Eiichiro Uchimura

In situ tissue regeneration using a novel tissue-engineered, small-caliber vascular graft without cell seeding

OBJECTIVE Various types of natural and synthetic scaffolds with arterial tissue cells or differentiated stem cells have recently attracted interest as potential small-caliber vascular grafts. It was thought that the synthetic graft with the potential to promote autologous tissue regeneration without any seeding would be more practical than a seeded graft. In this study, we investigated in situ tissue regeneration in small-diameter arteries using a novel tissue-engineered biodegradable vascular graft that did not require ex vivo cell seeding. METHODS Small-caliber vascular grafts (4 mm in diameter) were fabricated by compounding a collagen microsponge with a biodegradable woven polymer tube that was constructed in a plain weave pattern with a double layer of polyglycolic acid (core) and poly-L-lactic acid (sheath) fibers. We implanted these tissue-engineered vascular grafts bilaterally into the carotid arteries of mongrel dogs (body weight, 20-25 kg). No anticoagulation regimen was used after implantation. We sacrificed the dogs 2, 4, 6, and 12 months (n = 4 in each group) after implantation and evaluated the explants histologically and biochemically. RESULTS All of the tissue-engineered vascular grafts were patent with no signs of thrombosis or aneurysm at any time. Histologic and biochemical examinations showed excellent in situ tissue regeneration with an endothelial cell monolayer, smooth muscle cells, and a reconstructed vessel wall with elastin and collagen fibers. CONCLUSION Our study indicated that this novel tissue-engineered vascular graft promoted in situ tissue regeneration and did not require ex vivo cell seeding, thereby conferring better patency on small-caliber vascular prostheses.

In-Situ Visualization and Quantification of Mineralization of Cultured Osteogenetic Cells

An osteoblastic cell line (HOS cells) produces a prominent osteoid matrix with mineralization. Fibroblasts, on the other hand, do not exhibit this mineralization. To evaluate the degree of mineralization, we added calcein to the culture medium and then observed the culture wells by using an image analyzer. The calcein uptake into the cell/matrix layer was detected in the HOS cells but not in the fibroblasts. The calcein uptake was also quantified in situ by using an image analyzer, which revealed high levels in the HOS cells, which correlated well with the calcium content of the mineralized matrix. Rat marrow cells were also cultured in media containing calcein, fetal bovine serum, β-glycerophosphate, L-ascorbic acid 2-phosphate, and with or without dexamethasone. With the dexamethasone, the cells exhibited osteogenic differentiation that resulted in mineralized matrix formation after about 10 days. The matrix formation coincided with the appearance of calcein uptake into the cell/matrix layer, with the amount of calcein uptake increasing with time. By contrast, the culture without the dexamethasone did not exhibit matrix formation and the calcein uptake was negligible. In the case of both HOS cell and rat marrow cell cultures in vitro, calcein did not affect expressions of their alkaline phosphatase activity or osteocalcin production. Furthermore, histologic observation revealed that rat marrow cells subcultured with calcein could show osteogenic ability after in vivo implantation. These results suggest that the current method of detecting calcein uptake in a culture allows the monitoring of the osteogenic capacity of cultured cells, as well as the measurement of the amount of mineralization produced by the osteogenic cells. Given that osteogenic cultured cells/mineralized matrices are used in bone reconstruction surgery, the in situ monitoring method is invaluable in that it allows us to evaluate the osteogenic capacity of in vitro constructs.

On-chip transfection of PC12 cells based on the rational understanding of the role of ECM molecules: efficient, non-viral transfection of PC12 cells using collagen IV

Transfection microarrays (TMA) are important emerging tools for the study of genetic events in living cells in a high-throughput fashion and with significant material economy. However, the difficulty to transfect various relevant cell types on-chip hinders the use of TMAs. Herein we present the realization of a transfection microarray applicable to PC12 cells that heavily relies on the use of ECM molecules. Collagen IV and at a lesser extent laminin or collagen I, but not fibronectin or poly-l-lysine were found to significantly increase the solution-phase as well as on-chip transfection efficiency of PC12 cells. The highest transfection efficiency obtained was consistently above 60%. The observed correlations between the transfection efficiencies and the differential adhesion-induced events triggered by the studied ECMs provides the basis for the rationalization of the role of ECMs on the transfection process.

Newly Developed Tissue-Engineered Material for Reconstruction of Vascular Wall Without Cell Seeding

BACKGROUND We have developed a tissue-engineered patch for cardiovascular repair. Tissue-engineered patches facilitated site-specific in situ recellularization and required no pretreatment with cell seeding. This study evaluated the patches implanted into canine pulmonary arteries. METHODS Tissue-engineered patches are biodegradable sheets woven with double-layer fibers. The fiber is composed of polyglycolic acid and poly-L-lactic acid, and compounding collagen microsponges. The patches (20- x 25-mm) were implanted into the canine pulmonary arterial trunks. At 1, 2, and 6 months after implantation (n = 4), they were explanted and characterized by histologic and biochemical analyses. Commercially available patches served as the control. No anticoagulant therapy was administered postoperatively. RESULTS No aneurysm or thrombus was present within the patch area in all groups. The remodeled tissue predominantly consisted of elastic and collagen fibers, and the endoluminal surface was covered with a monolayer of endothelial cells and multilayers of smooth muscle cells beneath the endothelial layer. The elastic and collagen fibers and smooth muscle cells kept increasing with a maximum at 6 months, while a monolayer of endothelial cells was preserved. The expression levels of messenger RNA of several growth factors in the tissue-engineered patches were higher than those of native tissue at 1 and 2 months and decreased to normal level at 6 months. No regenerated tissue was found on the endoluminal surface in the control group. CONCLUSIONS The novel tissue-engineered patches showed in situ repopulation of host cells without prior ex vivo cell seeding. This is promising material for repair of the cardiovascular system.

Reverse transfection using gold nanoparticles.

Reverse transfection from a solid surface has the potential to deliver genes into various types of cell and tissue more effectively than conventional methods of transfection. We present a method for reverse transfection using a gold colloid (GC) as a nanoscaffold by generating nanoclusters of the DNA/reagent complex on a glass surface, which could then be used for the regulation of the particle size of the complex and delivery of DNA into nuclei. With this method, we have found that the conjugation of gold nanoparticles (20 nm in particle size) to the pEGFP-N1/Jet-PEI complex resulted in an increase in the intensity of fluorescence of enhanced green fluorescent protein (EGFP) (based on the efficiency of transfection) from human mesenchymal stem cells (hMSCs), as compared with the control without GC. In this manner, we constructed a method for reverse transfection using GC to deliver genes into the cells effectively.

Area-based analyzing technique at cell array experiment using neuronal cell line

We propose a simple procedure for the identification and quantitative analysis of neurite outgrowth in neuronal cell lines that were uniformly differentiated. Upon stimulation most neuronal cell lines extend neurites in the differentiation process, resulting, according to our observation, in the increase of cell surface area. This increase is dependent on the length and the number of extended neurites. Furthermore, we use this method for the phenotype analysis of cell array experiments to perform large-scale functional evaluation of genes involved in the neurite outgrowth during neuronal differentiation.