Effects of Leaflet Design on Transvalvular Gradients of Bioprosthetic Heart Valves.

Bioprosthetic aortic valves (BAVs) are becoming the prostheses of choice in heart valve replacement. The objective of this paper is to assess the effects of leaflet geometry on the mechanics and hemodynamics of BAVs in a fluid structure interaction model. The curvature and angle of leaflets were varied in 10 case studies whereby the following design parameters were altered: a circular arch, a line, and a parabola for the radial curvature, and a circular arch, a spline, and a parabola for the circumferential curvature. Six different leaflet angles (representative of the inclination of the leaflets toward the surrounding aortic wall) were analyzed. The 3-dimensional geometry of the models were created using SolidWorks, Pointwise was used for meshing, and Comsol Multiphysics was used for implicit finite element calculations. Realistic loading was enforced by considering the time-dependent strongly-coupled interaction between blood flow and leaflets. Higher mean pressure gradients as well as von Mises stresses were obtained with a parabolic or circular curvature for radial curvature or a parabolic or spline curvature for the circumferential curvature. A smaller leaflet angle was associated with a lower pressure gradient, and, a lower von Mises stress. The leaflet curvature and angle noticeably affected the speed of valve opening, and closing. When a parabola was used for circumferential or radial curvature, leaflets displacements were asymmetric, and they opened and closed more slowly. A circular circumferential leaflet curvature, a linear leaflet radial curvature, and leaflet inclination toward the surrounding aortic wall were associated with superior BAVs mechanics.

Quicker yet safe: skeletonization of 1640 internal mammary arteries with harmonic technology in 965 patients.

OBJECTIVES: Skeletonization of the internal mammary artery (IMA) facilitates arterial grafting and has been shown to reduce deep sternal infection but is more time-consuming and tedious than pedicle harvest. We wished to determine if use of harmonic technology (HT) facilitates skeletonization of the IMA and is as safe as the conventional technique of skeletonization. METHODS: In a consecutive series of 1057 patients with isolated coronary artery bypass graft (CABG) surgery from 2003 to 2013, adverse events and recorded harvest times were compared between harmonic (965 patients) and non-harmonic patients (86 patients). RESULTS: HT was used to harvest 1640 IMAs in 965 (91%) of 1057 consecutive CABG patients and skeletonization with the traditional technique (use of an electrocautery tip as a dissector) was used to harvest 147 IMAs in 86 patients. Six patients had no IMA harvested with this surgery (4 patients had an IMA used from a previous CABG, 1 had no disease of the left anterior descending coronary artery and 1 patient was in cardiogenic shock precluding IMA use). Excluding patients with single-vessel disease, 730/987 (74%) of patients received bilateral IMAs. Demographics of patients with and without harmonic skeletonization, respectively, were the following: mean age: 64.7 vs 67.7 years; diabetes: 33 vs 34%; women: 21 vs 26% and median European System for Cardiac Operative Risk Evaluation: 2.9 vs 3.2. The mean harvest time for 77 non-harmonic skeletonized mammary arteries (49 surgeries) was 32.2 min (95% confidence interval (CI): 30.1, 34.3), for harmonic skeletonized arteries after 450 surgeries was 28.4 min, (95% CI: 27.8, 29.1) and in the last 100 IMAs harvested for the isolated harmonic device use/mammary was 15.4 min (95% CI: 14.0, 16.7). Major adverse events for patients with and without harmonic skeletonization, respectively, were: reoperation for bleeding: 2.7 vs 3.5% (difference = 0.8%, 95% CI: -3.2, 4.8); damaged mammaries: 0.4 vs 0.7% (difference = 0.3%, 95% CI: -1.0, 1.7); deep sternal infection: 1.6 vs 1.2% (difference = -0.4%, 95% CI: -2.8, 2.0) and perioperative infarction: 1.7 vs 2.3% (difference = 0.7%, 95% CI: -2.6, 4.0). CONCLUSION: In this largest series to date of harmonic IMA skeletonization, this technique results in rare damage, is quicker and with a comparable adverse event rate compared with the non-harmonic method.

Canadian cardiac surgeons' perspectives on biomedical innovation.

