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.
Clinically approved replacement valves for use in pediatric patients are associated with a high frequency of reoperations, thus limiting their effectiveness.1 This limitation has led us to undertake a tissue-engineering (TE) approach toward the development of replacement heart valves by seeding autologous cells onto porous biodegradable polymer scaffolds.2–7 Initial animal studies have produced encouraging results. Outstanding issues still remain, however, including optimal cell source, in vitro conditioning, in vivo maturation, and scaffold materials with appropriate mechanical and chemical characteristics as well as proper scaffold-cell interactions to promote extracellular matrix (ECM) production and regulate cellular behavior.
We investigated noninvasively isolated cell types for pediatric applications, including bone marrow–derived mesenchymal stem cells and peripheral blood–derived endothelial progenitor cells (EPC).6,7 Evidence suggests that subsets of endothelial cells (EC) can transdifferentiate to a mesenchymal phenotype.8–11 Furthermore, preliminary studies have demonstrated that circulating EPC can potentially provide interstitial and EC functions required to produce ECM on PGA/P4HB nonwoven scaffolds, suggesting their use in constructing autologous tissue engineering of heart valves (TEHV) (V.L.S., et al, unpublished data, 2004).
Scaffolds based on a biocompatible and biodegradable elastomer elastomeric poly (glycerol sebacate) (PGS) have recently been developed that offer desirable mechanical properties such as of low tissue-like stiffness and rubber-like elasticity, thereby allowing for large deformations.12,13 Coating of surfaces with cell-adhesion mediators like fibronectin (FN), laminin (LM), or other ECM components enhances cell attachment, proliferation, differentiation, and migration.14 In an attempt to differentiate the mechanochemical characteristics of scaffold materials and understand the cell-surface interactions, we precoated the PGS scaffold with ECM proteins and assessed the cellular behavior of the EPC.
Given the importance of EPC plasticity and its potential role in ECM synthesis, organization, and remodeling, we hypothesized that precoating PGS scaffolds with ECM proteins could promote EPC differentiation, proliferation, and ECM formation, and that the levels of expression are dependent on ECM polymeric coating.
Materials and Methods
Animal care procedures were approved by the Animal Care Committee of the Children’s Hospital Boston.
Isolation and Culture of EPC
EPC were isolated from 30 mL of heparinized (1000 IU/mL) ovine (age 4 weeks, weight 10 to 15 kg) peripheral blood as described.7,15 Histopaque gradient (density 1.077 g/mL; Sigma) centrifugation was used to obtain leukocyte fraction. Isolated mononuclear cells were transferred to EBM-2 medium supplemented with EGM-2 SingleQuots (Clonetics), antibiotics, and 20% FBS (Hyclone) but without hydrocortisone, and placed on a 6-well plate coated with 1% gelatin (Sigma) for 24 hours then transferred to new gelatin-coated plates. After 4 days in culture, nonadherent cells were removed and the culture maintained through days 7 to 10. Ovine vascular EC and SMC were prepared as previously described.2 At ≈80% confluence, cells were harvested with 0.05% trypsin-EDTA (GIBCO) and replated in 150 cm2 polystyrene dishes (Corning) at 104 cells/cm2. After 3 to 4 weeks, significant cell counts necessary for seeding were obtained. Cells were characterized before seeding.
Preparation of the Tissue-Engineered Constructs
Flat PGS scaffolds were synthesized and fabricated (pore size 75 to 150 μm, porosity 90%, thickness 1.0 mm, specific gravity 69 mg/cm3) by salt fusion method as previously described.13 PGS scaffolds were sterilized in graded series of ethanol (70%, 50%, and 25%), followed by gently washing twice with PBS and autoclaved water.
Precoating With ECM Proteins
Kinetic studies were performed with incubation times ranging from 1 hour to 5 days to determine the duration to reach equilibrium. The PGS scaffold pieces were coated with ECM substrates in PBS, incubated, and washed multiple times with PBS. Concentration-dependent experiments were carried out to quantify the amount of bound proteins on the PGS scaffolds and the amount of proteins in the supernatants spectrophotometrically. The measured protein content bound to the scaffold was compared with the actual original precoated protein solution, and the effective coating concentration of each substrate was established.
