Three Dimensional OCT in the Engineering of Tissue Constructs: A Potentially Powerful Tool for Assessing Optimal Scaffold Structure.

Optical Coherence Tomography (OCT) provides detailed, real-time information on the structure and composition of constructs used in tissue engineering. The focus of this work is the OCT three-dimensional assessment of scaffolding architecture and distribution of cells on it. PLGA scaffolds were imaged in two and three-dimensions, both seeded and unseeded with cells. Then two types of scaffolds were reconstructed in three dimensions. Both scaffolding types were examined at three different seeding densities. The importance of three-dimensional assessments was evident, particularly with respect to porosity and identification of asymmetrical cell distribution.

Three-dimensional visualization of microvessel architecture of whole-mount tissue by confocal microscopy.

The three-dimensional architecture of the nascent microvascular network is a critical determinant of vascular perfusion in the setting of regenerative growth, vasculopathies and cancer. Current methods for microvessel visualization are limited by insufficient penetration and instability of endothelial immunolabels, inadequate vascular perfusion by the high-viscosity polymers used for vascular casting, and destruction of tissue stroma during the processing required for scanning electron microscopy. The aim of this study was to develop whole-mount tissue processing methods for 3D in situ visualization of the microvasculature that were also compatible with supplementary labeling for other structures of interest in the tissue microenvironment. Here, we present techniques that allow imaging of the microvasculature by confocal microscopy, to depths of up to 1500 mum below the specimen surface. Our approach includes labeling luminal surfaces of endothelial cells by i.v. injection of fluorescently conjugated lectin and filling the microvasculature with carbon or fluorescent nanoparticles/Mercox, followed by optical clearing of thick tissue sections to reduce light scatter and permit 3D visualization of microvessel morphology deep into the sample. Notably, tissue stroma is preserved, allowing simultaneous labeling of other structures by immunohistochemistry or nuclear dyes. Results are presented for various murine tissues including fat, muscle, heart and brain under conditions of normal health, as well as in the setting of a glioma model growing in the subcutaneous space or orthotopically in the brain parenchyma.

A novel immunoadsorption device for removing beta2-microglobulin from whole blood.

BACKGROUND: High plasma levels of beta2-microglobulin (beta2m) have been implicated in the formation of the severely destructive and potentially fatal amyloid deposits that are characteristic of dialysis-related amyloidosis (DRA). Conventional renal replacement technologies remove insufficient quantities of beta2m to normalize plasma levels. This limitation arises because of nonspecific adsorptive qualities and reliance on size exclusion, which can also remove other middle molecular weight proteins. These nonspecific approaches also make it difficult to evaluate the role and contribution of middle molecular weight molecules to the pathology of DRA and other morbidities of end-stage renal disease. A high-affinity and biologically specific approach could target a protein, prevent a significant loss of other important molecules, and improve the apparent adsorption rate within an extracorporeal device. METHODS: Agarose-immobilized murine anti-human beta2m monoclonal antibodies were used in a Vortex Flow Plasmapheretic Reactor (VFPR) to remove donor baseline and controlled amounts of recombinant beta2m from human blood in vitro. The extracorporeal circuit was hemoperfused at 200 mL/min for two hours. RESULTS: The immunoadsorptive media had a binding site density of 30 microg beta2m per mL of settled gel. The VFPR cleared baseline quantities of donor beta2m below detectable limits of the assay. The experiments with higher initial beta2m concentrations reached an equilibrium concentration within 20 minutes, corresponding to a 92% clearance. No deleterious hemocompatibility issues were observed (complete blood count, total protein, and plasma free hemoglobin). CONCLUSIONS: The adsorptive kinetics of the VFPR are optimal for the conditions used and support the use of immunoadsorption for the removal of beta2m.

Cardiac tissue engineering: cell seeding, cultivation parameters, and tissue construct characterization.

