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Robert T Tranquillo

Publications and source records attributed to Robert T Tranquillo.

14 recordsLinked to original sources
PubMed2006

Cytokine-induced differentiation of multipotent adult progenitor cells into functional smooth muscle cells.

Smooth muscle formation and function are critical in development and postnatal life. Hence, studies aimed at better understanding SMC differentiation are of great importance. Here, we report that multipotent adult progenitor cells (MAPCs) isolated from rat, murine, porcine, and human bone marrow demonstrate the potential to differentiate into cells with an SMC-like phenotype and function. TGF-beta1 alone or combined with PDGF-BB in serum-free medium induces a temporally correct expression of transcripts and proteins consistent with smooth muscle development. Furthermore, SMCs derived from MAPCs (MAPC-SMCs) demonstrated functional L-type calcium channels. MAPC-SMCs entrapped in fibrin vascular molds became circumferentially aligned and generated force in response to KCl, the L-type channel opener FPL64176, or the SMC agonists 5-HT and ET-1, and exhibited complete relaxation in response to the Rho-kinase inhibitor Y-27632. Cyclic distention (5% circumferential strain) for 3 weeks increased responses by 2- to 3-fold, consistent with what occurred in neonatal SMCs. These results provide evidence that MAPC-SMCs are phenotypically and functionally similar to neonatal SMCs and that the in vitro MAPC-SMC differentiation system may be an ideal model for the study of SMC development. Moreover, MAPC-SMCs may lend themselves to tissue engineering applications.

Animals↗
PubMed2006

Endothelialization and flow conditioning of fibrin-based media-equivalents.

It is generally accepted that endothelialization and subsequent development of a functional endothelium are of paramount importance to the success of any bioartificial artery. In this study, we aimed to assess the ability of smooth muscle cell-remodeled, fibrin-based media-equivalents (MEs) to be endothelialized, examine the morphological changes of endothelial cells (ECs) associated with exposure to physiologically-relevant shear stress in a custom-built bioreactor, and determine if adherent ECs are capable of withstanding average physiological shear stresses. It was found that MEs could be readily endothelialized with surface coverages of 98.8 +/- 0.9% after two days, and the ECs expressed von Willebrand factor. Furthermore, EC retention remained high (steady: 96.5 +/- 4.4%, pulsatile: 94.3 +/- 4.3%) under exposure to physiologically relevant shear stresses for 48 h. The results indicate that these MEs are conducive to generating an EC monolayer, with the ECs possessing adhesion strength sufficient to withstand physiological shear stress and maintain a normal phenotype.

Adaptation, Physiological↗
PubMed2006

Small-diameter artificial arteries engineered in vitro.

Although the need for a functional arterial replacement is clear, the lower blood flow velocities of small-diameter arteries like the coronary artery have led to the failure of synthetic materials that are successful for large-diameter grafts. Although autologous vessels remain the standard for small diameter grafts, many patients do not have a vessel suitable for use because of vascular disease, amputation, or previous harvest. As a result, tissue engineering has emerged as a promising approach to address the shortcomings of current therapies. Investigators have explored the use of arterial tissue cells or differentiated stem cells combined with various types of natural and synthetic scaffolds to make tubular constructs and subject them to chemical and/or mechanical stimulation in an attempt to develop a functional small-diameter arterial replacement graft with varying degrees of success. Here, we review the progress in all these major facets of the field.

Animals↗
PubMed2006

Cell sourcing and culture conditions for fibrin-based valve constructs.

