George C. Engelmayr

Accordion-like honeycombs for tissue engineering of cardiac anisotropy.

Tissue engineered grafts may be useful in myocardial repair, however previous scaffolds have been structurally incompatible with recapitulating cardiac anisotropy. Utilizing microfabrication techniques, a novel accordion-like honeycomb microstructure was rendered in poly(glycerol sebacate) to yield porous, elastomeric 3-D scaffolds with controllable stiffness and anisotropy. Accordion-like honeycomb scaffolds with cultured neonatal rat heart cells demonstrated utility via: (1) closely matched mechanical properties compared to native adult rat right ventricular myocardium, with stiffnesses controlled by polymer curing time; (2) heart cell contractility inducible by electric field stimulation with directionally-dependent electrical excitation thresholds (p<0.05); and (3) greater heart cell alignment (p<0.0001) than isotropic control scaffolds. Prototype bilaminar scaffolds with 3-D interconnected pore networks yielded electrically excitable grafts with multi-layered neonatal rat heart cells. Accordion-like honeycombs can thus overcome principal structural-mechanical limitations of previous scaffolds, promoting the formation of grafts with aligned heart cells and mechanical properties more closely resembling native myocardium.

From Stem Cells to Viable Autologous Semilunar Heart Valve

Background—An estimated 275 000 patients undergo heart valve replacement each year. However, existing solutions for valve replacement are complicated by the morbidity associated with lifelong anticoagulation of mechanical valves and the limited durability of bioprostheses. Recent advances in tissue engineering and our understanding of stem cell biology may provide a lifelong solution to these problems. Methods and Results—Mesenchymal stem cells were isolated from ovine bone marrow and characterized by their morphology and antigen expression through immunocytochemistry, flow cytometry, and capacity to differentiate into multiple cell lineages. A biodegradable scaffold was developed and characterized by its tensile strength and stiffness as a function of time in cell-conditioned medium. Autologous semilunar heart valves were then created in vitro using mesenchymal stem cells and the biodegradable scaffold and were implanted into the pulmonary position of sheep on cardiopulmonary bypass. The valves were evaluated by echocardiography at implantation and after 4 months in vivo. Valves were explanted at 4 and 8 months and examined by histology and immunohistochemistry. Valves displayed a maximum instantaneous gradient of 17.2±1.33 mm Hg, a mean gradient of 9.7±1.3 mm Hg, an effective orifice area of 1.35±0.17 cm2, and trivial or mild regurgitation at implantation. Gradients changed little over 4 months of follow-up. Histology showed disposition of extracellular matrix and distribution of cell phenotypes in the engineered valves reminiscent of that in native pulmonary valves. Conclusions—Stem-cell tissue-engineered heart valves can be created from mesenchymal stem cells in combination with a biodegradable scaffold and function satisfactorily in vivo for periods of >4 months. Furthermore, such valves undergo extensive remodeling in vivo to resemble native heart valves.

Advanced Material Strategies for Tissue Engineering Scaffolds

Tissue engineering seeks to restore the function of diseased or damaged tissues through the use of cells and biomaterial scaffolds. It is now apparent that the next generation of functional tissue replacements will require advanced material strategies to achieve many of the important requirements for long-term success. Here we provide representative examples of engineered skeletal and myocardial tissue constructs in which scaffolds were explicitly designed to match native tissue mechanical properties as well as to promote cell alignment. We discuss recent progress in microfluidic devices that can potentially serve as tissue engineering scaffolds, since mass transport via microvascular-like structures will be essential in the development of tissue engineered constructs on the length scale of native tissues. Given the rapid evolution of the field of tissue engineering, it is important to consider the use of advanced materials in light of the emerging role of genetics, growth factors, bioreactors, and other technologies.

