addition, fibroblasts may also regulate angiogenesis through alterations to the mechanical environment via myocardial remodeling, including matrix degradation and deposition, and tissue contraction. Changes to the extracellular mechanical enviroment may lead to changes in basic cell functions such as proliferation, apoptosis, and growth factor expression.Copyright © 2008 by ASME

Tissue engineering offers an attractive alternative for treatment of chronic nonhealing diabetic ulcers, which account for more than 27% of the $10.9 billion total diabetic health care costs in the US annually [1]. The harsh environment of a diabetic ulcer is characterized by reduced expression of angiogenic factors, insufficient vascularization, excess protease activity, matrix degradation and hyperglycemia-induced cell apoptosis [2]. A major factor contributing to insufficient neovascularization in diabetic nonhealing wounds may be deficiency in the recruitment of endothelial cells (ECs) and endothelial precursor cells (EPCs) to the wound site [3]. Recent studies focusing on altering the wound’s cellular and molecular environment using bone-marrow-derived stem cells, growth factors (delivered either directly or using gene or cell therapy), bioengineered skin constructs, and biological matrices, such as collagen and hyaluronic acid gels had promising wound healing outcomes [4]. These studies suggest that strategies aimed at modifying the extracellular environment of the diabetic wound to enhance cell survival and angiogenesis are promising for development of new therapies for diabetic wound healing.Copyright © 2008 by ASME

Mandibular hypoplasia is a debilitating pediatric condition caused by under-development of bone in young children. Current treatment options are expensive and morbid. The focus of our research is to elucidate the biophysical mechanisms of how mechanical strain can regulate bone growth via osteoblasts, and apply this knowledge to new therapies. As a first step, we have developed a novel non-invasive approach to quantify cellular strain in vivo by utilizing transfected tensile sensors in the form of Forster Resonance Electron Transfer (FRET) cassettes, and applied this tool to determine live cell responses to externally applied stretch. Stretching was controlled through a custom made device consisting of a micromanipulator and electric motor, mounted on the Nikon A1R multi-photon microscope stage. Adult mouse fibroblasts were transfected with FRET cassettes containing Venus (yellow) and Cerulean (blue) fluorescent proteins and an α-helix amino acid linker, followed by cell seeding onto a silicone membrane and stretching at 0.4% per minute. Cell images were recorded and analyzed to quantify stretch-induced change in FRET ratio (blue/yellow) and cell responses, including changes in cell morphology, dimensions and area. The results demonstrate a significant correlation between change in FRET emission ratio and cell stretch (R2=0.6). The trends shows that as strain increases from 0% to 3% in a cell, the ratio of acceptor(yellow)/donor(blue) emission linearly decreases by ∼50%, indicating a consistent loss in FRET efficiency and presenting a reliable non-invasive method for quantification of intracellular strain in live cells. Atomic force microscopy was utilized to confirm that the addition of a FRET cassette did not change the mechanical properties of the cell cytoskeleton. Overall, these results validate the potential of a FRET-based sensor for in vivo biomechanical cell studies.

Background— Promoting survival of transplanted cells or endogenous precursors is an important goal. We hypothesized that a novel approach to promote vascularization would be to create injectable microenvironments within the myocardium that recruit endothelial cells and promote their survival and organization. Methods and Results— In this study we demonstrate that self-assembling peptides can be injected and that the resulting nanofiber microenvironments are readily detectable within the myocardium. Furthermore, the self-assembling peptide nanofiber microenvironments recruit progenitor cells that express endothelial markers, as determined by staining with isolectin and for the endothelial-specific protein platelet–endothelial cell adhesion molecule-1. Vascular smooth muscle cells are recruited to the microenvironment and appear to form functional vascular structures. After the endothelial cell population, cells that express α-sarcomeric actin and the transcription factor Nkx2.5 infiltrate the peptide microenvironment. When exogenous donor green fluorescent protein–positive neonatal cardiomyocytes were injected with the self-assembling peptides, transplanted cardiomyocytes in the peptide microenvironment survived and also augmented endogenous cell recruitment. Conclusions— These experiments demonstrate that self-assembling peptides can create nanofiber microenvironments in the myocardium and that these microenvironments promote vascular cell recruitment. Because these peptide nanofibers may be modified in a variety of ways, this approach may enable injectable tissue regeneration strategies.