1. Cells Tissues Organs. 2011;194(1):13-24. Epub 2011 Jan 19.

Engineered vascular tissue fabricated from aggregated smooth muscle cells. Gwyther TA, Hu JZ, Christakis AG, Skorinko JK, Shaw SM, Billiar KL, Rolle MW. Department of Biomedical Engineering, Worcester Polytechnic Institute, Worcester, Mass., USA. The goal of this study was to develop a system to rapidly generate engineered tissue constructs from aggregated cells and cell-derived extracellular matrix (ECM) to enable evaluation of cell-derived tissue structure and function. Rat aortic smooth muscle cells seeded into annular agarose wells (2, 4 or 6 mm inside diameter) aggregated and formed thick tissue rings within 2 weeks of static culture (0.76 mm at 8 days; 0.94 mm at 14 days). Overall, cells appeared healthy and surrounded by ECM comprised of glycosoaminoglycans and collagen, although signs of necrosis were observed near the centers of the thickest rings. Tissue ring strength and stiffness values were superior to those reported for engineered tissue constructs cultured for comparable times. The strength (100-500 kPa) and modulus (0.5-2 MPa) of tissue rings increased with ring size and decreased with culture duration. Finally, tissue rings cultured for 7 days on silicone mandrels fused to form tubular constructs. Ring margins were visible after 7 days, but tubes were cohesive and mechanically stable, and histological examination confirmed fusion between ring subunits. This unique system provides a versatile new tool for optimization and functional assessment of cell-derived tissue, and a new approach to creating tissue-engineered vascular grafts. Copyright 2011 S. Karger AG, Basel. PMCID: PMC3128156 PMID: 21252472 [PubMed - indexed for MEDLINE]

2. Tissue Eng Part A. 2010 Jun;16(6):1901-12. Scaffold-free in vitro arterial mimetics: the importance of smooth muscle-endothelium contact. Chaterji S, Park K, Panitch A. Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana 47907, USA. We have developed an in vitro endothelial cell (EC)-smooth muscle cell (SMC) coculture platform that can mimic either the healthy or diseased state of blood vessels. Transforming growth factor-beta1 (TGF-beta1) and heparin were introduced to the SMC cultures to upregulate the SMC differentiation markers, alpha-smooth

muscle actin (alpha-SMA) and calponin (homotypic model). Interestingly, seeding of near-confluent concentrations of ECs on the SMCs (heterotypic model) induced higher levels of alpha-SMA and calponin expression in the SMC cultures than did the addition of heparin and TGF-beta1 alone. The expression levels increased further on pretreating the SMCs with TGF-beta1 and heparin before adding a near-confluent monolayer of ECs. In contrast, seeding of sparse concentrations of ECs forced the SMCs into a more hyperplastic state as determined by alpha-SMA and calponin expression. This study highlights the importance of both soluble factors and EC seeding densities when considering culture conditions; in vivo SMCs are in close proximity with and interact with a monolayer of ECs. Our study suggests that this architecture is important for healthy vascular tissue function. In addition, it shows that disruption of this architecture can be used to mimic diseased states. As the EC-SMC coculture model can mimic either a diseased or a healthy blood vessel it may be useful as a test bed for evaluating cardiovascular therapeutics. PMCID: PMC2949266 PMID: 20088699 [PubMed - indexed for MEDLINE]

3. Regen Med. 2010 Jan;5(1):107-20. Biomaterials for vascular tissue engineering. Ravi S, Chaikof EL. Department of Surgery, Emory University, Atlanta, GA 30332, USA. Cardiovascular disease is the leading cause of mortality in the USA. The limited availability of healthy autologous vessels for bypass grafting procedures has led to the fabrication of prosthetic vascular conduits. While synthetic polymers have been extensively studied as substitutes in vascular engineering, they fall short of meeting the biological challenges at the blood-material interface. Various tissue engineering strategies have emerged to address these flaws and increase long-term patency of vascular grafts. Vascular cell seeding of scaffolds and the design of bioactive polymers for in situ arterial regeneration have yielded promising results. This article describes the advances made in biomaterials design to generate suitable materials that not only match the mechanical properties of native vasculature, but also promote cell growth, facilitate extracellular matrix production and inhibit thrombogenicity. PMCID: PMC2822541 PMID: 20017698 [PubMed - indexed for MEDLINE]

