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Survival of living tumor cells underlies many influences such as nutrient saturation, oxygen level, drug concentrations or mechanical forces. Data-supported mathematical modeling can be a powerful tool to get a better understanding of cell behavior in different settings. However, under consideration of numerous environmental factors mathematical modeling can get challenging. We present an approach to model the separate influences of each environmental quantity on the cells in a collective manner by introducing the "environmental stress level". It is an immeasurable auxiliary variable, which quantifies to what extent viable cells would get in a stressed state, if exposed to certain conditions. A high stress level can inhibit cell growth, promote cell death and influence cell movement. As a proof of concept, we compare two systems of ordinary differential equations, which model tumor cell dynamics under various nutrient saturations respectively with and without considering an environmental stress level. Particle-based Bayesian inversion methods are used to quantify uncertainties and calibrate unknown model parameters with time resolved measurements of in vitro populations of liver cancer cells. The calibration results of both models are compared and the quality of fit is quantified. While predictions of both models show good agreement with the data, there is indication that the model considering the stress level yields a better fitting. The proposed modeling approach offers a flexible and extendable framework for considering systems with additional environmental factors affecting the cell dynamics.

Understanding the effects of inflammation and cirrhosis on the regulation of drug metabolism during the progression of hepatocellular carcinoma (HCC) is critical for developing patient-specific treatment strategies. In this work, we created novel three-dimensional vascularized HCC-on-a-chips (HCCoC), composed of HCC, endothelial, stellate, and Kupffer cells tuned to mimic normal or cirrhotic liver stiffness. HCC inflammation was controlled by tuning Kupffer macrophage numbers, and the impact of cytochrome P450-3A4 (CYP3A4) was investigated by culturing HepG2 HCC cells transfected with CYP3A4 to upregulate expression from baseline. This model allowed for the simulation of chemotherapeutic delivery methods such as intravenous injection and transcatheter arterial chemoembolization (TACE). We showed that upregulation of metabolic activity, incorporation of cirrhosis and inflammation, increase vascular permeability due to upregulated inflammatory cytokines leading to significant variability in chemotherapeutic treatment efficacy. Specifically, we show that further modulation of CYP3A4 activity of HCC cells by TACE delivery of doxorubicin provides an additional improvement to treatment response and reduces chemotherapy-associated endothelial porosity increase. The HCCoCs were shown to have utility in uncovering the impact of the tumor microenvironment (TME) during cancer progression on vascular properties, tumor response to therapeutics, and drug delivery strategies.Statement of SignificanceRegulation of drug metabolism during the cancer progression of hepatocellular carcinoma (HCC) can be influential to develop personalized treatment strategies. We created novel vascularized hepatocellular carcinoma-chip (HCCoC) composed of tunable collagen and four main liver-specific cell lines to be used as a preclinical tool. In this model, we found cancer evolution states such as inflammation and cirrhosis increases vascular permeability progressively as a result of increased inflammatory cytokines. Furthermore, delivery of doxorubicin only with embolization improved treatment efficacy by decreasing CYP3A4 activity, which can modulate treatment outcome. Overall, we found different disease states can be influential on CYP3A4, thus its targeting can improve HCC treatment outcome.

Inflammatory breast cancer (IBC), a rare form of breast cancer associated with increased angiogenesis and metastasis, is largely driven by tumor-stromal interactions with the vasculature and the extracellular matrix (ECM). However, there is currently a lack of understanding of the role these interactions play in initiation and progression of the disease. In this study, we developed the first three-dimensional, in vitro, vascularized, microfluidic IBC platform to quantify the spatial and temporal dynamics of tumor-vasculature and tumor-ECM interactions specific to IBC. Platforms consisting of collagen type 1 ECM with an endothelialized blood vessel were cultured with IBC cells, MDA-IBC3 (HER2+) or SUM149 (triple negative), and for comparison to non-IBC cells, MDA-MB-231 (triple negative). Acellular collagen platforms with endothelialized blood vessels served as controls. SUM149 and MDA-MB-231 platforms exhibited a significantly (p < .05) higher vessel permeability and decreased endothelial coverage of the vessel lumen compared to the control. Both IBC platforms, MDA-IBC3 and SUM149, expressed higher levels of vascular endothelial growth factor (p < .05) and increased collagen ECM porosity compared to non-IBCMDA-MB-231 (p < .05) and control (p < .01) platforms. Additionally, unique to the MDA-IBC3 platform, we observed progressive sprouting of the endothelium over time resulting in viable vessels with lumen. The newly sprouted vessels encircled clusters of MDA-IBC3 cells replicating a key feature of in vivo IBC. The IBC in vitro vascularized platforms introduced in this study model well-described in vivo and clinical IBC phenotypes and provide an adaptable, high throughput tool for systematically and quantitatively investigating tumor-stromal mechanisms and dynamics of tumor progression.

The goal of this study is to calibrate a multiscale model of tumor angiogenesis with time-resolved data to allow for systematic testing of mathematical predictions of vascular sprouting. The multi-scale model consists of an agent-based description of tumor and endothelial cell dynamics coupled to a continuum model of vascular endothelial growth factor concentration. First, we calibrate ordinary differential equation models to time-resolved protein expression data to estimate the rates of secretion and consumption of vascular endothelial growth factor by endothelial and tumor cells, respectively. These parameters are then input into the multiscale tumor angiogenesis model, and the remaining model parameters are then calibrated to time resolved confocal microscopy images obtained within a 3D vascularized microfluidic platform. The microfluidic platform mimics a functional blood vessel with a surrounding collagen matrix seeded with inflammatory breast cancer cells, which induce tumor angiogenesis. Once the multi-scale model is fully parameterized, we forecast the spatiotemporal distribution of vascular sprouts at future time points and directly compare the predictions to experimentally measured data. We assess the ability of our model to globally recapitulate angiogenic vasculature density, resulting in an average relative calibration error of 17.7% ± 6.3% and an average prediction error of 20.2% ± 4% and 21.7% ± 3.6% using one and four calibrated parameters, respectively. We then assess the model’s ability to predict local vessel morphology (individualized vessel structure as opposed to global vascular density), initialized with the first time point and calibrated with two intermediate time points. To the best of our knowledge, this represents the first study to integrate well-controlled, experimental data into a mechanism-based, multiscale, mathematical model of angiogenic sprouting to make specific, testable predictions.

Variations in tumor biology from patient to patient combined with the low overall survival rate of hepatocellular carcinoma (HCC) present significant clinical challenges. During the progression of chronic liver diseases from inflammation to the development of HCC, microenvironmental properties, including tissue stiffness and oxygen concentration, change over time. This can potentially impact drug metabolism and subsequent therapy response to commonly utilized therapeutics, such as doxorubicin, multi-kinase inhibitors (e.g., sorafenib), and other drugs, including immunotherapies. In this study, we utilized four common HCC cell lines embedded in 3D collagen type-I gels of varying stiffnesses to mimic normal and cirrhotic livers with environmental oxygen regulation to quantify the impact of these microenvironmental factors on HCC chemoresistance. In general, we found that HCC cells with higher baseline levels of cytochrome p450-3A4 (CYP3A4) enzyme expression, HepG2 and C3Asub28, exhibited a cirrhosis-dependent increase in doxorubicin chemoresistance. Under the same conditions, HCC cell lines with lower CYP3A4 expression, HuH-7 and Hep3B2, showed a decrease in doxorubicin chemoresistance in response to an increase in microenvironmental stiffness. This differential therapeutic response was correlated with the regulation of CYP3A4 expression levels under the influence of stiffness and oxygen variation. In all tested HCC cell lines, the addition of sorafenib lowered the required doxorubicin dose to induce significant levels of cell death, demonstrating its potential to help reduce systemic doxorubicin toxicity when used in combination. These results suggest that patient-specific tumor microenvironmental factors, including tissue stiffness, hypoxia, and CYP3A4 activity levels, may need to be considered for more effective use of chemotherapeutics in HCC patients.

