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1.
Nat Biomed Eng ; 5(8): 926-940, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34373601

RESUMO

Current protocols for the differentiation of human pluripotent stem cells (hPSCs) into chondrocytes do not allow for the expansion of intermediate progenitors so as to prospectively assess their chondrogenic potential. Here we report a protocol that leverages PRRX1-tdTomato reporter hPSCs for the selective induction of expandable and ontogenetically defined PRRX1+ limb-bud-like mesenchymal cells under defined xeno-free conditions, and the prospective assessment of the cells' chondrogenic potential via the cell-surface markers CD90, CD140B and CD82. The cells, which proliferated stably and exhibited the potential to undergo chondrogenic differentiation, formed hyaline cartilaginous-like tissue commensurate to their PRRX1-expression levels. Moreover, we show that limb-bud-like mesenchymal cells derived from patient-derived induced hPSCs can be used to identify therapeutic candidates for type II collagenopathy and we developed a method to generate uniformly sized hyaline cartilaginous-like particles by plating the cells on culture dishes coated with spots of a zwitterionic polymer. PRRX1+ limb-bud-like mesenchymal cells could facilitate the mass production of chondrocytes and cartilaginous tissues for applications in drug screening and tissue engineering.


Assuntos
Proteínas de Homeodomínio/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Pluripotentes/citologia , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Condrócitos/citologia , Condrócitos/metabolismo , Condrócitos/transplante , Condrogênese , Doenças do Colágeno/terapia , Meios de Cultura/química , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Pluripotentes/metabolismo , Polímeros/química , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Antígenos Thy-1/metabolismo , Engenharia Tecidual
2.
J Biosci Bioeng ; 2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34420897

RESUMO

Generally, the thickness of tubular tissues formed from silicone rods through encapsulation of the foreign-body reaction is less than approximately 0.2 mm. On the other hand, it is unclear how hollow cylindrical molds can provide thick tubular tissues, known as Biotubes, with a thickness exceeding 1 mm, during in-body tissue architecture (iBTA) using encapsulation. In this study, histological and structural analyses were performed to understand the reason for the formation of thick mold-based Biotubes. Molds were assembled with a gap between a silicone rod and a stainless-steel cylinder and were embedded into the dorsal subcutaneous pouches of beagles for 2 or 4 weeks. Thick Biotubes were obtained from the harvested mold. The histological analysis showed that the lumen side of the thick Biotubes consisted primarily of type I collagen fibers and α-smooth muscle actin-positive cells, similar to the original rod-based thin Biotubes formed only from silicone rods. Interestingly, the outer region of the thick Biotubes was an immature connective tissue consisting of type III collagen, including primitive somatic stem cells expressing CD90 and SSEA4. These stem cells may have contributed to the formation of the thick-walled Biotubes by differentiating into other cell types and through growth factor production. Because of the potential tissue-repair ability of these stem cells, iBTA could be useful for elucidating the regeneration process, remodeling the physiology/pathology of tissue defects/damage, and cell acquisition. This technology can provide autologous stem cells without in vitro cell culture. Moreover, thick-walled Biotubes may be useful as an alternative stem cell-containing material in regenerative medicine.

3.
J Artif Organs ; 23(4): 358-364, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32656667

RESUMO

The cultivation of cells on microcarriers (MCs) in stirred suspension system is useful method for the large-scale culture of human mesenchymal stem cells (hMSCs) for allogenic transplantation. To harvest hMSCs from MCs without using proteolytic enzyme treatment but by lowering temperature, polystyrene MCs coated with a copolymer called CAT having zwitterionic and thermoresponsive characteristics, which has a lower critical solution temperature (LCST) in the range of 28-32 â„ƒ, were developed and compared with those coated with poly(N-isopropylacrylamide) (PNIPAM), which has an LCST almost the same as that of the CAT copolymer. A preliminary study using polystyrene dishes coated with the CAT copolymer at various densities showed superior adhesion efficiency and cell growth compared with those coated with PNIPAM; however, the rate of cell recovery by lowering the temperature to 24 â„ƒ was only about 80% in both cases. Although cells grew on polystyrene MCs coated with PNIPAM (0.64-16 µg/cm2) and on those coated with CAT (0.0050-1.0 µg/cm2), the cell recovery rate at 24 â„ƒ was lower than 20%. The decrease in recovery temperature from 24 to 4 â„ƒ resulted in about 50% cell recovery from CAT-coated (0.010-0.10 µg/cm2) MC, whereas the rate of cell recovery from PNIPAM-coated MC remained at about 20%. CAT (0.20 µg/cm2) coating after treatment of polystyrene MCs with oxygen plasma discharge increased the cell recovery rate to 72% at 4 â„ƒ. Consequently, the combination of oxygen plasma discharge treatment and CAT coating of polystyrene MCs might provide not only adhesion efficiency and growth of MSCs comparable to those on polystyrene MCs without any treatment but also a high cell recover rate of more than 70%.