4. Tissue Eng Part A. 2009 Dec;15(12):3951-60. Generating elastin-rich small intestinal submucosa-based smooth muscle constructs

utilizing exogenous growth factors and cyclic mechanical stimulation. Heise RL, Ivanova J, Parekh A, Sacks MS. Engineered Tissue Mechanics and Mechanobiology Laboratory, Department of Bioengineering and McGowan Institute, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, Pennsylvania 15219, USA. Successful approaches to tissue engineering smooth muscle tissues utilize biodegradable scaffolds seeded with autologous cells. One common problem in using biological scaffolds specifically is the difficulty of inducing cellular penetration and controlling de novo extracellular matrix deposition/remodeling in vitro. Our hypothesis was that small intestinal submucosa (SIS) exposed to specific mechanical stimulation regimes would modulate the synthesis of de novo collagen and elastin by bladder smooth muscle cells (BSMC) within the SIS matrix. We further hypothesized that the cytokines vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2), two key growth factors involved in epithelial mesenchymal signaling, will promote the cellular penetration into SIS necessary for mechanical stimulation. BSMC were seeded at 0.5 x 10(6) cells/cm(2) onto the luminal side of SIS specimens. VEGF (10 ng/mL) and FGF-2 (5 ng/mL) were added to each insert in the media every other day for up to 7 days in static culture. Following static culture, specimens were stretched strip-biaxially under 15% peak strain at either 0.5 or 0.1 Hz for an additional 7 days. Following the culture period, specimens were assayed histologically and biochemically for cellular penetration, proliferation, elastin, collagen, and protease activity. Histological analyses demonstrated that in standard culture media, BSMC remained on the surface of the SIS while both FGF-2 and VEGF profoundly promoted ingrowth of the BSMC into the SIS. The penetration of the cells in response to these cytokines was confirmed using a Transwell assay. Following cellular penetration, BSMC produced significant amounts of elastic fibers under cyclic mechanical stretching at 0.1 Hz under 15% stretch, as evidenced by colorimetric assay and histology using a Verhoeff-Van Gieson stain. Protease activity was assessed in the media and found to be statistically increased in static culture following FGF-2 treatment. These findings demonstrate, for the first time, the capability of BSMC to produce histologically apparent elastin fibers in vitro. Moreover, our results suggest that a strategy involving growth factors and controlled mechanical stimulation may be used to engineer functional, elastin-rich tissue replacements using decellularized biologically derived scaffolds. PMCID: PMC2792073 PMID: 19569874 [PubMed - indexed for MEDLINE]

5. Tissue Eng Part A. 2009 Nov;15(11):3331-40. A novel cylindrical biaxial computer-controlled bioreactor and biomechanical testing device for vascular tissue engineering.

Zaucha MT, Raykin J, Wan W, Gauvin R, Auger FA, Germain L, Michaels TE, Gleason RL Jr. The George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332-0405, USA. It is becoming evident that tissue-engineered constructs adapt to altered mechanical loading, and that specific combinations of multidirectional loads appear to have a synergistic effect on the remodeling. However, most studies of mechanical stimulation of engineered vascular tissue engineering employ only uniaxial stimulation. Here we present a novel computer-controlled bioreactor and biomechanical testing device designed to precisely and simultaneously control mean and cyclic values of transmural pressure (at rates up to 1 Hz and ranges of 40 mmHg), luminal flow rate, and axial length (or load) applied to gel-derived, scaffold-derived, and self-assembly-derived tissue-engineered blood vessels during culture, while monitoring vessel geometry with a resolution of 6.6 mum. Intermittent monitoring of the extracellular matrix and cells is accomplished on live tissues using multi-photon confocal microscopy under unloaded and loaded conditions at multiple time-points in culture (on the same vessel) to quantify changes in cell and extracellular matrix content and organization. This same device is capable of performing intermittent cylindrical biaxial biomechanical testing at multiple time-points in culture (on the same vessel) to quantify changes in the mechanical behavior during culture. Here we demonstrate the capabilities of this new device on self-assembly-derived and collagen-gel-derived tissue-engineered blood vessels. PMCID: PMC2792052 PMID: 19385725 [PubMed - indexed for MEDLINE]

6. Proc Natl Acad Sci U S A. 2009 Oct 20;106(42):17675-80. Epub 2009 Oct 1. Image-based multiscale modeling predicts tissue-level and network-level fiber reorganization in stretched cell-compacted collagen gels. Sander EA, Stylianopoulos T, Tranquillo RT, Barocas VH. 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. PMCID: PMC2764876 PMID: 19805118 [PubMed - indexed for MEDLINE]