Single-walled carbon nanohorns (SWNHs) have been shown to be effective photothermal enhancers and drug delivery agents for potential cancer therapy, particularly for the eradication of bladder cancer lesions. In this study, the potential for SWNHs to serve as nanotheranostic vehicles is demonstrated through simultaneous delivery of the chemotherapeutic cisplatin from SWNH cone interiors and imaging of the nanoparticle transport to tumor cells using conjugated quantum dots (QDs). Following the formation of cisplatin-modified SWNH-QD (SWNH-QD + cis) hybrids, their characterization by scanning and transmission electron microscopy (STEM, TEM), energy dispersive spectroscopy (EDS), and dynamic light scattering (DLS) were performed to characterize the composite nanoparticles. Drug release profiles and 50% inhibitory concentration (IC50) determination showed that QDs do not hinder the therapeutic ability of SWNHs. In addition, the hybrids were trackable over the course of a 3 day period, which indicates that internalized SWNHs can continue to deliver therapy after removal of the nano-agents from the cell culture. This unique SWNH hybrid can successfully be used as an imaging agent to study nanoparticle transport and consequently the delivery of a therapeutic to the targeted cancer cells.

Introduction: Inflammatory breast cancer (IBC) is an aggressive and rare disease with poor prognosis, accounting for 10% of breast cancer mortality [1]. A primary factor contributing to the bleak prognosis is the lack of IBC specific treatments. There are currently no IBC specific therapies due to a lack of IBC specific diagnostic and targeting markers. Efforts focused on identifying driver mutations and tumor targets have implicated tumor stroma including stromal cells such as macrophages in mediating IBC-like symptoms. This highlights the significance of understanding the interactions of tumor cells with the tumor stroma in greater detail and the knowledge would enable determination of targetable biology from these interactions which would facilitate development of IBC specific treatments and therapeutics. What is needed is a model to capture the complexity of IBC, identify critical spatial hetero-cellular interactions and target them successfully in a physiologically relevant and high-throughput manner. Approach: To address this need, we developed a 3D IBC microfluidic platform, unique in its simultaneous integration of functional blood vessels, tumor cells, macrophages, and type I collagen whose density, stiffness, and porosity mimics cancerous breast stroma. The platform will be used to study the influence of macrophage-tumor-endothelial interactions on 2 key critical features of IBC: vascular sprouting and formation of IBC emboli surrounded by vascular sprouts. Results: The 3D IBC microfluidic platform composed of MDA-IBC3 cells and a functional endothelial blood vessel demonstrated both vascular sprouting and emboli formation, key features of IBC tumors seen in IBC patient derived xenograft (PDX) models. Additionally, we observed vascular nesting of MDA-IBC3 emboli, recreating a characteristic IBC phenomenon observed in Mary-X PDX models. Incorporation of macrophages significantly increased the number of new vascular sprouts, sprouting rate and resulted in sprouts forming at earlier time points. Additionally, the presence of macrophages resulted in the formation of a significantly more porous collagen matrix (p Conclusion: IBC is an aggressive and invasive breast cancer with a poor prognosis linked to tumor-stroma interactions. Current preclinical to study IBC consist primarily of PDX models where determining the influence of specific signaling pathways and microenvironmental stimuli on tumor progression is challenging. Here we present a novel 3D microfluidic IBC platform to study tumor stromal interactions in a controlled manner. The MDA-IBC3 breast tumor platform demonstrated both vascular sprouting and emboli formation, key features of IBC seen in PDX models and the presence of macrophages increased both angiogenic sprouting and remodeling of the collagen matrix. The stark differences in the tumor platform response associated with macrophage presence strengthens the hypothesis of tumor stroma as a key player driving the aggressive nature of IBC and reveals a potential target for IBC therapeutics. [1] Fernandez, S.V., et al., Breast cancer research and treatment, 140(1): p. 23-33, 2013 Citation Format: Manasa Gadde, Caleb Phillips, Omar Rahal, Wendy Woodward, Marissa Rylander, Thomas Yankeelov. An in vitro microfluidic tumor platform for modeling and investigating tumor stromal interactions in inflammatory breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-06-02.

This paper presents the development of a vascularized breast tumor and healthy or tumorigenic liver microenvironments-on-a-chip connected in series. This is the first description of a vascularized multi tissue-on-a-chip microenvironment for modeling cancerous breast and cancerous/healthy liver microenvironments, to allow for the study of dynamic and spatial transport of particles. This device enables the dynamic determination of vessel permeability, the measurement of drug and nanoparticle transport, and the assessment of the associated efficacy and toxicity to the liver. The platform is utilized to determine the effect of particle size on the spatiotemporal diffusion of particles through each microenvironment, both independently and in response to the circulation of particles in varying sequences of microenvironments. The results show that when breast cancer cells were cultured in the microenvironments they had a 2.62-fold higher vessel porosity relative to vessels within healthy liver microenvironments. Hence, the permeability of the tumor microenvironment increased by 2.35- and 2.77-fold compared with a healthy liver for small and large particles, respectively. The extracellular matrix accumulation rate of larger particles was 2.57-fold lower than smaller particles in a healthy liver. However, the accumulation rate was 5.57-fold greater in the breast tumor microenvironment. These results are in agreement with comparable in vivo studies. Ultimately, the platform could be utilized to determine the impact of the tissue or tumor microenvironment, or drug and nanoparticle properties, on transport, efficacy, selectivity, and toxicity in a dynamic, and high-throughput manner for use in treatment optimization.

Two of the central challenges of using mathematical models for predicting the spatiotemporal development of tumors is the lack of appropriate data to calibrate the parameters of the model, and quantitative characterization of the uncertainties in both the experimental data and the modeling process itself. We present a sequence of experiments, with increasing complexity, designed to systematically calibrate the rates of apoptosis, proliferation, and necrosis, as well as mobility, within a phase-field tumor growth model. The in vitro experiments characterize the proliferation and death of human liver carcinoma cells under different initial cell concentrations, nutrient availabilities, and treatment conditions. A Bayesian framework is employed to quantify the uncertainties in model parameters. The average difference between the calibration and the data, across all time points is between 11.54% and 14.04% for the apoptosis experiments, 7.33% and 23.30% for the proliferation experiments, and 8.12% and 31.55% for the necrosis experiments. The results indicate the proposed experiment-computational approach is generalizable and appropriate for step-by-step calibration of multi-parameter models, yielding accurate estimations of model parameters related to rates of proliferation, apoptosis, and necrosis.