Cardiac tissue engineering has been motivated by the need to create functional tissue equivalents for scientific studies and cardiac tissue repair. We previously demonstrated that contractile cardiac cell-polymer constructs can be cultivated using isolated cells, 3-dimensional scaffolds, and bioreactors. In the present work, we examined the effects of (1) cell source (neonatal rat or embryonic chick), (2) initial cell seeding density, (3) cell seeding vessel, and (4) tissue culture vessel on the structure and composition of engineered cardiac muscle. Constructs seeded under well-mixed conditions with rat heart cells at a high initial density ((6-8) x 10(6) cells/polymer scaffold) maintained structural integrity and contained macroscopic contractile areas (approximately 20 mm(2)). Seeding in rotating vessels (laminar flow) rather than mixed flasks (turbulent flow) resulted in 23% higher seeding efficiency and 20% less cell damage as assessed by medium lactate dehydrogenase levels (p < 0.05). Advantages of culturing constructs under mixed rather than static conditions included the maintenance of metabolic parameters in physiological ranges, 2-4 times higher construct cellularity (p &le 0.0001), more aerobic cell metabolism, and a more physiological, elongated cell shape. Cultivations in rotating bioreactors, in which flow patterns are laminar and dynamic, yielded constructs with a more active, aerobic metabolism as compared to constructs cultured in mixed or static flasks. After 1-2 weeks of cultivation, tissue constructs expressed cardiac specific proteins and ultrastructural features and had approximately 2-6 times lower cellularity (p < 0.05) but similar metabolic activity per unit cell when compared to native cardiac tissue.

The antiangiogenic agents TNP-470 and 2-methoxyestradiol inhibit the growth of angiosarcoma in mice.

BACKGROUND: Endothelial malignancies, such as angiosarcoma and hemangioendothelioma, are often resistant to chemotherapy and surgery, and may result in death. Improved means of therapy are needed for these disorders. OBJECTIVE: We wanted to determine whether angiosarcoma can be treated with angiogenesis inhibitors in mice. METHODS: Mice were inoculated with a cell line that gives rise to angiosarcoma and were treated with the angiogenesis inhibitors 2-methoxyestradiol and TNP-470. Response to therapy was monitored by measurement of tumors. RESULTS: TNP-470 caused an 84% reduction in tumor size, and 2-methoxyestradiol caused a 68% reduction in tumor size. CONCLUSION: Angiogenesis inhibitors are highly effective in treatment of angiosarcoma in mice. Clinical trials of these agents in humans with angiosarcoma and hemangioendothelioma are warranted.

Microvessel count and cerebrospinal fluid basic fibroblast growth factor in children with brain tumours.

Tumour growth is angiogenesis-dependent; brain tumours have more intense neovascularisation than other tumours and produce basic fibroblast growth factor, a potent angiogenic mediator. Because little is known about the release of basic fibroblast growth factor from brain tumours into extracellular fluids, we tested cerebrospinal fluid (CSF) from 26 children and young adults with brain tumours and 18 controls for basic fibroblast growth factor and for proliferative activity on cultured capillary endothelial cells. We also measured the density of microvessels in tumours by immunohistochemical staining. Basic fibroblast growth factor was detected in the CSF of 62% (16 of 26) patients with brain tumours but in none of the controls. Specimens with basic fibroblast growth factor stimulated DNA synthesis of capillary endothelial cells in vitro. Endothelial proliferative activity was blocked by neutralising antibodies to basic fibroblast growth factor. Basic fibroblast growth factor correlated with mitogenic activity in CSF in vitro (p < or = 0.0001), and with density of microvessels in histological sections (p < or = 0.005). A microvessel count of > or = 68 per 200 x field was associated with tumour recurrence (p = 0.005) and with mortality (p = 0.02). Basic fibroblast growth factor in brain tumours may mediate angiogenesis as measured by microvessel density in histological sections, so has potential as both a marker for neoplasia and a target for tumour treatments. Furthermore, evaluation of cerebrospinal fluid basic fibroblast growth factor, along with microvessel quantitation in biopsied tumours, may provide improved prognostic information for the management of patients with brain tumours.

Chemotaxis of human microvessel endothelial cells in response to acidic fibroblast growth factor.