7. J Biomech Eng. 2009 Oct;131(10):101016. A phenomenological model for mechanically mediated growth, remodeling, damage, and plasticity of gel-derived tissue engineered blood vessels. Raykin J, Rachev AI, Gleason RL Jr. Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, 30332, USA. Mechanical stimulation has been shown to dramatically improve mechanical and functional properties of gel-derived tissue engineered blood vessels (TEBVs). Adjusting factors such as cell source, type of extracellular matrix, cross-linking, magnitude, frequency, and time course of mechanical stimuli (among many other factors) make interpretation of experimental results challenging. Interpretation of data from such multifactor experiments requires modeling. We present a modeling framework and simulations for mechanically mediated growth, remodeling, plasticity, and damage of gel-derived TEBVs that merge ideas from classical plasticity, volumetric growth, and continuum damage mechanics. Our results are compared with published data and suggest that this model framework can predict the evolution of geometry and material behavior under common experimental loading scenarios. PMCID: PMC3093136 PMID: 19831486 [PubMed - indexed for MEDLINE]

8. Philos Transact A Math Phys Eng Sci. 2009 Sep 13;367(1902):3339-62. Mechanical strain enhances survivability of collagen micronetworks in the presence of collagenase: implications for load-bearing matrix growth and stability.

Bhole AP, Flynn BP, Liles M, Saeidi N, Dimarzio CA, Ruberti JW. Department of Mechanical and Industrial Engineering, Northeastern, University, , 360 Huntington Avenue, Boston, MA 02139, USA. There has been great interest in understanding the methods by which collagen-based load-bearing tissue is constructed, grown and maintained in vertebrate animals. To date, the responsibility for this process has largely been placed with mesenchymal fibroblastic cells that are thought to fully control the morphology of load-bearing extracellular matrix (ECM). However, given clear limitations in the ability of fibroblastic cells to precisely place or remove single collagen molecules to sculpt tissue, we have hypothesized that the material itself must play a critical role in the determination of the form of structural ECM. We here demonstrate directly, using live, dynamic, differential interference contrast imaging, that mechanically strained networks of collagen fibrils, exposed to collagenase (Clostridium histolyticum), degrade preferentially. Specifically, unstrained fibrils are removed 'quickly', while strained fibrils persist significantly longer. The demonstration supports the idea that collagen networks are mechanosensitive in that they are stabilized by mechanical strain. Thus, collagen molecules (together with their complement enzymes) may comprise the basis of a smart, load-adaptive, structural material system. This concept has the potential to drastically simplify the assumed role of the fibroblast, which would need only to provide ECM molecules and mechanical force to sculpt collagenous tissue. PMCID: PMC2865878 PMID: 19657003 [PubMed - indexed for MEDLINE]

9. Biomaterials. 2009 Sep;30(25):4078-84. Epub 2009 May 26. Controlled cyclic stretch bioreactor for tissue-engineered heart valves. Syedain ZH, Tranquillo RT. 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. PMCID: PMC2762550 PMID: 19473698 [PubMed - indexed for MEDLINE]

10. Ann Biomed Eng. 2009 Jul;37(7):1263-72. Epub 2009 May 5. Quantification of the temporal evolution of collagen orientation in mechanically conditioned engineered cardiovascular tissues. Rubbens MP, Driessen-Mol A, Boerboom RA, Koppert MM, van Assen HC, TerHaar Romeny BM, Baaijens FP, Bouten CV. Soft Tissue Biomechanics & Engineering, Department of Biomedical Engineering, Eindhoven University of Technology, WH 4.107, Den Dolech 2, P.O. Box 513, 5600 MB, Eindhoven, The Netherlands. m.p.rubbens@tue.nl Load-bearing soft tissues predominantly consist of collagen and exhibit anisotropic, non-linear visco-elastic behavior, coupled to the organization of the collagen fibers. Mimicking native mechanical behavior forms a major goal in cardiovascular tissue engineering. Engineered tissues often lack properly organized collagen and consequently do not meet in vivo mechanical demands. To improve collagen architecture and mechanical properties, mechanical stimulation of the tissue during in vitro tissue growth is crucial. This study describes the evolution of collagen fiber orientation with culture time in engineered tissue constructs in response to mechanical loading. To achieve this, a novel technique for the quantification of collagen fiber orientation is used, based on 3D vital imaging using multiphoton microscopy combined with image analysis. The engineered tissue constructs consisted of cell-seeded biodegradable rectangular scaffolds, which were either constrained or intermittently strained in longitudinal direction. Collagen fiber orientation analyses revealed that mechanical loading induced collagen alignment. The alignment shifted from oblique at the surface of the construct towards parallel to the straining direction in deeper tissue layers. Most importantly, intermittent straining improved and accelerated the alignment of the collagen fibers, as compared to constraining the constructs. Both the method and the results are relevant to create and monitor load-bearing tissues with an organized anisotropic collagen network. PMCID: PMC2690830 PMID: 19415496 [PubMed - indexed for MEDLINE]