Precoated PGS scaffolds were prepared in a disposable scintillation vial (Wheaton, 20 mL) using the following substrates: calf skin Coll I (30 μg/mL), bovine plasma FN (20 μg/mL) and human placenta LM (10 μg/mL, Sigma), recombinant protein collagen type III (Coll III: 100 μg/mL, Novus biologicals), aortic elastin (EL; 220 μg/mL, Elastin Products), and fibrin (FB): plasma fibrinogen (5 mg/mL, Sigma) and thrombin (Calbiochem, 14 IE/mL). Vials were rotated at 2 rpm at 37°C for 1.5 hours with FN, 24 hours with Coll I, LM, and FB, 2 day with Coll III, and 5 days with EL.
Seeding and Culturing
Dynamic rotational seeding and culturing were performed as described.15 EPC were seeded in a single procedure, and the number of cells were controlled by counting using a hemocytometer before seeding on the PGS scaffold. EPC (15×106 per cm2; passages 4 to 6) were seeded onto ECM precoated scaffolds and rotated at 2 rpm at 37°C for 3 days. Culture medium (10 mL) was changed after 4 hours then every 8 to 12 hours thereafter. Seeded scaffolds were cultured in a polystyrene roller bottle (850 cm2, Corning) and rotated at 0.2 cycles/min at 37°C for 14 days (media pH was maintained at pH >7).
Analysis of EPC Cultures and Tissue-Engineered Constructs
First passage and seeded cells were plated and methanol-fixed as described.7,15 Slides were blocked with 1% BSA in PBS, incubated with primary antibodies against CD31 (1:1000, Santa Cruz), vWF (1:500, DAKO), and α-SMA Clone 1A4 (1:2000, Sigma) followed by fluorescein-conjugated secondary antibodies (Vector Laboratories). Slides were examined and photographed under a fluorescence microscope (Nikon Eclipse TE2000).
Cells were lysed with 4 mol/L urea, 0.5% sodium dodecyl sulfate, 0.5% Nonidet P-40, 100 mmol/L Tris, and 5 mmol/L ethylenediaminetetraacetic acid, pH 7.4, containing protease inhibitors (Roche). Protein concentrations were determined (BCA Assay Kit, Pierce). Immunoblotting of cells and seeded scaffold extracts using α-Tubulin Clone DM 1A (Sigma), CD31 (1:500), and α-SMA (1:1000) were performed as described.15
Histology and Immunohistochemistry
Histological analysis and characterization of cell phenotypes were performed as described.15 Representative portions of scaffolds were formalin-fixed and paraffin-embedded. Serial sections (6 μm) were stained with H&E for morphology and antibodies specific for α-SMA, vWF, CD31, and FN (Novus Biologicals). Immunostaining was performed by the ABC method with biotin-labeled secondary antibodies (Vector Laboratories). Total nuclei, vWF+, CD31+, and α-SMA+ cells per cm2 of scaffolds were evaluated microscopically at x400 in histological sections and counted by 2 blinded observers independently.
Scanning Electron Microscopy
Samples were fixed in 2.5% (v/v) glutaraldehyde and 1% (wt/vol) osmium tetraoxide, dehydrated in graded series of ethanol, dried, and sputter coated with gold-palladium alloy (≈3 nm thickness; Cressington Scientific Instruments). Each scaffold sample was imaged at 5 kV on a JEOL JSM-6330F field emission SEM.16
Measurement of Cellular Proliferation and Apoptosis
Seeded scaffolds were labeled in vitro with BrdU (16 ng/mL/d, Sigma) for 3 days as described.15,17 TUNEL staining was performed and evaluated as described.15 Cells containing dark nuclear BrdU staining were considered to be BrdU+ and to have undergone DNA synthesis during labeling. Overall proliferation and apoptosis data were obtained by counting the number of BrdU+ and TUNEL+ cells out of the total cell number in histological sections.
Quantification and Evaluation of Fibronectin Content
Evaluation of FN content was performed by use of cross-sectional sections of TE constructs. The percent positive area was stained for FN. Captured images were analyzed twice independently with MetaMorph Imaging System (Molecular Devices) by a blinded observer. A manual threshold process was used to determine the area of positive immunoreactivity. The percentage of total area with positive color was recorded for each TE construct.