In vivo monitoring of function of autologous engineered pulmonary valve

OBJECTIVES Clinical translation of tissue-engineered heart valves requires valve competency and lack of stenosis in the short and long term. Early studies of engineered valves showed promise, although lacked complete definition of valve function. Building on prior experiments, we sought to define the in vivo changes in structure and function of autologous engineered pulmonary valved conduits. METHODS Mesenchymal stem cells were isolated from neonatal sheep bone marrow and seeded onto a bioresorbable scaffold. After 4 weeks of culture, valved conduits were implanted. Valve function, cusp, and conduit dimensions were evaluated at implantation (echocardiography), at the experimental midpoint (magnetic resonance imaging), and at explant, at 1 day, and 1, 6, 12, or 20 weeks postoperatively (direct measurement, echocardiography). Histologic evaluation was performed. RESULTS Nineteen animals underwent autologous tissue-engineered valved conduit replacement. At implantation, valved conduit function was excellent; maximum transvalvular pressure gradient by Doppler echocardiography was 17 mm Hg; most valved conduits showed trivial pulmonary regurgitation. At 6 postoperative weeks, valve cusps appeared less mobile; pulmonary regurgitation was mild to moderate. At 12 weeks or more, valved conduit cusps were increasingly attenuated and regurgitant. Valved conduit diameter remained unchanged over 20 weeks. Dimensional measurements by magnetic resonance imaging correlated with direct measurement at explant. CONCLUSIONS We demonstrate autologous engineered tissue valved conduits that function well at implantation, with subsequent monitoring of dimensions and function in real time by magnetic resonance imaging. In vivo valves undergo structural and functional remodeling without stenosis, but with worsening pulmonary regurgitation after 6 weeks. Insights into mechanisms of in vivo remodeling are valuable for future iterations of engineered heart valves.

Biomimetic engineered muscle with capacity for vascular integration and functional maturation in vivo.

Significance Engineering of highly functional skeletal muscle tissues can provide accurate models of muscle physiology and disease and aid treatment of various muscle disorders. Previous tissue-engineering efforts have fallen short of recreating structural and contractile properties of native muscle in vitro. Here, we describe the creation of biomimetic skeletal muscle tissues with structural, functional, and myogenic properties characteristic of native muscle and contractile stress values that surpass those of neonatal rat muscle. When implanted and real-time imaged in live animals, engineered muscle grafts undergo robust vascularization and perfusion, exhibit continued myogenesis, and show further improvements in intracellular calcium handling and contractile function. This process is significantly enhanced by myogenic predifferentiation and formation of aligned muscle architecture in vitro. Tissue-engineered skeletal muscle can serve as a physiological model of natural muscle and a potential therapeutic vehicle for rapid repair of severe muscle loss and injury. Here, we describe a platform for engineering and testing highly functional biomimetic muscle tissues with a resident satellite cell niche and capacity for robust myogenesis and self-regeneration in vitro. Using a mouse dorsal window implantation model and transduction with fluorescent intracellular calcium indicator, GCaMP3, we nondestructively monitored, in real time, vascular integration and the functional state of engineered muscle in vivo. During a 2-wk period, implanted engineered muscle exhibited a steady ingrowth of blood-perfused microvasculature along with an increase in amplitude of calcium transients and force of contraction. We also demonstrated superior structural organization, vascularization, and contractile function of fully differentiated vs. undifferentiated engineered muscle implants. The described in vitro and in vivo models of biomimetic engineered muscle represent enabling technology for novel studies of skeletal muscle function and regeneration.

THE ROLE OF ORGAN LEVEL CONDITIONING ON THE PROMOTION OF ENGINEERED HEART VALVE TISSUE DEVELOPMENT IN-VITRO USING MESENCHYMAL STEM CELLS