11. Physiol Rev. 2009 Jul;89(3):957-89. Vascular extracellular matrix and arterial mechanics. Wagenseil JE, Mecham RP. Department of Biomedical Engineering, Saint Louis University, Washington University School of Medicine, St. Louis, Missouri 63110, USA. An important factor in the transition from an open to a closed circulatory system was a change in vessel wall structure and composition that enabled the large arteries to store and release energy during the cardiac cycle. The component of the arterial wall in vertebrates that accounts for these properties is the elastic fiber network organized by medial smooth muscle. Beginning with the onset of pulsatile blood flow in the developing aorta, smooth muscle cells in the vessel wall produce a complex extracellular matrix (ECM) that will ultimately define the mechanical properties that are critical for proper function of the adult vascular system. This review discusses the structural ECM proteins in the vertebrate aortic wall and will explore how the choice of ECM components has changed through evolution as the cardiovascular system became more advanced and pulse pressure increased. By correlating vessel mechanics with physiological blood pressure across animal species and in mice with altered vessel compliance, we show that cardiac and vascular development are physiologically coupled, and we provide evidence for a universal elastic modulus that controls the parameters of ECM deposition in vessel wall development. We also discuss mechanical models that can be used to design better tissue-engineered vessels and to test the efficacy of clinical treatments. PMCID: PMC2775470 PMID: 19584318 [PubMed - indexed for MEDLINE]

12. Vascular. 2009 May-Jun;17 Suppl 1:S45-54. Polymeric materials for tissue engineering of arterial substitutes. Ravi S, Qu Z, Chaikof EL. Department of Surgery, Emory University, Atlanta, GA 30322, USA. Cardiovascular disease is the leading cause of mortality in the United States. The limited availability of healthy autologous vessels for bypass grafting procedures has led to the fabrication of prosthetic vascular conduits. Synthetic polymeric materials, while providing the appropriate mechanical strength, lack the compliance and biocompatibility that bioresorbable and naturally occurring protein polymers offer. Vascular tissue engineering approaches have emerged in order to meet the challenges of designing a vascular graft with long-term patency. In vitro culture techniques that have been explored with vascular cell

seeding of polymeric scaffolds and the use of bioactive polymers for in situ arterial regeneration have yielded promising results. This review describes the development of polymeric materials in various tissue engineering strategies for the improvement in the mechanical and biological performance of an arterial substitute. PMCID: PMC2714487 PMID: 19426609 [PubMed - indexed for MEDLINE]

13. Biomaterials. 2009 Mar;30(7):1401-12. Epub 2008 Dec 12. Engineered skeletal muscle tissue networks with controllable architecture. Bian W, Bursac N. Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA. The engineering of functional skeletal muscle tissue substitutes holds promise for the treatment of various muscular diseases and injuries. However, no tissue fabrication technology currently exists for the generation of a relatively large and thick bioartificial muscle made of densely packed, uniformly aligned, and differentiated myofibers. In this study, we describe a versatile cell/hydrogel micromolding approach where polydimethylsiloxane (PDMS) molds containing an array of elongated posts were used to fabricate relatively large neonatal rat skeletal muscle tissue networks with reproducible and controllable architecture. By combining cell-mediated fibrin gel compaction and precise microfabrication of mold dimensions including the length and height of the PDMS posts, we were able to simultaneously support high cell viability, guide cell alignment along the microfabricated tissue pores, and reproducibly control the overall tissue porosity, size, and thickness. The interconnected muscle bundles within the porous tissue networks were composed of densely packed, aligned, and highly differentiated myofibers. The formed myofibers expressed myogenin, developed abundant cross-striations, and generated spontaneous tissue contractions at the macroscopic spatial scale. The proliferation of non-muscle cells was significantly reduced compared to monolayer cultures. The more complex muscle tissue architectures were fabricated by controlling the spatial distribution and direction of the PDMS posts. PMCID: PMC2726993 PMID: 19070360 [PubMed - indexed for MEDLINE]

14. Proc Natl Acad Sci U S A. 2008 May 6;105(18):6537-42. Epub 2008 Apr 24. Cyclic distension of fibrin-based tissue constructs: evidence of adaptation during growth of engineered connective tissue.