Assuntos
Técnicas de Cultura de Células , Proliferação de Células/fisiologia , Células-Tronco Mesenquimais/citologia , Resinas Acrílicas , Humanos , Polímeros , Temperatura
4.
Surg Today ; 49(11): 958-964, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31098758

RESUMO

PURPOSE: Although various artificial tracheas have been developed, none have proven satisfactory for clinical use. In-body tissue architecture (IBTA) has enabled us to produce collagenous tissues with a wide range of shapes and sizes to meet the needs of individual recipients. In the present study, we investigated the long-term outcomes of patch tracheoplasty using an IBTA-induced collagenous tissue membrane ("biosheet") in a beagle model. METHODS: Nine adult female beagles were used. Biosheets were prepared by embedding cylindrical molds assembled with a silicone rod and a slitting pipe into dorsal subcutaneous pouches for 2 months. The sheets were then implanted by patch tracheoplasty. An endoscopic evaluation was performed after 1, 3, or 12 months. The implanted biosheets were harvested for a histological evaluation at the same time points. RESULTS: All animals survived the study. At 1 month after tracheoplasty, the anastomotic parts and internal surface of the biosheets were smooth with ciliated columnar epithelium, which regenerated into the internal surface of the biosheet. The chronological spread of chondrocytes into the biosheet was observed at 3 and 12 months. CONCLUSIONS: Biosheets showed excellent performance as a scaffold for trachea regeneration with complete luminal epithelium and partial chondrocytes in a 1-year beagle implantation model of patch tracheoplasty.


Assuntos
Materiais Biocompatíveis , Membranas Artificiais , Procedimentos Cirúrgicos Reconstrutivos/métodos , Engenharia Tecidual , Traqueia/cirurgia , Estenose Traqueal/cirurgia , Animais , Modelos Animais de Doenças , Cães , Feminino , Fatores de Tempo , Resultado do Tratamento
5.
Cytotechnology ; 71(3): 743-750, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31123912

RESUMO

To obtain a large number of human mesenchymal stem cell (hMSCs) for allograft, nonwoven fabrics (NWF) were used as a cell culture scaffold. NWF are three-dimensional fiber aggregates formed by heat bonding and have a high surface area for cell adhesion and elongation. Inoculation hMSC was done to a center of NWF disc (diameter, 15.1 mm; depth, 0.1 mm). A cell suspension inoculum had a volume of 10 µL, which was close to the void volume of the disc, and resulted in a high initial (24 h) cell adhesion efficiency. Use of green fluorescent protein expressing rat MSCs and fluorescence microscopy revealed that adding an additional 10 µL of medium at 0-2 h after the cell inoculation made the initial horizontal distribution of cells in the NWF disc more uniform. Addition of 10 µL of the medium after 1 and 2 h of hMSC inoculation (0.15 × 103 cells/cm2 NWF-fiber) markedly increased the final cell density (21 days) from 2.48 to 7.45 × 103 cells/cm2 NWF-fiber and fold increase in cell density by 16-48-fold. In conclusion, the addition of an additional medium after inoculation made the initial cells distribution in NWF more uniform, which might result in higher final cell density.

6.
Biomaterials ; 185: 232-239, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30248647

RESUMO

In-body tissue architecture (iBTA), a cell-free, in vivo tissue engineering technology that can produce autologous implantable tissues of the desired shape by subcutaneously embedding specially designed molds, was used to develop long tubular collagenous tissues called Biotubes. Spiral molds for long Biotubes were assembled with an outer pipe-shaped spiral shell and an inner spiral mandrel, and embedded into subcutaneous pouches of beagle dogs or goats for 1 or 2 months. Tubular collagenous tissues were formed at the space between the shell and the mandrel of the mold. Depending on the spiral turn number in the mold, Biotubes of 25 cm or 50 cm (internal diameter 4 mm or 5 mm) were prepared with nearly homogeneous mechanical and histological properties over their entire length. Biotubes stored in 70% ethanol were allogenically implanted into beagle dogs or goats to evaluate their in vivo performance. The 25-cm Biotubes functioned as arterial grafts with no need for luminal modification or mechanical support, and demonstrated vascular reconstruction within 3 months after implantation into dogs. The 50-cm Biotubes functioned as arteriovenous shunt grafts in the neck region of goats without thrombus formation and vascular deformation for 1 month. Thus, the world's longest tissue-engineered vascular grafts with small diameter could be developed using iBTA.