Solute transport is modeled using mixture theory, applied to the nanoparticle accumulation and concentration decay in the tissue space for different vascular configurations. A comparison of a single capillary configuration (SBC) with two parallel cylindrical blood vessels (2 BC) and a lymph vessel parallel to a blood vessel (BC_LC) embedded in the tissue cylinder is performed for five solute molecular weights between 0.1 kDa and 70 kDa. We found that the presence of a second capillary reduces the extravascular concentration compared to a single capillary and this reduction is enhanced by the presence of a lymph vessel. Co-current flow direction between two adjacent vessels led to nonhomogeneous nanoparticle distribution for larger particle sizes in the tissue space, while smaller particles (0.1 kDa and 3 kDa) showed the propensity to get trapped locally in the tissue during counter-current flow. Varying the intercapillary distance with respect to vessel diameter shows a deviation of 10-30 % concentration for 2 BC and 45-60% concentration for BC_LC configuration compared to the reference SBC configuration. Finally, we introduce a non-dimensional time scale that captures the concertation as a function of the transport and geometric parameters. We find that the peak solute concentration in the tissue space occurs at a non-dimensional time, T_peak^* = 0.027+/-0.018, irrespective of the solute size, tissue architecture, and microvessel flow direction. This suggests that if indeed such a universal time scale holds, the knowledge of this time would allow estimation of the time window at which solute concentration in tissue peaks. Hence this can aid in the design of future therapeutic efficacy studies as an example for triggering drug release or laser excitation in the case of photothermal therapies., Comment: 26 pages, 9 figures

New directions in medical and biomedical sciences have gradually emerged over recent years that will change the way diseases are diagnosed and treated and are leading to the redirection of medicine toward patient-specific treatments. We refer to these new approaches for studying biomedical systems as predictive medicine, a new version of medical science that involves the use of advanced computer models of biomedical phenomena, high-performance computing, new experimental methods for model data calibration, modern imaging technologies, cutting-edge numerical algorithms for treating large stochastic systems, modern methods for model selection, calibration, validation, verification, and uncertainty quantification, and new approaches for drug design and delivery, all based on predictive models. The methodologies are designed to study events at multiple scales, from genetic data, to sub-cellular signaling mechanisms, to cell interactions, to tissue physics and chemistry, to organs in living human subjects. The present document surveys work on the development and implementation of predictive models of vascular tumor growth, covering aspects of what might be called modeling-and-experimentally based computational oncology. The work described is that of a multi-institutional team, centered at ICES with strong participation by members at M. D. Anderson Cancer Center and University of Texas at San Antonio. This exposition covers topics on signaling models, cell and cell-interaction models, tissue models based on multi-species mixture theories, models of angiogenesis, and beginning work of drug effects. A number of new parallel computer codes for implementing finite-element methods, multi-level Markov Chain Monte Carlo sampling methods, data classification methods, stochastic PDE solvers, statistical inverse algorithms for model calibration and validation, models of events at different spatial and temporal scales is presented. Importantly, new methods for model selection in the presence of uncertainties fundamental to predictive medical science, are described which are based on the notion of Bayesian model plausibilities. Also, as part of this general approach, new codes for determining the sensitivity of model outputs to variations in model parameters are described that provide a basis for assessing the importance of model parameters and controlling and reducing the number of relevant model parameters. Model specific data is to be accessible through careful and model-specific platforms in the Tumor Engineering Laboratory. We describe parallel computer platforms on which large-scale calculations are run as well as specific time-marching algorithms needed to treat stiff systems encountered in some phase-field mixture models. We also cover new non-invasive imaging and data classification methods that provide in vivo data for model validation. The study concludes with a brief discussion of future work and open challenges.

Traditional experimental platforms to study cancer biology consist of two-dimensional (2D) cell culture systems and animal models. Although 2D cell cultures have yielded fundamental insights into cancer biology, they do not provide a physiologically representative three-dimensional (3D) volume for cell attachment and infiltration. These systems also cannot recapitulate critical features of the tumor microenvironment including hemodynamics, matrix mechanics, cellular crosstalk, and matrix interactions in a dynamic manner, or impose chemical and mechanical gradients. While animal models provide physiologic fidelity, they can be highly variable and cost prohibitive for extensive biological investigation and therapeutic optimization. Furthermore, the interplay of many different microenvironmental variables, such as growth factors, immune reaction, and stromal interactions, make it difficult to isolate the effect of a specific stimulus on cell response using animal models. Due to these limitations, 3D in vitro tumor models have recently emerged as valuable tools for the study of cancer progression as these systems have the ability to overcome many of the limitations of static 2D monolayers and mammalian systems. Initial 3D in vitro models have consisted of static 3D co-culture platforms and have been successful in providing a deeper insight compared to animal and static 2D systems. However, the majority of these existing systems lack the presence of physiological flow, a pivotal stimuli in tumor growth and metastasis and important consideration for transport of diagnostic or therapeutic agents. In order to consider the influence of flow on cancer progression microfluidic platforms are being widely used. The integration of microfluidic technology and microfabrication techniques with tumor biology has resulted in complex 3D microfluidic platforms capable of investigating various key stages in cancer evolution including angiogenesis and metastasis. 3D microfluidic platforms are able to provide a physiologically representative tumor environment while allowing for dynamic monitoring and simultaneous control of multiple factors such as cellular and extracellular matrix composition, fluid velocity and wall shear stress, and both biochemical and mechanical gradients.

Endothelial cells lining blood vessels are exposed to various hemodynamic forces associated with blood flow. These include fluid shear, the tangential force derived from the friction of blood flowing across the luminal cell surface, tensile stress due to deformation of the vessel wall by transvascular flow, and normal stress caused by the hydrodynamic pressure differential across the vessel wall. While it is well known that these fluid forces induce changes in endothelial morphology, cytoskeletal remodeling, and altered gene expression, the effect of flow on endothelial organization within the context of the tumor microenvironment is largely unknown. Using a previously established microfluidic tumor vascular model, the objective of this study was to investigate the effect of normal (4 dyn/cm(2)), low (1 dyn/cm(2)), and high (10 dyn/cm(2)) microvascular wall shear stress (WSS) on tumor-endothelial paracrine signaling associated with angiogenesis. It is hypothesized that high WSS will alter the endothelial phenotype such that vascular permeability and tumor-expressed angiogenic factors are reduced. Results demonstrate that endothelial permeability decreases as a function of increasing WSS, while co-culture with tumor cells increases permeability relative to mono-cultures. This response is likely due to shear stress-mediated endothelial cell alignment and tumor-VEGF-induced permeability. In addition, gene expression analysis revealed that high WSS (10 dyn/cm(2)) significantly down-regulates tumor-expressed MMP9, HIF1, VEGFA, ANG1, and ANG2, all of which are important factors implicated in tumor angiogenesis. This result was not observed in tumor mono-cultures or static conditioned media experiments, suggesting a flow-mediated paracrine signaling mechanism exists with surrounding tumor cells that elicits a change in expression of angiogenic factors. Findings from this work have significant implications regarding low blood velocities commonly seen in the tumor vasculature, suggesting high shear stress-regulation of angiogenic activity is lacking in many vessels, thereby driving tumor angiogenesis.

Nanoparticle research has seen advances in many fields, including the imaging and treatment of cancer. Specifically, nanotechnology has been investigated for its potential to be used as a tool to deliver well-tested drugs in potentially safer concentrations through both passive and active tumor targeting, while additionally providing means for a secondary therapy or imaging contrast. In particular, the use of light in conjunction with nanoparticle-based imaging and therapies has grown in popularity in recent years due to advances in utilizing light energy. In this review, we will first discuss nanoparticle platforms that can be used for optical imaging of cancer, such as fluorescence generation with quantum dots and surface-enhanced Raman scattering with plasmonic nanoparticles. We then analyze nanoparticle therapies, including photothermal therapy, photodynamic therapies, and photoacoustic therapy and their differences in exploiting light for cancer treatment. For photothermal therapies in particular, we have aggregated data on key variables in gold nanoparticle treatment protocols, such as exposure energy and nanoparticle concentration, and hope to highlight the need for normalization of variable reporting across varying experimental conditions and energy sources. We additionally discuss the potential to co-deliver chemotherapeutic drugs to the tumor using nanoparticles and how light can be harnessed for multifunctional approaches to cancer therapy. Finally, current in vitro methods of testing these therapies is discussed as well as the potential to improve on clinical translatability through 3D tissue phantoms. This review is focused on presenting, for the first time, a comprehensive comparison on a wide variety of photo based nanoparticle interactions leading to novel treatments and imaging tools from a basic science to clinical aspects and future directions.