Syedain ZH, Weinberg JS, Tranquillo RT. 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. PMCID: PMC2373356 PMID: 18436647 [PubMed - indexed for MEDLINE]

15. Ann Biomed Eng. 2008 Feb;36(2):244-53. Epub 2007 Dec 8. Effect of strain magnitude on the tissue properties of engineered cardiovascular constructs. Boerboom RA, Rubbens MP, Driessen NJ, Bouten CV, Baaijens FP. Department of Biomedical Engineering, Soft Tissue Biomechanics and Engineering, Eindhoven University of Technology, PO Box 513, 5600 MB, Eindhoven, The Netherlands. r.a.boerboom@tue.nl Mechanical loading is a powerful regulator of tissue properties in engineered cardiovascular tissues. To ultimately regulate the biochemical processes, it is essential to quantify the effect of mechanical loading on the properties of engineered cardiovascular constructs. In this study the Flexercell FX-4000T (Flexcell Int. Corp., USA) straining system was modified to simultaneously apply

various strain magnitudes to individual samples during one experiment. In addition, porous polyglycolic acid (PGA) scaffolds, coated with poly-4-hydroxybutyrate (P4HB), were partially embedded in a silicone layer to allow long-term uniaxial cyclic mechanical straining of cardiovascular engineered constructs. The constructs were subjected to two different strain magnitudes and showed differences in biochemical properties, mechanical properties and organization of the microstructure compared to the unstrained constructs. The results suggest that when the tissues are exposed to prolonged mechanical stimulation, the production of collagen with a higher fraction of crosslinks is induced. However, straining with a large strain magnitude resulted in a negative effect on the mechanical properties of the tissue. In addition, dynamic straining induced a different alignment of cells and collagen in the superficial layers compared to the deeper layers of the construct. The presented model system can be used to systematically optimize culture protocols for engineered cardiovascular tissues. PMCID: PMC2211363 PMID: 18066665 [PubMed - indexed for MEDLINE]

16. J Biomech. 2008;41(4):762-9. Epub 2008 Jan 28. Combined effects of microtopography and cyclic strain on vascular smooth muscle cell orientation. Houtchens GR, Foster MD, Desai TA, Morgan EF, Wong JY. Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA. Cellular alignment studies have shown that cell orientation has a large effect on the expression and behavior of cells. Cyclic strain and substrate microtopography have each been shown to regulate cellular alignment. This study examined the combined effects of these two stimuli on the alignment of bovine vascular smooth muscle cells (VSMCs). Cells were cultured on substrates with microgrooves of varying widths oriented either parallel or perpendicular to the direction of an applied cyclic tensile strain. We found that microgrooves oriented parallel to the direction of the applied strain limited the orientation response of VSMCs to the mechanical stimulus, while grooves perpendicular to the applied strain enhanced cellular alignment. Further, the extent to which parallel grooves limited cell alignment was found to be dependent on the groove width. It was found that for both a small (15microm) and a large (70microm) groove width, cells were better able to reorient in response to the applied strain than for an intermediate groove width (40microm). This study indicates that microtopographical cues modulate the orientation response of VSMCs to cyclic strain. The results suggest that there is a range of microgroove dimensions that is most effective at maintaining the orientation of the cells in the presence of an opposing stimulus induced by cyclic strain.

PMCID: PMC2365919 PMID: 18222460 [PubMed - indexed for MEDLINE]

17. Tissue Eng. 2007 Nov;13(11):2601-13. Review: advances in vascular tissue engineering using protein-based biomaterials. Stegemann JP, Kaszuba SN, Rowe SL. Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, New York, NY 12180, USA. stegemann@rpi.edu The clinical need for improved blood vessel substitutes, especially in small-diameter applications, drives the field of vascular tissue engineering. The blood vessel has a well-characterized structure and function, but it is a complex tissue, and it has proven difficult to create engineered tissues that are suitable for widespread clinical use. This review is focused on approaches to vascular tissue engineering that use proteins as the primary matrix or "scaffold" material for creating fully biological blood vessel replacements. In particular, this review covers four main approaches to vascular tissue engineering: 1) cell-populated protein hydrogels, 2) cross-linked protein scaffolds, 3) decellularized native tissues, and 4) self-assembled scaffolds. Recent advances in each of these areas are discussed, along with advantages of and drawbacks to these approaches. The first fully biological engineered blood vessels have entered clinical trials, but important challenges remain before engineered vascular tissues will have a wide clinical effect. Cell sourcing and recapitulating the biological and mechanical function of the native blood vessel continue to be important outstanding hurdles. In addition, the path to commercialization for such tissues must be better defined. Continued progress in several complementary approaches to vascular tissue engineering is necessary before blood vessel substitutes can achieve their full potential in improving patient care. PMCID: PMC2257983 PMID: 17961004 [PubMed - indexed for MEDLINE]