Assuntos
Prótese Vascular , Animais , Bioprótese/efeitos adversos , Prótese Vascular/efeitos adversos , Artérias Carótidas/cirurgia , Artérias Carótidas/ultraestrutura , Cães , Desenho de Equipamento , Feminino , Cabras , Engenharia Tecidual/métodos
7.
Biophys Physicobiol ; 15: 165-172, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30250775

RESUMO

The replica-exchange molecular dynamics (REMD) method has been used for conformational sampling of various biomolecular systems. To maximize sampling efficiency, some adjustable parameters must be optimized. Although it is agreed that shorter intervals between the replica-exchange attempts enhance traversals in the temperature space, details regarding the artifacts caused by these short intervals are controversial. In this study, we revisit this problem by performing REMD simulations on an alanine octapeptide in an implicit solvent. Fifty different sets of conditions, which are a combination of five replica-exchange periods, five different numbers of replicas, and two thermostat coupling time constants, were investigated. As a result, although short replica-exchange intervals enhanced the traversals in the temperature space, they led to artifacts in the ensemble average of the temperature, potential energy, and helix content. With extremely short replica-exchange intervals, i.e., attempted at every time step, the ensemble average of the temperature deviated from the thermostat temperature by ca. 7 K. Differences in the ensembles were observed even for larger replica-exchange intervals (between 100 and 1,000 steps). In addition, the shorter thermostat coupling time constant reduced the artifacts found when short replica-exchange intervals were used, implying that these artifacts are caused by insufficient thermal relaxation between the replica-exchange events. Our results will be useful to reduce the artifacts found in REMD simulations by adjusting some key parameters.

8.
PeerJ ; 6: e4769, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29780670

RESUMO

Elucidating the molecular mechanism of helix-coil transitions of short peptides is a long-standing conundrum in physical chemistry. Although the helix-coil transitions of poly-glutamic acid (PGA) have been extensively studied, the molecular details of its unfolding process still remain unclear. We performed all-atom canonical molecular dynamics simulations for a 20-residue PGA, over a total of 19 µs, in order to investigate its helix-unfolding processes in atomic resolution. Among the 28 simulations, starting with the α-helical conformation, all showed an unfolding process triggered by the unwinding of terminal residues, rather than by kinking and unwinding of the middle region of the chain. The helix-coil-helix conformation which is speculated by the previous experiments was not observed. Upon comparison between the N- and C-termini, the latter tended to be unstable and easily unfolded. While the probabilities of helix elongation were almost the same among the N-terminal, middle, and C-terminal regions of the chain, unwinding of the helix was enriched at the C-terminal region. The turn and 310-helix conformations were kinetic intermediates in the formation and deformation of α-helix, consistent with the previous computational studies for Ala-based peptides.

9.
Eur J Vasc Endovasc Surg ; 55(6): 882-887, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29661648

RESUMO

OBJECTIVE: The world's smallest calibre "microbiotube" vascular graft was recently developed, with an inner diameter of 0.6 mm. It was formed using in-body tissue architecture (iBTA) and has a high degree of patency and capacity for regeneration in the acute phase, 1 month after implantation. This consecutive study investigated the compatibility and stability of microbiotubes in the chronic phase of implantation for 12 months for potential application in microsurgery. METHODS: This was an in vivo experimental study. The microbiotubes were prepared by embedding the mould subcutaneously in rats for 2 months. Allogenic microbiotubes (n = 16) were implanted into the bilateral femoral arteries (inner diameter 0.5 mm) of eight Wistar rats in an end to end anastomosis manner for 12 months. Follow up 7-Tesla magnetic resonance angiograms were performed every 3 months. Histological observation was performed 12 months after implantation. RESULTS: All patent grafts (n = 12, patency 75%) one month after implantation maintained their patency up to 12 months without any abnormal morphological changes or calcification. Histological observation at 12 months showed that layered α-smooth muscle actin positive cells with a monolayer luminal covering of endothelial cells had formed from the proximal to the distal anastomoses. A thin elastic fibre layer formed in the luminal area. After implantation, all components of the microbiotube were similar to those of a native artery. CONCLUSIONS: This study suggests that microbiotubes have high compatibility, stability, and durability as replacement grafts over the short to mid-term period.