Bioengineered vascular grafts provide a promising alternative to autografts for replacing diseased or damaged arteries, but necessitate scaffold designs capable of supporting a confluent endothelium that resists endothelial cell (EC) detachment under fluid flow. To this end, we investigated whether tuning electrospun topography (i.e. fiber diameter and orientation) could impact EC morphology, alignment, and structural protein organization with the goal of forming a confluent and well-adhered endothelium under fluid flow. To test this, a composite polymer blend of Poly(e-caprolactone) (PCL) and type I collagen was electrospun to form scaffolds with controlled fiber diameters ranging from approximately 100 nm to 1200 nm and with varying degrees of fiber alignment. ECs were seeded onto scaffolds, and cell morphology and degree of alignment were quantified using image analysis of fluorescently stained cells. Our results show that ECs form confluent monolayers on electrospun scaffolds, with cell alignment systematically increasing with a larger degree of fiber orientation. Additionally, cells on aligned electrospun scaffolds display thick F-actin bundles parallel to the direction of fiber alignment and strong VE-cadherin expression at cell-cell junctions. Under fluid flow, ECs on highly aligned scaffolds had greater resistance to detachment compared to cells cultured on randomly oriented and semi-aligned scaffolds. These results indicate that scaffolds with aligned topographies may be useful in forming a confluent endothelium with enhanced EC adhesion for vascular tissue engineering applications.

Background and Objective Regenerative medicine involves the bioengineering of a functional tissue or organ by seeding living cells on a biodegradable scaffold cultured in a bioreactor. A major barrier to creating functional tissues, however, has been the inability to monitor the dynamic and complex process of scaffold maturation in real time, making control and optimization extremely difficult. Current methods to assess maturation of bioengineered constructs, such as histology or organ bath physiology, are sample-destructive. Optical coherence tomography (OCT) has recently emerged as a key modality for structural assessment of native blood vessels as well as engineered vessel mimics. The objective of this study was to monitor and assess in real time the development of a bioengineered blood vessel using a novel approach of combining both free-space and catheter-based OCT imaging in a new quartz-walled bioreactor. Development of the blood vessel was characterized by changes in thickness and scattering coefficient over a 30-day period. Materials and Methods We constructed a novel blood vessel bioreactor utilizing a rotating cylindrical quartz cuvette permitting free-space OCT imaging of an installed vessel's outer surface. A vascular endoscopic OCT catheter was used to image the lumen of the vessels. The quartz cuvette permits 360 degree, free-space OCT imaging of the blood vessel. Bioengineered blood vessels were fabricated using biodegradable polymers (15% PCL/collagen, ∼300 µm thick) and seeded with CH3 10t1/2 mesenchymal stem cells. A swept-source OCT imaging system comprised of a 20 kHz tunable laser (Santec HSL2000) with 1,300 nm central wavelength and 110 nm FWHM bandwidth was used to assess the vessels. OCT images were obtained at days 1, 4, 7, 14, 21, and 30. Free-space (exterior surface) OCT images were co-registered with endoscopic OCT images to determine the vessel wall thickness. DAPI-stained histological sections, acquired at same time point, were evaluated to quantify wall thickness and cellular infiltration. Non-linear curve fitting of free-space OCT data to the extended Huygen–Fresnel model was performed to determine optical scattering properties. Results Vessel wall thickness increased from 435 ± 15 µm to 610 ± 27 µm and Vessel scattering coefficient increased from 3.73 ±0.32 cm−1 to 5.74 ± 0.06 cm−1 over 30 days. Histological studies showed cell migration from the scaffold surface toward the lumen and cell proliferation over the same time course. The imaging procedure did not have any significant impact on scaffold dimensions, cell migration, or cell proliferation. Conclusions This study suggests that combination of free-space and catheter-based OCT for blood vessel imaging provides accurate structural information of the developing blood vessel. We determined that free-space OCT images could be co-registered with catheter-based OCT images to monitor structural features such as wall thickness or delamination of the developing tissue-engineered blood vessel within a bioreactor. Structural parameters and optical properties obtained from OCT imaging correlate with histological sections of the blood vessel and could potentially be used as markers to non-invasively and non-destructively assess regeneration of engineered tissues in real time. Lasers Surg. Med. 45:391–400, 2013. © 2013 Wiley Periodicals, Inc.

The goal of drug delivery is to ensure that therapeutic molecules reach the intended target organ or tissue, such that the effectiveness of the drug is maximized. The efficiency of a drug delivery system greatly depends on the choice of drug carrier. Recently, there has been growing interest in using micro- and nanofibers for this purpose. The reasons for this growing interest include these materials' high surface area to volume ratios, ease of fabrication, high mechanical properties, and desirable drug release profile. Here, we review developments in using these materials made by the most prevalent methods of fiber fabrication: electrospinning, micro fluidics, wet spinning, rotary spinning, and self-assembly for drug delivery purposes. Additionally, we discuss the potential to use these fiber based systems in research and clinical applications.

Irreversible electroporation (IRE) is emerging as a powerful tool for tumor ablation that utilizes pulsed electric fields to destabilize the plasma membrane of cancer cells past the point of recovery. The ablated region is dictated primarily by the electric field distribution in the tissue, which forms the basis of current treatment planning algorithms. To generate data for refinement of these algorithms, there is a need to develop a physiologically accurate and reproducible platform on which to study IRE in vitro. Here, IRE was performed on a 3D in vitro tumor model consisting of cancer cells cultured within dense collagen I hydrogels, which have been shown to acquire phenotypes and respond to therapeutic stimuli in a manner analogous to that observed in in vivo pathological systems. Electrical and thermal fluctuations were monitored during treatment, and this information was incorporated into a numerical model for predicting the electric field distribution in the tumors. When correlated with Live/Dead staining of the tumors, an electric field threshold for cell death (500 V/cm) comparable to values reported in vivo was generated. In addition, submillimeter resolution was observed at the boundary between the treated and untreated regions, which is characteristic of in vivo IRE. Overall, these results illustrate the advantages of using 3D cancer cell culture models to improve IRE-treatment planning and facilitate widespread clinical use of the technology. National Science Foundation CBET-1055913, CBET-0955072

In vitro arterial flow bioreactor systems are widely used in tissue engineering to investigate response of endothelial cells to shear. However, the assumption that such models reproduce physiological flow has not been experimentally tested. Furthermore, shear stresses experienced by the endothelium are generally calculated using a Poiseuille flow assumption. Understanding the performance of flow bioreactor systems is of great importance, since interpretation of biological responses hinges on the fidelity of such systems and the validity of underlying assumptions. Here we test the physiologic reliability of arterial flow bioreactors and the validity of the Poiseuille assumption for a typical system used in tissue engineering. A particle image velocimetry system was employed to experimentally measure the flow within the vessel with high spatial and temporal resolution. Two types of vessels were considered: first, fluorinated ethylene propylene (FEP) tubing representative of a human artery without cells; and second, FEP tubing with a confluent layer of endothelial cells on the vessel lumen. Instantaneous wall shear stress (WSS), time-averaged WSS, and oscillatory shear index were computed from velocity field measurements and compared between cases. The flow patterns and resulting wall shear were quantitatively determined to not accurately reproduce physiological flow, and that the Poiseuille flow assumption was found to be invalid. This work concludes that analysis of cell response to hemodynamic parameters using such bioreactors should be accompanied by corresponding flow measurements for accurate quantification of fluid stresses.