18. Biomaterials. 2007 Sep;28(26):3824-33. Epub 2007 May 21. The role of ERK signaling in protein hydrogel remodeling by vascular smooth muscle cells. Hong H, McCullough CM, Stegemann JP. Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY, USA.

Collagen type I and fibrin hydrogels have been used for cell-based therapies and tissue engineering. These matrices can be broken down and remodeled by cells, but the effects that these proteins have on cell function are not completely understood. We examined activation of the extracellular signal-regulated kinase (ERK) signaling pathway by vascular smooth muscle cells (VSMC) in response to 2D and 3D matrices of type I collagen, fibrin, or a 1:1 composite mixture of these proteins. After 3 days of culture, ERK phosphorylation, osteopontin secretion, and MMP-2 activation were all markedly increased in 3D matrices, compared with 2D substrates. A strong positive correlation existed between these protein markers of the synthetic phenotype and phosphorylated ERK levels, and this relationship persisted across matrix geometries and compositions. Cell proliferation in 3D matrices was inversely correlated to ERK activation, while on 2D substrates a modest positive correlation was observed. Pharmacologic inhibition of ERK signaling confirmed that this pathway was involved in the observed phenotype shifts. This study suggests that contextual activation of the ERK pathway results in different effects on cell phenotype, depending on the geometry and composition of the ECM. These findings add to our understanding of cell function and remodeling in protein-based hydrogel biomaterials. PMCID: PMC2001258 PMID: 17544501 [PubMed - indexed for MEDLINE]

19. J Clin Invest. 2006 Dec;116(12):3139-49. Epub 2006 Nov 9. Cytokine-induced differentiation of multipotent adult progenitor cells into functional smooth muscle cells. Ross JJ, Hong Z, Willenbring B, Zeng L, Isenberg B, Lee EH, Reyes M, Keirstead SA, Weir EK, Tranquillo RT, Verfaillie CM. Stem Cell Institute, University of Minnesota Medical School, Minneapolis, MN, USA. Erratum in J Clin Invest. 2007 Jul;117(7):2014. 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. PMCID: PMC1635164 PMID: 17099777 [PubMed - indexed for MEDLINE]

20. Ann Biomed Eng. 2006 Nov;34(11):1678-90. Epub 2006 Oct 11. Cellular and matrix mechanics of bioartificial tissues during continuous cyclic stretch. Wille JJ, Elson EL, Okamoto RJ. Department of Biomedical Engineering, Washington University, St. Louis, MO 63130, USA. Bioartificial tissues are useful model systems for studying cell and extra-cellular matrix mechanics. These tissues provide a 3D environment for cells and allow tissue components to be easily modified and quantified. In this study, we fabricated bioartificial tissue rings from a 1 ml solution containing one million cardiac fibroblasts and 1 mg collagen. After 8 days, rings compacted to <1% of original volume and cell number increased 2.4 fold. We initiated continuous cyclic stretching of the rings after 2, 4, or 8 days of incubation, while monitoring the tissue forces. Peak tissue force during each cycle decreased rapidly after initiating stretch, followed by further slow decline. We added 2 microM Cytochalasin-D to some rings prior to initiation of stretch to determine the force contributed by the matrix. Cell force was estimated by subtracting matrix force from tissue force. After 12 h, matrix force-strain curves were highly nonlinear. Cell force-strain curves were linear during loading and showed hysteresis indicating viscoelastic behavior. Cell stiffness increased with stretching frequency from 0.001-0.25 Hz. Cell stiffness decreased with stretch amplitude (5-25%) at 0.1 Hz. The trends in cell stiffness do not fit simple viscoelastic models previously proposed, and suggest possible strain-amplitude related changes during cyclic stretch. PMCID: PMC1705520 PMID: 17033741 [PubMed - indexed for MEDLINE]