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.

Biodegradable collagen patch with covalently immobilized VEGF for myocardial repair.

Vascularization of engineered tissues in vitro and in vivo remains a key problem in translation of engineered tissues to clinical practice. Growth factor signalling can be prolonged by covalent tethering, thus we hypothesized that covalent immobilization of vascular endothelial growth factor (VEGF-165) to a porous collagen scaffold will enable rapid vascularization in vivo. Covalent immobilization may be preferred over controlled release or cell transfection if the effects are desired within the biomaterial rather than the surrounding tissue. Scaffolds were prepared with 14.5 ± 1.4 ng (Low) or 97.2 ± 8.0 ng (High) immobilized VEGF, or left untreated (control), and used to replace a full right ventricular free wall defect in rat hearts. In addition to rapid vascularization, an effective cardiac patch should exhibit neither thinning nor dilatation upon implantation. In vitro, VEGF enhanced the growth of endothelial and bone marrow cells seeded onto scaffolds. In vivo, High VEGF patches had greater blood vessel density (p < 0.01) than control at Day 7 and 28 due to increased cell recruitment and proliferation (p < 0.05 vs. control). At Day 28, VEGF-treated patches were significantly thicker (p < 0.05) than control, and thickness correlated positively with neovascularization (r = 0.67, p = 0.023). Importantly, angiogenesis in VEGF scaffolds contributed to improved cell survival and tissue formation.

c-Kit function is necessary for in vitro myogenic differentiation of bone marrow hematopoietic cells.

In recent years, the differentiation of bone marrow cells (BMCs) into myocytes has been extensively investigated, but the findings remain inconclusive. The purpose of this study was to determine the conditions necessary to induce myogenic differentiation in short-term cultures of adult BMCs, and to identify the BMC subpopulation responsible for this phenomenon. We report that high-density cultures of murine hematopoietic BMCs gave rise to spontaneous beating cell clusters in the presence of vascular endothelial and fibroblast growth factors. These clusters originated from c-kit(pos) cells. The formation of the clusters could be completely blocked by adding a c-kit/tyrosine kinase inhibitor, Gleevec (imatinib mesylate; Novartis International, Basel, Switzerland, http://www.novartis.com), to the culture. Cluster formation was also blunted in BMCs from c-kit-deficient (Kit(W)/Kit(W-v)) mice. Clustered cells expressed cardiomyocyte-specific transcription factor genes Gata-4 and Nkx2.5, sarcomeric proteins beta-MHC and MLC-2v, and ANF and connexin-43. Immunostaining revealed alpha-sarcomeric actinin expression in more than 90% of clustered cells. Under electron microscopy, the clustered cells exhibited a sarcomeric myofiber arrangement and z-bands. This study defines the microenvironment required to achieve a reproducible in vitro model of beating, myogenic cell clusters. This model could be used to examine the mechanisms responsible for the postnatal myogenic differentiation of BMCs. Our results identify c-kit(pos) bone marrow hematopoietic cells as the source of the myogenic clusters.

The MRL mouse heart does not recover ventricular function after a myocardial infarction.

INTRODUCTION: Murphy Roth Large (MRL) mice have a remarkable regenerative capacity. A recent report demonstrated rapid cardiac healing in these mice following cryogenically induced right ventricular injury, suggesting the potential for new regenerative therapies to restore cardiac function in mammals. We therefore evaluated the cardiac regenerative wound-healing response and functional recovery of MRL mice in response to a clinically relevant left ventricular coronary ligation. METHODS: Female MRL/MpJ+/+ and C57BL/6 mice underwent left coronary artery ligation. Cardiac function was evaluated by echocardiography at Days 0, 5, 15, and 60. At Day 96, invasive hemodynamics were assessed by pressure-volume loops using a Millar catheter. Hearts were perfusion fixed for histomorphometric analysis at Days 5, 15, and 96. Some hearts were fresh frozen at Days 5 and 15 for immunohistochemical analysis and digital quantitation of blood vessel density (CD31) and cellular proliferation (Ki67). RESULTS: MRL mice healed ear punch wounds (89% reduction in area) more extensively than C57BL/6 mice (28% reduction in area) but did not differ functionally from C57BL/6 animals before or after coronary ligation. In addition, blood vessel density and cell proliferation were similar between the two strains. CONCLUSIONS: Although MRL mice rapidly healed ear injury, they did not exhibit regeneration of the left ventricle or enhanced functional improvement in response to coronary ligation. The prospect of cardiac regeneration after myocardial infarction will require further studies designed to elucidate the possible mechanisms of functional restoration.