We have previously shown that combined flexure and flow (CFF) augment engineered heart valve tissue formation using bone marrow-derived mesenchymal stem cells (MSC) seeded on polyglycolic acid (PGA)/poly-L-lactic acid (PLLA) blend nonwoven fibrous scaffolds (Engelmayr, et al., Biomaterials 2006; vol. 27 pp. 6083-95). In the present study, we sought to determine if these phenomena were reproducible at the organ level in a functional tri-leaflet valve. Tissue engineered valve constructs (TEVC) were fabricated using PGA/PLLA nonwoven fibrous scaffolds then seeded with MSCs. Tissue formation rates using both standard and augmented (using basic fibroblast growth factor [bFGF] and ascorbic acid-2-phosphate [AA2P]) media to enhance the overall production of collagen were evaluated, along with their relation to the local fluid flow fields. The resulting TEVCs were statically cultured for 3 weeks, followed by a 3 week dynamic culture period using our organ level bioreactor (Hildebrand et al., ABME, Vol. 32, pp. 1039-49, 2004) under approximated pulmonary artery conditions. Results indicated that supplemented media accelerated collagen formation (approximately 185% increase in collagen mass/MSC compared to standard media), as well as increasing collagen mass production from 3.90 to 4.43 pg/cell/week from 3 to 6 weeks. Using augmented media, dynamic conditioning increased collagen mass production rate from 7.23 to 13.65 pg/cell/week (88.8%) during the dynamic culture period, along with greater preservation of net DNA. Moreover, when compared to our previous CFF study, organ level conditioning increased the collagen production rate from 4.76 to 6.42 pg/cell/week (35%). Newly conducted CFD studies of the CFF specimen flow patterns suggested that oscillatory surface shear stresses were surprisingly similar to a tri-leaflet valve. Overall, we found that the use of simulated pulmonary artery conditions resulted in substantially larger collagen mass production levels and rates found in our earlier CFF study. Moreover, given the fact that the scaffolds underwent modest strains (approximately 7% max) during either CFF or physiological conditioning, the oscillatory surface shear stresses estimated in both studies may play a substantial role in eliciting MSC collagen production in the highly dynamic engineered heart valve fluid mechanical environment.

Protein Precoating of Elastomeric Tissue-Engineering Scaffolds Increased Cellularity, Enhanced Extracellular Matrix Protein Production, and Differentially Regulated the Phenotypes of Circulating Endothelial Progenitor Cells

Background— Optimal cell sources and scaffold-cell interactions remain unanswered questions for tissue engineering of heart valves. We assessed the effect of different protein precoatings on a single scaffold type (elastomeric poly (glycerol sebacate)) with a single cell source (endothelial progenitor cells). Methods and Results— Elastomeric poly (glycerol sebacate) scaffolds were precoated with laminin, fibronectin, fibrin, collagen types I/III, or elastin. Characterized ovine peripheral blood endothelial progenitor cells were seeded onto scaffolds for 3 days followed by 14 days incubation. Endothelial progenitor cells were CD31+, vWF+, and α-SMA- before seeding confirmed by immunohistochemistry and immunoblotting. Both precoated and uncoated scaffolds demonstrated surface expression of CD31+ and vWF+, α-SMA+ cells and were found in the “interstitium” of the scaffold. Protein precoating of elastomeric poly (glycerol sebacate) scaffolds revealed significantly increased cellularity and altered the phenotypes of endothelial progenitor cells, which resulted in changes in cellular behavior and extracellular matrix production. Moreover, mechanical flexure testing demonstrated decreased effective stiffness of the seeded scaffolds compared with unseeded controls. Conclusions— Scaffold precoating with extracellular matrix proteins can allow more precise “engineering” of cellular behavior in the development of tissue engineering of heart valves constructs by altering extracellular matrix production and cell phenotype.

Transforming Growth Factor-β1 Modulates Extracellular Matrix Production, Proliferation, and Apoptosis of Endothelial Progenitor Cells in Tissue-Engineering Scaffolds

