Publications by authors named "Robert T Tranquillo"

72 Publications

In vitro models of angiogenesis and vasculogenesis in fibrin gel.

Exp Cell Res 2013 Oct 22;319(16):2409-17. Epub 2013 Jun 22.

Departments of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA.

In vitro models of endothelial assembly into microvessels are useful for the study of angiogenesis and vasculogenesis. In addition, such models may be used to provide the microvasculature required to sustain engineered tissues. A large range of in vitro models of both angiogenesis and vasculogenesis have utilized fibrin gel as a scaffold. Although fibrin gel is conducive to endothelial assembly, its ultrastructure varies substantially based on the gel formulation and gelation conditions, making it challenging to compare between models. This work reviews existing models of endothelial assembly in fibrin gel and posits that differerences between models are partially caused by microstructural differences in fibrin gel.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.yexcr.2013.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3919069PMC
October 2013

A multiscale approach to modeling the passive mechanical contribution of cells in tissues.

J Biomech Eng 2013 Jul;135(7):71007

Department of Chemical Engineering and Materials Science, University of Minnesota–Twin Cities, Minneapolis, MN 55455, USA.

In addition to their obvious biological roles in tissue function, cells often play a significant mechanical role through a combination of passive and active behaviors. This study focused on the passive mechanical contribution of cells in tissues by improving our multiscale model via the addition of cells, which were treated as dilute spherical inclusions. The first set of simulations considered a rigid cell, with the surrounding ECM modeled as (1) linear elastic, (2) Neo-Hookean, and (3) a fiber network. Comparison with the classical composite theory for rigid inclusions showed close agreement at low cell volume fraction. The fiber network case exhibited nonlinear stress-strain behavior and Poisson's ratios larger than the elastic limit of 0.5, characteristics similar to those of biological tissues. The second set of simulations used a fiber network for both the cell (simulating cytoskeletal filaments) and matrix, and investigated the effect of varying relative stiffness between the cell and matrix, as well as the effect of a cytoplasmic pressure to enforce incompressibility of the cell. Results showed that the ECM network exerted negligible compression on the cell, even when the stiffness of fibers in the network was increased relative to the cell. Introduction of a cytoplasmic pressure significantly increased the stresses in the cell filament network, and altered how the cell changed its shape under tension. Findings from this study have implications on understanding how cells interact with their surrounding ECM, as well as in the context of mechanosensation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1115/1.4024350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3705800PMC
July 2013

Influence of cyclic mechanical stretch and tissue constraints on cellular and collagen alignment in fibroblast-derived cell sheets.

Tissue Eng Part C Methods 2013 May 8;19(5):386-95. Epub 2013 Jan 8.

Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA.

Mechanical forces play an important role in shaping the organization of the extracellular matrix (ECM) in developing and mature tissues. The resulting organization gives the tissue its unique functional properties. Understanding how mechanical forces influence the alignment of the ECM is important in tissue engineering, where recapitulating the alignment of the native tissue is essential for appropriate mechanical anisotropy. In this work, a novel method was developed to create and stretch tubular cell sheets by seeding neonatal dermal fibroblasts onto a rotating silicone tube. We show the fibroblasts proliferated to create a confluent monolayer around the tube and a collagenous, isotropic tubular tissue over 4 weeks of static culture. These silicone tubes with overlying tubular tissue constructs were mounted into a cyclic distension bioreactor and subjected to cyclic circumferential stretch at 5% strain, 0.5 Hz for 3 weeks. We found that the tissue subjected to cyclic stretch compacted axially over the silicone tube in comparison to static controls, leading to a circumferentially aligned tissue with higher membrane stiffness and maximum tension. In a subsequent study, the tissue constructs were constrained against axial compaction during cyclic stretching. The resulting alignment of fibroblasts and collagen was perpendicular (axial) to the stretch direction (circumferential). When the cells were devitalized with sodium azide before stretching, similarly constrained tissue did not develop strong axial alignment. This work suggests that both mechanical stretching and mechanical constraints are important in determining tissue organization, and that this organization is dependent on an intact cytoskeleton.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/ten.TEC.2012.0423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3603568PMC
May 2013

Decellularized tissue-engineered heart valve leaflets with recellularization potential.

Tissue Eng Part A 2013 Mar 10;19(5-6):759-69. Epub 2012 Dec 10.

Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota, USA.

