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.

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.

Identification of twinfilin-2 as a factor involved in neurite outgrowth by RNAi-based screen.

Using RNA interference (RNAi) to suppress gene expression, we attempted to identify tyrosine kinases involved in the extension of neurites from SH-SY5Y cells. A comprehensive analysis of gene "knock-down" profiles with small interfering RNAs (siRNAs) revealed candidate proteins that might control neurite extension. Phenotype-based screening of differentiating SH-SY5Y cells following retinoic acid (RA) stimulation indicated that twinfilin-2 is a protein that is involved in neurite outgrowth, as confirmed by morphological analysis of twinfilin-2-overexpressing cells.

Reverse transfection using antibodies against a cell surface antigen in mammalian adherent cell lines.

Reverse transfection from a solid surface has the potential to deliver genes to various cells more efficiently than conventional methods. However, the effective gene delivery from a solid surface requires an optimized extracellular matrix (ECM) for the coating of glass slides, dependent on the nature of the cells. In a search for an appropriate substrate for the universal application to multiple types of cell, we focused on cell surface antigens and examined the effects of antibodies raised against them on gene transfer from an antibody-coated surface. We found that a coating of CD29-specific antibody allowed the most effective delivery of genes by reverse transfection in every type of cell that we examined. Our results suggest that reverse transfection with antibodies against CD29 might provide a universal tool for gene delivery and cell array-based analyses.

Novel method of decellularization of porcine valves using polyethylene glycol and gamma irradiation.

BACKGROUND: Recent tissue-engineered valves are in need of a breakthrough to overcome several limitations against clinical applications. We have developed a new method of decellularization using polyethylene glycol and gamma irradiation. METHODS: Fresh porcine aortic valves were decellularized using polyethylene glycol and gamma irradiation. These were evaluated by histologic, biochemical (DNA, solubilized protein and collagen content), mechanical (strength test, transmission electron microscopy) and immunologic (porcine endogenous retrovirus and the alpha-1.3 galactosyl epitope) analyses. Implantations into the subcutaneous tissue of rats (1 week, n = 10; 2 months, n = 10) and into the descending aorta of dogs (2 months, n = 6; 6 months, n = 3) were used as in vivo studies. RESULTS: Complete decellularization was confirmed by histologic examination and by determining the DNA and solubilized protein content. The decellularized valve showed no significant differences in its mechanical strength or collagen content compared with native porcine tissues. The ultrastructure was well preserved in transmission electron microscope images. The DNA sequence of a porcine endogenous retrovirus and the alpha-1.3 galactosyl epitope were eliminated after the decellularizing process. No acute rejection and little calcification was noted in the rat model. In the dog model at 2 months, the surface of the graft was completely covered with a monolayer of endothelial cells. In addition, several layers of vimentin-positive cells lay under the endothelial cells. At 6 months after implantation, many smooth muscle cells, monolayer endothelial cells, and some vasculogenesis were seen. CONCLUSIONS: The decellularizing method provided low immunogenicity, low risk of unknown infections, and was little subject to calcification. The decellularized tissues showed acceptable durability and recellularization.

Method for reverse transfection using gold colloid as a nano-scaffold.

DNA microarray of non-viral reverse transfection in cell engineering allows drastic downsizing of large-scale functional screening of genes and siRNAs. However the control of localizability and efficiency of the microarray is still considered as a critical barrier in practical use. One of the major breakthrough to increase the transfection efficiency may be control in the condition of DNA/transfection reagent complex on the microarray surface. In this paper, we showed that negatively charged gold colloid (GC) is successfully used to control the DNA/reagent complex on a glass surface. The conjugation of gold nanoparticles (20 nm in diameter) to the pEGFP-N1/Jet-PEI complex resulted in a more than 2.5-fold 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 to the control without GC. Our method for reverse transfection should be useful not only for cell array-based analyses but also as a novel gene-delivery method for gene therapy in regenerative medicine.

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.

Biodegradable polymer with collagen microsponge serves as a new bioengineered cardiovascular prosthesis.