Cell sourcing for tissue-engineered heart valves remains a critical issue. In this work, human dermal fibroblasts (HDF) or porcine valve interstitial cells (PVIC) were entrapped in adherent fibrin disk constructs and harvested at 3 and 5 weeks. We compared the fibrin remodeling abilities of each cell type in Dulbecco's Modified Eagle's Medium (DMEM) and DMEM/F12 supplemented with transforming growth factor beta (TGF), and the response of PVIC to DMEM/F12 supplemented with fibroblast growth factor (FGF), a combination of FGF and TGF, and TGF with varying ascorbic acid (AA) concentrations. Culture media were supplemented with serum, insulin, AA, a fibrinolysis inhibitor, and antibiotics. DMEM maximized collagen and elastin deposition by HDF, while DMEM/F12 with FGF yielded the highest fibrin remodeling response by PVIC. HDF degraded fibrin slower than PVIC, and PVIC constructs had higher cellularity than HDF constructs in DMEM and DMEM/F12 at 3 weeks. FGF addition increased collagen content, collagen deposited per cell, and collagen as percentage of total protein compared to medium supplemented with TGF or TGF and FGF. AA addition increased collagen deposition by PVIC, but there was no dose dependence between 50 and 150 microg/mL AA. These results collectively show that PVIC are able to remodel fibrin faster and exhibit greater mechanical stiffening compared to HDF. Conditions for increased collagen deposition are also identified toward the engineering of valve constructs.

Animals↗
PubMed2006

Tissue-engineered valves with commissural alignment.

We present a novel approach to producing bioartificial valves using the tissue-equivalent method of entrapping cells within a biopolymer gel and using a mold design that presents appropriate mechanical constraints to the cell-induced gel compaction to yield both the fibril alignment and the geometry of a native valve. Bileaflet valves were fabricated from bovine collagen and neonatal human dermal fibroblasts as proof of principle. The resultant valves possessed both commissure-tocommissure alignment of collagen fibers in the leaflets and circumferential alignment in the root. While this alignment was manifested in planar biaxial tensile mechanical properties, histology of the leaflets revealed an aligned collagen matrix but lacking other extracellular matrix (ECM) components present in the native valve. The apparent lack of ECM production by the fibroblasts after contracting and aligning the collagen fibrils is consistent with peak loads during biaxial testing being only approximately 10% of native leaflet values and a 0:1 coupling index that was only approximately 50% of native leaflet values despite exhibiting comparable values for the anisotropy index.

Animals↗
PubMed2003

Temporal variations in cell migration and traction during fibroblast-mediated gel compaction.

Current models used in our laboratory to assess the migration and traction of a population of cells within biopolymer gels are extended to investigate temporal changes in these parameters during compaction of mechanically constrained gels. The random cell migration coefficient, micro (t) is calculated using a windowing technique by regressing the mean-squared displacement of cells tracked at high magnification in three dimensions with a generalized least squares algorithm for a subset of experimental time intervals, and then shifting the window interval-by-interval until all time points are analyzed. The cell traction parameter, tau(0)(t), is determined by optimizing the solution of our anisotropic biphasic theory to tissue equivalent compaction. The windowing technique captured simulated sinusoidal and step changes in cell migration superposed on a persistent random walk in simulated cell movement. The optimization software captured simulated time dependence of compaction on cell spreading. Employment of these techniques on experimental data using rat dermal fibroblasts (RDFs) and human foreskin fibroblasts (HFFs) demonstrated that these cells exhibit different migration-traction relationships. Rat dermal fibroblast migration was negatively correlated to traction, suggesting migration was not the driving force for compaction with these cells, whereas human foreskin fibroblast migration was positively correlated to traction.

Algorithms↗
PubMed2003

Elastic fiber production in cardiovascular tissue-equivalents.

Elastic fiber incorporation is critical to the success of tissue-engineered arteries and heart valves. Elastic fibers have not yet been observed in tissue-engineered replacements fabricated in vitro with smooth muscle cells. Here, rat smooth muscle cells (SMC) or human dermal fibroblasts (HDF) remodeled collagen or fibrin gels for 4 weeks as the basis for a completely biological cardiovascular tissue replacement. Immunolabeling, alkaline extraction and amino acid analysis identified and quantified elastin. Organized elastic fibers formed when neonatal SMC were cultured in fibrin gel. Fibrillin-1 deposition occurred but elastin was detected in regions without fibrillin-1, indicating that a microfibril template is not required for elastic fiber formation within fibrin. Collagen did not support substantial elastogenesis by SMC. The quantity of crosslinked elastic fibers was enhanced by treatment with TGF-beta1 and insulin, concomitant with increased collagen production. These additives overcame ascorbate's inhibition of elastogenesis in fibrin. The elastic fibers that formed in fibrin treated with TGF-beta1 and insulin contained crosslinks, as evidenced by the presence of desmosine and an altered elastin labeling pattern when beta-aminopropionitrile (BAPN) was added. These findings indicate that in vitro elastogenesis can be achieved in tissue engineering applications, and they suggest a physiologically relevant model system for the study of three-dimensional elastic structures.