Tissue-engineered heart valves (TEHV) have been proposed as a promising solution for the clinical needs of pediatric patients. In vivo studies have shown TEHV leaflet contraction and regurgitation after several months of implantation. This has been attributed to contractile cells utilized to produce the extracellular matrix (ECM) during TEHV culture. Here, we utilized such cells to develop a mature ECM in a fibrin-based scaffold that generates commissural alignment in TEHV leaflets and then removed these cells using detergents. Further, we evaluated recellularization with potentially noncontractile cells. A tissue-engineered leaflet model was developed with mechanical anisotropy and tensile properties comparable to an ovine pulmonary valve leaflet. No change in tensile properties occurred after decellularization using 1% sodium dodecyl sulfate and 1% Triton detergent treatment. Cell removal was verified by DNA quantitation and western blot analysis for cellular proteins. Histological and scanning electron microscope imaging showed no significant change in the ECM organization and microstructure. We further tested the recellularization potential of decellularized leaflets by seeding human mesenchymal stem cells (hMSC) on the surface of the leaflets and evaluated them at 1 and 3 weeks in two culture conditions. One medium (M1) was chosen to maintain the MSC phenotype while a second medium (M2) was used to potentially differentiate cells to an interstitial cell phenotype. Cellular quantitation showed that the engineered leaflets were recellularized to the highest concentration with M2 followed by M1, with minimum cell invasion of decellularized native leaflets. Histology showed cellular invasion throughout the thickness of the leaflets in M2 and partial invasion in M1. hMSC stained positive for MSC markers, but also for α-smooth muscle actin in both media at 1 week, with no presence of MSC markers at 3 weeks with the exception of CD90. These results show that engineered leaflets, while having similar tensile properties and collagen content compared to native leaflets, have better recellularization potential.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/ten.TEA.2012.0365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3566676PMC
March 2013

Microstructural and mechanical differences between digested collagen-fibrin co-gels and pure collagen and fibrin gels.

Acta Biomater 2012 Nov 22;8(11):4031-42. Epub 2012 Jul 22.

Department of Chemical Engineering and Materials Science, University of Minnesota-Twin Cities, 421 Washington Avenue South East, Minneapolis, MN 55455, USA.

Collagen and fibrin are important extracellular matrix (ECM) components in the body, providing structural integrity to various tissues. These biopolymers are also common scaffolds used in tissue engineering. This study investigated how co-gelation of collagen and fibrin affected the properties of each individual protein network. Collagen-fibrin co-gels were cast and subsequently digested using either plasmin or collagenase; the microstructure and mechanical behavior of the resulting networks were then compared with the respective pure collagen or fibrin gels of the same protein concentration. The morphologies of the collagen networks were further analyzed via three-dimensional network reconstruction from confocal image z-stacks. Both collagen and fibrin exhibited a decrease in mean fiber diameter when formed in co-gels compared with the pure gels. This microstructural change was accompanied by an increased failure strain and decreased tangent modulus for both collagen and fibrin following selective digestion of the co-gels. In addition, analysis of the reconstructed collagen networks indicated the presence of very long fibers and the clustering of fibrils, resulting in very high connectivities for collagen networks formed in co-gels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.actbio.2012.07.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3462241PMC
November 2012

Mechanical behavior of collagen-fibrin co-gels reflects transition from series to parallel interactions with increasing collagen content.

J Biomech Eng 2012 Jan;134(1):011004

Department of Chemical Engineering and Materials Science, University of Minnesota - Twin Cities, Minneapolis, MN 55455, USA.

Fibrin and collagen, biopolymers occurring naturally in the body, are biomaterials commonly-used as scaffolds for tissue engineering. How collagen and fibrin interact to confer macroscopic mechanical properties in collagen-fibrin composite systems remains poorly understood. In this study, we formulated collagen-fibrin co-gels at different collagen-to-fibrin ratios to observe changes in the overall mechanical behavior and microstructure. A modeling framework of a two-network system was developed by modifying our micro-scale model, considering two forms of interaction between the networks: (a) two interpenetrating but noninteracting networks ("parallel"), and (b) a single network consisting of randomly alternating collagen and fibrin fibrils ("series"). Mechanical testing of our gels show that collagen-fibrin co-gels exhibit intermediate properties (UTS, strain at failure, tangent modulus) compared to those of pure collagen and fibrin. The comparison with model predictions show that the parallel and series model cases provide upper and lower bounds, respectively, for the experimental data, suggesting that a combination of such interactions exists between the collagen and fibrin in co-gels. A transition from the series model to the parallel model occurs with increasing collagen content, with the series model best describing predominantly fibrin co-gels, and the parallel model best describing predominantly collagen co-gels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1115/1.4005544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3717315PMC
January 2012

Hypoxic culture and insulin yield improvements to fibrin-based engineered tissue.

Tissue Eng Part A 2012 Apr 5;18(7-8):785-95. Epub 2011 Dec 5.

Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota 55455, USA.

We examined the effect of insulin supplementation and hypoxic culture (2% vs. 20% oxygen tension) on collagen deposition and mechanical properties of fibrin-based tubular tissue constructs seeded with neonatal human dermal fibroblasts. The results presented here demonstrate that constructs cultured under hypoxic conditions with insulin supplementation increased in collagen density by approximately five-fold and both the ultimate tensile strength (UTS) and modulus by more than three-fold compared with normoxic (20% oxygen tension), noninsulin supplemented controls. In addition, collagen deposited on a per-cell basis increased by approximately four-fold. Interaction was demonstrated for hypoxia and insulin in combination in terms of UTS and collagen production on a per-cell basis. This interaction resulted from two distinct processes involved in collagen fibril formation. Western blot analysis showed that insulin supplementation alone increased Akt phosphorylation and the combined treatment increased collagen prolyl-4-hydroxylase. These molecules are distinct regulators of collagen deposition, having an impact at both the transcriptional and posttranslational modification stages of collagen fibril formation that, in turn, increase collagen density in the tissue constructs. These findings highlight the potential of utilizing insulin supplementation and hypoxic culture in combination to increase the mechanical strength and stiffness of fibrin-based engineered tissues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/ten.TEA.2011.0017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3313606PMC
April 2012

Guided sprouting from endothelial spheroids in fibrin gels aligned by magnetic fields and cell-induced gel compaction.