Barriers to successful innovation can be identified and potentially addressed by exploring the perspectives of key stakeholders in the innovation process. Cardiac surgeons in Canada were surveyed for personal perspectives on biomedical innovation. Quantitative data was obtained by questionnaire and qualitative data via interviews with selected survey participants. Surgeons were asked to self-identify into 1 of 3 categories: "innovator," "early adopter," or "late adopter," and data were compared between groups. Most surgeons viewed innovation favourably and this effect was consistent irrespective of perceived level of innovativeness. Key barriers to the innovation pathway were identified: (1) support from colleagues and institutions; (2) Canada's health system; (3) sufficient investment capital; and (4) the culture of innovation within the local environment. Knowledge of the innovation process was perceived differently based on self-reported innovativeness. The majority of surgeons did not perceive themselves as having the necessary knowledge and skills to effectively translate innovative ideas to clinical practice. In general, responses indicate support for implementation of leadership and training programs focusing on the innovation process in an effort to prepare surgeons and enhance their ability to successfully innovate and translate new therapies. The perspectives of cardiac surgeons provide an intriguing portal into the challenges and opportunities for healthcare innovation in Canada.

Cell therapy limits myofibroblast differentiation and structural cardiac remodeling: basic fibroblast growth factor-mediated paracrine mechanism.

BACKGROUND: Experimental cell therapy attenuates maladaptive cardiac remodeling and improves heart function. Paracrine mechanisms have been proposed. The effect of cell therapy on post infarction cardiac fibroblast and extracellular matrix (ECM) regulation was examined. METHODS AND RESULTS: Vascular smooth muscle cells (VSMC) were injected into the border zone of subacute infarcted syngeneic Fischer rat hearts and compared with medium-injected controls. Twelve weeks post injection, cell-treated hearts showed preserved ECM content and attenuated structural chamber remodeling. Myofibroblast activation (α-smooth muscle actin expression) was decreased significantly, while basic fibroblast growth factor (bFGF) expression, a known inhibitor of transforming growth factor ß-1-induced fibroblast differentiation, was increased. Matrix metalloproteinase-2 expression and activation by gelatin zymography was unchanged between groups, while its endogenous inhibitor, tissue inhibitors of matrix metalloproteinase (TIMP)-2, showed both increased expression and enhanced inhibitory capacity in cell-treated hearts. To define paracrine mechanisms, in vitro effects of VSMC conditioned media on myofibroblast activation were assessed by 3-D collagen gel contraction assay. VSMC conditioned media significantly inhibited collagen contraction, while a specific bFGF inhibitor abolished this paracrine response. TIMP-2 induced collagen contraction, but the effect was suppressed in the presence of bFGF. CONCLUSIONS: Extracellular matrix dysregulation post myocardial infarction is improved by cell therapy. These data suggest that cell transplantation attenuates myofibroblast activation and subsequent maladaptive structural chamber remodeling through paracrine mechanisms involving bFGF and TIMP-2.

Kryptonite bone cement prevents pathologic sternal displacement.

BACKGROUND: Wire cerclage closure of sternotomy is the standard of care despite evidence of pathologic sternal displacement (> 2 mm) during physiologic distracting forces (coughing). Postoperative functional recovery, respiration, pain, sternal dehiscence, and infection are influenced by early bone stability. This translational research report provides proof-of-concept (part A) and first-in-man clinical data (part B) with use of a triglyceride-based porous adhesive to rapidly enhance the stability of conventional sternal closure. METHODS: In part A, fresh human cadaver blocks were subjected to midline sternotomy and either conventional wire closure or modified adhesive closure. After 24 hours at 37 degrees C, using a biomechanical test apparatus, a step-wise increase in lateral distracting force simulated physiologic stress. Sternal displacement was measured by microdisplacement sensors. In part B, a selected clinical case series was performed and sternal perfusion assessed by serial single photon emission computed tomography imaging. RESULTS: Wire closure resulted in measurable bony displacement with increasing load. Pathologic displacement (> or = 2 mm) was observed in all regional segments at loads 400 newton (N) or greater. In contrast, adhesive closure completely eliminated pathologic displacement at forces 600 N or less (p < 0.001). In patients, adhesive closure was not associated with adverse events such as adhesive migration, embolization, or infection. There was excellent qualitative correlation between cadaver and clinical computed tomographic images. Sternal perfusion was not compromised by adhesive closure. CONCLUSIONS: This first-in-man series provides proof-of-concept indicating that a novel biologic bone adhesive is capable of rapid sternal fixation and complete elimination of pathologic sternal displacement under physiologic loading conditions. A randomized clinical trial is warranted to further define the potential risks and benefits of this innovative technique.

