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ABSTRACT: During angiogenesis, endothelial cells (ECs) use both soluble and insoluble cues to expand the existing vascular network to meet changing trophic needs of the tissue. Fundamental to this expansion are physical interactions between ECs and extracellular matrix (ECM) that influence sprout migration, lumen formation, and stabilization. These physical interactions suggest that ECM mechanical properties may influence sprouting ECs and, therefore, angiogenic responses. In a 3D angiogenic model in which a monolayer of ECs is induced to invade an underlying collagen matrix, we measured angiogenic responses as a function of collagen matrix stiffness by inducing collagen crosslinking with microbial transglutaminase (mTG). With biaxial mechanical testing, we measured stiffer collagen matrices with both mTG treatment and incubation time. Using two-photon excited fluorescence (TPF) and second harmonic generation (SHG), we showed collagen TPF intensity increased with mTG treatment and the ratio of TPF/SHG correlated with biaxially tested mechanical stiffness. SHG and optical coherence microscopy (OCM) were further used to show that other ECM physical properties such as porosity and pore size did not change with mTG treatment, thus verifying matrix stiffness was tuned independently of matrix density. Our results showed that stiffer matrices promote more angiogenic sprouts that invade deeper. No differences in lumen size were observed between control and mTG stiffened matrices, but greater remodeling was revealed in stiffer gels using SHG and OCM. Results of this study showed angiogenic responses are influenced by stiffness and suggested that ECM properties may be useful in regenerative medicine applications to engineer angiogenesis.
Acta biomaterialia 04/2013; · 3.98 Impact Factor
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ABSTRACT: Abstract Background: Lymph flow depends on both the rate of lymph production by tissues and the extent of passive and active pumping. Here we aim to characterize the passive mechanical properties of a lymphangion in both mid-lymphangion and valve segments to assess regional differences along a lymphangion, as well as evaluating its structural composition. Methods and Results: Mesenteric lymphatic vessels were isolated and cannulated in a microchamber for pressure-diameter (P-D) testing. Vessels were inflated from 0 to 20 cmH(2)O at a rate of 4 cmH(2)O/min, and vessel diameter was continuously tracked, using an inverted microscope, video camera, and custom LabVIEW program, at both mid-lymphangion and valve segments. Isolated lymphatic vessels were also pressure-fixed at 2 and 7 cmH(2)O and imaged using a nonlinear optical microscope (NLOM) to obtain collagen and elastin structural information. We observed a highly nonlinear P-D response at low pressures (3-5 cmH(2)O), which was modeled using a three-parameter constitutive equation. No significant difference in the passive P-D response was observed between mid-lymphangion and valve regions. NLOM imaging revealed an inner elastin layer and outer collagen layer at all locations. Lymphatic valve leaflets were predominantly elastin with thick axially oriented collagen bands at the insertion points. Conclusions: We observed a highly nonlinear P-D response at low pressures (3-5 cmH(2)O) and developed the first constitutive equation to describe the passive P-D response for a lymphangion. The passive P-D response did not vary among regions, in agreement with the composition of elastin and collagen in the lymphatic wall.
Lymphatic Research and Biology 11/2012;
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ABSTRACT: The inverse relationship between two-photon excited fluorescence (TPEF) and laser pulse duration suggests that two-photon microscopy (TPM) performance may be improved by decreasing pulse duration. However, for ultrashort pulses of sub-10 femtosecond (fs) in duration, its spectrum contains the effective gain bandwidth of Ti:Sapphire and its central wavelength is no longer tunable. An experimental study was performed to explore this apparent tradeoff between untuned sub-10 fs transform-limited pulse (TLP) and tunable 140 fs pulse for TPEF. Enhancement factors of 1.6, 6.7, and 5.2 are measured for Indo-1, FITC, and TRITC excited by sub-10 fs TLP compared with 140 fs pulse tuned to the two-photon excitation (TPE) maxima at 730 nm, 800 nm, and 840 nm, respectively. Both degenerate (v(1) = v(2)) and nondegenerate (v(1) ≠ v(2)) mixing of sub-10 fs TLP spectral components result in its broad second-harmonic (SH) power spectrum and high spectral density, which can effectively compensate for the lack of central wavelength tuning and lead to large overlap with dye TPE spectra for TPEF enhancements. These pulse properties were also exploited for demonstrating its potential applications in multicolor imaging with TPM.