Cells cultured within a three-dimensional (3D) in vitro environment have the ability to acquire phenotypes and respond to stimuli analogous to in vivo biological systems. This approach has been utilized in tissue engineering and can also be applied to the development of a physiologically relevant in vitro tumor model. In this study, collagen I hydrogels cultured with MDA-MB-231 human breast cancer cells were bioengineered as a platform for in vitro solid tumor development. The cell–cell and cell-matrix interactions present during in vivo tissue progression were encouraged within the 3D hydrogel architecture, and the biocompatibility of collagen I supported unconfined cellular proliferation. The development of necrosis beyond a depth of ~150–200 μm and the expression of hypoxia-inducible factor (HIF)-1α were demonstrated in the in vitro bioengineered tumors. Oxygen and nutrient diffusion limitations through the collagen I matrix as well as competition for available nutrients resulted in growing levels of intra-cellular hypoxia, quantified by a statistically significant (p < 0.01) upregulation of HIF-1α gene expression. The bioengineered tumors also demonstrated promising angiogenic potential with a statistically significant (p < 0.001) upregulation of vascular endothelial growth factor (VEGF)-A gene expression. In addition, comparable gene expression analysis demonstrated a statistically significant increase of HIF-1α (p < 0.05) and VEGF-A (p < 0.001) by MDA-MB-231 cells cultured in the 3D collagen I hydrogels compared to cells cultured in a monolayer on two-dimensional tissue culture polystyrene. The results presented in this study demonstrate the capacity of collagen I hydrogels to facilitate the development of 3D in vitro bioengineered tumors that are representative of the pre-vascularized stages of in vivo solid tumor progression.

Background and Objectives Photothermal therapies have limited efficacy and application due to the poor penetration depth of light inside tissue. In earlier work, we described the development of novel fiberoptic microneedles to provide a means to mechanically penetrate dermal tissue and deliver light directly into a localized target area. This paper presents an alternate fiberoptic microneedle design with the capability of delivering more diffuse, but therapeutically useful photothermal energy. Laser lipolysis is envisioned as a future clinical application for this design. Materials and Methods A novel fiberoptic microneedle was developed using hydrofluoric acid etching of optical fiber to permit diffuse optical delivery. Microneedles etched for 10, 30, and 50 minutes, and an optical fiber control were compared with three techniques. First, red light delivery from the microneedles was evaluated by imaging the reflectance of the light from a white paper. Second, spatial temperature distribution of the paper in response to near-IR light (1,064 nm, 1 W CW) was recorded using infrared thermography. Third, ex vivo adipose tissue response during 1,064 nm, (5 W CW) irradiation was recorded with bright field microscopy. Results Acid etching exposed a 3 mm length of the fiber core, allowing circumferential delivery of light along this length. Increasing etching time decreased microneedle diameter, resulting in increased uniformity of red and 1,064 nm light delivery along the microneedle axis. For equivalent total energy delivery, thinner microneedles reduced carbonization in the adipose tissue experiments. Conclusions We developed novel microscale optical diffusers that provided a more homogeneous light distribution from their surfaces, and compared performance to a flat-cleaved fiber, a device currently utilized in clinical practice. These fiberoptic microneedles can potentially enhance clinical laser procedures by providing direct delivery of diffuse light to target chromophores, while minimizing undesirable photothermal damage in adjacent, non-target tissue. Lasers Surg. Med. 43:914-920, 2011. © 2011 Wiley Periodicals, Inc.

This study introduces the use of high-frequency pulsed electric fields for tissue electroporation. Through the development of finite element models and the use of analytical techniques, electroporation with rectangular, bipolar pulses is investigated. The electric field and temperature distribution along with the associated transmembrane potential development are considered in a heterogeneous skin fold geometry. Results indicate that switching polarity on the nanosecond scale near the charging time of plasma membranes can greatly improve treatment outcomes in heterogeneous tissues. Specifically, high-frequency fields ranging from 500 kHz to 1 MHz are best suited to penetrate epithelial layers without inducing significant Joule heating, and cause electroporation in underlying cells.

Developing combinatorial treatments involving laser irradiation and nanoparticles require an understanding of the effect of nanoparticle inclusion on tissue thermal properties, such as thermal conductivity. This information will permit a more accurate prediction of temperature distribution and tumor response following therapy, as well as provide additional information to aid in the selection of the appropriate type and concentration of nanoparticles. This study measured the thermal conductivity of tissue representative phantoms containing varying types and concentrations of carbon nanotubes (CNTs). Multi-walled carbon nanotubes (MWNTs, length of 900–1200 nm and diameter of 40–60 nm), single-walled carbon nanotubes (SWNTs, length of 900–1200 nm and diameter

Tissue engineering and the use of nanofibrous biomaterial scaffolds offer a unique perspective for studying cancer development in vitro. Current in vitro models of tumorigenesis are limited by the use of static, two-dimensional (2D) cell culture monolayers that lack the structural architecture necessary for cell-cell interaction and three-dimensional (3D) scaffolds that are too simplistic for studying basic pathological mechanisms. In this study, two nanofibrous biomaterials that mimic the structure of the extracellular matrix, bacterial cellulose and electrospun polycaprolactone (PCL)/collagen I, were investigated as potential 3D scaffolds for an in vitro cancer model. Multiple cancer cell lines were cultured on each scaffold material and monitored for cell viability, proliferation, adhesion, infiltration, and morphology. Both bacterial cellulose and electrospun PCL/collagen I, which have nanoscale structures on the order of 100-500 nm, have been used in many diverse tissue engineering applications. Cancer cell adhesion and growth were limited on bacterial cellulose, while all cellular processes were enhanced on the electrospun scaffolds. This initial analysis has demonstrated the potential of electrospun PCL/collagen I scaffolds toward the development of an improved 3D in vitro cancer model.

Nanoparticles have significant potential as selective photo-absorbing agents for laser based cancer treatment. This study investigates the use of single walled carbon nanohorns (SWNHs) as thermal enhancers when excited by near infrared (NIR) light for tumor cell destruction. Absorption spectra of SWNHs in deionized water at concentrations of 0, 0.01, 0.025, 0.05, 0.085, and 0.1 mg/ml were measured using a spectrophotometer for the wavelength range of 200-1,400 nm. Mass attenuation coefficients were calculated using spectrophotometer transmittance data. Cell culture media containing 0, 0.01, 0.085, and 0.333 mg/ml SWNHs was laser irradiated at 1,064 nm wavelength with an irradiance of 40 W/cm{sup 2} for 0-5 minutes. Temperature elevations of these solutions during laser irradiation were measured with a thermocouple 8 mm away from the incident laser beam. Cell viability of murine kidney cancer cells (RENCA) was measured 24 hours following laser treatment with the previously mentioned laser parameters alone or with SWNHs. Cell viability as a function of radial position was determined qualitatively using trypan blue staining and bright field microscopy for samples exposed to heating durations of 2 and 6 minutes alone or with 0.085 mg/ml SWNHs. A Beckman Coulter Vi-Cell instrument quantified cell viability of samples treated with varyingmore » SWNH concentration (0, 0.01, 0.085, and 0.333 mg/ml) and heating durations of 0-6 minutes. Spectrophotometer measurements indicated inclusion of SWNHs increased light absorption and attenuation across all wavelengths. Utilizing SWNHs with laser irradiation increased temperature elevation compared to laser heating alone. Greater absorption and higher temperature elevations were observed with increasing SWNH concentration. No inherent toxicity was observed with SWNH inclusion. A more rapid and substantial viability decline was observed over time in samples exposed to SWNHs with laser treatment compared with samples experiencing laser heating or SWNH treatment alone. Samples heated for 6 minutes with 0.085 mg/ml SWNHs demonstrated increasing viability as the radial distance from the incident laser beam increased. The significant increases in absorption, temperature elevation, and cell death with inclusion of SWNHs in laser therapy demonstrate the potential of their use as agents for enhancing photothermal tumor destruction.« less