Resident and bone marrow-derived mesenchymal stem cells in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a major healthcare problem worldwide affecting more than half a million patients each year. Despite considerable advances in the treatment of HNSCC, a high rate of recurrences aggravates the clinical situation and disease outcomes have only modestly improved. Recent insights show that cancer is not only a disease of the transformed epithelium but is also influenced and dependent on its stromal environment. In this review we suggest that resident and bone marrow (BM)-derived mesenchymal stem cells (MSCs) are precursors of the stroma associated with HNSCC and contribute to blood- and lymph angiogenesis, modulate the immune system and produce tumor-associated myofibroblasts. In addition, the impact of radiation therapy on the stromal reaction in HNSCC is discussed. Understanding the mechanisms of how MSCs promote invasive growth and metastasis in HNSCC and respond to cancer management strategies is of profound medical importance and will help us to design improved therapeutic protocols.

Sustained release and activation of the growth factor basic fibroblast growth factor from loaded scaffolds in heart valve tissue engineering.

OBJECTIVES: Loading of biological matrices offers an opportunity to induce specific cell behaviour. We previously reported the use of growth factors to promote cell invasion and proliferation in tissue valve engineering. We investigated biological matrices preloaded with heparin as an ionically attractive template for the binding, activation and sustained release of basic fibroblast growth factor (bFGF). METHODS: Heparin loading concentrations were evaluated and different incubation times were tested. Heparin and heparin-bound bFGF uptake and release were evaluated by (123)I radio-labelling. Biological activity of bFGF was evaluated in vitro. RESULTS: Maximum heparin uptake was observed for 2000 microg/ml at 2 h and stabilized thereafter. bFGF-loaded matrices showed an initial burst release of 15% within 4 h and thereafter sustained release reaching 21% at 24 h. Released bFGF was bioactive. CONCLUSIONS: This model would be useful in tissue engineering using porcine aortic matrices and could be applied using other growth factors or combinations.

Differential impact of TGF-beta and EGF on fibroblast differentiation and invasion reciprocally promotes colon cancer cell invasion.

Several studies indicate that cancer-associated fibroblasts play a critical role in cancer cell invasion and metastasis, the hallmarks of malignancy. To better understand the mechanisms underlying such effects, we established a heterotypic model of human fibroblasts (primary colon fibroblasts and immortalized human dermal fibroblasts) in co-culture with human colon cancer cells (HCT-8/E11), using three-dimensional collagen type-I and Matrigel matrices. We report that TGF-beta is the unique and dominant factor to provide pro-invasive signals to HCT-8/E11 colon cancer cells from TGF-beta-treated human fibroblasts in three-dimensional collagen type I and Matrigel matrices. These effects are not mimicked or reversed by EGF or bFGF, and are associated with the TGF-beta-mediated induction of myofibroblast differentiation and functional markers, such as alpha-SMA, the haptotactic matrix molecule TNC, collagen type 1 maturation enzyme P4H, serine protease FAP, and myofibroblast contractility. Accordingly, TGF-beta induced a strong activation of RhoA and stress fiber formation in fibroblasts, with no impact on Rac1-GTP levels. In contrast, EGF down-regulated Rho-GTP levels in fibroblasts, giving permissive signals for Rac1 activation, fibroblast polarization, and invasion. Taken together, our data imply that TGF-beta and EGF exert invasive growth-promoting actions in human colon tumors through a differential and cumulative impact on the stromal and cancer cell compartments. Our data predict that inhibitors directed at this reciprocal molecular and cellular crosstalk will have therapeutic applications for targeting the invasive growth of human primary tumors and their metastatic spread.

Genipin blues: an alternative non-toxic crosslinker for heart valves?

