Biomedical subjects

Stephen L Hilbert

Publications and source records attributed to Stephen L Hilbert.

4 recordsLinked to original sources

PubMed2004

Explant pathology study of decellularized carotid artery vascular grafts.

The purpose of this study was to evaluate the morphologic findings in small-diameter freeze-dried decellularized carotid artery grafts implanted in goats as carotid artery interposition grafts for 6-7 months. Unimplanted decellularized carotid artery grafts did not contain intact cells; however, remnants of smooth muscle cells were present in the media. The extracellular matrix was well preserved. All decellularized grafts were patent at explant, without significant dimensional changes or aneurysm formation. Their luminal surfaces were lined by a thin neointima, consisting of myofibroblasts, collagen, and a discontinuous layer of endothelial cells. Histologic evidence of calcification within the explants was not observed; however, electron microscopy showed calcification of minute remnants of cell membranes. Inflammatory cells were not present in the graft wall. Host cell migration was greatest in the adventitia along the length of the graft. Migration of host cells into the media was more apparent close to the anastomoses, forming cellular nests rich in extracellular proteoglycans, whereas cell migration into areas subjacent to the lumen was minimal. Ingrowth of host blood vessels was not observed. These results demonstrate satisfactory structural and morphologic features of a decellularized carotid artery small-diameter graft implanted for up to 7 months.

Animals↗

PubMed2004

Effects of constant static pressure on the biological properties of porcine aortic valve leaflets.

An understanding of how mechanical forces impact cells within valve leaflets would greatly benefit the development of a tissue-engineered heart valve. In this study, the effect of constant ambient pressure on the biological properties of heart valve leaflets was evaluated using a custom-designed pressure system. Native porcine aortic valve leaflets were exposed to static pressures of 100, 140, or 170 mmHg for 48 h. Collagen synthesis, DNA synthesis, sulfated glycoaminoglycan (sGAG) synthesis, alpha-SMC actin expression, and extracellular matrix (ECM) structure were examined. Results showed that elevated pressure caused an increase in collagen synthesis. This increase was not statistically significant at 100 mmHg, but at 140 mmHg and 170 mmHg collagen synthesis increased by 37.5 and 90%, respectively. No significant difference in DNA or sGAG synthesis was observed at elevated pressures, with the exception that DNA synthesis at 100 mmHg decreased. A notable decline in alpha-SMC actin was observed over the course of the experiments although no significant difference was observed between the pressure and control groups. It was concluded that elevated pressure caused a proportional increase in collagen synthesis of porcine aortic valve leaflets, but was unable to preserve alpha-SMC actin immunoreactive cells.

Actins↗

PubMed2004

Cyclic pressure affects the biological properties of porcine aortic valve leaflets in a magnitude and frequency dependent manner.

An understanding of how mechanical forces impact cells within valve leaflets would greatly benefit the development of a tissue-engineered heart valve. Previous studies by this group have shown that exposure to constant static pressure leads to enhanced collagen synthesis in porcine aortic valve leaflets. In this study, the effect of cyclic pressure was evaluated using a custom-designed pressure system. Different pressure magnitudes (100, 140, and 170 mmHg) as well as pulse frequencies (0.5, 1.167, and 2 Hz) were studied. Collagen synthesis, cell proliferation, sGAG synthesis, alpha-SMC actin expression, and extracellular matrix (ECM) structure were chosen as markers for valvular biological responses. Results showed that aortic valve leaflets responded to cyclic pressure in a magnitude and frequency-dependent manner. Increases in pressure magnitude (with the frequency fixed at 1.167 Hz) resulted in significant increases in both collagen and sGAG synthesis, while DNA synthesis remained unchanged. Responses to pulse frequency (with the magnitude fixed at 100 mmHg) were more complex. Collagen and sGAG synthesis were increased by 25 and 14% respectively at 0.5 Hz; but were not affected at 1.167 and 2 Hz. In contrast, DNA synthesis increased by 72% at 2 Hz, but not at 0.5 and 1.167 Hz. Under extreme pressure conditions (170 mmHg, 2 Hz), collagen and sGAG synthesis were increased but to a lesser degree than at 170 mmHg, and 1.167 Hz. Cell proliferation was not affected. A notable decline in a-SMC actin was observed over the course of the experiments, although no significant difference was observed between the cyclic pressure and control groups. It was concluded that cyclic pressure affected biosynthetic activity of aortic valve leaflets in a magnitude and frequency dependent manner. Collagen and sGAG synthesis were positively correlated and more responsive to pressure magnitude than pulse frequency. DNA synthesis was more responsive to pulse frequency than pressure magnitude. However, when combined, pressure magnitude and pulse frequency appeared to have an attenuating effect on each other. The number of alpha-SMC actin positive cells did not vary with cyclic pressure, regardless of pulse frequency and pressure magnitude.

Animals↗

PubMed2004

Prototype anionic detergent technique used to decellularize allograft valve conduits evaluated in the right ventricular outflow tract in sheep.

BACKGROUND AND AIM OF THE STUDY: Biodegradable polymeric materials or extracellular matrix scaffolds are used in tissue-engineered heart valve designs, with the expectation of replicating the anatomic, histological and biomechanical characteristics of semi-lunar valves. The study aim was to evaluate the extent of in-vivo recellularization and the explant pathology findings of a prototype anionic, non-denaturing detergent and endonuclease technique used to decellularize allograft (homograft) valve conduits implanted in the right ventricular outflow tract (RVOT) of sheep, and to identify possible risks associated with tissue-engineered heart valve conduits based on decellularized allograft semilunar valve scaffolds. METHODS: Valve conduits were decellularized using a solution of N-lauroylsarcosinate and endonucleases, rinsed in lactated Ringers solution, and stored in an antibiotic solution at 4 degrees C until implanted. Explanted valves and unimplanted controls were examined macroscopically, radiographically (for calcification) and histologically using immunohistochemistry (IHC), routine and special histological stains, transmission electron microscopy (TEM) and polarized light microscopy (evaluation of collagen crimp). RESULTS: Cells and cellular remnants were uniformly absent in the decellularized cusps, but occasional focal sites of arterial wall smooth muscle cells and to a greater extent subvalvular cardiac myocytes were variably retained. The trilaminar histological structure of the cusp was preserved. Valve conduit-related pathology consisted of intracuspal hematoma formation, collagen fraying, thinning of the conduit wall, and inflammatory cells associated with cardiac myocyte remnants. Cuspal calcification was not seen, but elastic fibers in the conduit wall and retained subvalvular cardiac myocyte remnants were liable to calcification. Fibrous sheath formation was present on the luminal surface of the conduit and extended over the cuspal surfaces to a variable extent. Myofibroblast-like cells repopulated the conduit wall and the basal region of the cusp. Re-endothelialization was variably present on the cuspal surfaces. CONCLUSION: Explant pathology findings showed that in-vivo recellularization occurred, but was focally limited to regions of the arterial wall and cusp base. Safety concerns related to detergent and endonuclease use were identified. Methods to eliminate the potential for structural deterioration and enhance the rate and extent of recellularization of valve conduit tissue are required. Pathology findings showed implantation of valve conduits in the RVOT of juvenile sheep for 20 weeks to be a reliable animal model for the initial in-vivo assessment of decellularized valves. A 20-week period may be insufficient however to evaluate the long-term safety and effectiveness of a tissue-engineered valve conduit, as these depend on effective and phenotypically appropriate recellularization accompanied by sustained cell viability and function.

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