Amino Acids↗
PubMed2003

Cryopreservation of collagen-based tissue equivalents. I. Effect of freezing in the absence of cryoprotective agents.

The effect of freezing on the viability and mechanical properties of tissue-equivalents (TEs) was determined under a variety of cooling conditions, with the ultimate aim of optimizing the cryopreservation process. TEs (a class of bioartificial tissues) were prepared by incubating entrapped human foreskin fibroblasts in collagen gels for a period of 2 weeks. TEs were detached from the substrate and frozen in phosphate-buffered saline using a controlled rate freezer (CRF) at various cooling rates (0.5, 2, 5, 20, and 40 degrees C/min to -80 or -160 degrees C) or in a directional solidification stage (DSS) (5 degrees C/min to -80 degrees C) or slam frozen (>1000 degrees C/min). Viability of the fibroblasts in the TEs was assessed by ethidium homodimer and Hoechst assays immediately after thawing. Uniaxial tension experiments were also performed on an MTS (Eden Prairie, MN) Micro Bionix system to assess the postthaw mechanical properties of the frozen-thawed TEs. Cooling rates of either 2 or 5 degrees C/min using the CRF were optimal for preserving both immediate cell viability and mechanical properties of the TEs, postthaw. By 72 h postthaw, TEs frozen in the CRF at 5 degrees C/min to -80 degrees C showed a slight decrease in cell viability, with a significant increase in tangent modulus and ultimate tensile stress suggesting a cell-mediated recovery mechanism. Both the postthaw mechanical properties and cell viability are adversely affected by freezing to the lower end temperature of -160 degrees C. Mechanical properties are adversely affected by freezing in the DSS.

Cell Culture Techniques↗
PubMed2003

Long-term cyclic distention enhances the mechanical properties of collagen-based media-equivalents.

In this study, we sought to identify the key parameters involved in long-term cyclic distension (CD) as they pertain to the development of collagen-based media-equivalents (MEs). By using only highly compacted, cross-linked constructs, we avoided the complicating issues of irrecoverable creep and transient alignment, and isolated the effects of cyclic mechanical loading on ME development. Our system allowed us to study this development over a wide range of parameters including strain amplitude, pulse frequency, pulse shape, and culture time. We found that in most cases involving cyclic distension, MEs were both stronger and stiffer than constructs that were grown under static conditions. The mechanical properties were not significantly different from static controls after two weeks of CD, however, five weeks of CD was sufficient to note significant increases in both stiffness and strength. The strain, stretch time, and relaxation time were all important variables in determining ME mechanical properties. While we were unable to detect a significant net change in the amount of total collagen, we observed significant deposition of insoluble elastin in our CDMEs, something that has never been previously reported using adult smooth muscle cells. Finally, these changes in ME development did not depend on the age of the MEs prior to the initiation of CD.

Adaptation, Physiological↗
PubMed2002

A novel implantable collagen gel assay for fibroblast traction and proliferation during wound healing.

