Elucidating the signal for contact guidance contained in aligned fibrils with a microstructural-mechanical model.

Despite its importance in physiological processes and tissue engineering, the mechanism underlying cell contact guidance in an aligned fibrillar network has defied elucidation due to multiple interdependent signals that such a network presents to cells, namely, anisotropy of adhesion, porosity and mechanical behaviour. A microstructural-mechanical model of fibril networks was used to assess the relative magnitudes of these competing signals in networks of varied alignment strength based on idealized cylindrical pseudopods projected into the aligned and orthogonal directions and computing the anisotropy of metrics chosen for adhesion, porosity and mechanical behaviour: cylinder-fibre contact area for adhesion, persistence length of pores for porosity and total force to displace fibres from the cylindrical volume as well as network stiffness experienced upon cylinder retraction for mechanical behaviour. The signals related to mechanical anisotropy are substantially higher than adhesion and porosity anisotropy, especially at stronger network alignments, although their signal to noise (S/N) values are substantially lower. The former finding is consistent with a recent report that fibroblasts can sense fibril alignment via anisotropy of network mechanical resistance, and the model reveals this can be due to either mechanical resistance to pseudopod protrusion or retraction given their signal and S/N values are similar.

Cell contact guidance via sensing anisotropy of network mechanical resistance.

Despite the ubiquitous importance of cell contact guidance, the signal-inducing contact guidance of mammalian cells in an aligned fibril network has defied elucidation. This is due to multiple interdependent signals that an aligned fibril network presents to cells, including, at least, anisotropy of adhesion, porosity, and mechanical resistance. By forming aligned fibrin gels with the same alignment strength, but cross-linked to different extents, the anisotropic mechanical resistance hypothesis of contact guidance was tested for human dermal fibroblasts. The cross-linking was shown to increase the mechanical resistance anisotropy, without detectable change in network microstructure and without change in cell adhesion to the cross-linked fibrin gel. This methodology thus isolated anisotropic mechanical resistance as a variable for fixed anisotropy of adhesion and porosity. The mechanical resistance anisotropy |Y*| -1 - |X*| -1 increased over fourfold in terms of the Fourier magnitudes of microbead displacement |X*| and |Y*| at the drive frequency with respect to alignment direction Y obtained by optical forces in active microrheology. Cells were found to exhibit stronger contact guidance in the cross-linked gels possessing greater mechanical resistance anisotropy: the cell anisotropy index based on the tensor of cell orientation, which has a range 0 to 1, increased by 18% with the fourfold increase in mechanical resistance anisotropy. We also show that modulation of adhesion via function-blocking antibodies can modulate the guidance response, suggesting a concomitant role of cell adhesion. These results indicate that fibroblasts can exhibit contact guidance in aligned fibril networks by sensing anisotropy of network mechanical resistance.

Evaluation of the probe burst test as a measure of strength for a biologically-engineered vascular graft.

Biologically-engineered vascular grafts have the potential to provide a viable alternative to donor vessels and synthetic grafts. In congenital heart defect patients, the need is even more dire since neither has the capacity to provide somatic growth. To ensure clinically-used grafts perform to accepted standards, mechanical strength is a crucial consideration, with burst testing being considered as one key metric. While ISO 7198 standards for prosthetic vascular grafts provide multiple choices for burst testing, most studies with tissue-engineered grafts have been performed with only pressure burst testing. Here, we compare the performance of a decellularized tube of collagenous matrix grown from dermal fibroblasts, possessing circumferential fiber alignment and anisotropic tensile properties, as determined from pressure and probe burst testing. The two burst tests showed a strong correlation with each other and with tensile strength. Further, relatively weak and strong batches of grafts showed commensurate differences in pressure and probe burst values. Both probe burst and tensile strength measurements in the central and edge regions of the grafts were similar in value, consistent with homogenous collagen content and microstructure throughout the grafts as indicated by histology, in contrast to ovine femoral and carotid arteries similarly tested. Finite element analysis of the probe burst test pre-failure for a homogeneous, isotropic approximation of the matrix constitutive behavior indicated dependence of the (inferred) effective failure stress achievable on probe diameter. The results indicate a probe burst test in a sampled edge region of this biologically-engineered graft provides a representative measure of burst strength of the entire graft.