Quantitative Biochemical Matrix Analysis
Quantification of total cellular DNA was performed as described.18 Representative portions of scaffolds were weighed and extracted in a solution of 0.125-mg/mL papain and 10 mmol/L L-cysteine dihydrochloride (Sigma) in phosphate buffered EDTA (PBE, Sigma). Total DNA was measured (Picogreen dsDNA Quantitation Kit; Molecular Probes). Total collagen was extracted in 0.5 mol/L acetic acid and pepsin (1 mg/mL Pepsin A); proteoglycans and S-GAG in 4 mol/L guanidine-HCl and 0.5 mol/L sodium acetate and measured (Sircol and Blyscan assay kits; Biocolor Ltd.). Total collagen and S-GAG content of the Coll I/ III-precoated scaffolds were calculated by subtracting the amount of Coll I/III precoated on the scaffold from the measured collagen and S-GAG content.
Effective Stiffness Measurement Methods
Measurement of the effective stiffness, E, of tissue samples was performed as described.16,18 In brief, rectangular samples were marked with 5 to 10 small black dots and subjected to 3-point flexure by a calibrated flexure bar of known stiffness. Displacements of sample markers, flexure bar marker, and reference marker were recorded. The program calculated the applied moment, M, and the resulting change in curvature, Δκ, of each sample. The flexural rigidity, EI, of each sample was calculated per the Bernoulli-Euler moment-curvature relationship: M=EIΔκ. The moment of inertia, I, was calculated in which t and w are the thickness and width of the sample, respectively: equation , t was measured using a calibrated digital image of each sample (SigmaScan Pro), and E was calculated by dividing EI by I.
All results are expressed as mean±SEM. Comparisons between groups were made with a Student t test (Sigma Stat, Jandel Scientific). If measurements failed the normality test, the nonparametric Mann-Whitney rank sum test was used. P<0.001 and <0.05 were considered significant.
Statement of Responsibility
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
Circulating EPC Demonstrate Endothelial Phenotype Before Seeding
Peripheral blood mononuclear cells were cultured for 7 days generating EPC as expected. Characterization of cells at 3 stages was performed: Day-7 attached cells, outgrowth EC (developing after 2 to 3 weeks), and isolated clonal and single-population amplified EPC cultures. Before seeding, each exhibited endothelial morphology and expressed endothelial antigens CD31 and vwF by immunofluorescence (Figure 1A). No immunoreactivity was observed for α-SMA in EPC or control IgG (data not shown). To further characterize the endothelial phenotype, immunoblots of cell lysates from pure cultures of EPC confirmed the expression of CD31 and not α-SMA (Figure 1B). Ovine carotid artery–derived EC served as controls, demonstrating appropriate presence of immunoreacitvity for CD31 and absence of α-SMA markers. Ovine carotid artery–derived SMC served as controls, demonstrating appropriate absence of immunoreactivity for CD31 and presence of α-SMA markers.
Protein Adsorption Studies
Adsorption kinetic studies identified the minimum coating time necessary to obtain a constant and maximum amount of adsorbed protein on the PGS scaffolds. The amount of bound protein on the PGS scaffolds increased proportionately with the protein solution concentration, reaching a plateau value (Figure 2). Fibrin clots were prepared with fibrinogen complex and thrombin components. Initially, we evaluated a dose-range of fibrin concentrations (2 to 5 mg/mL) with thrombin at 2.5 U/mL (Figure 2C, left). Subsequently, dose-dependent experiments on thrombin (5 to 20 U/mL) were carried out in the presence of fibrin (5 mg/mL). The plateau value of bound protein on the PGS scaffolds was reached at a fibrin and thrombin concentrations of 5 mg/mL and 14 U/mL, respectively (Figure 2C, right). Binding experiments using precoated ECM substrates demonstrated similar protein binding at ≈98% levels for all substrates examined.
Protein Precoating Enhances Cellularity in EPC-PGS Scaffolds
FN-precoated scaffolds were stained with H&E and demonstrated increased cellularity both in the luminal surface with ECM formation (arrows) and “interstitial” layer of the scaffolds as compared with the uncoated controls (Figure 3A). At 14 days of incubation, all precoated scaffolds demonstrated increased cellularity (FN: 49%, P<0.001; Coll III: 44%, FB: 36%, EL: 34%, and LM: 31%, P<0.05) versus those of uncoated controls except Coll I precoated scaffolds which had a 25% increase in cellularity (Figures 3B and 4⇓). FN-precoated scaffolds revealed substantial cellularity compared with uncoated controls (19610±812 versus 9938±1205 per cm2, P<0.001). SEM examination was performed on precoated, uncoated, seeded, and unseeded scaffolds (Figure 3C). Precoated scaffolds (FN, Coll I/III and FB) demonstrated smooth and confluent surface layer with clusters of EPC filling the interconnecting spaces in the polymer matrices. In contrast, less homogenous constructs with very sparse and incomplete cellular coverage were seen in LM- and EL-precoated scaffolds and uncoated controls.