Assuntos
Prótese Vascular , Engenharia Tecidual , Animais , Materiais Biocompatíveis/farmacologia , Implante de Prótese Vascular/métodos , Artéria Femoral/fisiologia , Artéria Femoral/cirurgia , Sobrevivência de Enxerto , Angiografia por Ressonância Magnética , Masculino , Microcirurgia/métodos , Microvasos/fisiologia , Microvasos/cirurgia , Desenho de Prótese , Ratos Wistar , Transplante Autólogo , Grau de Desobstrução Vascular/fisiologia
10.
ASAIO J ; 64(3): 395-405, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29517511

RESUMO

Tissue-engineered heart valves (TEHVs) are expected to be viable grafts. However, it is unknown whether they transit their histological structure after implantation. We developed a novel autologous TEHV (named stent biovalve) for transcatheter implantation, using in-body tissue engineering based on a tissue encapsulation phenomenon. In this study, a time-course histological transition of implanted biovalves was investigated in goats. Three types of stent biovalves were prepared by 2 month embedding of plastic molds mounted with metallic stents, in the subcutaneous spaces. After extracting the molds with tissue and removing the molds only, stent biovalves were constituted entirely from the connective tissues. Stent biovalves were implanted in the aortic or pulmonary valve position of other goats with transcatheter technique. In each animal, the stent biovalve was explanted at 1 month step (from 1 to 6 months) or as long as possible. Total 12 goats (five for aortic and seven for pulmonary) were successfully implanted. The maximum duration became 19 months as a result. Even then the leaflets of the biovalves kept their shape and elasticity, and neither calcification nor thrombi were observed in any cases and duration. Histology showed the recipients' cells covering the laminar surface of the leaflets like the endothelium even after 1 month. The cells have also migrated in the leaflets gradually and finally constructed characteristic 3 layered tissues like native leaflets. Implanted stent biovalves can adapt their histological structure to the environment. They have a potential as viable grafts keeping better function and biocompatibility.


Assuntos
Próteses Valvulares Cardíacas , Desenho de Prótese , Engenharia Tecidual , Animais , Cabras , Implante de Prótese de Valva Cardíaca , Valva Pulmonar
11.
J Biomed Mater Res B Appl Biomater ; 105(5): 1009-1015, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-26892839

RESUMO

We have developed inducible cell self-organization through weakly positively charged culture surfaces. In this study, a thermoresponsive and zwitterionic copolymer comprised of N,N-dimethylaminoethyl methacrylate (DMAEMA) and methacrylic acid (MA) (PDMAEMA-co-PMA; Mn: ∼9.7 × 104 g/mol; PDMAEMA/PMA ratio: 10) was designed for inducing cell self-organization. The copolymer formed single polymer-derived polyion complex (sPIC) nanoparticles following dissolution in an aqueous solution. The sPIC nanoparticles had a positive charge (ca. 25 mV). Self-organization occurred in adipose-derived vascular stromal cell monolayers cultivated on sPIC-deposited surfaces. There were dramatic morphological changes of these cells with the formation of capillary-like networks and single-cell aggregates with little cytotoxicity. This was a significant improvement compared with cells grown on previously developed surfaces deposited with PIC, a mixture of PDMAEMA and plasmid DNA. Thus, sPICs of PDMAEMA-co-PMA may allow for the accurate evaluation of a variety of cell behaviors with less cytotoxicity, and may facilitate additional potential medical applications such as cell-based therapy and drug discovery. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1009-1015, 2017.


Assuntos
Tecido Adiposo/metabolismo , Teste de Materiais , Nanopartículas/química , Ácidos Polimetacrílicos , Células-Tronco/metabolismo , Tecido Adiposo/citologia , Animais , Ácidos Polimetacrílicos/síntese química , Ácidos Polimetacrílicos/química , Ratos , Células-Tronco/citologia
12.
J Tissue Eng Regen Med ; 10(10): E518-E526, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-24668614

RESUMO

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.


Assuntos
Córnea/metabolismo , Córnea/cirurgia , Fibroblastos/metabolismo , Membranas Artificiais , Engenharia Tecidual , Animais , Coelhos
13.
J Biomed Mater Res A ; 104(1): 305-12, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26386244

RESUMO

Three-dimensional cell spheroids prepared without using any artificial scaffold materials are desirable for cell-based transplants. However, conventional cell culture systems are inefficient for rapid, large-scale and non-cytotoxic generation of size-controlled spheroids (>1 mm diameter) that are required for tissue regenerative therapy application. In this study, we prepared millimeter-order spheroids of adipose-derived mesenchymal stem cells (ADSCs) by controlling the spheroid size (diameter range: 0.4-2.5 mm). Notably, spheroid generation required only one day of culture on charged culture dishes. Almost all spheroid-derived ADSCs were viable and produced adhesion molecules and growth factors, which play an important role in tissue regeneration. Moreover, spheroid-derived ADSCs could infiltrate and recellularize collagenous tissue membranes in vitro. The ADSC spheroids developed in this study could be directly (without additional processing) used for cell-based tissue regeneration therapy. Furthermore, the rapid scale-up process and noncytotoxic generation of spheroids would also enable other applications such as use as screening models for drug discovery.