TIMP-3 deficiency accelerates cardiac remodeling after myocardial infarction.

The activity of TIMP-3, a natural tissue inhibitor of matrix metalloproteinases (MMPs), is decreased in the failing heart. This study evaluated the response to coronary ligation of cardiac structure, function, and matrix remodeling in wild-type (WT) mice, and those deficient in TIMP-3 (timp-3(-/-)). The coronary artery was ligated in timp-3(-/-) and age-matched WT mice. At various time points over the following 28-day period, left ventricular structure and function (by echocardiography, pressure-volume measurements and morphometry), MMP levels and activity, blood vessel density, cell proliferation, apoptosis, matrix structure, and inflammatory cytokine levels were assessed in both groups. After ligation, mortality was significantly greater in timp-3(-/-) than in WT mice. Morphometry and echocardiography demonstrated no difference in heart size or function prior to ligation; however, the progression of left ventricular systolic dysfunction was accelerated in timp-3(-/-) mice at 7, 14 and 28 days after infarction compared to WT controls. Left ventricular dilatation, gelatinase MMP activity, and TNF-alpha levels were significantly greater in timp-3(-/-) than in WT mice at different times after ligation. By histological evaluation, timp-3(-/-) mice exhibited significantly increased blood vessel density, cell proliferation, and apoptosis in the infarct area, and reduced collagen content in the viable remote myocardium compared to WT mice at 7 and 14 days after ligation. TIMP-3 deficiency accelerated maladaptive cardiac remodeling after a myocardial infarction by promoting matrix degradation and inflammatory cytokine expression. This study supports further investigations to determine whether such remodeling could be reduced by augmenting TIMP-3 expression in the infarcted myocardium.

c-kit dysfunction impairs myocardial healing after infarction.

BACKGROUND: We hypothesized that c-kit receptor function in the bone marrow is important for facilitating healing, leading to efficient cardiac repair after myocardial infarction (MI). METHODS AND RESULTS: We used Kit(W)/Kit(W-v) c-kit mutant mice and their wild-type littermates to assess the importance of c-kit function in cardiac remodeling after coronary ligation. We found that mutant mice developed 1.6-fold greater ventricular dilation (P=0.008) attributable to a 1.3-fold greater infarct expansion by day 14 after MI (P=0.01). The number of proliferating smooth muscle alpha-actin expressing cells was 1.8-fold lower in mutant mice at day 3 (P<0.01), resulting in a 1.6 to 1.8-fold reduction in total regional nonvascular smooth muscle alpha-actin expressing cells by both microscopy and flow cytometry (P<0.001 for both). This decrease was accompanied by a 1.4-fold reduction in the number of CD31 expressing blood vessels (P<0.05). Prior transplantation of wild-type bone marrow cells into mutant mice rescued the efficient establishment of vessel-rich repair tissue by inducing a 1.5-fold increase in nonvascular smooth muscle alpha-actin expressing cells and CD31 expressing blood vessels (P<0.05 for both). The increased recruitment of cells into the infarct region in the chimeric mice was associated with reduced infarct expansion (P<0.03) compared to wild-type levels. CONCLUSIONS: Bone marrow c-kit function critically impacts the myofibroblast repair response in infarcted hearts. Interventions that increase the infiltration of c-kit+ cells to the infarcted heart may potentiate this endogenous repair response, prevent infarct expansion, and improve the recovery of cardiac function after MI.

Cardioprotective c-kit+ cells are from the bone marrow and regulate the myocardial balance of angiogenic cytokines.