Biomaterials 2011 Sep 1;32(26):6111-8. Epub 2011 Jun 1.

Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA.

An aligned engineered microvascular network is critical to the culture of thick or highly metabolic tissue in vitro due to the need for inlet and outlet sides for perfusion of the network. Contact guidance may be a way to achieve aligned networks, but the relationship between the alignment of endothelial sprouts and the alignment of extracellular matrix fibers has yet to be fully elucidated. The data presented here show that sprouts from human blood outgrowth endothelial cell spheroids align with fibrin fibrils, and that the extent to which the sprouts align depends upon the strength of the fibril alignment. This was true for both magnetically-aligned fibrin and fibrin aligned via cell-induced gel compaction, although magnetically-aligned fibrin was more effective over the same culture period. The data also demonstrate that longer sprouts are grown when the fibrils, and thus the sprouts, are more strongly aligned. The formation of aligned endothelial sprouts using these methods can be an essential step in the generation of aligned microvascular networks.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biomaterials.2011.05.018DOI Listing
September 2011

Shear stress responses of adult blood outgrowth endothelial cells seeded on bioartificial tissue.

Tissue Eng Part A 2011 Oct 1;17(19-20):2511-21. Epub 2011 Jul 1.

Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota 55455, USA.

Human blood outgrowth endothelial cells (HBOECs) are expanded from circulating endothelial progenitor cells in peripheral blood and thus could provide a source of autologous endothelial cells for tissue-engineered vascular grafts. To examine the suitability of adult HBOECs for use in vascular tissue engineering, the shear stress responsiveness of these cells was examined on bioartificial tissue formed from dermal fibroblasts entrapped in tubular fibrin gels. HBOECs adhered to this surface, deposited collagen IV and laminin, and remained adherent when exposed to 15 dyn/cm(2) shear stress for 24 h. The shear stress responses of HBOECs were compared to human umbilical vein endothelial cells (HUVECs). As with HUVECs, HBOECs upregulated vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 when exposed to tumor necrosis factor (TNF)-α and shear stress decreased the expression of these adhesion molecules on TNF-α-activated monolayers. Nitric oxide production was elevated by shear stress, but did not vary between cell types. Both cell types decreased platelet adhesion to the bioartificial tissue, whereas pre-exposing the cells to flow decreased platelet adhesion further. These results illustrate the potential utility for HBOECs in vascular tissue engineering, as not only do the cells adhere to bioartificial tissue and remain adherent under physiological shear stress, they are also responsive to shear stress signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/ten.TEA.2011.0055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3179622PMC
October 2011

TGF-β1 diminishes collagen production during long-term cyclic stretching of engineered connective tissue: implication of decreased ERK signaling.

J Biomech 2011 Mar 20;44(5):848-55. Epub 2011 Jan 20.

Department of Chemical Engineering & Materials Science, University of Minnesota, MN 55455, USA.

Cyclic stretching and growth factors like TGF-β have been used to enhance extracellular matrix (ECM) production by cells in engineered tissue to achieve requisite mechanical properties. In this study, the effects of TGF-β1 were evaluated during long-term cyclic stretching of fibrin-based tubular constructs seeded with neonatal human dermal fibroblasts. Samples were evaluated at 2, 5, and 7 weeks for tensile mechanical properties and ECM deposition. At 2 weeks, +TGF-β1 samples had 101% higher collagen concentration but no difference in ultimate tensile strength (UTS) or modulus compared to -TGF-β1 samples. However, at weeks 5 and 7, -TGF-β1 samples had higher UTS/modulus and collagen concentration, but lower elastin concentration compared to +TGF-β1 samples. The collagen was better organized in -TGF-β1 samples based on picrosirius red staining. Western blot analysis at weeks 5 and 7 showed increased phosphorylation of ERK in -TGF-β1 samples, which correlated with higher collagen deposition. The TGF-β1 effects were further evaluated by western blot for αSMA and SMAD2/3 expression, which were 16-fold and 10-fold higher in +TGF-β1 samples, respectively. The role of TGF-β1 activated p38 in inhibiting phosphorylation of ERK was evaluated by treating samples with SB203580, an inhibitor of p38 activation. SB203580-treated cells showed increased phosphorylation of ERK after 1 hour of stretching and increased collagen production after 1 week of stretching, demonstrating an inhibitory role of activated p38 via TGF-β1 signaling during cyclic stretching. One advantage of TGF-β1 treatment was the 4-fold higher elastin deposition in samples at 7 weeks. Further cyclic stretching experiments were thus conducted with constructs cultured for 5 weeks without TGF-β1 to obtain improved tensile properties followed by TGF-β1 supplementation for 2 weeks to obtain increased elastin content, which correlated with a reduction in loss of pre-stress during preconditioning for tensile testing, indicating functional elastin. This study shows that a sequential stimulus approach - cyclic stretching with delayed TGF-β1 supplementation - can be used to engineer tissue with desirable tensile and elastic properties.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jbiomech.2010.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3061833PMC
March 2011

Ruthenium-catalyzed photo cross-linking of fibrin-based engineered tissue.

