X-ray phase-contrast computed tomography visualizes the microstructure and degradation profile of implanted biodegradable scaffolds after spinal cord injury.

Tissue engineering strategies for spinal cord repair are a primary focus of translational medicine after spinal cord injury (SCI). Many tissue engineering strategies employ three-dimensional scaffolds, which are made of biodegradable materials and have microstructure incorporated with viable cells and bioactive molecules to promote new tissue generation and functional recovery after SCI. It is therefore important to develop an imaging system that visualizes both the microstructure of three-dimensional scaffolds and their degradation process after SCI. Here, X-ray phase-contrast computed tomography imaging based on the Talbot grating interferometer is described and it is shown how it can visualize the polyglycolic acid scaffold, including its microfibres, after implantation into the injured spinal cord. Furthermore, X-ray phase-contrast computed tomography images revealed that degradation occurred from the end to the centre of the braided scaffold in the 28 days after implantation into the injured spinal cord. The present report provides the first demonstration of an imaging technique that visualizes both the microstructure and degradation of biodegradable scaffolds in SCI research. X-ray phase-contrast imaging based on the Talbot grating interferometer is a versatile technique that can be used for a broad range of preclinical applications in tissue engineering strategies.

Histological maturation of vascular smooth muscle cells in in situ tissue-engineered vasculature.

The goal of regenerative medicine is to achieve histological and functional recovery to the level of the original tissue. For this purpose, we have developed a biodegradable scaffold to create cell-free in-situ tissue-engineered vasculature (iTEV) with good long-term results. However, the regeneration process of vascular smooth muscle cells (VSMCs) over time has yet to be examined. To evaluate the regeneration ability of VSMCs, the inferior vena cava of experimental animals was replaced with iTEV, and tested at 1, 3, 6, 12, and 24 months (n = 6 each) after implantation. Six animals were enrolled to compare 24-month iTEV and native vasculature in single individual samples. There were no complications throughout the study. Immunohistology, protein expression analysis, and biochemical findings indicate that iTEV can gradually regenerate and develop into a mature vessel within 24 months using our biodegradable scaffold. These results provide a time course for the regeneration of VSMCs within the tissue-engineered vascular autograft constructed using a biodegradable scaffold.

Long-term results of cell-free biodegradable scaffolds for in situ tissue engineering of pulmonary artery in a canine model.

We previously developed a cell-free, biodegradable scaffold for in-situ tissue-engineering vasculature (iTEV) in a canine inferior vena cava (IVC) model. In this study, we investigated application of this scaffold for iTEV of the pulmonary artery (iTEV-PA) in a canine model. In vivo experiments were conducted to determine scaffold characteristics and long-term efficacy. Biodegradable scaffolds comprised polyglycolide knitted fibers and an l-lactide and ε-caprolactone copolymer sponge, with an outer glycolide and ε-caprolactone copolymer monofilament reinforcement. Tubular scaffolds (8 mm diameter) were implanted into the left pulmonary artery of experimental animals (n = 7) and evaluated up to 12 months postoperatively. Angiography of iTEV-PA after 12 months showed a well-formed vasculature without marked stenosis, aneurysmal change or thrombosis of iTEV-PA. Histological analysis revealed a vessel-like vasculature without calcification. However, vascular smooth muscle cells were not well-developed 12 months post-implantation. Biochemical analyses showed no significant difference in hydroxyproline and elastin content compared with native PA. Our long-term results of cell-free tissue-engineering of PAs have revealed the acceptable qualities and characteristics of iTEV-PAs. The strategy of using this cell-free biodegradable scaffold to create relatively small PAs could be applicable in pediatric cardiovascular surgery requiring materials.

Long-term results of cell-free biodegradable scaffolds for in situ tissue-engineering vasculature: in a canine inferior vena cava model.

We have developed a new biodegradable scaffold that does not require any cell seeding to create an in-situ tissue-engineering vasculature (iTEV). Animal experiments were conducted to test its characteristics and long-term efficacy. An 8-mm tubular biodegradable scaffold, consisting of polyglycolide knitted fibers and an L-lactide and ε-caprolactone copolymer sponge with outer glycolide and ε-caprolactone copolymer monofilament reinforcement, was implanted into the inferior vena cava (IVC) of 13 canines. All the animals remained alive without any major complications until euthanasia. The utility of the iTEV was evaluated from 1 to 24 months postoperatively. The elastic modulus of the iTEV determined by an intravascular ultrasound imaging system was about 90% of the native IVC after 1 month. Angiography of the iTEV after 2 years showed a well-formed vasculature without marked stenosis or thrombosis with a mean pressure gradient of 0.51 ± 0.19 mmHg. The length of the iTEV at 2 years had increased by 0.48 ± 0.15 cm compared with the length of the original scaffold (2-3 cm). Histological examinations revealed a well-formed vessel-like vasculature without calcification. Biochemical analyses showed no significant differences in the hydroxyproline, elastin, and calcium contents compared with the native IVC. We concluded that the findings shown above provide direct evidence that the new scaffold can be useful for cell-free tissue-engineering of vasculature. The long-term results revealed that the iTEV was of good quality and had adapted its shape to the needs of the living body. Therefore, this scaffold would be applicable for pediatric cardiovascular surgery involving biocompatible materials.

Development of an operator-independent method for seeding tissue-engineered vascular grafts.