BACKGROUND AND AIM OF THE STUDY: One approach in tissue-engineering involves the implantation of decellularized, xenogenic scaffolds, with the expectation of repopulation in vivo. However, a major limitation of this method is the propensity to induce a strong immune host response. The study aim was to mitigate this immunogenicity by employing a crosslinking treatment with genipin. METHODS: Porcine matrices were prepared using a detergent-enzymatic treatment and fixed in 0.01% or 0.001% aqueous genipin. The mechanical properties of the matrices were monitored by tensile strength testing. The survival of chicken fibroblasts was used to determine cell-friendliness of the matrices. Non-fixed, decellularized biological scaffolds (n = 3) were implanted in a sheep model and compared to an equal number of genipin-fixed scaffolds (n = 6). Matrices implanted in the pulmonary position were explanted after six weeks and examined using light and transmission electron microscopy. The antibody reaction against porcine tissue in sheep serum was also determined. RESULTS: Statistically significant differences were found between non-fixed leaflets, 0.001% genipin-and 0.6% glutaraldehyde (GA)-fixed leaflets for work to maximum load (non-fixed 0.00646 J; genipin-fixed 0.00509 J; GA-fixed 0.00543 J) and stiffness (non-fixed 9281 N/m; genipin-fixed 16214 N/m; GA-fixed 14401 N/m). Genipin-treated matrices were not cytotoxic. For all concentrations of genipin a high proportion of viable cells was present (79-100%). Low-dose GA (10 microg/ml) showed a distinct cytotoxicity (24.8% viability). At explant, an intense chronic inflammatory response was observed in non-fixed matrices, in contrast to genipin-fixed scaffolds. The sheep serum showed a marked decrease in IgG response in both 0.001% and 0.01% genipin-fixed matrices (IgG 30 and 20, respectively) when compared to non-fixed matrices (IgG 40). CONCLUSION: Genipin crosslinking of the matrices attenuated, but did not eliminate, the inflammatory host reaction. Whether genipin treatment might extend the durability of xenogenic scaffolds remains to be investigated.

Autologous glue: part of the sticky mystery unraveled.

OBJECTIVE: The aim of the study was to evaluate the safety and efficacy of an autologous surgical tissue adhesive. METHODS: Autologous glue was made out of canine concentrated plasma proteins mixed with 7.5% glutaraldehyde. Tensile strength and cytotoxicity of the autologous glue were tested. In a dog model, 8 transectioned iliac arteries were reanastomosed by using the animal's glue as the sole fixation method. After 120 days, all animals were angiographically controlled for patency and killed for histologic and immunohistochemical examination of the anastomosis. RESULTS: The autologous glue showed sufficient tensile strength (557 +/- 135 N/mm2). The elasticity of the glue is influenced by variations of concentrations in both proteins and glutaraldehyde. Glutaraldehyde remained cytotoxic, even at low concentrations of 2.5%. All operative procedures were successful. Angiographs performed before animal death showed all but 1 vessel to be patent and showed manifest compression signs in 3 anastomoses. Histological examination revealed only a foreign-body reaction adjacent to the surface of the glue. The autologous glue does not trigger any immune response on immunochemistry. Because fibroblastic neo-endothelial lining was near to normal, potential glutaraldehyde leaching does not seem too harmful for the vascular juncture in the dog model. CONCLUSIONS: Autologous glutaraldehyde glue has been used successfully as a vascular adhesive. In contrast to our previous studies with heterologous glue, we did not find a fierce acute inflammatory reaction indicating immune triggering. Nevertheless, glutaraldehyde remains a cytotoxic cross-linker. It is yet not known whether autologous glutaraldehyde glue can be used safely in clinical practice.

Readily available porcine aortic valve matrices for use in tissue valve engineering. Is cryopreservation an option?

The clinical use of acellular biological valves as scaffolds in tissue valve engineering would require them to be readily available. This study examines the feasibility of cryopreserving porcine aortic valve matrices for use in tissue valve engineering. Matrices prepared using an enzymatic-detergent decellularization protocol were examined before and after cryopreservation. The biochemical status of tissues were evaluated by collagen and uronic acid (proteoglycan) determination and their mechanical properties were determined using a burst test. The histological and ultrastructural properties were evaluated by light and electron microscopy. Cryopreservation did not significantly affect the collagen and uronic acid content of aortic leaflet matrices. Histological and ultrastructural sections, however, confirmed extensive disruption of the extracellular collagen matrix and inter-fibrillar proteoglycan associations following cryopreservation. Although neither the breakage force nor the maximum force at failure was significantly different in matrices before and after cryopreservation, the strain observed in matrices was significantly higher after cryopreservation. To our knowledge this is the first study to investigate the effects of cryopreservation on aortic valve matrices. Cryopreservation did not significantly alter the biochemical properties of porcine aortic valve matrices. Nevertheless, cryopreservation had significant adverse effects on the structural and mechanical properties of matrices. Cryopreserved matrices showed significantly higher strain when stressed compared to non cryopreserved matrices. While, theoretically matrices are only expected to be functional for a limited time until regenerated in vivo, further mechanical testing is necessary to evaluate the effects of these changes on the durability of porcine aortic valve matrices for use in tissue valve engineering.