Journal of Biomedical Optics 02/2012; 17(2):025003. · 3.16 Impact Factor
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ABSTRACT: Elastin is the primary component of elastic fibres in arteries, which contribute significantly to the structural integrity of the wall. Fibrillin-1 is a microfibrillar glycoprotein that appears to stabilize elastic fibres mechanically and thereby to delay a fatigue-induced loss of function due to long-term repetitive loading. Whereas prior studies have addressed some aspects of ageing-related changes in the overall mechanical properties of arteries in mouse models of Marfan syndrome, we sought to assess for the first time the load-carrying capability of the elastic fibres early in maturity, prior to the development of ageing-related effects, dilatation, or dissection.
We used elastase to degrade elastin in common carotid arteries excised, at 7-9 weeks of age, from a mouse model (mgR/mgR) of Marfan syndrome that expresses fibrillin-1 at 15-25% of normal levels. In vitro biaxial mechanical tests performed before and after exposure to elastase suggested that the elastic fibres exhibited a nearly normal load-bearing capability. Observations from nonlinear optical microscopy suggested further that competent elastic fibres not only contribute to load-bearing, they also increase the undulation of collagen fibres, which endows the normal arterial wall with a more compliant response to pressurization.
These findings support the hypothesis that it is an accelerated fatigue-induced damage to or protease-related degradation of initially competent elastic fibres that render arteries in Marfan syndrome increasingly susceptible to dilatation, dissection, and rupture.
Cardiovascular research 08/2011; 92(2):287-95. · 5.80 Impact Factor
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ABSTRACT: A combined nonlinear optical microscopy (NLOM) and optical coherence microscopy (OCM) imaging system has been assembled in order to simultaneously capture co-registered volumetric images of corneal morphology and biochemistry. Tracking of cell nuclei visible in the OCM volume enabled the calculation of strain depth profile in response to changes in intraocular pressure for rabbit cornea stroma. Results revealed nonlinear responses with a depth dependent strain distribution, exhibiting smaller strains in the anterior and larger strains in the posterior stroma. Cross-sectional images of collagen lamellae, visible in NLOM, showed inhomogeneous collagen structure along the anterior-posterior direction that correlated well with the noted heterogeneous corneal mechanical responses.
Biomedical Optics Express 01/2011; 2(5):1135-46. · 2.33 Impact Factor
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ABSTRACT: Recent studies have renewed interest in the effects of perivascular tethering on vascular mechanics, particularly growth and remodeling. We quantified effects of axial and circumferential tethering on rabbit pial arterioles from the ventral occipital lobe of the brain. The homeostatic axial pre-stretch, which is influenced by perivascular tethering, was measured in situ to be 1.24±0.04. Using a cannulated microvessel preparation, wall mechanics were then quantified in vitro for isolated arterioles at low (1.10) or normal (1.24) values of axial stretch and for tethered arterioles having perivascular support. Axial stretch did not significantly affect changes in circumferential stretch or stress upon pressurization, but circumferential tethering caused arteriolar geometry to change from a circular cross-section at normal pressure to an elliptical one at pressures above and below normal. Calculations suggested that the observed levels of ellipticity could cause a modest decrease in volumetric blood flow, but also a pronounced variation in shear stress around the circumference of the arteriole. An elliptical cross-section could thus increase vascular resistance or promote luminal remodeling at pressures different from normal. This characterization of effects of perivascular tethering on pial arterioles should prove useful in future studies of roles of perturbed cerebral blood flow on the propensity of the cerebral microcirculation to remodel.