The manual seeding of cells onto a biodegradable scaffold by pipetting is an effective method of cell seeding. However, the widespread use and ultimate clinical utility of this technique is limited by operator variability. This study was conducted to evaluate an operator-independent vacuum-seeding method for use in an upcoming clinical trial. Using bone marrow-derived mononuclear cells, we achieved seeding comparable to manually seeded scaffolds in terms of cellular attachment, distribution, and viability in vacuum-seeded grafts at vacuum pressures of -25 to -50 mmHg. In conclusion, we describe an operator-independent seeding method for use in the clinical setting.

Comparison of bioabsorbable materials for use in artificial tracheal grafts.

Limited information exists regarding the usefulness of bioabsorbable materials in the design of tracheal grafts. The aim of this study was to evaluate the feasibility of three bioabsorbable materials for use as artificial trachea. Three sets of grafts were prepared: Group 1 (n=6), knitted polyglactin 910 mesh; Group 2 (n=3), copolymer of L-lactide and epsilon-caprolactone sponge reinforced with polyglycoride fibers; and Group 3 (n=8), copolymer of L-lactide and epsilon-caprolactone sponge covered with knitted poly-L-lactide mesh. All grafts were internally reinforced with a titanium stent. A 10-cartilage-ring-length of canine mediastinal trachea was resected and replaced by a bioabsorbable prosthesis with the aid of an omental flap. In Groups 1 and 2, the patency rates decreased below 50% within two months after surgery. In Group 3, six of eight dogs maintained patency rates above 50% from 10 months to 2 years after surgery. Grafts prepared with a copolymer of L-lactide and epsilon-caprolactone sponge covered with knitted poly-l-lactide mesh (Group 3) can function for up to two years after surgery. These results provide evidence toward the feasibility of utilizing bioabsorbable materials as a tracheal prosthesis.

Evaluation of tissue-engineered vascular autografts.

This study evaluated the endothelial function and mechanical properties of tissue-engineered vascular autografts (TEVAs) constructed with autologous mononuclear bone marrow cells (MN-BMCs) and a biodegradable scaffold using a canine inferior vena cava (IVC) model. MN-BMCs were obtained from a dog and seeded onto a biodegradable tubular scaffold consisting of polyglycolide fiber and poly(L-lactide-co-epsilon-caprolactone) sponge. This scaffold was implanted in the IVC of the same dog on the day of surgery. TEVAs were analyzed biochemically, biomechanically, and histologically after implantation. When TEVAs were explanted and stimulated with acetylcholine at 1 month, they produced nitrates and nitrites dose dependently. N(G)-nitro-L-arginine methylester significantly inhibited these reactions. With stimulation by acetylcholine, factor VIII-positive cells of TEVAs produced endothelial nitric oxide synthase proteins, and the ratio of endothelial nitric oxide synthase/s17 mRNA was similar among native IVC and TEVAs 1 and 3 months after implantation. TEVAs had biochemical properties and wall thickness similar to those of native IVC at 6 months after implantation, and tolerated venous pressure well without any problems such as calcification. The number of inflammatory cells in TEVAs and the ratio of CD4/s17 mRNA decreased significantly with time. These results indicate that TEVAs are a biocompatible material with functional endothelial cells and biomechanical properties and do not have unwanted side effects.

Tensile properties and biological response of poly(L-lactic acid) felt graft: an experimental trial for rotator-cuff reconstruction.

Poly(L-lactic acid) felt (PLLA felt) was prepared for reconstruction of the rotator cuff in animal models. Small changes were found in the tensile strength of both the cultured PLLA felt and the PLLA felt implanted on the paravertebral muscle of rabbits up to 16 postoperative weeks. The stiffness of the felt implanted on the muscle from 6 to 16 weeks showed a statistically significant increase. When the infraspinatus tendons of beagle dog were reconstructed with the PLLA felt, the ultimate strength of PLLA felt increased threefold, and the stiffness increased fivefold by 16 postoperative weeks compared to that of the initial PLLA felt. They were statistically significant (p < 0.01). All the implanted specimens ruptured at the junction between the bone and the PLLA felt. Histological examination demonstrated infiltration of fibrous tissue into the interstices of the PLLA felt fibers. Connection between the infraspinatus tendon and the PLLA felt was tight with the formed scar tissue, but the connective tissue between the bone and PLLA felt fibers was sparse even at 16 and 32 postoperative weeks. A few deteriorated PLLA felt fibers were observed at 32 postoperative weeks. It was concluded that the degradation rate of PLLA felt was low and the tensile recovery of the PLLA felt graft in beagle dogs was excellent. Thus, PLLA felt might be a useful bioabsorbable material for rotator-cuff reconstruction.

Evaluation of the antiadhesion potential of UV cross-linked gelatin films in a rat abdominal model.

Among five kinds of rat adhesion models tested, the following model was selected. The epigastric vein 2.5 cm from the midline of the abdomen was cut by sharp scissors, and the lateral side of the cut epigastric vein was ligated using a 3-0 silk suture. This model could be easily prepared and gave a rate of adhesion formation of 90%, which was useful for screening antiadhesive materials. For the kinetic study of tissue adhesion in this model, an injured site was covered with a non-degradable poly(vinyl alcohol) (PVA) film. The incidence rate of adhesion was 18%, when the PVA film covered the injured site for 2 days. This suggests that an antiadhesive barrier should cover the injured site for at least 2 days. The antiadhesion efficacy of cross-linked gelatin films were evaluated using this adhesion model. The UV cross-linked gelatin film which was designed to exist for 2 days but to disappear at day 3 in the rat abdominal cavity showed the highest antiadhesion efficacy.