Pediatric tri-tube valved conduits made from fibroblast-produced extracellular matrix evaluated over 52 weeks in growing lambs.

There is a need for replacement heart valves that can grow with children. We fabricated tubes of fibroblast-derived collagenous matrix that have been shown to regenerate and grow as a pulmonary artery replacement in lambs and implemented a design for a valved conduit consisting of three tubes sewn together. Seven lambs were implanted with tri-tube valved conduits in sequential cohorts and compared to bioprosthetic conduits. Valves implanted into the pulmonary artery of two lambs of the first cohort of four animals functioned with mild regurgitation and systolic pressure drops <10 mmHg up to 52 weeks after implantation, during which the valve diameter increased from 19 mm to a physiologically normal ~25 mm. In a second cohort, the valve design was modified to include an additional tube, creating a sleeve around the tri-tube valve to counteract faster root growth relative to the leaflets. Two valves exhibited trivial-to-mild regurgitation at 52 weeks with similar diameter increases to ~25 mm and systolic pressure drops of <5 mmHg, whereas the third valve showed similar findings until moderate regurgitation was observed at 52 weeks, correlating to hyperincrease in the valve diameter. In all explanted valves, the leaflets contained interstitial cells and an endothelium progressing from the base of the leaflets and remained thin and pliable with sparse, punctate microcalcifications. The tri-tube valves demonstrated reduced calcification and improved hemodynamic function compared to clinically used pediatric bioprosthetic valves tested in the same model. This tri-tube valved conduit has potential for long-term valve growth in children.

Vascular grafts and valves that animate, made from decellularized biologically-engineered tissue tubes.

Biologically-engineered matrix - a tissue that is grown in vitro from donor cells, decellularized, and stored prior to use as off-the-shelf allografts - offers a promising alternative to current cardiovascular biomaterials. This perspective reviews preclinical studies and clinical trials of vascular grafts and valves comprising biologically-engineered matrix, with a focus on those based on donor dermal fibroblast remodeling of fibrin gel with the capacity to heal and grow following recellularization, via animation of the matrix. It concludes with a discussion of related key clinical considerations.

Biologically-engineered mechanical model of a calcified artery.

Vascular calcification is a commonly occurring pathological process and is recognized as an independent prognostic marker for cardiovascular morbidity and mortality. Recent progress in developing novel therapies to modify vascular calcification is critically hampered due to the lack of reliable in vitro experimental models that recapitulate the structural and mechanical attributes of calcified arteries. In this study, we show the ability to model the behavior of diffuse vascular calcification in vitro using biologically-engineered grafts approximating the composition, structure, and mechanical properties of arteries. Transmural calcification was achieved by exposing the acellular grafts of collagenous ECM to complete medium containing elevated Calcium (Ca) and Phosphate (P) concentrations. It was found that increasing the serum concentration from 2% to 10% increased the extent and degree of calcification based on histochemical, ultrastructural, chemical and thermal analyses. The presence of variably-sized spherical calcific deposits within the matrix further confirmed its morphological similarity to pathologic calcification. Mechanical testing demonstrated up to a 16-fold decrease in compliance due to the calcification, consistent with prior reports for calcified arteries. The model developed thus has potential to improve the design and development of interventional devices and therapies for the diagnosis and treatment of arterial calcification. STATEMENT OF SIGNIFICANCE: The presence of extensive vascular calcification makes angiographic/interventional procedures difficult due to reduced arterial compliance. Current attempts to develop safe and effective non-surgical adjunctive techniques to treat calcified arteries are largely limited by the lack of a physiologically relevant testing platform that mimics the structural and mechanical features of vascular calcification. Herein, we developed an off-the-shelf calcified artery model, with the goal to accelerate the pre-clinical development of novel therapies for the management of arterial calcification. To the extent of our knowledge, this is the first report of an in vitro tissue-engineered model of diffuse arterial calcification.

Tissue-engineered transcatheter vein valve.