Journal of biomechanics 10/2010; 43(14):2717-21. · 2.66 Impact Factor
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ABSTRACT: The microstructural orientation of vascular wall constituents is of interest to scientists and clinicians because alterations in their native states are associated with various cardiovascular diseases. In the arterial media, the orientation of these constituents is often described as circumferential. However, it has been noted that, just below the endothelial surface, the vascular wall constituents are oriented axially. To further study this reported change in orientation, and to resolve previous observations (which were made under conditions of no load), we used nonlinear optical microscopy to examine the orientation of collagen and elastin fibers in the inner medial region of bovine common carotid arteries. Images were obtained from this part of the arterial wall under varying degrees of mechanical strain: 0%, 10% axial, 10% circumferential, and 10% biaxial. We observed that close to the endothelium these components are aligned in the axial direction but abruptly change to a circumferential alignment at a depth of approximately 20 mum from the endothelial surface. The application of mechanical strain resulted in a significantly greater degree of fiber alignment, both collagen and elastin, in the strain direction, regardless of their initial unloaded orientation. Furthermore, variations in strain conditions resulted in an increase or a decrease in the overall degree of fiber alignment in the subendothelial layer depending on the direction of the applied strain. This high-resolution investigation adds more detail to existing descriptions of complex structure-function relationships in vascular tissue, which is essential for a better understanding of the pathophysiological processes resulting from injury, disease progression, and interventional therapies.
AJP Heart and Circulatory Physiology 02/2010; 298(5):H1537-45. · 3.71 Impact Factor
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ABSTRACT: Many investigators have engineered diverse connective tissues having good mechanical properties, yet few tools enable a global understanding of the associated formation of collagen fibers, the primary determinant of connective tissue stiffness. Toward this end, we developed a biomechanical model for collagenous tissues grown on polymer scaffolds that accounts for the kinetics of polymer degradation as well as the synthesis and degradation of multiple families of collagen fibers in response to cyclic strains imparted in a bioreactor. The model predicted well both overall thickness and stress-stretch relationships for tubular engineered vessels cultured for 8 weeks, and suggested that a steady state had not yet been reached. To facilitate future refinements of the model, we also developed bioreactors that enable intravital nonlinear optical microscopic imaging. Using these tools, we found that collagen fiber alignment was driven strongly by nondegraded polymer fibers at early times during culture, with subsequent mechano-stimulated dispersal of fiber orientations as polymer fibers degraded. In summary, mathematical models of growth and remodeling of engineered tissues cultured on polymeric scaffolds can predict evolving tissue morphology and mechanics after long periods of culture, and related empirical observations promise to further our understanding of collagen matrix development in vitro.
Proceedings of the National Academy of Sciences 12/2009; 107(8):3335-9. · 9.68 Impact Factor
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ABSTRACT: Functional optical characterization of disease progression and response to therapy suffers from loss of spatial resolution and imaging depth due to scattering. Here we report on the ability of dimethyl sulfoxide (DMSO) alone to reduce the optical scattering of skin. We observed a threefold reduction in the scattering of skin with topical DMSO application. With an in vivo window chamber model, we observed a threefold increase in light transmittance through the preparation and enhanced visualization of subsurface microvasculature. Collectively, our data demonstrate the potential of DMSO alone to mitigate effects of scattering, which we expect will improve molecular imaging studies.
Lasers in Surgery and Medicine 03/2009; 41(2):142-8. · 2.75 Impact Factor
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ABSTRACT: Little is known about the precise mechanical stimuli that cells sense and respond to as they maintain or refashion the extracellular matrix in multiaxially loaded native or bioengineered tissues. Such information would benefit many areas of research involving soft tissues, including tissue morphogenesis, wound healing, and tissue engineering. A custom tissue culture device has been constructed that can impart well-defined biaxial stretches on cruciform-shaped, fibroblast-seeded collagen gels and be mounted on the stage of a nonlinear optical microscopy (NLOM) system for microscopic characterization of matrix organization. The cruciform geometry permitted direct comparison of matrix (re-)organization within regions of the collagen gel exposed to either uniaxial or biaxial boundary conditions and examination by NLOM for up to 6 days. In addition, sequential NLOM measurements of collagen fiber orientations within gels while stretched, unloaded, or decellularized delineated contributions of applied stretches, cell-mediated tractions, and matrix remodeling on the measured distributions. The integration of intravital NLOM with novel bioreactors enables visualization of dynamic tissue properties in culture.