Background— Valvular endothelial cells and circulating endothelial progenitor cells (EPCs) can undergo apparent phenotypic change from endothelial to mesenchymal cell type. Here we investigated whether EPCs can promote extracellular matrix formation in tissue engineering scaffolds in response to transforming growth factor (TGF)-β1. Method and Results— Characterized ovine peripheral blood EPCs were seeded onto poly (glycolic acid)/poly (4-hydroxybutyrate) scaffolds for 5 days. After seeding at 2×106 cells/cm2, scaffolds were incubated for 5 days in a roller bottle, with or without the addition of TGF-β1. After seeding at 15×106 cells/cm2, scaffolds were incubated for 10 days in a roller bottle with or without the addition of TGF-β1 for the first 5 days. Using immunofluorescence and Western blotting, we demonstrated that EPCs initially exhibit an endothelial phenotype (ie, CD31+, von Willebrand factor+, and α–smooth muscle actin (SMA)−) and can undergo a phenotypic change toward mesenchymal transformation (ie, CD31+ and α-SMA+) in response to TGF-β1. Scanning electron microscopy and histology revealed enhanced tissue formation in EPC-TGF-β1 scaffolds. In both the 10- and 15-day experiments, EPC-TGF-β1 scaffolds exhibited a trend of increased DNA content compared with unstimulated EPC scaffolds. TGF-β1–mediated endothelial to mesenchymal transformation correlated with enhanced expression of laminin and fibronectin within scaffolds evidenced by Western blotting. Strong expression of tropoelastin was observed in response to TGF-β1 equal to that in the unstimulated EPC. In the 15-day experiments, TGF-β1–stimulated scaffolds revealed dramatically enhanced collagen production (types I and III) and incorporated more 5-bromodeoxyuridine and TUNEL staining compared with unstimulated controls. Conclusions— Stimulation of EPC-seeded tissue engineering scaffolds with TGF-β1 in vitro resulted in a more organized cellular architecture with glycoprotein, collagen, and elastin synthesis, and thus noninvasively isolated EPCs coupled with the pleiotropic actions of TGF-β1 could offer new strategies to guide tissue formation in engineered cardiac valves.

A Structural Model for the Flexural Mechanics of Nonwoven Tissue Engineering Scaffolds

The development of methods to predict the strength and stiffness of biomaterials used in tissue engineering is critical for load-bearing applications in which the essential functional requirements are primarily mechanical. We previously quantified changes in the effective stiffness (E) of needled nonwoven polyglycolic acid (PGA) and poly-L-lactic acid (PLLA) scaffolds due to tissue formation and scaffold degradation under three-point bending. Toward predicting these changes, we present a structural model for E of a needled nonwoven scaffold in flexure. The model accounted for the number and orientation of fibers within a representative volume element of the scaffold demarcated by the needling process. The spring-like effective stiffness of the curved fibers was calculated using the sinusoidal fiber shapes. Structural and mechanical properties of PGA and PLLA fibers and PGA, PLLA, and 50:50 PGA/PLLA scaffolds were measured and compared with model predictions. To verify the general predictive capability, the predicted dependence of E on fiber diameter was compared with experimental measurements. Needled nonwoven scaffolds were found to exhibit distinct preferred (PD) and cross-preferred (XD) fiber directions, with an E ratio (PD/XD) of approximately 3:1. The good agreement between the predicted and experimental dependence of E on fiber diameter (R2 = 0.987) suggests that the structural model can be used to design scaffolds with E values more similar to native soft tissues. A comparison with previous results for cell-seeded scaffolds (Engelmayr, G. C., Jr., et al., 2005, Biomaterials, 26(2), pp. 175-187) suggests, for the first time, that the primary mechanical effect of collagen deposition is an increase in the number of fiber-fiber bond points yielding effectively stiffer scaffold fibers. This finding indicated that the effects of tissue deposition on needled nonwoven scaffold mechanics do not follow a rule-of-mixtures behavior. These important results underscore the need for structural approaches in modeling the effects of engineered tissue formation on nonwoven scaffolds, and their potential utility in scaffold design.

Co-culture induces alignment in engineered cardiac constructs via MMP-2 expression

Cardiac tissue engineering has been limited by the inability to recreate native myocardial structural features. We hypothesized that heart cell elongation and alignment in 3D engineered cardiac constructs would be enhanced by using physiologic ratios of cardiomyocytes (CM) and cardiac fibroblasts (CF) via matrix metalloprotease (MMP)-dependent mechanisms. Co-cultured CM and CF constructs were compared to CM-enriched constructs using either basal media or media with a general MMP inhibitor for 8 days. Co-cultured constructs exhibited significantly increased cell alignment (p<0.0002), which was eliminated by MMP inhibition. Co-cultured constructs expressed substantial active MMP-2 protein that was not present in CM-enriched constructs, increased pro-MMP-2 (p<0.001), and reduced pro-MMP-9 (p<0.001) expression. Apoptosis was decreased by co-culture (p<0.05), independent of MMP inhibition. These results demonstrated that co-culture of CF in physiologic ratios within engineered cardiac constructs improved cell elongation and alignment via increased MMP-2 expression and activation, and also improved viability independent of MMP activity.