Chronic venous insufficiency affects over 2 million patients in the US alone, with severe cases involving thousands of patients with chronic leg ulcers and potential amputation. Current treatment options are limited, with surgical repair of vein valves being the most effective but challenging solution. A transcatheter vein valve made from a biologically-engineered matrix possessing the ability to regenerate has the potential to provide both valve function and long-term hemocompatibility and durability because the matrix becomes endothelialized and populated with host tissue cells. We have developed a novel tissue-engineered transcatheter vein valve (TEVV) on a Nitinol stent and demonstrated function and durability in vitro. Tissue was grown from fibroblasts in fibrin gel so as to embed the stent, with a tubular extension of the engineered tissue from one end of the stent that was stitched along opposite sides and everted into the stent to form a bileaflet valve. Following decellularization, to create an "off-the-shelf" TEVV comprised of the resulting collagenous matrix, it was tested in a pulse duplicator to evaluate hydrodynamic properties for a range of flow rates. The TEVV was shown to have forward pressure drops in the range of 2-4 mmHg, low closing volume, and nil regurgitation. Further hydrodynamic tests were performed after crimping and then again after 1 million cycle durability testing, showing no degradation of valve performance or any visual damage to the matrix. The TEVV held over 600 mmHg backpressure after the durability testing, ensuring the valve would withstand pressure spikes well outside of the normal in vivo range. Catheter-based delivery into the ovine iliac vein demonstrated TEVV closing 2 weeks p.o. and endothelialization without thrombosis 8 weeks p.o.

Shear Conditioning of Adipose Stem Cells for Reduced Platelet Binding to Engineered Vascular Grafts.

Conferring antithrombogenicity to tissue-engineered vascular grafts remains a major challenge, especially for urgent bypass grafting that excludes approaches based on expanding autologous endothelial cells (ECs) that requires weeks of cell culture. Adipose-derived stem cells (ASCs) are available from most patients in sufficient number for coronary bypass graft seeding and may be effective as allogeneic cells. We thus compared the adhesion and platelet binding of human ASCs that were shear conditioned with constant and pulsatile shear stress (SS) after seeding the cells on a biologically engineered matrix suitable for arterial grafts. A monolayer of cells was maintained up to 15 dyn/cm2 constant SS and up to 15 dyn/cm2 mean pulsatile SS for 6 days of shear flow. Platelet binding was reduced from 83% to 6% of surface area and nitric oxide production was increased 23-fold with 7.5-15 dyn/cm2 constant SS, but not pulsatile SS, relative to cells cultured statically on the matrix for 6 days. The reduction in platelet binding varied from no reduction to maximum reduction over a constant shear range of ∼2 to 4 dyn/cm2, respectively. Collectively, the study supports the potential use of ASCs to seed the luminal surface of a vascular graft made from this biologically engineered matrix to confer an antithrombogenic surface during the development of an endothelium from the seeded cells or the surrounding blood and tissue.

A cardiac patch from aligned microvessel and cardiomyocyte patches.

Cardiac tissue engineering aims to produce replacement tissue patches in the lab to replace or treat infarcted myocardium. However, current patches lack preformed microvascularization and are therefore limited in thickness and force production. In this study, we sought to assess whether a bilayer patch composed of a layer made from human induced pluripotent stem cell-derived cardiomyocytes and a microvessel layer composed of self-assembled human blood outgrowth endothelial cells and pericytes was capable of engrafting on the epicardial surface of a nude rat infarct model and becoming perfused by the host 4 weeks after acute implantation. The bilayer configuration was found to increase the twitch force production, improve human induced pluripotent stem cell-derived cardiomyocyte survival and maturation, and increase patent microvessel lumens compared with time-matched single layer controls after 2 weeks of in vitro culture. Upon implantation, the patch microvessels sprouted into the cardiomyocyte layer of the patch and inosculated with the host vasculature as evidenced by species-specific perfusion labels and erythrocyte staining. Our results demonstrate that the added microvessel layer of a bilayer patch substantially improves in vitro functionality and that the bilayer patch is capable of engraftment with rapid microvessel inosculation on injured myocardium. The bilayer format will allow for scaling up in size through the addition of layers to obtain thicker tissues generating greater force in the future.

A completely biological "off-the-shelf" arteriovenous graft that recellularizes in baboons.