Hydrodynamic evaluation of kangaroo aortic valve matrices for tissue valve engineering.

We evaluated the hydrodynamic performance of kangaroo aortic valve matrices (KMs) (19, 21, and 23 mm), as potential scaffolds in tissue valve engineering using a pulsatile left heart model at low and high cardiac outputs (COs) and heart rates (HRs) of 60 and 90 beats/min. Data were measured in two samples of each type, pooled in two CO levels (2.1 +/- 0.7 and 4.2 +/- 0.6 L/min; mean +/- standard errors on the mean), and analyzed using analysis of variance with CO level, HR, and valve type as fixed factors and compared to similar porcine matrices (PMs). Transvalvular pressure gradient (DeltaP) was a function of HR (P < 0.001) and CO (P < 0.001) but not of valve type (P = 0.39). DeltaP was consistently lower in KMs but not significantly different from PMs. The effective orifice area and performance index of kangaroo matrices was statistically larger for all sizes at both COs and HRs.

Acellular porcine and kangaroo aortic valve scaffolds show more intense immune-mediated calcification than cross-linked Toronto SPV valves in the sheep model.

AIM OF THE STUDY: A major limitation of currently available bioprosthetic valves is their propensity to calcify. At present, one approach in tissue-engineering, uses decellularized, xenogenic scaffolds that are implanted, with the expectation of complete matrix repopulation in vivo. Whether or not such a decellularized matrix will be sufficiently endowed to prevent calcification is unknown. MATERIALS AND METHODS: This study examines the calcification potential of xenogenic biological scaffolds from two species, namely pigs (n=3) and kangaroos (n=3) in the sheep model and compared them to a commercially available glutaraldehyde treated porcine bioprosthetic valve (Toronto SPV) (n=3). RESULTS: Valves and matrices were explanted after 120 days. Histologically (H&E and Von Kossa stain) more calcium was found in the acellular matrices. The mean calcium content (mg/g-dw) of the Toronto SPV valve leaflets was 2.63 mg/g-dw compared to 43.81 mg/g-dw (P=0.12) in kangaroo and 105.08 mg/g-dw (P=0.004) in porcine matrices. On electron microscopy calcific deposits were located between as well as in close association with the collagen fibers in all tissue. In contrast to the cross-linked gluteraldehyde fixed bioprostheses both matrices showed strong immune IgG reaction. CONCLUSION: Toronto SPV valves calcified significantly less than the tested biological matrices irrespective of species of origin. Surprisingly, xenogenic decelullarized scaffolds are inherently prone to calcification due to a strong immunogenicity.

Growth factor modulation of fibroblast proliferation, differentiation, and invasion: implications for tissue valve engineering.

We have previously shown that transforming growth factor-beta1 (TGF-beta1) stimulates transdifferentiation of fibroblasts into smooth muscle alpha-actin (alpha-SMA) positive myofibroblasts. However, TGF-beta, as such, is unsuitable for effective population of a heart valve matrix, because it dose-dependently inhibits growth of fibroblasts. The aim of this study was to investigate combinations of other growth factors with TGF-beta to stimulate the proliferation of suitably differentiated cells and to enhance their invasion into aortic valve matrices. Human dermal mesenchymal cells (hDMC1.1) were treated with combinations of growth factors to stimulate these cells to trans-differentiate into myofibroblasts, to proliferate, and to invade. Growth factors were chosen after expression of their respective receptors was confirmed in hDMC1.1 using reverse transcriptase polymerase chain reaction. We combined TGF-beta with several growth factors such as insulin-like growth factor (IGF-1, IGF-2), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF-AA, PDGF-BB, and PDGFAB). Nuclear Ki67 staining, MTT assay, and cell counting revealed that only EGF and bFGF were capable of overcoming TGF-beta-induced growth inhibition. However, bFGF but not EGF inhibited TGF-beta-induced alpha-SMA expression, as evidenced by immuno-cytochemistry and Western blotting. A growth factor cocktail (TGF-beta, EGF, bFGF) has been established that maintains TGF-beta-induced trans-differentiation but overcomes TGF-beta-induced growth inhibition while stimulating fibroblast proliferation and invasion.

Scanning electron microscopic surface topography of ablation catheter perforations and calcific tear in an explanted bioprosthetic heart valve.