OBJECTIVE: Biodegradable materials with autologous cell seeding have attracted much interest as potential cardiovascular grafts. However, pretreatment of these materials requires a complicated and invasive procedure that carries the risk of infection. To avoid these problems, we sought to develop a biodegradable graft material containing collagen microsponge that would permit the regeneration of autologous vessel tissue. The ability of this material to accelerate in situ cellularization with autologous endothelial and smooth muscle cells was tested with and without precellularization. METHODS: Poly(lactic-co-glycolic acid) as a biodegradable scaffold was compounded with collagen microsponge to form a vascular patch material. These poly(lactic-co-glycolic acid)-collagen patches with (n = 10) or without (n = 10) autologous vessel cellularization were used to patch the canine pulmonary artery trunk. Histologic and biochemical assessments were performed 2 and 6 months after the implantation. RESULTS: There was no thrombus formation in either group, and the poly(lactic-co-glycolic acid) scaffold was almost completely absorbed in both groups. Histologic results showed the formation of an endothelial cell monolayer, a parallel alignment of smooth muscle cells, and reconstructed vessel wall with elastin and collagen fibers. The cellular and extracellular components in the patch had increased to levels similar to those in native tissue at 6 months. CONCLUSIONS: The poly(lactic-co-glycolic acid)-collagen microsponge patch with and without precellularization showed good histologic findings and durability. This patch shows promise as a bioengineered material for promoting in situ cellularization and the regeneration of autologous tissue in cardiovascular surgery.

Transfection microarray of human mesenchymal stem cells and on-chip siRNA gene knockdown.

The transfection efficiency of primary cells is the bottleneck for their use with miniaturized formats for gene validation assays. We have found that when formulations containing various reporter plasmids were microarrayed on glass slides (chips), hMSCs cultivated on the chip incorporated and expressed the microarrayed plasmid DNAs with high efficiency and virtually total spatial resolution. Fibronectin, as the key formulation component, was found to significantly increase the on-chip transfection efficiency in hMSCs as well as many other cells. Further, we have conclusively proven that when siRNA was co-arrayed with the target plasmid DNA, a concentration-dependent gene knockdown was observed. Thus, massively miniaturized RNAi gene knockdown experiments can now be performed in primary cells, previously unusable with transfection microarrays (TMA).

Novel method of preparing acellular cardiovascular grafts by decellularization with poly(ethylene glycol).

We have developed a new method of preparing acellular vascular grafts. Cellular components, including cell membranes and proteins in cytosol, were efficiently extracted from the vessels in a concentrated aqueous solution of poly(ethylene glycol), an amphiphilic biocompatible polymer. The residual DNA was digested by deoxyribonuclease I treatment after extraction with poly(ethylene glycol). The two-step extraction process proved quite effective at removing the cellular components while causing little damage to the extracellular matrices. We did not use any detergent that would damage the extracellular matrices. Therefore, vascular endothelial cells grew well on the acellular vessels after recellularization, promising longi-patent cardiovascular grafts.

Anomalous binding profile of phenylboronic acid with N-acetylneuraminic acid (Neu5Ac) in aqueous solution with varying pH.

Borates are known to interact with carbohydrate moieties expressed on the surface of biological membranes of a variety of cells, viruses, bacteria, and fungi. This study revealed the anomalous binding profile of borate in aqueous solution with N-acetylneuraminic acid (Neu5Ac, sialic acid) as a potential receptor site on the surfaces of biological membranes using (11)B, (1)H, (13)C, and (15)N nuclear magnetic resonance spectroscopies. 3-(Propionamido)phenylboronic acid (PAPBA) was chosen as the model borate compound. The equilibrium constant (K) for Neu5Ac binding to PAPBA was compared with those for glucose, mannose, and galactose, which are the major carbohydrate constituents of glycoproteins and glycolipids expressed on biological membranes. In the Neu5Ac/PAPBA system, the unusual pH dependency of the K values, a decrease in K with increasing pH, was observed, suggesting the formation of a trigonal-formed complex stabilized by the coordination of an amide group of Neu5Ac at the C-5 position to the boron atom, forming intramolecular B-N or B-O bonding. Furthermore, the anomalously high complexing ability at physiological pH 7.4 was confirmed for this system, with the K value 37.6 which is approximately 7 times higher than that for glucose. This exceptionally high value of K at physiological pH, compared to those of other sugars, strongly suggests that the boronic acid selectively recognizes the Neu5Ac residues of the glycosylated components including glycoproteins and gangliosides existing on the surface of the biological membranes.