This study demonstrates the capability of multiwalled carbon nanotubes (MWNTs) coupled with laser irradiation to enhance treatment of cancer cells through enhanced and more controlled thermal deposition, increased tumor injury, and diminished heat shock protein (HSP) expression. We also explored the potential promise of MWNTs as drug delivery agents by observing the degree of intracellular uptake of these nanoparticles. To determine the heat generation capability of MWNTs, the absorption spectra and temperature rise during heating were measured. Higher optical absorption was observed for MWNTs in water compared with water alone. For identical laser parameters, MWNT-containing samples produced a significantly greater temperature elevation compared to samples treated with laser alone. Human prostate cancer (PC3) and murine renal carcinoma (RENCA) cells were irradiated with a 1,064-nm laser with an irradiance of 15.3 W/cm2 for 2 heating durations (1.5 and 5 minutes) alone or in combination with MWNT inclusion. Cytotoxicity and HSP expression following laser heating was used to determine the efficacy of laser treatment alone or in combination with MWNTs. No toxicity was observed for MWNTs alone. Inclusion of MWNTs dramatically decreased cell viability and HSP expression when combined with laser irradiation. MWNT cell internalization was measured using fluorescence and transmission electron microscopy following incubation of MWNTs with cells. With increasing incubation duration, a greater number of MWNTs were observed in cellular vacuoles and nuclei. These findings offer an initial proof of concept for the application of MWNTs in cancer therapy. Cancer Res; 70(23); 9855–64. ©2010 AACR.

Hyperthermia can induce heat shock protein (HSP) expression in tumours, which will cause enhanced tumour viability and increased resistance to additional thermal, chemotherapy, and radiation treatments. The study objective was to determine the relationship of hyperthermia protocols with HSP expression kinetics and cell death and develop corresponding computational predictive models of normal and cancerous prostate cell response.HSP expression kinetics and cell viability were measured in PC3 prostate cancer and RWPE-1 normal prostate cells subjected to hyperthermia protocols of 44° to 60°C for 1 to 30 min. Hsp27, Hsp60, and Hsp70 expression kinetics were determined by western blotting and visualised with immunofluorescence and confocal microscopy. Based on measured HSP expression data, a mathematical model was developed for predicting thermally induced HSP expression. Cell viability was measured with propidium iodide staining and flow cytometry to quantify the injury parameters necessary for predicting cell death following hyperthermia.Significant Hsp27 and Hsp70 levels were induced in both cell types with maximum HSP expression occurring at 16 h post-heating, and diminishing substantially after 72 h. PC3 cells were slightly more sensitive to thermal stress than RWPE-1 cells. Arrhenius analysis of injury data suggested a transition between injury mechanisms at 54°C. HSP expression and injury models were effective at predicting cellular response to hyperthermia.Measurement of thermally induced HSP expression kinetics and cell viability associated with hyperthermia enabled development of thermal dosimetry guidelines and predictive models for HSP expression and cell injury as a function of thermal stress to investigate and design more effective hyperthermia therapies.

The direct correlation between levels of heat shock protein expression and efficiency of its tissue protection function motivates this study of how thermal doses can be used for an optimal stress protocol design. Heat shock protein 70 (HSP70) expression kinetics were visualized continuously in cultured bovine aortic endothelial cells (BAECs) on a microscope heating stage using green fluorescent protein (GFP) as a reporter. BAECs were transfected with a DNA vector, HSP(p)-HSP70-GFP which expresses an HSP70-GFP fusion protein under control of the HSP70 promoter. Expression levels were validated by western blot analysis. Transfected cells were heated on a controlled temperature microscope stage at 42 degrees C for a defined period, then shifted to 37 degrees C for varied post-heating times. The expression of HSP70-GFP and its sub-cellular localization were visualized via fluorescence microscopy. The progressive expression kinetics were measured by quantitative analysis of serial fluorescence images captured during heating protocols from 1 to 2 h and post-heating times from 0 to 20 h. The results show two sequential peaks in HSP70 expression at approximately 3 and 12 h post-heat shock. A progressive translocation of HSP70 from the cytoplasm to the nucleus was observed from 6 to 16 h. We conclude that we have successfully combined molecular cloning and optical imaging to study HSP70 expression kinetics. The kinetic profile for HSP70-GFP fusion protein is consistent with the endogenous HSP70. Furthermore, information on dynamic intracellular translocation of HSP70 was extracted from the same experimental data.

Lumen endothelialization of bioengineered vascular scaffolds is essential to maintain small-diameter graft patency and prevent thrombosis postimplantation. Unfortunately, nondestructive imaging methods to visualize this dynamic process are lacking, thus slowing development and clinical translation of these potential tissue-engineering approaches. To meet this need, a fluorescence imaging system utilizing a commercial optical coherence tomography (OCT) catheter was designed to visualize graft endothelialization.C7 DragonFly™ intravascular OCT catheter was used as a channel for delivery and collection of excitation and emission spectra. Poly-dl-lactide (PDLLA) electrospun scaffolds were seeded with endothelial cells (ECs). Seeded cells were exposed to Calcein AM before imaging, causing the living cells to emit green fluorescence in response to blue laser. By positioning the catheter tip precisely over a specimen using high-fidelity electromechanical components, small regions of the specimen were excited selectively. The resulting fluorescence intensities were mapped on a two-dimensional digital grid to generate spatial distribution of fluorophores at single-cell-level resolution. Fluorescence imaging of endothelialization on glass and PDLLA scaffolds was performed using the OCT catheter-based imaging system as well as with a commercial fluorescence microscope. Cell coverage area was calculated for both image sets for quantitative comparison of imaging techniques. Tubular PDLLA scaffolds were maintained in a bioreactor on seeding with ECs, and endothelialization was monitored over 5 days using the OCT catheter-based imaging system.No significant difference was observed in images obtained using our imaging system to those acquired with the fluorescence microscope. Cell area coverage calculated using the images yielded similar values. Nondestructive imaging of endothelialization on tubular scaffolds showed cell proliferation with cell coverage area increasing from 15 ± 4% to 89 ± 6% over 5 days.In this study, we showed the capability of an OCT catheter-based imaging system to obtain single-cell resolution and to quantify endothelialization in tubular electrospun scaffolds. We also compared the resulting images with traditional microscopy, showing high fidelity in image capability. This imaging system, used in conjunction with OCT, could potentially be a powerful tool for in vitro optimization of scaffold cellularization, ensuring long-term graft patency postimplantation.

Type I collagen hydrogels have been used successfully as three-dimensional substrates for cell culture and have shown promise as scaffolds for engineered tissues and tumors. A critical step in the development of collagen hydrogels as viable tissue mimics is quantitative characterization of hydrogel properties and their correlation with fabrication parameters, which enables hydrogels to be tuned to match specific tissues or fulfill engineering requirements. A significant body of work has been devoted to characterization of collagen I hydrogels; however, due to the breadth of materials and techniques used for characterization, published data are often disjoint and hence their utility to the community is reduced. This review aims to determine the parameter space covered by existing data and identify key gaps in the literature so that future characterization and use of collagen I hydrogels for research can be most efficiently conducted. This review is divided into three sections: (1) relevant fabrication parameters are introduced and several of the most popular methods of controlling and regulating them are described, (2) hydrogel properties most relevant for tissue engineering are presented and discussed along with their characterization techniques, (3) the state of collagen I hydrogel characterization is recapitulated and future directions are proposed. Ultimately, this review can serve as a resource for selection of fabrication parameters and material characterization methodologies in order to increase the usefulness of future collagen-hydrogel-based characterization studies and tissue engineering experiments.