Tissue Engineering Part A 01/2009; 15(7):1553-64. · 4.64 Impact Factor
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ABSTRACT: Ultrashort pulse, multispectral nonlinear optical microscopy (NLOM) is developed and used to image, simultaneously, a mixed population of cells expressing different fluorescent protein mutants in a 3D tissue model of angiogenesis. Broadband, sub-10-fs pulses are used to excite multiple fluorescent proteins and generate second harmonic in collagen simultaneously. A 16-channel multispectral detector is used to delineate the multiple nonlinear optical signals, pixel by pixel, in NLOM. The ability to image multiple fluorescent protein mutants and collagen, simultaneously, enables serial measurements of cell-cell and cell-matrix interactions in our 3D tissue model and characterization of fundamental processes in angiogenic morphogenesis.
Journal of innovative optical health sciences. 01/2009; 2(1):27-35.
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ABSTRACT: The interactions between endothelial cells (ECs) and the extracellular matrix (ECM) are fundamental in mediating various steps of angiogenesis, including cell adhesion, migration and sprout formation. Here, we used a noninvasive and non-destructive nonlinear optical microscopy (NLOM) technique to optically image endothelial sprouting morphogenesis in three-dimensional (3D) collagen matrices. We simultaneously captured signals from collagen fibers and endothelial cells using second harmonic generation (SHG) and two-photon excited fluorescence (TPF), respectively. Dynamic 3D imaging revealed EC interactions with collagen fibers along with quantifiable alterations in collagen matrix density elicited by EC movement through and morphogenesis within the matrix. Specifically, we observed increased collagen density in the area between bifurcation points of sprouting structures and anisotropic increases in collagen density around the perimeter of lumenal structures, but not advancing sprout tips. Proteinase inhibition studies revealed membrane-associated matrix metalloproteinase were utilized for sprout advancement and lumen expansion. Rho-associated kinase (p160ROCK) inhibition demonstrated that the generation of cell tension increased collagen matrix alterations. This study followed sprouting ECs within a 3D matrix and revealed that the advancing structures recognize and significantly alter their extracellular environment at the periphery of lumens as they progress.
Experimental Cell Research 12/2008; 315(3):396-410. · 3.58 Impact Factor
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ABSTRACT: Broadband, sub-10-fs pulses, can be propagated through polarization-maintaining single mode fiber (PMF) for use in nonlinear optical microscopy (NLOM). We demonstrate delivery of near transform-limited, 1 nJ pulses from a Ti:Al(2)O(3) (75 MHz repetition rate) oscillator via PMF to the NLOM focal plane while maintaining 120 nm of bandwidth. Negative group delay dispersion (GDD) introduced to pre-compensate normal dispersion of the optical fiber and microscope optics ensured linear pulse propagation through the PMF. The minimized time-bandwidth product of the laser pulses at the NLOM focus allowed the nonlinear excitation of multiple fluorophores simultaneously without central wavelength tuning. Polarization sensitive NLOM imaging using second harmonic generation in collagen was demonstrated using PMF delivered pulses. Two-photon excited fluorescence spectra and second harmonic images taken with and without the fiber indicates that the fiber based system is capable of generating optical signals that are within a factor of two to three of our traditional NLOM.