Prosthetic arteriovenous grafts (AVGs) conventionally used for hemodialysis are associated with inferior primary patency rates and increased risk of infection compared with autogenous vein grafts. We tissue-engineered an AVG grown from neonatal human dermal fibroblasts entrapped in bovine fibrin gel that is then decellularized. This graft is both "off-the-shelf" (nonliving) and completely biological. Grafts that are 6 mm in diameter and about 15 cm in length were evaluated in a baboon model of hemodialysis access in an axillary-cephalic or axillary-brachial upper arm AVG construction procedure. Daily antiplatelet therapy was given. Grafts underwent both ultrasound assessment and cannulation at 1, 2, 3, and 6 months and were then explanted for analysis. Excluding grafts with cephalic vein outflow that rapidly clotted during development of the model, 3- and 6-month primary patency rates were 83% (5 of 6) and 60% (3 of 5), respectively. At explant, patent grafts were found to be extensively recellularized (including smoothelin-positive smooth muscle cells with a developing endothelium on the luminal surface). We observed no calcifications, loss of burst strength, or outflow stenosis, which are common failure modes of other graft materials. There was no overt immune response. We thus demonstrate the efficacy of an off-the-shelf AVG that is both acellular and completely biological.

Implantation of a Tissue-Engineered Tubular Heart Valve in Growing Lambs.

Current pediatric heart valve replacement options are suboptimal because they are incapable of somatic growth. Thus, children typically have multiple surgeries to replace outgrown valves. In this study, we present the in vivo function and growth potential of our tissue-engineered pediatric tubular valve. The valves were fabricated by sewing two decellularized engineered tissue tubes together in a prescribed pattern using degradable sutures and subsequently implanted into the main pulmonary artery of growing lambs. Valve function was monitored using periodic ultrasounds after implantation throughout the duration of the study. The valves functioned well up to 8 weeks, 4 weeks beyond the suture strength half-life, after which their insufficiency index worsened. Histology from the explanted valves revealed extensive host cell invasion within the engineered root and commencing from the leaflet surfaces. These cells expressed multiple phenotypes, including endothelial, and deposited elastin and collagen IV. Although the tubes fused together along the degradable suture line as designed, the leaflets shortened compared to their original height. This shortening is hypothesized to result from inadequate fusion at the commissures prior to suture degradation. With appropriate commissure reinforcement, this novel heart valve may provide the somatic growth potential desired for a pediatric valve replacement.

Tissue engineering of acellular vascular grafts capable of somatic growth in young lambs.

Treatment of congenital heart defects in children requiring right ventricular outflow tract reconstruction typically involves multiple open-heart surgeries because all existing graft materials have no growth potential. Here we present an 'off-the-shelf' vascular graft grown from donor fibroblasts in a fibrin gel to address this critical unmet need. In a proof-of-concept study, the decellularized grafts are implanted as a pulmonary artery replacement in three young lambs and evaluated to adulthood. Longitudinal ultrasounds document dimensional growth of the grafts. The lambs show normal growth, increasing body weight by 366% and graft diameter and volume by 56% and 216%, respectively. Explanted grafts display physiological strength and stiffness, complete lumen endothelialization and extensive population by mature smooth muscle cells. The grafts also show substantial elastin deposition and a 465% increase in collagen content, without signs of calcification, aneurysm or stenosis. Collectively, our data support somatic growth of this completely biological graft.

Swelling of Collagen-Hyaluronic Acid Co-Gels: An In Vitro Residual Stress Model.

Tissue-equivalents (TEs), simple model tissues with tunable properties, have been used to explore many features of biological soft tissues. Absent in most formulations however, is the residual stress that arises due to interactions among components with different unloaded levels of stress, which has an important functional role in many biological tissues. To create a pre-stressed model system, co-gels were fabricated from a combination of hyaluronic acid (HA) and reconstituted Type-I collagen (Col). When placed in solutions of varying osmolarity, HA-Col co-gels swell as the HA imbibes water, which in turn stretches (and stresses) the collagen network. In this way, co-gels with residual stress (i.e., collagen fibers in tension and HA in compression) were fabricated. When the three gel types tested here were immersed in hypotonic solutions, pure HA gels swelled the most, followed by HA-Col co-gels; no swelling was observed in pure collagen gels. The greatest swelling rates and swelling ratios occurred in the lowest salt concentration solutions. Tension on the collagen component of HA-Col co-gels was calculated from a stress balance and increased nonlinearly as swelling increased. The swelling experiment results were in good agreement with the stress predicted by a fibril network + non-fibrillar interstitial matrix computational model.