Migration of microvessel endothelial cells (MEC) in response to angiogenic stimuli is a key aspect of angiogenesis, whether in physiologic or pathologic situations. In this work, we provide a rigorous quantitative assessment of the chemokinetic and chemotactic responses of human MEC to acidic fibroblast growth factor (aFGF). A uniform concentration of 1 micrograms/ml of heparin was included in most experiments to exploit heparin's potentiating effect on aFGF activity. The migration is measured in an under-agarose assay with a linear geometry, and evaluated in terms of the random motility and chemotaxis coefficients, mu and chi, which are defined in a mathematical model. The change in value of mu with changes in aFGF concentration provides a quantitative description of the stimulated random motility response, a process known as chemokinesis. This allows the true directional response in gradients to be quantified by the chemotaxis coefficient, chi, and its variation with attractant concentration. The effect of aFGF on MEC random motility is relatively small, with the random motility coefficient ranging from 4.6 +/- 0.4 x 10(-9) to 9.9 +/- 0.3 x 10(-9) cm2/second (mean +/- SE) over four orders of magnitude of aFGF concentration (10(-11) to 10(-8) M). On the other hand, the magnitude of the chemotaxis coefficient at optimal concentrations is quite large (2600 +/- 750 cm2/second-M around 10(-10) M aFGF), demonstrating a significant degree of MEC directional sensitivity to aFGF gradients. The chemotaxis coefficient shows a biphasic dependence on aFGF concentration, suggestive of a receptor-mediated response in which apparent differences in receptor occupancy govern directional bias. These results provide support for the hypothesis that MEC chemotaxis accounts for the directed microvessel growth observed in angiogenesis.

Endothelialization of vascular prosthetic surfaces after seeding or sodding with human microvascular endothelial cells.

The rapid establishment of an endothelial cell (EC) monolayer on the luminal surface of small-diameter vascular grafts may be necessary to prevent early thrombosis and failure. We have studied procedures used to promote EC coverage of vascular grafts and have compared preclotting prosthetic surfaces with ECs in platelet-rich plasma (seeding) with plating ECs onto a preestablished clot (sodding). We evaluated the rate of monolayer formation, the subsequent resistance to shear stress, and the effects of EC growth factors (ECGF and heparin) on these functions. Woven Dacron was seeded or sodded at a density of 2 x 10(5) cells/cm2 with human adult microvessel ECs derived from adipose tissue. In the presence of ECGF-heparin, the immediate establishment of an EC layer after sodding was observed, whereas seeded grafts required almost 48 hours for cells to reach the surface. In the absence of ECGF-heparin, sodded grafts still exhibited a complete monolayer of EC, whereas ECs were not observed at the surface of seeded grafts after 48 hours. After exposure to shear stress (up to 20 dynes/cm2) for 2 hours, most freshly sodded EC remained attached; however, the loss of loosely adherent cells did occur. EC seeded grafts remained covered with fibrin matrix after exposure to shear stress. We conclude that the use of a microvessel sodding technique as an alternative to previously reported seeding techniques is necessary for the immediate formation of an EC monolayer before implantation.

Quantitative analysis of random motility of human microvessel endothelial cells using a linear under-agarose assay.

Angiogenesis is a multistep process intimately involved in embryonic development and subsequent cardiovascular homeostasis and pathology. A major event in the process of angiogenesis is endothelial cell migration. Common in vitro assays (filter, under-agarose, phagokinetic track) used for the evaluation of migration are interpreted by measurements such as leading front distance, total cells migrated, and total area of migration. However, these quantities depend very heavily upon the physical aspects of the assay such as geometry, chemoattractant concentration and diffusivity, and observation time. Thus, while these common cell motility measurements are convenient, they do not represent solely the intrinsic cell response to an attractant. Alternatively, cell motility responses can be described by parameters which do not depend on the physical aspects of the assay system. Such parameters, termed phenomenologic parameters, have been defined for cell migration in a mathematical model derived by others. This model defines two parameters, the random motility coefficient, mu, and the chemotaxis coefficient, chi, which describe the migration responses to uniform concentrations and to gradients of stimulant, respectively. We have used this approach to evaluate the random motility response of human microvessel endothelial cells isolated from omental fat. Human microvessel endothelial cell random motility was measured in uniform concentrations of heparin (10(-3) to 10(3) micrograms/ml) using an under-agarose assay with linear geometry. The value of mu was found to remain constant at 8.2 x 10(-9) cm2/second for all concentrations tested and without heparin. These data indicate that heparin at these concentrations does not significantly stimulate random migration of human microvessel endothelial cell. These results suggest that the potentiating effect of heparin on angiogenesis may not be mediated through a direct affect on endothelial cell migration. Because the random motility coefficient and chemotaxis coefficient are representative of intrinsic cell motility behavior, their use should provide more specific information on endothelial cell migration than other commonly used measurements.