Irreversible electroporation (IRE) is a new technology for ablating aberrant tissue that utilizes pulsed electric fields (PEFs) to kill cells by destabilizing their plasma membrane. When treatments are planned correctly, the pulse parameters and location of the electrodes for delivering the pulses are selected to permit destruction of the target tissue without causing thermal damage to the surrounding structures. This allows for the treatment of surgically inoperable masses that are located near major blood vessels and nerves. In select cases of high-dose IRE, where a large ablation volume is desired without increasing the number of electrode insertions, it can become challenging to design a pulse protocol that is inherently nonthermal. To solve this problem we have developed a new electrosurgical device that requires no external equipment or protocol modifications. The design incorporates a phase change material (PCM) into the electrode core that melts during treatment and absorbs heat out of the surrounding tissue. Here, this idea is reduced to practice by testing hollow electrodes filled with gallium on tissue phantoms and monitoring temperature in real time. Additionally, the experimental data generated are used to validate a numerical model of the heat transfer problem, which is then applied to investigate the cooling performance of other classes of PCMs. The results indicate that metallic PCMs, such as gallium, are better suited than organics or salt hydrates for thermal management, because their comparatively higher thermal conductivity aids in heat dissipation. However, the melting point of the metallic PCM must be properly adjusted to ensure that the phase transition is not completed before the end of treatment. When translated clinically, phase change electrodes have the potential to continue to allow IRE to be performed safely near critical structures, even in high-dose cases.

As solid tumors develop, a variety of physical stresses arise including growth induced compressive force, matrix stiffening due to desmoplasia, and increased interstitial fluid pressure and altered flow patterns due to leaky vasculature and poor lymphatic drainage [1]. These microenvironmental stresses likely contribute to the abnormal cell behavior that drives tumor progression, and have become an increasingly significant area of cancer research. Of particular importance, is the role of flow shear stress on tumor-endothelial signaling, vascular function, and angiogenesis. Compared to normal vasculature, blood vessels in tumors are poorly functional due to dysregulated expression of angiogenic growth factors, such as vascular endothelial growth factor (VEGF) or the angiopoietins. Also, because of the abnormal vessel structure, blood velocities can be an order of magnitude lower than that of normal microvessels. Recently published work utilizing intravital microscopy to measure blood velocities in mouse mammary fat pad tumors, demonstrated for the first time that shear rate gradients in tumors may help guide branching and growth of new vessels [2]. However, much still remains unknown about how shear stress regulates endothelial organization, permeability, or expression of growth factors within the context of the tumor microenvironment.Copyright © 2013 by ASME

A new image analysis method called the spatial phantom evaluation of cellular thermal response in layers (SPECTRL) is presented for assessing spatial viability response to nanoparticle enhanced photothermal therapy in tissue representative phantoms. Sodium alginate phantoms seeded with MDA-MB-231 breast cancer cells and single-walled nanohorns were laser irradiated with an ytterbium fiber laser at a wavelength of 1064 nm and irradiance of 3.8 W cm(-2) for 10-80 s. SPECTRL quantitatively assessed and correlated 3D viability with spatiotemporal temperature. Based on this analysis, kill and transition zones increased from 3.7 mm(3) and 13 mm(3) respectively to 44.5 mm(3) and 44.3 mm(3) as duration was increased from 10 to 80 s. SPECTRL provides a quantitative tool for measuring precise spatial treatment regions, providing information necessary to tailor therapy protocols.

Regenerative medicine involves the bioengineering of a functional tissue or organ by seeding living cells on a biodegradable scaffold cultured in a bioreactor. A major barrier to creating functional tissues, however, has been the inability to monitor the dynamic and complex process of scaffold maturation in real time, making control and optimization extremely difficult. Current methods to assess maturation of bioengineered constructs, such as histology or organ bath physiology, are sample-destructive. Optical coherence tomography (OCT) has recently emerged as a key modality for structural assessment of native blood vessels as well as engineered vessel mimics. The objective of this study was to monitor and assess in real time the development of a bioengineered blood vessel using a novel approach of combining both free-space and catheter-based OCT imaging in a new quartz-walled bioreactor. Development of the blood vessel was characterized by changes in thickness and scattering coefficient over a 30-day period.We constructed a novel blood vessel bioreactor utilizing a rotating cylindrical quartz cuvette permitting free-space OCT imaging of an installed vessel's outer surface. A vascular endoscopic OCT catheter was used to image the lumen of the vessels. The quartz cuvette permits 360 degree, free-space OCT imaging of the blood vessel. Bioengineered blood vessels were fabricated using biodegradable polymers (15% PCL/collagen, ∼300 µm thick) and seeded with CH3 10t1/2 mesenchymal stem cells. A swept-source OCT imaging system comprised of a 20 kHz tunable laser (Santec HSL2000) with 1,300 nm central wavelength and 110 nm FWHM bandwidth was used to assess the vessels. OCT images were obtained at days 1, 4, 7, 14, 21, and 30. Free-space (exterior surface) OCT images were co-registered with endoscopic OCT images to determine the vessel wall thickness. DAPI-stained histological sections, acquired at same time point, were evaluated to quantify wall thickness and cellular infiltration. Non-linear curve fitting of free-space OCT data to the extended Huygen-Fresnel model was performed to determine optical scattering properties.Vessel wall thickness increased from 435 ± 15 µm to 610 ± 27 µm and Vessel scattering coefficient increased from 3.73 ± 0.32 cm⁻¹ to 5.74 ± 0.06 cm⁻¹ over 30 days. Histological studies showed cell migration from the scaffold surface toward the lumen and cell proliferation over the same time course. The imaging procedure did not have any significant impact on scaffold dimensions, cell migration, or cell proliferation.This study suggests that combination of free-space and catheter-based OCT for blood vessel imaging provides accurate structural information of the developing blood vessel. We determined that free-space OCT images could be co-registered with catheter-based OCT images to monitor structural features such as wall thickness or delamination of the developing tissue-engineered blood vessel within a bioreactor. Structural parameters and optical properties obtained from OCT imaging correlate with histological sections of the blood vessel and could potentially be used as markers to non-invasively and non-destructively assess regeneration of engineered tissues in real time.

Although successful remission has been achieved when cancer is diagnosed and treated during its earliest stages of development, a tumor that has established neovascularization poses a significantly greater risk of mortality. The inability to recapitulate the complexities of a maturing in vivo tumor microenvironment in an in vitro setting has frustrated attempts to identify and test anti-angiogenesis therapies that are effective at permanently halting cancer progression. We have established an in vitro tumor angiogenesis model driven solely by paracrine signaling between MDA-MB-231 breast cancer cells and telomerase-immortalized human microvascular endothelial (TIME) cells co-cultured in a spatially relevant manner. The bilayered bioengineered tumor model consists of TIME cells cultured as an endothelium on the surface of an acellular collagen I hydrogel under which MDA-MB-231 cells are cultured in a separate collagen I hydrogel. Results showed that TIME cells co-cultured with the MDA-MB-231 cells demonstrated a significant increase in cell number, rapidly developed an elongated morphology, and invasively sprouted into the underlying acellular collagen I layer. Comparatively, bioengineered tumors cultured with less aggressive MCF7 breast cancer cells did not elicit an angiogenic response. Angiogenic sprouting was demonstrated by the formation of a complex capillary-like tubule network beneath the surface of a confluent endothelial monolayer with lumen formation and anastomosing branches. In vitro angiogenesis was dependent on vascular endothelial growth factor secretion, matrix concentration, and duration of co-culture. Basic fibroblast growth factor supplemented to the co-cultures augmented angiogenic sprouting. The development of improved preclinical tumor angiogenesis models, such as the one presented here, is critical for accurate evaluation and refinement of anti-angiogenesis therapies.