Protein Precoating Promotes Proliferation and Decreases the Number of Apoptotic Cells in EPC-PGS Scaffolds
BrdU and TUNEL staining identified multiple cells undergoing proliferation (Figure 5A) and apoptosis (Figure 5B), respectively of the seeded precoated scaffolds. Immunohistochemistry (IHC) analysis revealed a 1.8- to 2.9-fold (P<0.05) increase in BrdU incorporation (a) and decrease in the number of apoptotic cells (b) on precoated scaffolds compared with uncoated controls (Figure 5C). To examine the relationship between proliferation and apoptosis, total nuclei counts were replotted as a function of cell growth (proliferation minus apoptosis) (Figure 5C-c). Precoated scaffolds demonstrated a 30% to 60% cell growth with increasing cellularity except in LM-precoated scaffolds at 16% cell growth (Figure 4).
Differential Effect of Different Protein Coatings on Cell Phenotype
Immunohistochemistry (IHC) studies revealed the presence of CD31+, vwF+, and α-SMA+ cells in FN-precoated scaffold (Figure 6A). Both endothelial and α-SMA cell types were detected on the luminal surface (top), and in the “interstitial” layer of the seeded scaffolds (bottom). Both cell types were observed in all precoated and uncoated scaffolds (data not shown). Differences in the number of cell phenotypes were detected within precoated scaffolds as well as between precoated and uncoated scaffolds (Figure 6B). IHC analysis revealed a significant fold decrease in the predominance of VWF+ over α-SMA+ cells (FN: 2.6; Coll III: 4.2; FB: 2.8; Coll I: 3.7, P<0.001) in precoated scaffolds compared with uncoated controls. There was no significant difference between EL and LM-precoated and uncoated scaffolds (Figure 6B, left). A similar trend was observed in the distribution of CD31+ and α-SMA+ cells in the precoated and uncoated scaffolds (Figures 6B, right, and 4⇑).
Protein Precoating Increases Fibronectin Content in EPC-PGS Scaffolds
Based on our observations we investigated whether other protein precoating substrates promote FN production. Immunostaining of both precoated and uncoated scaffolds with anti-human FN antibody demonstrated ubiquitous expression throughout the scaffold (data not shown). Immunostaining of LM-precoated scaffolds (d-e) revealed enhanced FN expression compared with uncoated controls (a-b) and comparable to the native valve (c) (Figure 7A). Quantitative analysis in precoated LM and Coll III scaffolds revealed increased FN content by 68% (P<0.001) and 28% (P<0.05), respectively compared with uncoated scaffolds. Increased FN content verses control was observed in other protein precoated scaffolds with the exception of FB-precoated scaffolds (Figures 7B and 4⇑).
Protein Precoating Affects DNA and Collagen Content but Not GAG Production
Precoated scaffolds revealed a 23% to 63% increase in DNA content compared with the uncoated controls (FN: 57%, P<0.05; Coll III: 63%, P<0.001; FB: 23%, P<0.05). Furthermore, the collagen content in all precoated scaffolds was >2-fold that in uncoated controls except for EL-pre-coated scaffolds (FN: 34-fold, Coll III: 8-fold, FB: 13-fold, P<0.001; LM and Coll I: 3-fold, P<0.05). In contrast, S-GAG content fell by 16% to 47% compared with uncoated controls in all precoated scaffolds (FN: 32%, FB: 16%, P<0.05; EL: 47%, P<0.001 and Coll I: 32% P<0.05) except for Coll III- and LM-precoated scaffolds (Figures 4 and 8⇓).
Seeding EPC Accelerates the Degradation of PGS Scaffold
After 14 days of incubation, the effective stiffness of the seeded constructs, regardless of whether the PGS was precoated or not, decreased by 33% (P<0.001) from that of unseeded PGS scaffolds. Stiffness of the PGS scaffolds, regardless whether it was seeded or not, approached that of the native pulmonary valve (PV; 5.49±0.25 and 8.14±0.12, respectively versus 25.86±6.18 kPa; Figures 4 and 9⇓).