Biomaterials 2011 Apr 31;32(10):2479-88. Epub 2010 Dec 31.

Department of Biomedical Engineering, University of Minnesota, 7-105 Nils Hasselmo Hall, 312 Church St., Minneapolis, MN 55455, United States.

Most cross-linking methods utilize chemistry or physical processes that are detrimental to cells and tissue development. Those that are not as harmful often do not provide a level of strength that ultimately meets the required application. The purpose of this work was to investigate the use of a ruthenium-sodium persulfate cross-linking system to form dityrosine in fibrin-based engineered tissue. By utilizing the tyrosine residues inherent to fibrin and cell-deposited proteins, at least 3-fold mechanical strength increases and 10-fold stiffness increases were achieved after cross-linking. This strengthening and stiffening effect was found to increase with culture duration prior to cross-linking such that physiologically relevant properties were obtained. Fibrin was not required for this effect as demonstrated by testing with collagen-based engineered tissue. Cross-linked tissues were implanted subcutaneously and shown to have minimal inflammation after 30 days, similar to non-cross-linked controls. Overall, the method employed is rapid, non-toxic, minimally inflammatory, and is capable of increasing strength and stiffness of engineered tissues to physiological levels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biomaterials.2010.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3791330PMC
April 2011

Implantable arterial grafts from human fibroblasts and fibrin using a multi-graft pulsed flow-stretch bioreactor with noninvasive strength monitoring.

Biomaterials 2011 Jan 8;32(3):714-22. Epub 2010 Oct 8.

Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA.

Tissue-engineered arteries based on entrapment of human dermal fibroblasts in fibrin gel yield completely biological vascular grafts that possess circumferential alignment characteristic of native arteries and essential to their mechanical properties. A bioreactor was developed to condition six grafts in the same culture medium while being subjected to similar cyclic distension and transmural flow resulting from pulsed flow distributed among the graft lumens via a manifold. The lumenal pressure and circumferential stretch were noninvasively monitored and used to calculate stiffness in the range of 80-120 mmHg and then to successfully predict graft burst strength. The length of the graft was incrementally shortened during bioreactor culture to maintain circumferential alignment and achieve mechanical anisotropy comparable to native arteries. After 7-9 weeks of bioreactor culture, the fibrin-based grafts were extensively remodeled by the fibroblasts into circumferentially-aligned tubes of collagen and other extracellular matrix with burst pressures in the range of 1400-1600 mmHg and compliance comparable to native arteries. The tissue suture retention force was also suitable for implantation in the rat model and, with poly(lactic acid) sewing rings entrapped at both ends of the graft, also in the ovine model. The strength achieved with a biological scaffold in such a short duration is unprecedented for an engineered artery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biomaterials.2010.09.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3042747PMC
January 2011

The matrix-binding domain of microfibril-associated glycoprotein-1 targets active connective tissue growth factor to a fibroblast-produced extracellular matrix.

Macromol Biosci 2010 Nov;10(11):1338-44

Department of Biomedical Engineering, University of Minnesota, 7-105 Hasselmo Hall, 312 Church St. SE, Minneapolis, MN 55455, USA.

It is advantageous to use biomaterials in tissue engineering that stimulate extracellular matrix (ECM) production by the cellular component. Connective tissue growth factor (CTGF) stimulates type I collagen (COL1A1) transcription, but is functionally limited as a free molecule. Using a matrix-binding domain (MBD) from microfibril-associated glycoprotein-1, the fusion protein MBD-CTGF was targeted to the ECM and tested for COL1A1 transcriptional activation. MBD-CTGF produced by the ECM-synthesizing fibroblasts, or provided exogenously, localized to the elastic fiber ECM. MBD-CTGF, but not CTGF alone, led to a two-fold enhancement of COL1A1 expression. This study introduces a targeting technology that can be used to elevate collagen transcription in engineered tissues and thereby improve tissue mechanics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/mabi.201000121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3157317PMC
November 2010

Fibrin degradation enhances vascular smooth muscle cell proliferation and matrix deposition in fibrin-based tissue constructs fabricated in vitro.

Tissue Eng Part A 2010 Oct;16(10):3261-70

Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota 55455, USA.