The authors report a triplet of leaflet destruction in a bioprosthetic aortic valve explanted at 12 years after iatrogenic ablation catheter perforation in a patient who underwent coronary artery bypass surgery and multiple ablative procedures in the interim. Lesions were examined topographically by scanning electron microscopy. Calcium content was evaluated by mass spectrometry and Von Kossa staining. Leaflets exhibited little calcification, except at the commissures of the valve. Scanning electron microscopy revealed distinct lesion topography. The authors present the scanning electron microscopic characteristics of these lesions and of an incision into the valve made for comparison using a pair of scissors. This is believed to be the first report of scanning electron micrographs of ablation catheter perforations and a calcific tear in the same explanted valve. The findings provide a source for comparison in the etiological determination of explanted bioprosthetic valve lesions using scanning electron microscopy.

Bovine glue (BioGlue) is catabolized by enzymatic reaction in the vascular dog model.

PURPOSE: The aim of the study is to explore the feasibility, patency, and histologic changes of a sutureless vascular anastomotic technique using biological glue as sole fixation method. DESCRIPTION: Eight mongrel dogs (+/-15 kg) underwent direct reanastomosis of their transsected iliac arteries. Both ends were placed on a 5-mm balloon and the anastomosis was secured with biological glue (BioGlue, Cryolife, Kennesaw, GA). No intravascular suture material was used. All survivors were angiographically controlled for patency after 6 weeks and 3 months. Then the animals were euthanized and tissues were obtained for histologic and pathologic examination by light and electron microscopy. EVALUATION: All procedures were successful except for 1 animal that died of uncontrollable bleeding at the anastomotic site. All first-time angiographically controlled grafts except three were patent. One animal showed manifest signs of fungal infection. Histology detected early granulocyte infiltration with an important enzymatic reaction adjacent to the surface of glue. Later on, the glue gradually regressed to disappear completely. Fibroblastic neointimal lining was noticed in most of the anastomoses, with some marked differences in the endothelium compared with normal. CONCLUSIONS: Good permeability (57%) was observed in this new sutureless anastomotic technique in the canine model. In contrast to previous reported studies we noticed a clear enzymatic breakdown of the glue before total disappearance. It is not yet known to what extend use of the bovine glue was responsible for this phenomenon.

Transforming growth factor-beta-induced transition of fibroblasts: a model for myofibroblast procurement in tissue valve engineering.

BACKGROUND AND AIM OF THE STUDY: The selection of a suitable cell type for scaffold seeding, its isolation and adequate expansion in vitro remains a major challenge in tissue valve engineering. The study aim was to establish a model for efficient procurement of myofibroblasts for in-vitro seeding using fibroblasts as progenitor cells. METHODS: Dermal and arterial mesenchymal cells from human (hDMC1.1 and hAMC1.1) and sheep (sDMC1.1 and sAMC1.1) were isolated by sequential culture. Cell isolates were characterized by stringent criteria based on morphology, immunocytochemistry using antibodies to vimentin, cytokeratin, prolyl 4-hydroxylase, smooth muscle alpha-actin (alpha-SMA) and smooth muscle myosin, and by Western blotting for alpha-SMA and N-cadherin. Cultures with less than 10-20% alpha-SMA-positive cells were considered to be fibroblastic. Cells were subsequently transdifferentiated with the cytokine transforming growth factor-beta1 (TGF-beta1) during five days, and then evaluated morphotypically, by immunocytochemistry, and by Western blotting. The metabolic and functional properties of TGF-beta1-treated and untreated colonies were compared by measuring the expression of extracellular proteins (collagen type 1 and tenascin-C) and by a collagen matrix contraction assay. RESULTS: TGF-beta1 successfully transformed both human and sheep fibroblasts to metabolically active and functional myofibroblasts based on stringent criteria for myofibroblast characterization. Alpha-SMA positivity of 100% was obtained in all cases (hDMC1.1, hAMC1.1, sDMC1.1, and sAMC1.1) after transformation compared to less than 50% in the non-transformed state (hAMC1.1, 17%; hDMC1.1, 10%; sAMC1.1, 43%; and sDMC1.1, 30%). This observation was further supported by increased contractility and an up-regulation of extracellular protein production in transdifferentiated cells. CONCLUSION: Untreated arterial cell isolates were, at best, less than 50% alpha-SMA-positive. By allowing procurement of high densities of myofibroblasts in a relatively short time, the model was seen to be a potentially useful tool in tissue valve engineering, at least in investigations using autologous cells in the sheep model.