Force generation of different human cardiac valve interstitial cells: relevance to individual valve function and tissue engineering.

BACKGROUND AND AIM OF THE STUDY: Cardiac valves perform highly sophisticated functions that depend upon the specific characteristics of the component interstitial cells (ICs). The ability of valve ICs to contribute to these functions may be related to the generation of different types of tension within the valve structure. The study aim was to characterize cellular morphology and the forces generated by valve ICs and to compare this with morphology and forces generated by other cell types. METHODS: Cultured human valve ICs, pericardial fibroblasts and vascular smooth muscle cells were seeded in 3-D collagen gels and placed in a device that accurately measures the forces generated. Cell morphology was determined in seeded gels fixed in glutaraldehyde, stained with toluidine blue and visualized using a high-definition stereo light microscope. RESULTS: Valve ICs generated an average peak force of 30.9 +/- 10.4 dynes over a 24-h period which, unlike other cell types tested, increased as cell density decreased (R = 0.67, p <0.0001). The temporal pattern of force generation in mitral valve cells was significantly faster than in aortic or tricuspid cells (p <0.05). Microscopic examination revealed the formation of cellular processes establishing a cell/cell and cell/matrix network. When externally induced changes in matrix tension occurred, the valve ICs unlike the other cell types - did not respond to restore the previous level of tension. CONCLUSION: Human cardiac valve ICs produce a specific pattern of force generation that may be related to the individual function of each heart valve. The specialized function of these cells may serve as a guide for the choice of candidate cells for tissue engineering heart valves.

Human cardiac valve interstitial cells in collagen sponge: a biological three-dimensional matrix for tissue engineering.

BACKGROUND AND AIM OF STUDY: The use of a biological, biodegradable scaffold remodeled by cells to resemble a valve leaflet is an attractive approach to tissue engineering. The study aim was to evaluate the suitability of a three-dimensional biodegradable collagen sponge for maintenance of cell viability, proliferation and phenotype of cultured human cardiac valve interstitial cells (ICs). METHODS: Pieces of valve leaflets were snap-frozen, sectioned and stained by immunoperoxidase. Interstitial cells were cultured from cardiac valves and plated onto glass coverslips or seeded in collagen sponge, then stained by immunofluorescence or immunoperoxidase. A panel of antibodies was used to determine cell phenotype. Cell viability was assessed using a dye-based cell proliferation assay, and cell death by lactate dehydrogenase measurement. RESULTS: ICs variably expressing the phenotypic markers were found throughout the native valve leaflet, but particularly on the ventricular side. Cultured ICs either on coverslips or in collagen sponge expressed vimentin, a fibroblast surface antigen and variable amounts of smooth muscle (SM) alpha-actin. Expression of the other phenotypic markers, SM myosin, desmin and prolyl 4-hydroxylase differed: interestingly, the ratio of cells in collagen sponge expressing these markers reflected that found in the native valve leaflet. Confocal microscopy of ICs in the collagen sponge revealed the presence of cells with long interconnecting extensions indicating cell communication. Cell proliferation and cell death assays established that cells were not only viable after four weeks in the sponge, but were also proliferating. CONCLUSION: This study demonstrates that collagen sponge is a suitable biodegradable scaffold that can maintain viable valve ICs and appears to enhance the capacity of the cell to express its original phenotype.

Profile and localization of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in human heart valves.

BACKGROUND AND AIMS OF THE STUDY: Tissue turnover is one of many factors involved in the operational longevity of heart valves. An understanding of how valves remodel their matrix in response to the hemodynamic environment in health and disease is crucial to the design and biological responsiveness of tissue-engineered valve substitutes. Matrix metalloproteinases (MMPs) are proteolytic enzymes involved in matrix remodeling in several tissues, and include interstitial collagenase (MMP-1, MMP-13), the gelatinases (MMP-2, MMP-9) and stromelysin (MMP-3). METHODS: Expression of MMPs and their tissue inhibitors (TIMPs) in human aortic, mitral, tricuspid and pulmonary valves from unused donor or transplant recipient hearts was determined by immunohistochemical staining using antibodies against human MMP-1, MMP-2, MMP-3 and MMP-9 and their inhibitors TIMP-1, TIMP-2, TIMP-3. Cell identification was achieved using antibodies against CD31(endothelial cells), smooth muscle alpha-actin (microfilaments) and CD68 (macrophages). RESULTS: MMP-1 was expressed in all valves, whereas MMP-2 was only expressed in aortic and pulmonary leaflets. MMP-3 and MMP-9 were not expressed. TIMP-1 and TIMP-2 were expressed in all leaflets, whereas TIMP-3 was observed only in tricuspid leaflets. CONCLUSION: Valves have a specific pattern of expression of MMPs and TIMPs, which appears to vary in different heart valves. The functional implications and central mechanisms responsible require further study. These findings have important implications in understanding the dynamic nature of valve remodeling and in aiding the development of tissue-engineered valves.