Clinical trials of bone marrow stem/progenitor cell therapy after myocardial infarction (MI) have shown promising results, but the mechanism of benefit is unclear. We examined the nature of endogenous myocardial repair that is dependent on the function of the c-kit receptor, which is expressed on bone marrow stem/progenitor cells and on recently identified cardiac stem cells. MI increased the number of c-kit+ cells in the heart. These cells were traced back to a bone marrow origin, using genetic tagging in bone marrow chimeric mice. The recruited c-kit+ cells established a proangiogenic milieu in the infarct border zone by increasing VEGF and by reversing the cardiac ratio of angiopoietin-1 to angiopoietin-2. These oscillations potentiated endothelial mitogenesis and were associated with the establishment of an extensive myofibroblast-rich repair tissue. Mutations in the c-kit receptor interfered with the mobilization of the cells to the heart, prevented angiogenesis, diminished myofibroblast-rich repair tissue formation, and led to precipitous cardiac failure and death. Replacement of the mutant bone marrow with wild-type cells rescued the cardiomyopathic phenotype. We conclude that, consistent with their documented role in tumorigenesis, bone marrow c-kit+ cells act as key regulators of the angiogenic switch in infarcted myocardium, thereby driving efficient cardiac repair.

Radial artery as an autologous cell source for valvular tissue engineering efforts.

To create a viable tissue-engineered aortic valve, it is important to identify suitable autologous cell sources that may be seeded onto a biocompatible scaffold. This study focused on the radial artery (RA) as one possible source, investigated optimal culture conditions, and determined the usefulness of small intestinal submucosa (SIS) as a scaffold for tissue-engineering. Porcine RA cells were cultured on either two-dimensional (2D) 100-mm dishes or three-dimensional (3D) 1-cm(2) SIS sheets, producing cell-scaffold composites (CSCs). Both 2D and 3D cultures were maintained in either Medium 199 (M199) or endothelial growth media (EGM) to determine optimal growth conditions. Cellular phenotype and matrix metalloproteinase (MMP) profiles were determined by immunoblotting of cell lysates and zymography of conditioned media, respectively. Cellular invasion was analyzed immunohistochemically on CSC tissue sections. We show that the RA contains phenotypes consistent with those found in the normal aortic valve. EGM, compared with M199, promotes the invasion and remodeling of SIS by RA cells, which is crucial in the process of replacing the foreign tissue scaffold prior to implantation. To our knowledge, this is the first study to show that the RA is a suitable source for the generation of a tissue-engineered valve.

Dermal fibroblasts cultured on small intestinal submucosa: Conditions for the formation of a neotissue.

Small intestinal submucosa (SIS) is a naturally occurring, acellular biomaterial that has been used extensively as a soft tissue replacement, as a scaffold for tissue engineering, and as a substrate for the study of cells in 3D culture. The aim of this study is to define culture parameters that promote neotissue formation with the use of dermal fibroblasts and SIS. SIS sheets were seeded with dermal fibroblasts and cultured for 4 weeks. The resultant cell-scaffold composites (CSCs) were cultured with media alone, media supplemented with ascorbic acid, or fibronectin-pretreated SIS and ascorbic acid. CSCs were analyzed for cellular invasion into the scaffold, the rate of type I collagen production, MMP gelatinolytic activity, thickness, and ultrastructural morphology. CSCs treated with fibronectin and ascorbate showed an increase in Type I collagen production, no change in the MMP gelatinolytic activity, an increase in CSC thickness, and an organized neotissue on the surface of the SIS. Minimal cellular invasion was noted, suggesting that fibroblasts use the SIS as a template for neotissue growth rather than as a scaffold. These results indicate that fibronectin-treated SIS cultured with dermal fibroblasts in the presence of ascorbic acid will promote true neotissue formation for future cardiovascular tissue engineering efforts.

Development of aortic valve sclerosis in a rabbit model of atherosclerosis: an immunohistochemical and histological study.

