ABSTRACT
BACKGROUND: Studies on tissue-engineered vascular scaffold construction mostly focus on biodegradable scaffold and acellular allogenic or xenogenlc vascular scaffold. However, there are some problems to be urgently solved, such as control of degradable speed of biodegradable scaffold, and donor-sourced bacterial virus infecting recipients during the implantation of acellular natural vascular scaffold.OBJECTIVE: This study was designed to treat allogenic blood vessels by ultrahigh pressure in conjunction with nuclease washing (decellularization) to observe the decellularization effects and porcine endogenous retroviras (PERV) removal.DESIGN: A controlled observation.SETTING: National Cardiovascular Center, Japan.MATERIALS: This study was performed at the National Cardiovascular Center, Japan from April 2004 to April 2005.Young healthy male 1-3-month-old minipigs, weighing 3-5 kg, were provided by Japanese Farm. The protocol was performed in accordance with ethical guidelines for the use and care of animals. The main reagents and equipments used in the present study were as follows: Hoechst 33258 (Dojindo Laboratories, Kumamoto, Japan), ultrahigh pressure device (KOBELCO, Kobe Steel, Ltd, Japan), and PCR (GENEAMP PCR SYSTEM 9700).METHODS: Porcine descending aorta vessels were isolated under a sterile condition and treated by cold isostatic pressing (981 MPa, 4 ℃) for disruption of donor cells. The cell debris was digested by nuclease and washed out by phosphate buffered saline for vascular scaffold.MAIN OUTCOME MEASURES: After processing of decellularization by ultrahigh pressure treatment, vascular DNA levels were quantitatively determined by a fluorescent probe (Hoechst 33258); Removal of cell components from vascular tissue and retention of scaffold fibers were observed by a transmission electron microscope (JEM 100 cx); Scaffold ultrastructure was observed via a scanning electron microscope (JBM 5200); The morphological structure of vascular wall was observed via an optical microscope (100 augmentation) . All these were performed to evaluate the antigen-removal effects of decellularization by ultrahigh pressure treatment from histological, molecular biological, and immunohistochemical standpoints. Proviral DNA levels of acellular PERV were measured by PCR to evaluate the effects of decellularization by ultrahigh pressure treatment on killing PERV, a typical pathogenic microorganism.RESULTS: After decellularization by ultrahigh pressure treatment, the wavy structure of fibers was completely retained, and tissues were thoroughly cell free. Transmission electron microscope results demonstrated that collagen fibers and elastic fibers, but not cell components were detectable. Scanning electron microscope results demonstrated that only acellular scaffold was found. There was no PERV detected in the treated tissues. However, the PERV could not be inactivated in the tissues treated by surface active agent. Intravascular DNA levels significantly altered from (31.7±3.5 ) mg/L pre-decelhilarization by ultrahigh pressure treatment to (1.16±0.23) mg/L post- decellularization by ultrahigh pressure treatment(P＜0.01). Results demonstrated that decellularization by ultrahigh pressure treatment ridded of cellular nucleus and contents mostly.CONCLUSION: The study demonstrated that decellularization by ultrahigh pressure treatment could fundamentally rid cell components of scaffold, and concomitantly inactivate PERV successfully.