In-body tissue-engineered collagenous connective tissue membranes (BIOSHEETs) for potential corneal stromal substitution.

There is a severe shortage of donor cornea for transplantation in many countries. Collagenous connective tissue membranes, named BIOSHEETs, grown in vivo were successfully implanted in rabbit corneal stroma for in vivo evaluation of their suitability as a corneal stromal substitute to solve this global donor shortage. BIOSHEETs were prepared by embedding silicone moulds into dorsal subcutaneous pouches in rabbits for 1 month and stored in glycerol. After re-swelling in saline and trephining, disk-shaped BIOSHEETs (4 mm diameter) were allogeneically implanted into stromal pockets prepared in the right cornea of seven rabbits. Clinical tests for corneal thickness and transparency, and tissue analyses were performed. Because the BIOSHEETs (thickness, 131 ± 14 µm) obtained were opaque immediately after implantation, the transparency of the cornea decreased. The total thickness of the BIOSHEET-implanted cornea increased from 364 ± 21.0 µm to 726 ± 131 µm. After 4 weeks' implantation, the thickness of the cornea stabilized (493 ± 80 µm at 4 weeks and 447 ± 46 µm at 8 weeks). The transparency of the cornea increased progressively with time of implantation. The random orientation of collagen fibrils in the original BIOSHEETs tended to be homogeneous, similar to that of the native stroma. No inflammatory cells accumulated and fibroblast-like cells infiltrated the implant. The BIOSHEETs showed high biocompatibility with stromal tissues; however, further studies are needed to test its functional aspects. Although this research is only intended as a proof of concept, BIOSHEETs may be considered a feasible corneal stromal replacement, especially for treating visual impairment caused by stromal haze. Copyright © 2013 John Wiley & Sons, Ltd.

Preparation of an autologous heart valve with a stent (stent-biovalve) using the stent eversion method.

We designed a novel method for constructing an autologous heart valve with a stent, called a stent-biovalve. In constructing completely autologous heart valves, named biovalves, which used in-body tissue architecture technology, tissues for leaflets were formed via ingrowths into narrow apertures in the preparation molds, frequently leading to delayed or incomplete biovalve preparation. In this technique, self-expandable nitinol stents after everting were mounted on an acrylic column-shaped part and partially covered with an acrylic cylinder-shaped part with three slits. This assembled mold was placed into subcutaneous abdominal pouches in beagles or goats for 4 weeks. Upon removing the acrylic parts after harvesting and trimming of capsulated tissues, a tubular hollow structure with three pocket-flaps of membranous tissue rigidly fixed to the stent's outer surface was obtained. Then, the stent was turned inside out to the original form, thus moving the pocket-flaps from outside to the inside. Stent-biovalves with a sufficient coaptation area were thus obtained with little tissue damage in all cases. The valve opened smoothly, and high aperture ratio was noted. This novel technique was thus highly effective in constructing a robust, completely autologous stent-biovalve with adequate valve function.

Post-mortem evaluation of expanded polytetrafluoroethylene (ePTFE) used in mitral valve repair in dogs.

Mitral valve repair is one of the treatment options for mitral regurgitation. Expanded polytetrafluoroethylene (ePTFE) is a polymer that has been widely used in cardiovascular surgery. In this case series, we report the autopsy and histological findings in 6 dogs that underwent cardiopulmonary bypass for mitral annuloplasty using ePTFE sheets and chordoplasty using ePTFE sutures. From May 2005 to October 2009, 3 female and 3 male dogs with severe mitral regurgitation underwent mitral valve repair. This case series included 3 Cavalier King Charles spaniels, 2 Maltese, and 1 Shih Tzu. The survival period after surgery was 19-72 (35 ± 19) months. In all the cases, autopsy revealed that the ePTFE sheets and sutures were not damaged and well integrated into the surrounding highly differentiated, connective tissues. Low-power microscopy revealed that in all cases, the tissues surrounding the ePTFE sheet in the mitral valve annulus had almost completely been covered by granulation tissue. No inflammatory infiltrate or thrombogenesis was observed around the ePTFE in any of the cases. There was no evidence of reactive changes in the region surrounding the ePTFE. These results suggest that ePTFE has excellent tissue compatibility and durability and can be effectively used for canine mitral valve repair.

Water-soluble argatroban for antithrombogenic surface coating of tissue-engineered cardiovascular tissues.

Argatroban is a powerful synthetic anticoagulant, but due to its water-insoluble nature, it is unsuitable for use as a coating material to reduce the thrombogenic potential of natural or tissue-engineered blood-contacting cardiovascular tissues. On the other hand, anionic compounds could adsorb firmly onto connective tissues. Therefore, in this study, an anionic form of argatroban was prepared by neutralization from its alkaline solution, dialysis, and freeze-drying. The subsequently obtained argatroban derivative could be easily dissolved in water. Analysis of the surface chemical composition showed that the water-soluble argatroban (WSA) could be adsorbed on the entire surface of tissue-engineered connective tissue sheets composed mainly of collagen. Adsorption was achieved on immersion of the tissue-engineered connective tissue sheet in a saline/WSA solution for only 30 s without any change in the mechanical properties of the tissue-engineered sheets. Complete surface adsorption (ca., 1 mg/cm(2) ) was obtained at WSA concentrations of over 5 mg/mL. WSA adsorption was maintained for at least 7 days with rinsing. Blood coagulation was significantly prevented on the WSA-adsorbed surfaces in acute in vitro experiments. The coating was applied to in vivo tissue-engineered vascular grafts (biotubes) or tri-leaflet tissues (biovalves) under development, ensuring a high likelihood of nonthrombogenicity of their blood-contacting surfaces with high patency, at least in the subchronic phase. It appears that WSA satisfies the initial requirements for a biocompatible aqueous coating material for use in natural or tissue-engineered tissues.