Optics Express 10/2008; 16(19):14723-30. · 3.59 Impact Factor
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ABSTRACT: Optical microscopy encompasses high-resolution imaging techniques that can be used to non-destructively investigate and characterize living biological systems and engineered tissue constructs in culture. In particular, nonlinear optical microscopy (NLOM) is well suited for the visualization and quantification of processes involved in cell-extracellular matrix interactions in vivo. Current NLOM technology enables concomitant molecular imaging and visualization of microstructural organization that could provide a direct link between signal transduction and biological effect at microscopic length scales that culminate into tissue macroscopic properties and function. This review highlights the fundamentals of nonlinear optical interactions between light and tissue and presents a direction for future technology development to better complement quantitative, high-throughput assays of the modern life sciences.
Tissue Engineering Part B Reviews 04/2008; 14(1):119-31. · 4.64 Impact Factor
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ABSTRACT: To characterize the microstructural response of the rabbit cornea to changes in intraocular pressure (IOP) by using nonlinear optical microscopy (NLOM).
Isolated rabbit corneas were mounted on an artificial anterior chamber in series with a manometer and were hydrostatically pressurized by a reservoir. The chamber was mounted on an upright microscope stage of a custom-built NLOM system for corneal imaging without using exogenous stains or dyes. Second harmonic generation in collagen was used to image through the full thickness of the central corneal stroma at IOPs between 5 and 20 mm Hg. Microstructural morphology changes as a function of IOP were used to characterize the depth-dependent response of the central cornea.
Regional collagen lamellae architecture through the full thickness of the stroma was specifically imaged as a function of IOP. Hypotensive corneas showed gaps between lamellar structures that decreased in size with increasing IOP. These morphologic features appear to result from interwoven lamellae oriented along the anterior-posterior axis and parallel to the cornea surface. They appear throughout the full thickness and disappear with tension in the anterior but persist in the posterior central cornea, even at hypertensive IOP.
NLOM reveals interwoven collagen lamellae sheets through the full thickness of the rabbit central cornea oriented along the anterior-posterior axis and parallel to the surface. The nondestructive nature of NLOM allows 3-dimensional imaging of stromal architecture as a function of IOP in situ. Collagen morphologic features were used as an indirect measure of depth-dependent mechanical response to changes in IOP.
Cornea 03/2008; 27(2):202-8. · 1.73 Impact Factor
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ABSTRACT: The effects of combined photodynamic therapy (PDT) and ionizing radiation are studied in a human glioma spheroid model. The degree of interaction between the two modalities depends in a complex manner on factors such as PDT irradiation fluence, fluence rate and dose of ionizing radiation. It is shown that gamma radiation and PDT interact in a synergistic manner only if both light fluence and gamma radiation dose exceed approximately 25 J cm−2 and 8 Gy, respectively. Synergistic interactions are observed only for the lower fluence rate (25 mW cm−2) investigated. The degree of interaction appears to be independent of both sequence and the PDT or ionizing radiation time intervals investigated (1 and 24 h). Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling assays show that low-fluence rate PDT is very efficient at inducing apoptotic cell death, whereas neither high-fluence rate PDT nor ionizing radiation produces significant apoptosis. Although the mechanisms remain to be elucidated, the data imply that the observed synergism is likely not due to gamma-induced cell cycle arrest or to PDT-induced inhibition of DNA repair.
Photochemistry and Photobiology 04/2007; 76(4):411 - 416. · 2.41 Impact Factor
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ABSTRACT: Light scattering from collagen within skin limits light-based therapeutics while increasing the risk of epidermal thermal injury. Specific chemicals show the ability to reduce light scattering by reversibly altering the optical properties of skin. This study examines the correlation between collagen solubility and the optical clearing potential (OCP) of sugars and sugar-alcohols using in vitro rodent skin.
Collagen solubility in dextrose, fructose, sucrose, and sorbitol was measured using near-UV spectroscopy. Light transmittance, reflectance, and rodent skin thickness were measured (giving skin reduced scattering coefficient) before and after exposure of the dermal surface to sugars and sugar-alcohols. OCP was calculated as the ratio of reduced scattering coefficients before and after exposures.