The integration of tissue engineering strategies with microfluidic technologies has enabled the design of in vitro microfluidic culture models that better adapt to morphological changes in tissue structure and function over time. These biomimetic microfluidic scaffolds accurately mimic native 3D microenvironments, as well as permit precise and simultaneous control of chemical gradients, hydrodynamic stresses, and cellular niches within the system. The recent application of microfluidic in vitro culture models to cancer research offers enormous potential to aid in the development of improved therapeutic strategies by supporting the investigation of tumor angiogenesis and metastasis under physiologically relevant flow conditions. The intrinsic material properties and fluid mechanics of microfluidic culture models enable high-throughput anti-cancer drug screening, permit well-defined and controllable input parameters to monitor tumor cell response to various hydrodynamic conditions or treatment modalities, as well as provide a platform for elucidating fundamental mechanisms of tumor physiology. This review highlights recent developments and future applications of microfluidic culture models to study tumor progression and therapeutic targeting under conditions of hydrodynamic stress relevant to the complex tumor microenvironment.

Bioengineering of vascular grafts holds great potential to address the shortcomings associated with autologous and conventional synthetic vascular grafts used for small diameter grafting procedures. Lumen endothelialization of bioengineered vascular grafts is essential to provide an antithrombogenic graft surface to ensure long-term patency after implantation. Conventional methods used to assess endothelialization in vitro typically involve periodic harvesting of the graft for histological sectioning and staining of the lumen. Endpoint testing methods such as these are effective but do not provide real-time information of endothelial cells in their intact microenvironment, rather only a single time point measurement of endothelium development. Therefore, nondestructive methods are needed to provide dynamic information of graft endothelialization and endothelium maturation in vitro. To address this need, we have developed a nondestructive fiber optic based (FOB) imaging method that is capable of dynamic assessment of graft endothelialization without disturbing the graft housed in a bioreactor. In this study we demonstrate the capability of the FOB imaging method to quantify electrospun vascular graft endothelialization, EC detachment, and apoptosis in a nondestructive manner. The electrospun scaffold fiber diameter of the graft lumen was systematically varied and the FOB imaging system was used to noninvasively quantify the affect of topography on graft endothelialization over a 7-day period. Additionally, results demonstrated that the FOB imaging method had a greater imaging penetration depth than that of two-photon microscopy. This imaging method is a powerful tool to optimize vascular grafts and bioreactor conditions in vitro, and can be further adapted to monitor endothelium maturation and response to fluid flow bioreactor preconditioning.

Aim: Nanoparticle-enhanced photothermal therapy is a promising alternative to tumor resection. However, quantitative measurements of cellular response to these treatments are limited. This article introduces a Bimodal Enhanced Analysis of Spatiotemporal Temperature (BEAST) algorithm to rapidly determine the viability of cancer cells in vitro following photothermal therapy alone or in combination with nanoparticles. Materials & methods: To illustrate the capability of the BEAST viability algorithm, single wall carbon nanohorns were added to renal cancer (RENCA) cells in vitro and time-dependent spatial temperature maps measured with an infrared camera during laser therapy were correlated with post-treatment cell viability distribution maps obtained by cell-staining fluorescent microscopy. Conclusion: The BEAST viability algorithm accurately and rapidly determined the cell viability as a function of time, space and temperature. Original submitted 13 July 2011; Revised submitted 12 March 2012; Published online 20 July 2012

Although bone has the intrinsic ability to “self-heal”, there are circumstances in which its regenerative capacity is limited or compromised, such as in critical bone defects. In these cases, the lack of osteogenic proteins at the wound site can prevent healing and external stimuli may be necessary to encourage bone growth [1]. Exogenous delivery of proteins and growth factors directly to the wound has been successful in bone regeneration, but is limited by the instability of the proteins and short half-lives. As a result, administration of multiple large doses of protein is necessary to retain a beneficial protein level. Due to these disadvantages, additional methods have been investigated to supply essential proteins to the bone defect [2].Copyright © 2012 by ASME

While research has shown that the fluid mechanics of the tumor vasculature reduce transport and uptake of therapeutics, the underlying role of these stresses in regulating tumor-endothelial cell signaling and neovascularization are not well understood. Understanding the reciprocal interaction between endothelial and tumor cells to mediate angiogenesis, and the effect of fluid shear on this process, may offer insight into the development of improved treatment modalities to control highly vascularized tumors. We have previously shown that breast cancer cells cultured under 2D, static conditions with endothelial cells significantly increase expression of pro-angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietin 2 (ANG2) [1]. These preliminary results motivated the investigation of tumor-endothelial cross-talk under 3D, dynamic co-culture conditions.Copyright © 2012 by ASME

Irreversible electroporation (IRE) is a non-thermal tissue ablation modality that is gaining momentum as a viable treatment option for tumors and other non-cancerous pathologies [1]. The protocol consists of delivering a series of short (∼ 100 μs) and intense (∼ 1000 V/cm) pulsed electric fields through electrodes inserted directly into or around a targeted tissue. The pulses induce a rapid buildup of charge across the plasma membrane of cells comprising the tissue that results in the creation of permanent membrane defects, ultimately leading to cell death. Because the extent of cell death relies predominately on the extent of charge buildup and not thermal processes, extracellular matrix components are spared, including major nerve and blood vessel architecture. Additionally, the ablation volume is predictable based on the electric field distribution and visible in real-time via MRI, CT, and ultrasound.

Despite the 200 billion dollars invested in cancer therapy research and development since 1971, only 5% of new drugs entering clinical trials successfully obtain FDA approval [1, 2]. There is a growing concern in the cancer research community that this slow movement in progress stems from the need for improved preclinical models for testing new therapeutic agents [1]. A burgeoning interface between cancer research and tissue engineering is transforming how tumor development is being studied in vitro. As a result, complex 3D cancer cell culture models are beginning to be developed with phenotypes representative of in vivo cancer progression [3].Copyright © 2012 by ASME

Cancer is one of the most deadly diseases and leading cause of death. Laser based photothermal therapy can provide a minimally invasive alternative to surgical resection. The selectivity and effectiveness of laser therapy can be greatly enhanced when photoabsorbing nanoparticles such as nanoshells, single walled carbon nanotubes, multi-walled carbon nanotubes, or single wall carbon nanohorns (SWNHs) are introduced into the tissue[1]. Quantitative methods for measuring tumor response to nanoparticle enhanced laser therapies are critical for determining appropriate laser parameters and nanoparticle properties needed to achieve maximum therapeutic benefit. We have previously reported a new method for measuring two dimensional (2D) spatial viability distributions in cell monolayers in response to laser irradiation and nanoparticles. This method has been refined to allow determination of cell viability in three dimensions (3D) within a more physiologically representative tumor volume. This refined method was used to determine the viability of breast cancer cells suspended within sodium alginate tissue phantoms following treatment with SWNHs and external laser irradiation. The tumor treatment volume was accurately quantified in response to varying laser treatment parameters and nanoparticle concentrations. Spatial cellular viability was also measured in ex vivo pig bladders in response to SWNHs and laser irradiation to provide a more anatomically relevant environment. These new measurement methods enable quantification of spatial viability and therapeutic effectiveness, using 3D tumor environments which are more representative than cell monolayers.Copyright © 2012 by ASME

The clinical need for alternatives to autologous vein and artery grafts for small-diameter vascular reconstruction have led researches to a tissue-engineering approach. Bioengineered vascular grafts provide a mechanically robust conduit for blood flow while implanted autologous cells remodel the construct to form a fully functional vessel [1]. A typical tissue-engineering approach involves fabricating a vascular scaffold from natural or synthetic materials, seeding the lumen of a vessel with endothelial cells (EC) and the vessel wall with smooth muscle cells or fibroblasts to mimic the functional properties of a native vessel. The cell-seeded vascular scaffold is then preconditioned in vitro using a pulsatile bioreactor to mimic in vivo conditions to enhance vessel maturation before implantation (Fig. 1).Copyright © 2012 by ASME