BACKGROUND AND AIM OF THE STUDY: It has been suggested that aortic valve sclerosis (AVS) is an atherosclerotic disease process that can proceed to aortic stenosis. The absence of reports studying an animal model of the early stages of this disease has precluded the development of preventive therapeutic strategies. A cholesterol-fed (0.25% cholesterol in chow) rabbit model of atherosclerosis that is characterized by a moderate level of hypercholesterolemia was studied to determine its efficacy as a model of early AVS. Cellular, structural and morphological changes in the aortic valves of these rabbits were studied. METHODS: Twenty rabbits were assigned randomly to four experimental groups: Group 1 received normal chow for 40 weeks; group 2 received 0.25% cholesterol-supplemented chow for 20 weeks; group 3 received 0.25% cholesterol-supplemented chow for 40 weeks; and group 4 received 0.25% cholesterol-supplemented chow for 20 weeks followed by normal chow for an additional 20 weeks. The aortas and aortic valves were analyzed using immunohistochemical and histological methods to detect cellular and structural components of the developing lesions. RESULTS: All rabbits in groups 2, 3 and 4 developed atherosclerotic lesions in their aortas. Aortic valves from these animals demonstrated thickening, lipid deposition, a change in collagen content and organization, a reorganization of elastin, and the presence of both macrophage infiltrate and osteopontin. CONCLUSION: These findings were consistent with the suggestion of a link between atherosclerosis and AVS. Results were also similar to changes reported in human sclerotic aortic valves, suggesting the suitability of this rabbit model of atherosclerosis as a model for AVS.

Current status of cellular therapy for ischemic heart disease.

Cellular therapy for acute myocardial infarction and ischemic cardiomyopathy has entered clinical trials across the globe. Early promising results have now provided the justification for larger randomized and blinded trials to address the efficacy of cellular therapy. A variety of fresh or cultured autologous cells have been delivered by catheter-guided endocardial, catheter-guided intracoronary, catheter-guided transvenous, and direct epicardial routes. This review will summarize the clinical data and highlight salient basic science data that support the ongoing efforts to identify the optimal cellular therapy both for acute myocardial infarction and chronic ischemic cardiomyopathy patients.

Smoothelin-positive cells in human and porcine semilunar valves.

Our aim was to further characterize the interstitial cell phenotypes of normal porcine and human semilunar valves, information necessary for the design of bioengineered valves and for the understanding of valve disease processes such as aortic valve sclerosis. Existence of fibroblasts, myofibroblasts, and smooth muscle-like cells within semilunar heart valves has been established. However, the nature of the smooth muscle cell population has been controversial. We used immunochemical and western blotting methods to determine the status of smoothelin and smooth muscle alpha-actin in the valve. Our examination of valve interstitial cells confirmed the presence of terminally differentiated, contractile smooth muscle cells in situ. They were arranged in small bundles of 5-35 cells within the ventricularis or as individual cells scattered throughout the valvular layers in vivo, and were present in cells explanted from the valves in vitro. Colocalization of these proteins in semilunar heart valves was achieved with double-labeling experiments. Protein extraction, followed by coimmunoprecipitation, electrophoresis, and western blotting confirmed the immunochemical analysis and suggested that smooth muscle alpha-actin and smoothelin interact, as has been previously postulated. The presence of contractile smooth muscle within the valve may be an important factor in understanding valve pathology and in the design of tissue engineering efforts.

Aortic valve interstitial cells: an evaluation of cell viability and cell phenotype over time.

BACKGROUND AND AIM OF THE STUDY: In investigating the mechanisms of aortic valve disease processes and to accurately construct tissue-engineered heart valve prostheses, a complete understanding of the native interstitial cell population is required. Previously, autopsy samples have been deemed unsuitable for immunocytochemical studies due to the ischemic time before harvesting. In this study, the viability and phenotypic profile of cells explanted from normal porcine heart valves up to 120 h post mortem was examined. METHODS: The aortic valve leaflets of porcine hearts were excised at 0, 6, 24, 48, 72, 96 and 120 h post mortem; one half of the tissue was used to explant cells, and the other half was fixed in 3.7% formalin for sectioning. Samples taken at each time point were cultured and cell viability was determined using a trypan blue exclusion assay. Immunocytochemical and immunohistochemical analyses, using specific markers for fibroblasts, myofibroblasts and smooth muscle cells, were used to compare cell phenotypes both in vitro and in situ. RESULTS: Absolute numbers of cells obtained from each leaflet decreased significantly over time; however, cell viability in culture was unaffected up to 96 h. At each time point, explanted cell populations expressed similar phenotypes when compared with histological samples prepared from the same valves. CONCLUSION: Porcine aortic valve interstitial cells may be explanted up to and including 96 h post mortem, with no statistically significant change in cell viability in vitro, and with a population that phenotypically resembles aortic valve interstitial cells in situ. These data suggest that human aortic valve interstitial cells may be successfully harvested at autopsy for in vitro studies.