Dextrose, fructose, sucrose, and sorbitol had at least twice the collagen solubility and twice the OCP as compared to glycerol. In general, collagen solubility correlated with each agent's ability to optically clear rodent skin.
These results demonstrate that sugar and sugar-alcohol interaction with collagen are a primary event in tissue optical clearing.
Lasers in Surgery and Medicine 03/2007; 39(2):140-4. · 2.75 Impact Factor
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ABSTRACT: An all-mirror dispersion-compensation setup is used to correct for quadratic and cubic phase distortions induced within a custom nonlinear optical microscope. Mouse tail tendon is used to characterize sub-10-fs pulses by interferometric autocorrelation. This is an ideal method for characterizing dispersion from the optical system, immersion medium, and wet biological sample. The generation of very short autocorrelations demonstrates the ability to compensate for phase distortions within the imaging system and efficient second-harmonic upconversion of the ultrashort pulse spectrum within collagen. Compensated autocorrelation traces are presented for biologically relevant objective lenses.
Optics Letters 07/2006; 31(11):1681-3. · 3.40 Impact Factor
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ABSTRACT: Clinical procedures wherein supraphysiologic temperatures must be achieved in deep layers of tissue via light are often compromised by optical scattering and absorption. Optical clearing of tissue superficial to the target improves the efficacy of such procedures. Glycerol is an attractive chemical agent for achieving dramatic reductions in tissue turbidity, but its net effects on healthy tissue are not fully understood. In this paper, we investigate possible alterations of biaxial mechanical properties in a model collagenous tissue, bovine epicardium, induced by glycerol. Furthermore, we examine the effects of glycerol on the biaxial thermomechanical properties of epicardium constrained at near-physiologic length. It is seen that mechanical changes induced by glycerol are fully reversed upon rehydration in normal saline. Moreover, glycerol protects cleared tissue by increasing its thermal stability and minimizing thermal alterations of mechanical properties.
IEEE Transactions on Biomedical Engineering 05/2006; 53(4):747-53. · 2.28 Impact Factor
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ABSTRACT: Keloids are disfiguring, proliferative scars that are a pathologic response to cutaneous injury. An organotypic tissue culture system (the Raft model 1-10) was used to investigate the feasibility of using photodynamic therapy (PDT) as an adjunctive therapy to treat keloids following surgical excision. The Raft co-culture system mimics skin by layering keratinocytes on top of fibroblasts embedded in a collagen matrix. PDT uses drugs that produce singlet oxygen in situ when irradiated by light, and may lead to a number of effects in living tissues varying from the modulation of growth to apoptosis. PDT is already used to treat several benign and malignant diseases in organs such as the skin, retina, and esophagus.
Normal adult, neonatal, and keloid fibroblasts and keratinocytes were isolated from skin obtained from patients undergoing elective procedures and used to construct the Rafts. Mature Rafts (after 4 days) were incubated with 5-amino levulinic acid (5-ALA), a photosensitizer, for 3 hours and were laser-irradiated (635 nm) for total energy delivery of 5 J/cm2, 10 J/cm2, or 20 J/cm2. Rafts were examined 24 hours and 14 days later. Cell viability was determined using confocal imaging combined with live-dead fluorescent dyes. Multi-photon microscope (MPM) imaged collagen structure and density. As Rafts contract over time, surface area was measured using optical micrometry daily.
At 10 and 20 J/cm2, near-total cell death was observed in all constructs, while at 5 J/cm2 cell viability was comparable to controls. Cell viability in keloid and neonatal Rafts was greater than that observed in normal adult Rafts. Treated Rafts contracted less over the 14-day period compared to controls. Contraction and collagen density were greatest in keloid and neonatal Rafts.
A PDT dosimetry range was established, which reduces tissue contraction and collagen density while minimizing injury to fibroblasts.
Lasers in Surgery and Medicine 10/2005; 37(3):231-44. · 2.75 Impact Factor