Completely biological tissue replacements can be fabricated by entrapping cells in a molded fibrin gel. Over time, the fibrin is degraded and replaced with cell-produced extracellular matrix. However, the relationship between fibrin degradation and matrix deposition has not been elucidated. We developed techniques to quantify fibrin degradation products (FDP) and examine plasmin activity in the conditioned medium from fibrin-based constructs. Fibrin-based tissue constructs fabricated with vascular smooth muscle cells (vSMC) were cultured for 5 weeks in the presence of varied concentrations of the fibrinolysis inhibitor -aminocaproic acid and cellularity, and deposited collagen and elastin were measured weekly. These data revealed that increasing concentrations of -aminocaproic acid led to delayed and diminished FDP production, lower vSMC proliferation, and decreased collagen and elastin deposition. FDP were shown to have a direct biological effect on vSMC cultures and vSMC within the fibrin-based constructs. Supplementing construct cultures with 250 or 500μg/mL FDP led to 30% higher collagen deposition than the untreated controls. FDP concentrations as high as 250μg/mL were estimated to exist within the constructs, indicating that FDP generation during remodeling of the fibrin-based constructs exerted direct biological activity. These results help explain many of the positive outcomes reported with fibrin-based tissue constructs in the literature, as well as demonstrate the importance of regulating plasmin activity during their fabrication.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/ten.tea.2009.0708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2947425PMC
October 2010

Fusion of concentrically layered tubular tissue constructs increases burst strength.

Ann Biomed Eng 2010 Jun 30;38(6):2226-36. Epub 2010 Apr 30.

Department of Biomedical Engineering, University of Minnesota, 7-114 Nils Hasselmo Hall, 312 Church Street SE, Minneapolis, MN 55455, USA.

Tubular tissue constructs prepared from neonatal human dermal fibroblasts entrapped in fibrin gel were incubated on a mandrel for three weeks to allow for initial fibrin remodeling into tissue before being concentrically layered and incubated for an additional three weeks on the mandrel. Upon harvest, double layer constructs were not statistically different from single layer control constructs in terms of length, collagen density, cell density, tensile modulus, or ultimate tensile strength. However, the thickness and burst pressure were both approximately twice the single layer control values. Metabolically active cells were detected at the interface, and scanning electron microscopy revealed fiber structures bridging the two layers, co-localizing with the cells, which exhibited minimal migration across the layers. In contrast, double layer constructs where tissue fusion was prohibited by mechanical distraction of the layers showed no increase in burst pressure despite having increased thickness and the same collagen and cell densities of the single layer control constructs; moreover, the burst failure occurred sequentially in the layers in contrast to simultaneous failure for the fused double layer constructs. This study provides insight into the nature of the interface and the role of cell behavior when tissue fusion occurs between two layers of bioartificial tissue in vitro. It also suggests a method for improving the burst strength of fibrin-based tubular tissue constructs by increasing the construct thickness via concentrically layering and fusing two constructs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10439-010-0045-zDOI Listing
June 2010

Image-based multiscale structural models of fibrous engineered tissues.

Annu Int Conf IEEE Eng Med Biol Soc 2009 ;2009:4270-2

University of Minnesota, Minneapolis, MN, USA.

While it is firmly established that the mechanical behavior of most biological tissues, including bioengineered tissues, is governed by an underlying network of protein fibers, it is still not clear how best to obtain and utilize structural information to predict mechanical response. In this paper, methods are presented to (1) quantify the fiber arrangement in a tissue from different imaging tools, (2) incorporate that structure into a multiscale model, and (3) solve the model equations to predict both the microscopic and the macroscopic tissue response. In principle these concepts could be applied to any tissue (incorporating the specific tissue components as needed), but for demonstration purposes, the focus of the current work is on cell-compacted collagen gel, a model engineered tissue.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1109/IEMBS.2009.5334586DOI Listing
April 2010

Image-based multiscale modeling predicts tissue-level and network-level fiber reorganization in stretched cell-compacted collagen gels.

Proc Natl Acad Sci U S A 2009 Oct 1;106(42):17675-80. Epub 2009 Oct 1.

Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA.

The mechanical environment plays an important role in cell signaling and tissue homeostasis. Unraveling connections between externally applied loads and the cellular response is often confounded by extracellular matrix (ECM) heterogeneity. Image-based multiscale models provide a foundation for examining the fine details of tissue behavior, but they require validation at multiple scales. In this study, we developed a multiscale model that captured the anisotropy and heterogeneity of a cell-compacted collagen gel subjected to an off-axis hold mechanical test and subsequently to biaxial extension. In both the model and experiments, the ECM reorganized in a nonaffine and heterogeneous manner that depended on multiscale interactions between the fiber networks. Simulations predicted that tensile and compressive fiber forces were produced to accommodate macroscopic displacements. Fiber forces in the simulation ranged from -11.3 to 437.7 nN, with a significant fraction of fibers under compression (12.1% during off-axis stretch). The heterogeneous network restructuring predicted by the model serves as an example of how multiscale modeling techniques provide a theoretical framework for understanding relationships between ECM structure and tissue-level mechanical properties and how microscopic fiber rearrangements could lead to mechanotransductive cell signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0903716106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2764876PMC
October 2009

Controlled compaction with ruthenium-catalyzed photochemical cross-linking of fibrin-based engineered connective tissue.