In an attempt to develop clinically feasible TEHV biomaterials for pediatric applications, we investigated the ability of noninvasively isolated EPC to differentiate, proliferate, and secrete ECM on elastomeric PGS scaffolds and determined the effect of precoating PGS scaffolds with various ECM proteins on cellular behavior.
Protein adsorption has profound impact on the interactions between a biomaterial and cells and tissues. In this study, significant amount of ECM protein adsorbed on PGS scaffolds. The amount of EL and Coll III were significantly higher than the other ECM molecules and they both took longer time to reach the steady state. The difference in the amount of adsorbed proteins and the equilibrium time is likely caused by different adhesion mechanisms and different affinities of the proteins toward PGS.
EPC have been shown to undergo an apparent phenotypic change from endothelial toward a mesenchymal cell type in response to TGF-β1 on scaffolds.15,19 The current study confirmed that EPC can undergo spontaneous phenotypic change on PGS scaffolds to a cell type which has characteristics of valvular interstitial cells, with EC and α-SMA–like cells throughout the surface and “interstitial” layer of the scaffold.20 Precoating PGS scaffolds with various ECM proteins resulted in increased cellularity and ECM protein production and regulated differentially the phenotypes of EPC. Moreover, the seeding and subsequent cultivation of EPC on PGS scaffolds led to a decrease in the overall stiffness of the construct in contrast to previous TEHV design-scaffolds which tend to be too stiff on initial cell seeding compared with native PV tissue.2–6
The finding that ECM production and cell phenotypes was strongly dependent on the different protein coatings whereas GAG production did not appear to be substrate- specific raised further hypothesis that there may be affinity of different types of integrin that regulates the production of EPC. Because native PV-and TEHV leaflets are subjected to local hemodynamic forces, responding by undergoing large anisotropic stretching and flexure, further investigations are warranted to determine the effects of biomechanical stimulation on regulating binding of integrins and nonintegrins to ECM precoated substrates, in particular FN, thus providing mechanistic insights and implication for EPC signaling on TE constructs.21
As EPC begin to undergo phenotypic changes on seeding and cultivation on PGS scaffolds, it is important to consider what cell type contributes to ECM formation in vitro. Immunostaining with cell-type specific antibodies demonstrated collagen expression in VWF+ and α-SMA+ cells within the seeded constructs (data not shown), but whether the presence of collagen and FN in these TE constructs stems directly from EC and α-SMA-like cells or indirectly from the paracrine effect of precoating on these cells remains unclear.
There are several limitations of the study. First, we did not explore other possibilities of cell fate processes such as cell adhesion, migration, differentiation, and other specific functions to further understand the molecular interactions between the ECM substrates and EPC on the elastomeric scaffolds. The LM precoated group had a lowest cell growth of 16% attributable to higher apoptosis than its proliferation rate at a moderately high total nuclei count. In contrast, EL precoated group had a higher cell growth at 35% attributable to higher proliferation than its apoptosis rate. However, at a similar total nuclei count as the LM-precoated scaffolds, it yielded a nadir point as shown in the graph (Figure 5C). This could be probably attributable to decreased cell adhesion or migration in the EL-precoated scaffolds. When the ECM substrate is passively absorbed, it can adopt a range of orientations, some of which may be favorable for binding site recognition and cell adhesion. The reduction in functional recognition or cell attachment on the absorbed EL-precoated surface may be attributable to high absorbed ECM density and concentration (220 μg/mL) or to changes in binding site accessibility or conformation.22 It is also noteworthy to define cell signaling cascade(s) by which EPC undergo appropriate molecular education as dictated by the ECM constituents to guide functional tissue development in an organized, predictable and controlled manner. The question of how the right quantity of a signaling molecule will be detected by EPC and or mesenchymal cells at the right time remains to be investigated.23 Second, we did not perform multiple time points studies to elucidate the role of EPC proliferation, apoptosis, differentiation