Inosculation and perfusion of pre-vascularized tissue patches containing aligned human microvessels after myocardial infarction.

A major goal of tissue engineering is the creation of pre-vascularized tissues that have a high density of organized microvessels that can be rapidly perfused following implantation. This is especially critical for highly metabolic tissues like myocardium, where a thick myocardial engineered tissue would require rapid perfusion within the first several days to survive transplantation. In the present work, tissue patches containing human microvessels that were either randomly oriented or aligned were placed acutely on rat hearts post-infarction and for each case it was determined whether rapid inosculation could occur and perfusion of the patch could be maintained for 6 days in an infarct environment. Patches containing self-assembled microvessels were formed by co-entrapment of human blood outgrowth endothelial cells and human pericytes in fibrin gel. Cell-induced gel contraction was mechanically-constrained resulting in samples with high densities of microvessels that were either randomly oriented (with 420 ± 140 lumens/mm(2)) or uniaxially aligned (with 940 ± 240 lumens/mm(2)) at the time of implantation. These patches were sutured onto the epicardial surface of the hearts of athymic rats following permanent ligation of the left anterior descending artery. In both aligned and randomly oriented microvessel patches, inosculation occurred and perfusion of the transplanted human microvessels was maintained, proving the in vivo vascularization potential of these engineered tissues. No difference was found in the number of human microvessels that were perfused in the randomly oriented (111 ± 75 perfused lumens/mm(2)) and aligned (173 ± 97 perfused lumens/mm(2)) patches. Our results demonstrate that tissue patches containing a high density of either aligned or randomly oriented human pre-formed microvessels achieve rapid perfusion in the myocardial infarct environment - a necessary first-step toward the creation of a thick, perfusable heart patch.

Effects of Intermittent and Incremental Cyclic Stretch on ERK Signaling and Collagen Production in Engineered Tissue.

Intermittent cyclic stretching and incrementally increasing strain amplitude cyclic stretching were explored to overcome the reported adaptation of fibroblasts in response to constant amplitude cyclic stretching, with the goals of accelerating collagen production and understanding the underlying cell signaling. The effects of constant amplitude, intermittent, and incremental cyclic stretching regimens were investigated for dermal fibroblasts entrapped in a fibrin gel by monitoring the extracellular signal-regulated kinase (ERK1/2) and p38 pathways, collagen transcription, and finally the deposited collagen protein. Activation of ERK1/2, which has been shown to be necessary for stretch-induced collagen transcription, was maximal at 15 min and decayed by 1 h. ERK1/2 was reactivated by an additional onset of stretching or by an increment in the strain amplitude 6 h after the initial stimulus, which was approximately the lifetime of activated p38, a known ERK1/2 inhibitor. While both intermittent and incremental regimens reactivated ERK1/2, only incremental stretching increased collagen production compared to samples stretched with constant amplitude, resulting in a 37% increase in collagen per cell after 2 weeks. This suggests that a regimen with small, frequent increments in strain amplitude is optimal for this system and should be used in bioreactors for engineered tissues requiring high collagen content.

Cells for tissue engineering of cardiac valves.

Heart valve tissue engineering is a promising alternative to prostheses for the replacement of diseased or damaged heart valves, because tissue-engineered valves have the ability to remodel, regenerate and grow. To engineer heart valves, cells are harvested, seeded onto or into a three-dimensional (3D) matrix platform to generate a tissue-engineered construct in vitro, and then implanted into a patient's body. Successful engineering of heart valves requires a thorough understanding of the different types of cells that can be used to obtain the essential phenotypes that are expressed in native heart valves. This article reviews different cell types that have been used in heart valve engineering, cell sources for harvesting, phenotypic expression in constructs and suitability in heart valve tissue engineering. Natural and synthetic biomaterials that have been applied as scaffold systems or cell-delivery platforms are discussed with each cell type. Copyright © 2015 John Wiley & Sons, Ltd.

Tissue Contraction Force Microscopy for Optimization of Engineered Cardiac Tissue.