Biomaterials 2009 Dec 25;30(35):6695-701. Epub 2009 Sep 25.

Department of Chemical Engineering, University of Minnesota, 421 Washington Ave SE, Minneapolis, MN 55455, USA.

Tissue engineering utilizing fibrin gel as a scaffold has the advantage of creating a completely biological replacement. Cells seeded in a fibrin gel can induce fibril alignment by traction forces when subjected to appropriate mechanical constraints. While gel compaction is key to successful tissue fabrication, excessive compaction can result due to low gel stiffness. This study investigated using ruthenium-catalyzed photo-cross-linking as a method to increase gel stiffness in order to minimize over-compaction. Cross-links between the abundant tyrosine molecules that comprise fibrin were created upon exposure to blue light. Cross-linking was effective in increasing the stiffness of the fibrin gel by 93% with no adverse effects on cell viability. Long-term culture of cross-linked tubular constructs revealed no detrimental effects on cell proliferation or collagen deposition due to cross-linking. After 4 weeks of cyclic distension, the cross-linked samples were more than twice as long as non-cross-linked controls, with similar cell and collagen contents. However, the cross-linked samples required a longer incubation period to achieve a UTS and modulus comparable to controls. This study shows that photo-cross-linking is an attractive option to stiffen the initial fibrin gel and thereby reduce cell-induced compaction, which can allow for longer incubation periods and thus more tissue growth without compaction below a useful size.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biomaterials.2009.08.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2853233PMC
December 2009

Monitoring collagen transcription by vascular smooth muscle cells in fibrin-based tissue constructs.

Tissue Eng Part C Methods 2010 Jun;16(3):459-67

Department of Biomedical Engineering, University of Minnesota , Minneapolis, MN, USA.

Current methods for measuring collagen content in engineered tissues are incompatible with monitoring of collagen production because they require destruction of the tissue. We have implemented a luciferase-based strategy to monitor collagen production noninvasively. Fibrin-based tissue constructs made using vascular smooth muscle cells stably transfected with a collagen I promoter/luciferase transgene developed with collagen content comparable to control cells, but could be imaged noninvasively to follow collagen transcription during tissue growth in vitro. We showed that these cells reported collagen I production at the transcriptional level in response to the growth factor transforming growth factor-beta1 and fibrinolytic inhibition by epsilon-aminocaproic acid and that these changes were consistent with changes at the mRNA and protein levels. As these cells report collagen changes instantly and without tissue destruction, they will facilitate construct optimization using multiple stimuli to produce functional engineered tissues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/ten.TEC.2009.0112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2945866PMC
June 2010

Planar biaxial mechanical behavior of bioartificial tissues possessing prescribed fiber alignment.

J Biomech Eng 2009 Aug;131(8):081006

Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA.

Though it is widely accepted that fiber alignment has a great influence on the mechanical anisotropy of tissues, a systematic study of the influence of fiber alignment on the macroscopic mechanical behavior by native tissues is precluded due to their predefined microstructure and heterogeneity. Such a study is possible using collagen-based bioartificial tissues that allow for alignment to be prescribed during their fabrication. To generate a systemic variation of strength of fiber alignment, we made cruciform tissue constructs in Teflon molds that had arms of different aspect ratios. We implemented our anisotropic biphasic theory of tissue-equivalent mechanics to simulate the compaction by finite element analysis. Prior to tensile testing, the construct geometry was standardized by cutting test samples with a 1:1 cruciform punch after releasing constructs from the molds. Planar biaxial testing was performed on these samples, after stretching them to their in-mold dimensions to recover in-mold alignment, to observe the macroscopic mechanical response with simultaneous fiber alignment imaging using a polarimetry system. We found that the strength of fiber alignment of the samples prior to release from the molds linearly increased with anisotropy of the mold. In testing after release, modulus ratio (modulus in fiber direction/modulus in normal direction) was greater as the initial strength of fiber alignment increased, that is, as the aspect ratio increased. We also found that the fiber alignment strength and modulus ratio increased in a hyperbolic fashion with stretching for a sample of given aspect ratio.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1115/1.3148194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3717317PMC
August 2009

Transmural flow bioreactor for vascular tissue engineering.

Biotechnol Bioeng 2009 Dec;104(6):1197-206

Department of Biomedical Engineering, University of Minnesota 55455, USA.