and other processes in the degradation and remodeling of precoated TE constructs. In the present study, the mechanical flexure test results suggest that the presence of EPC on the PGS scaffold may accelerate the scaffold’s hydrolytic degradation, further highlighting the importance of accounting for the influence of cell seeding in considering the mechanical properties of PGS. Previous studies support our outcomes that PGS scaffolds are elastomeric and exhibit desirable mechanical properties with low modulus and large elongation ratio.12 Such scaffolds are important for soft tissue applications and could be specifically tailored for TEHV development. We feel that pursuit of the multiple time points is beyond the scope our study. However, further temporal studies on the direct modulatory role of EPC-ECM substrates on the biodegradation properties of PGS scaffolds are warranted. Third, this study is not intended for optimization of our future TEHV design. Our study suggests that using single or multiple protein precoating substrates will allow us to build and adjust a particular biological environment to obtain cell- and tissue-specificity. Collectively, we demonstrated that using single or multiple protein precoating substrates could predetermine cellular phenotypes and differentiation as well as enhance ECM formation on scaffolds. Scaffold precoating with ECM proteins allows more precise engineering of cellular behavior in the development of TEHV constructs by altering ECM production and cell phenotype. This offers a unique strategy for designing TEHV by gaining precise control over the biomaterial as well as the cellular behavior it induces.23 We predict that we will be applying different ECM precoating substrates depending on the final specialized tissue type and structure that we sought to engineer.24
The authors acknowledge important advice and contribution from Drs Joyce Bischoff, Marsha Moses and Rosalyn Adam and Joseph Khoury (Children’s Hospital Boston) and David Martin, PhD (Tepha, Inc), and excellent technical assistance of Tonora Archibald, Jill Wylie-Sears, MS and Kaityln Llewylln, BS and Sadakatsu Ikeda, MD (Children’s Hospital Boston), and editorial expertise of Michael Blaze, PhD.
Sources of Funding
This work was supported by NIH grants HL-60463 (J.E.M), HL-68816 (M.S.S) and HL-68816-01 (M.S.S/ J.E.M). J.E.M is a recipient of TEPHA/ National Institutes of Standards and Technology (NIST NANB2H3053), Gross Cardiovascular and Tissue Engineering Donor Funds and Center for Integration of Medicine and Innovative Technology (CIMIT). Y.W. is a recipient of the National Science Foundation grant EEC-9731643. V.L.S. is the recipient of the American Heart Association National Scientist Development Grant 0635620T. G.C.E. was supported by American Heart Association Predoctoral Fellowship 0415406U, PA/DE Affiliate. J.A.J. is the recipient of a Summer Fellowship from the American Heart Association, Northeast Affiliate.
Presented at the American Heart Association Scientific Sessions, Chicago, Ill, November 12–15, 2006.
Castaneda AR Jr, Mayer JE, Hanley FL. Cardiac Surgery of the Neonate and Infant. New York: W.B. Saunders; 994.
Sutherland FW, Perry TE, Yu Y, Sherwood MC, Rabkin E, Masuda Y, Garcia GA, McLellan DL, Engelmayr GC Jr, Sacks MS, Schoen FJ, Mayer JE Jr. From stem cells to viable autologous semilunar heart valve. Circulation. 2005; 111: 2783–2791.
Armstrong EJ, Bischoff J. Heart valve development: endothelial cell signaling and differentiation. Circ Res. 2004; 95: 459–470.
Ishisaki A, Hayashi H, Li AJ, Imamura T. Human umbilical vein endothelium-derived cells retain potential to differentiate into smooth muscle-like cells. J Biol Chem. 2003; 278: 1303–1309.
Paruchuri S, Yang JH, Aikawa E, Melero-Martin JM, Khan ZA, Loukogeorgakis S, Schoen FJ, Bischoff J. Human pulmonary valve progenitor cells exhibit endothelial/mesenchymal plasticity in response to vascular endothelial growth factor-A and transforming growth factor-beta2. Circ Res. 2006; 99: 861–869.
Dvorin EL, Wylie-Sears J, Kaushal S, Martin DP, Bischoff J. Quantitative evaluation of endothelial progenitors and cardiac valve endothelial cells: proliferation and differentiation on poly-glycolic acid/poly-4-hydroxybutyrate scaffold in response to vascular endothelial growth factor and transforming growth factor beta1. Tissue Eng. 2003; 9: 487–493.
Rabkin E, Hoerstrup SP, Aikawa M, Mayer JE Jr, Schoen FJ. Evolution of cell phenotype and extracellular matrix in tissue-engineered heart valves during in-vitro maturation and in-vivo remodeling. J Heart Valve Dis. 2002; 11: 308–314;discussion 314.