We developed a high-throughput screening assay that allows for relative comparison of the twitch force of millimeter-scale gel-based cardiac tissues. This assay is based on principles taken from traction force microscopy and uses fluorescent microspheres embedded in a soft polydimethylsiloxane (PDMS) substrate. A gel-forming cell suspension is simply pipetted onto the PDMS to form hemispherical cardiac tissue samples. Recordings of the fluorescent bead movement during tissue pacing are used to determine the maximum distance that the tissue can displace the elastic PDMS substrate. In this study, fibrin gel hemispheres containing human induced pluripotent stem cell-derived cardiomyocytes were formed on the PDMS and allowed to culture for 9 days. Bead displacement values were measured and compared to direct force measurements to validate the utility of the system. The amplitude of bead displacement correlated with direct force measurements, and the twitch force generated by the tissues was the same in 2 and 4 mg/mL fibrin gels, even though the 2 mg/mL samples visually appear more contractile if the assessment were made on free-floating samples. These results demonstrate the usefulness of this assay as a screening tool that allows for rapid sample preparation, data collection, and analysis in a simple and cost-effective platform.

Cyclic Stretch and Perfusion Bioreactor for Conditioning Large Diameter Engineered Tissue Tubes.

A cyclic stretch and perfusion bioreactor was designed to culture large diameter engineered tissue tubes for heart valve applications. In this bioreactor, tubular tissues consisting of dermal fibroblasts in a sacrificial fibrin gel scaffold were placed over porated latex support sleeves and mounted in a custom bioreactor. Pulsatile flow of culture medium into the system resulted in cyclic stretching as well as ablumenal, lumenal, and transmural flow (perfusion). In this study, lumenal remodeling, composition, and mechanical strength and stiffness were compared for tissues cyclically stretched in this bioreactor on either the porated latex sleeves or solid latex sleeves, which did not permit lumenal or transmural flow. Tissues cyclically stretched on porated sleeves had regions of increased lumenal remodeling and cellularity that were localized to the columns of pores in the latex sleeve. A CFD model was developed with COMSOL Multiphysics(®) to predict flow of culture medium in and around the tissue, and the predictions suggest that the enhanced lumenal remodeling was likely a result of elevated shear stresses and transmural velocity in these regions. This work highlights the beneficial effects of increased nutrient transport and flow stimulation for accelerating in vitro tissue remodeling.

Functional Effects of a Tissue-Engineered Cardiac Patch From Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in a Rat Infarct Model.

UNLABELLED: A tissue-engineered cardiac patch provides a method to deliver cardiomyoctes to the injured myocardium with high cell retention and large, controlled infarct coverage, enhancing the ability of cells to limit remodeling after infarction. The patch environment can also yield increased survival. In the present study, we sought to assess the efficacy of a cardiac patch made from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to engraft and limit left ventricular (LV) remodeling acutely after infarction. Cardiac patches were created from hiPSC-CMs and human pericytes (PCs) entrapped in a fibrin gel and implanted acutely onto athymic rat hearts. hiPSC-CMs not only remained viable after in vivo culture, but also increased in number by as much as twofold, consistent with colocalization of human nuclear antigen, cardiac troponin T, and Ki-67 staining. CM+PC patches led to reduced infarct sizes compared with myocardial infarction-only controls at week 4, and CM+PC patch recipient hearts exhibited greater fractional shortening over all groups at both 1 and 4 weeks after transplantation. However, a decline occurred in fractional shortening for all groups over 4 weeks, and LV thinning was not mitigated. CM+PC patches became vascularized in vivo, and microvessels were more abundant in the host myocardium border zone, suggesting a paracrine mechanism for the improved cardiac function. PCs in a PC-only control patch did not survive 4 weeks in vivo. Our results indicate that cardiac patches containing hiPSC-CMs engraft onto acute infarcts, and the hiPSC-CMs survive, proliferate, and contribute to a reduction in infarct size and improvements in cardiac function. SIGNIFICANCE: In the present study, a cardiac patch was created from human induced pluripotent stem cell-derived cardiomyocytes and human pericytes entrapped in a fibrin gel, and it was transplanted onto infarcted rat myocardium. It was found that a patch that contained both cardiomyocytes and pericytes survived transplantation and resulted in improved cardiac function and a reduced infarct size compared with controls.