Nutrient transport limitation remains a fundamental issue for in vitro culture of engineered tissues. In this study, perfusion bioreactor configurations were investigated to provide uniform delivery of oxygen to media equivalents (MEs) being developed as the basis for tissue-engineered arteries. Bioreactor configurations were developed to evaluate oxygen delivery associated with complete transmural flow (through the wall of the ME), complete axial flow (through the lumen), and a combination of these flows. In addition, transport models of the different flow configurations were analyzed to determine the most uniform oxygen profile throughout the tissue, incorporating direct measurements of tissue hydraulic conductivity, cellular O(2) consumption kinetics, and cell density along with ME physical dimensions. Model results indicate that dissolved oxygen (DO) uniformity is improved when a combination of transmural and axial flow is implemented; however, detrimental effects could occur due to lumenal pressure exceeding the burst pressure or damaging interstitial shear stress imparted by excessive transmural flow rates or decreasing hydraulic conductivity due to ME compaction. The model was verified by comparing predicted with measured outlet DO concentrations. Based on these results, the combination of a controlled transmural flow coupled with axial flow presents an attractive means to increase the transport of nutrients to cells within the cultured tissue to improve growth (increased cell and extracellular matrix concentrations) as well as uniformity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/bit.22475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2944401PMC
December 2009

The impact of biomechanics in tissue engineering and regenerative medicine.

Tissue Eng Part B Rev 2009 Dec;15(4):477-84

Biomedical Engineering, University of Cincinnati , Cincinnati, Ohio, USA.

Biomechanical factors profoundly influence the processes of tissue growth, development, maintenance, degeneration, and repair. Regenerative strategies to restore damaged or diseased tissues in vivo and create living tissue replacements in vitro have recently begun to harness advances in understanding of how cells and tissues sense and adapt to their mechanical environment. It is clear that biomechanical considerations will be fundamental to the successful development of clinical therapies based on principles of tissue engineering and regenerative medicine for a broad range of musculoskeletal, cardiovascular, craniofacial, skin, urinary, and neural tissues. Biomechanical stimuli may in fact hold the key to producing regenerated tissues with high strength and endurance. However, many challenges remain, particularly for tissues that function within complex and demanding mechanical environments in vivo. This paper reviews the present role and potential impact of experimental and computational biomechanics in engineering functional tissues using several illustrative examples of past successes and future grand challenges.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/ten.TEB.2009.0340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3192183PMC
December 2009

Controlled cyclic stretch bioreactor for tissue-engineered heart valves.

Biomaterials 2009 Sep 26;30(25):4078-84. Epub 2009 May 26.

Department of Chemical Engineering & Materials Science, University of Minnesota, USA.

A tissue-engineered heart valve (TEHV) represents the ultimate valve replacement, especially for juvenile patients given its growth potential. To date, most TEHV bioreactors have been developed based on pulsed flow of culture medium through the valve lumen to induce strain in the leaflets. Using a strategy for controlled cyclic stretching of tubular constructs reported previously, we developed a controlled cyclic stretch bioreactor for TEHVs that leads to improved tensile and compositional properties. The TEHV is mounted inside a latex tube, which is then cyclically pressurized with culture medium. The root and leaflets stretch commensurately with the latex, the stretching being dictated by the stiffer latex and thus controllable. Medium is also perfused through the lumen at a slow rate in a flow loop to provide nutrient delivery. Fibrin-based TEHVs prepared with human dermal fibroblasts were subjected to three weeks of cyclic stretching with incrementally increasing strain amplitude. The TEHV possessed the tensile stiffness and stiffness anisotropy of leaflets from sheep pulmonary valves and could withstand cyclic pulmonary pressures with similar distension as for a sheep pulmonary artery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biomaterials.2009.04.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2762550PMC
September 2009

Image-based biomechanics of collagen-based tissue equivalents.

IEEE Eng Med Biol Mag 2009 May-Jun;28(3):10-8

Department of Biomedical Engineering, University of Minnesota, Minneapolis, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1109/MEMB.2009.932486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2762792PMC
June 2011

Planar biaxial behavior of fibrin-based tissue-engineered heart valve leaflets.

Tissue Eng Part A 2009 Oct;15(10):2763-72

Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota 55455, USA.

To design more effective tissue-engineered heart valve replacements, the replacement tissue may need to mimic the biaxial stress-strain behavior of native heart valve tissue. This study characterized the planar biaxial properties of tissue-engineered valve leaflets and native aortic valve leaflets. Fibrin-based valve equivalent (VE) and porcine aortic valve (PAV) leaflets were subjected to incremental biaxial stress relaxation testing, during which fiber alignments were measured, over a range of strain ratios. Results showed that VE leaflets exhibited a modulus and fiber reorientation behavior that correlated with strain ratio. In contrast, PAV leaflets maintained their relaxed modulus and fiber alignment when exposed to nonequibiaxial strain, but exhibited changes in stress relaxation. In uniaxial and equi-biaxial tension, there were few observed differences in relaxation behavior between VE and PAV leaflets, despite differences in the modulus and fiber reorientation. Likewise, in both tissues there was similar relaxation response in the circumferential and radial directions in biaxial tension, despite different moduli in these two directions. This study presents some fundamental differences in the mechanical response to biaxial tension of fibrin-based tissue-engineered constructs and native valve tissue. It also highlights the importance of using a range of strain ratios when generating mechanical property data for valvular and engineered tissues. The data presented on the stress-strain, relaxation, and fiber reorientation of VE tissue will be useful in future efforts to mathematically model and improve fibrin-based tissue-engineered constructs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/ten.tea.2008.0426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2792051PMC
October 2009

Cell-induced alignment augments twitch force in fibrin gel-based engineered myocardium via gap junction modification.