Phenotypic diversity in cultured cerebral microvascular endothelial cells.

Diversity exists in both the structure and function of the endothelial cells (EC) that comprise the microvasculature of different organs. Studies of EC have been aided by our ability to first isolate and subsequently establish cultures from microvascularized tissue. After the isolation of microvessel endothelial cells (MEC) derived from rat cerebrum, we observed morphologic differences in colonies of cells that grew in primary cultures. The morphologies ranged from a cobblestone phenotype considered typical of EC in culture to elongated and stellate cell appearances. Serially passaged cell lines were established based on two parameters: initially by growth and, seconds, on differences in primary colony morphology using selective weeding techniques. Each culture was examined for the presence of EC-characteristic markers which include Factor-VIII-related antigen, angiotensin-I-converting enzyme activity, collagen type IV synthesis, and PGI2 production. Variable expression of each of these characteristics among the established EC lines was observed. Growth curves established for each of the EC cultures demonstrated differences in both population doubling rates and cell densities at confluence. The endocytic capacity of each EC line was also evaluated. Our ability to isolate and establish a number of morphologically distinct EC cultures indicates that diversity exists within the EC that comprise the cerebral microvasculature. Diversity in the established cell lines suggests either the EC that line the brain microvasculature exist as a mosaic or that morphologically distinct cultures may originate from different microanatomical origins (arteriolar, true capillary, or venular) or may have resulted from cells at different points in their in vitro life spans at the time of isolation.

Kinetics of endothelial cell-surface attachment forces.

Physical and biochemical forces exist that are necessary for the persistent attachment and function of ECs on native and prosthetic blood vessels. The optimization of conditions that permit regeneration of these attachment forces may allow rapid establishment of a durable, biocompatible EC monolayer. We examined the effects of three major factors, protein substrate, EC incubation time, and shear stress, on the attachment kinetics of human adult ECs to two different polymers. ECs were incubated up to 30 minutes on polymers (PS or PET) coated with extracellular matrix proteins: collagen I/III, fibronectin, collagen IV/V, laminin, gelatin, or saline control. After incubation, continued attachment in the presence of shear stress (created in a rotating disc device) between zero and 90 dynes/cm2 for 30 minutes was evaluated. Maximal adherence was observed on all substrates by 30 minutes. Therefore, after a 30-minute incubation, the percentage of cells attached (postshear ECs/preshear ECs/preshear ECs X 100) was measured as a function of shear stress. ECs attached to a matrix of fibronectin or collagen I/III demonstrated shear-resistant adherence after as little as 5 minutes of static incubation before initial shear exposure. By 30 minutes, more than 90% of the ECs on both matrices demonstrated the ability to remain attached in the presence of 90 dynes/cm2 of shear stress. We conclude that forces that attach ECs to surfaces are affected by temporal factors (incubation time) and substrate composition and may be quantified with a defined shear stress detachment assay. Understanding and manipulating these temporal physiochemical parameters should allow one to re-create an optimal EC monolayer on a blood-contacting surface.

Rat brain microvessel extracellular matrix modulates the phenotype of cultured rat type 1 astroglia.

Astroglia from immature rat cerebral white matter which were plated on the insoluble extracellular matrix (ECM) secreted by rat cerebral microvessel endothelial cells (RCMEC) and maintained in a defined medium were induced to become stellate and to express glutamine synthetase. These effects were not elicited by RCMEC-conditioned medium. ECM secreted by rat pleural mesothelium elicited a lesser proportion of stellate astroglia and did not induce glutamine synthetase.