BACKGROUND: A novel implantable assay for studying cellular behavior in the wound environment was developed. The assay is unique in that it combines the more quantitative nature of in vitro assays with the greater physiological relevance of in vivo wound healing models. MATERIALS AND METHODS: Cells were seeded in a physiologically relevant biological matrix, a collagen gel, contained within a semipermeable tube, and then exposed to soluble factors of the wound environment at different stages of the wound healing response. Gels were harvested at prescribed time points, and cell proliferation rates and gel compaction were measured. These data were combined with our theory for cell-matrix mechanical interactions to estimate the cell traction exerted by the cells leading to gel compaction. Cell morphology and alpha-smooth muscle actin expression were also characterized. RESULTS: The proliferation of and traction exerted by fibroblasts exposed to the soluble wound environment were different from those in similar collagen gels maintained in culture in complete medium. Proliferation and traction also varied over the course of the wound healing response. Traction was higher and proliferation lower in day 1-5 wounds compared to day 7-11 wounds. Recovered cells no longer stained for alpha-smooth muscle actin, in contrast to cells maintained in culture. CONCLUSIONS: Changes in the soluble wound environment that occur as the wound healing response proceeds alter fibroblast traction and migration. We have developed a new assay that employs a physiologically relevant biological matrix and allows the effects of the dynamic soluble wound environment on cellular traction, proliferation, and other phenomena such as protein expression to be quantified.

Actins↗
PubMedDate not supplied

Cryopreservation of collagen-based tissue equivalents. II. Improved freezing in the presence of cryoprotective agents.

In Part I of this study we determined an optimal cooling rate for cryopreservation of collagen-based tissue equivalents (TEs) that preserves both the postthaw cell viability and mechanical properties, but results in tissue contraction and an overall loss of opacity. The empirically determined optimal cooling rate (5 degrees C/min) was obtained in a freezing medium consisting solely of phosphate-buffered saline (PBS) at physiological concentration (1x). In the present study we report the effect of freezing on TEs in the presence of PBS and two cryoprotective agents (CPAs) (glycerol and dimethyl sulfoxide [Me(2)SO]), at two different concentrations (0.5 and 1.0 M), to two different end temperatures (-80 and -160 degrees C), at a cooling rate of 5 degrees C/min. The controlled rate freezing experiments, postthaw cell viability, and mechanical property measurements were performed as described in Part I of this study. In addition to studying the effect of CPAs on the postthaw properties of TEs, we also investigated (1). the effect of freezing TEs attached to the substrate (as opposed to detached and floating in medium) to determine differences when freezing TEs subject to static mechanical stress via a mechanical constraint to contraction; (2). the effect of freezing glutaraldehyde-fixed TEs to determine differences in freezing-mediated damage to the microstructure; and (3). the effect of freezing more mature TEs that were incubated for 4 weeks in growth factor-supplemented medium as opposed to 2 weeks in basal medium. All TEs frozen at 5 degrees C/min to -80 degrees C in the presence of 0.5 M glycerol or Me(2)SO in PBS were found to be optimally cryopreserved in terms of maintaining opacity and structure as well as cell viability and mechanical properties as compared with unfrozen TEs. The postthaw mechanical properties were adversely affected by freezing to the lower end temperature of -160 degrees C in the presence of CPAs, with the samples frozen in the 1.0 M concentration of CPAs exhibiting a total loss of structural integrity on thawing. Furthermore, TEs frozen attached to the substrate showed decreased opacity and significant contraction as compared with TEs frozen detached from the substrate, as did cross-linked samples frozen without CPA.

Collagen↗
PubMedDate not supplied

Fiber alignment imaging during mechanical testing of soft tissues.

A method to image fiber alignment during mechanical testing of soft tissues was developed based on quantitative polarized light microscopy. Images were acquired after passing light through a rotating polarizer, a tissue sample, and an effective circular analyzer at multiple polarizer positions during uniaxial mechanical testing. The image set was analyzed offline using harmonic analysis to generate an alignment image, which contains the direction and strength of alignment at each image pixel. Alignment images of the entire tissue sample were generated every 3-5 s during the mechanical test allowing stress-strain behavior to be correlated with fiber alignment. Loading of fresh tissue-equivalent samples in the direction normal to the initial direction of fiber alignment revealed a spatially inhomogeneous realignment into the loading direction, with most realignment occurring near the free edges undergoing maximum lateral contraction and prior to significant load developing. Glutaraldehyde-fixed samples, in contrast, showed little realignment until yielding occurred.

Animals↗