Tissue Eng Part A 2009 Oct;15(10):3099-108

Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota 55455, USA.

A high-potential therapy for repairing the heart post-myocardial infarction is the implantation of tissue-engineered myocardium. While several groups have developed constructs that mimic the aligned structure of the native myocardium, to date no one has investigated the particular functional benefits conferred by alignment. In this study we created myocardial constructs in both aligned and isotropic configurations by entrapping neonatal rat cardiac cells in fibrin gel. Constructs were cultured statically for 2 weeks, and then characterized. Histological staining showed spread cells that express typical cardiac cell markers in both configurations. Isotropic constructs had higher final cell and collagen densities, but lower passive mechanical properties than aligned constructs. Twitch force associated with electrical pacing, however, was 181% higher in aligned constructs, and this improvement was greater than what would be expected from merely aligning the cells in the isotropic constructs in the force measurement direction. Our hypothesis was that this was due to improved gap junction formation/function facilitated by cell alignment, and further analyses of the twitch force data, as well as Western blot results of connexin 43 expression and phosphorylation state, support this hypothesis. Regardless of the specific mechanism, the results presented in this study underscore the importance of recapitulating the anisotropy of the native tissue in engineered myocardium.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/ten.TEA.2008.0502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2792050PMC
October 2009

Cyclic distension of fibrin-based tissue constructs: evidence of adaptation during growth of engineered connective tissue.

Proc Natl Acad Sci U S A 2008 May 24;105(18):6537-42. Epub 2008 Apr 24.

Departments of Chemical Engineering and Materials Science and Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA.

Tissue engineering provides a means to create functional living tissue replacements. Here, we examine the effects of 3 weeks of cyclic distension (CD) on fibrin-based tubular tissue constructs seeded with porcine valve interstitial cells. CD with circumferential strain amplitude ranging from 2.5% to 20% was applied to evaluate the effects of CD on fibrin remodeling into tissue. We hypothesized that during long-term CD cells adapt to cyclic strain of constant strain amplitude (constant CD), diminishing tissue growth. We thus also subjected constructs to CD with strain amplitude that was incremented from 5% to 15% over the 3 weeks of CD [incremental CD (ICD)]. For constant CD, improvement occurred in construct mechanical properties and composition, peaking at 15% strain: ultimate tensile strength (UTS) and tensile modulus increased 47% and 45%, respectively, over statically incubated controls (to 1.1 and 4.7 MPa, respectively); collagen density increased 29% compared with controls (to 27 mg/ml). ICD further improved outcomes. UTS increased 98% and modulus increased 62% compared with the largest values with constant CD, and collagen density increased 34%. Only in the case of ICD was the ratio of collagen content to cell number greater (70%) than controls, consistent with increased collagen deposition per cell. Studies with human dermal fibroblasts showed similar improvements, generalizing the findings, and revealed a 255% increase in extracellular signal-regulated kinase signaling for ICD vs. constant CD. These results suggest cell adaptation may limit conventional strategies of stretching with constant strain amplitude and that new approaches might optimize bioreactor operation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0711217105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2373356PMC
May 2008

Functional tissue-engineered valves from cell-remodeled fibrin with commissural alignment of cell-produced collagen.

Tissue Eng Part A 2008 Jan;14(1):83-95

Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota 55455, USA.

Heart valve replacements composed of living tissue that can adapt, repair, and grow with a patient would provide a more clinically beneficial option than current inert replacements. Bioartificial valves were produced by entrapping human dermal fibroblasts within a fibrin gel. Using a mold design that presents appropriate mechanical constraints to the cell-induced fibrin gel compaction, gross fiber alignment (commissure-to-commissure alignment in the leaflets and circumferential alignment in the root) and the basic geometry of a native aortic valve were obtained. After static incubation on the mold in complete medium supplemented with transforming growth factor beta 1, insulin, and ascorbate, collagen fibers produced by the entrapped cells were found to coalign with the fibrin based on histological analyses. The resultant tensile mechanical properties were anisotropic. Ultimate tensile strength and tensile modulus of the leaflets in the commissural direction were 0.53 and 2.34 MPa, respectively. The constructs were capable of withstanding backpressure commensurate with porcine aortic valves in regurgitation tests (330 mmHg) and opened and closed under physiological pressure swings of 10 and 20 mmHg, respectively. These data support proof of principle of using cell-remodeled fibrin gel to produce tissue-engineered valve replacements.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/ten.a.2007.0148DOI Listing
January 2008

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

J Clin Invest 2006 Dec 9;116(12):3139-49. Epub 2006 Nov 9.

Stem Cell Institute, University of Minnesota Medical School, Minneapolis, MN, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI28184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1635164PMC
December 2006

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

Tissue Eng 2006 Jun;12(6):1489-502

Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/ten.2006.12.1489DOI Listing
June 2006
-->