Assuntos
Tecido Adiposo/citologia , Tamanho Celular , Células-Tronco Mesenquimais/citologia , Esferoides Celulares/citologia , Animais , Apoptose , Marcação In Situ das Extremidades Cortadas , Ratos Endogâmicos Lew
14.
J Artif Organs ; 18(4): 322-9, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26130007

RESUMO

Biotubes, prepared using in-body tissue architecture (IBTA) technology, have adequate mechanical properties and excellent biocompatibility for vascular grafts. However, they have thin walls, lack vascular constructing cells, and are composed of subcutaneous connective tissues consisting mainly of collagen and fibroblasts. This study aimed to prepare Biotubes with a vascular-like structure including an endothelial cell lining and a smooth muscle cell by IBTA using adipose-derived vascular stromal cell (ADSCs)-exuding specially designed multiporous tubes (outer diameter 5 mm, length 24 mm, pore size 500 µm, pore number 180, cell number/tube >3.0 × 10(6)). ADSCs were separated from rat subcutaneous fat, suspended in a Matrigel™ solution at 4 °C, and then filled into the tubes. After the tubes were embedded into dorsal subcutaneous pouches of the same rats for 2 weeks, robust Biotubes with a wall thickness of >600 µm were formed surrounding the tubes. The luminal layer of the obtained Biotubes was dominated by the cells positive for an endothelial marker. Almost the entire intima, with a thickness of about 400 µm, was occupied with cells positive for a smooth muscle marker. Both cells were derived from ADSCs. Biotube walls were constructed by fusing ADSC-derived vascular constructing cells exuded from the tubes and fibroblasts and collagen from the surrounding connective tissue. A robust Biotubes with vascular cells component, were formed after only 2 weeks of subcutaneous incubation of ADSCs-exuding multiporous tubes.


Assuntos
Bioprótese , Prótese Vascular , Engenharia Tecidual/instrumentação , Tecidos Suporte , Tecido Adiposo/citologia , Animais , Endotélio Vascular , Ratos , Ratos Endogâmicos Lew , Ratos Wistar , Células Estromais , Enxerto Vascular
15.
J Vet Cardiol ; 17(1): 54-61, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25697493

RESUMO

OBJECTIVES: To evaluate the functionality of an autologous heart valve with stent (Stent-biovalve or SBV) after implantation in the pulmonic valve position in beagle dogs. ANIMALS: Five beagle dogs. METHODS: A mold with an aperture of a tri-leaflet structure was constructed from a pair of concave and convex rods to which a nitinol (NiTi) stent was mounted. This mold was embedded in a dorsal subcutaneous pouch in beagle dogs for 4 weeks. At the time of the removal, the surfaces of the molds were completely covered with connective tissues, tri-leaflet valves were formed and the NiTi stent was tightly connected to the structure. RESULTS: The mean burst strength of the SBV leaflet was 2710 mmHg (range 2280-3116 mmHg), which was approximately equal to that of the native pulmonic valve leaflet. After implantation in the pulmonary position, the SBV showed good functionality as a pulmonic valve. At 84 days after implantation, the SBV was replaced with autologous fibroblasts and collagenous tissues, and showed organization similar to that of native heart valves. CONCLUSION: Stent-Biovalves achieved good valvular function with laminar flow in the pulmonic valve position of beagle dogs.


Assuntos
Bioengenharia/métodos , Bioprótese , Cães , Teste de Materiais , Stents/veterinária , Animais , Fenômenos Biomecânicos , Tecido Conjuntivo
16.
J Artif Organs ; 18(1): 48-54, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25320016

RESUMO

The use of stent grafts for endovascular aortic repair has become an important treatment option for aortic aneurysms requiring surgery. This treatment has achieved excellent outcomes; however, problems like type 1 endoleaks and stent graft migration remain. Bio stent grafts (BSGs), which are self-expanding stents covered with connective tissue, were previously developed using "in-body tissue architecture" technology. We assessed their early adaptation to the aorta after transcatheter implantation in a beagle model. BSGs were prepared by subcutaneous embedding of acryl rods mounted with self-expanding nitinol stents in three beagles for 4 weeks (n = 3/dog). The BSGs were implanted as allografts into infrarenal abdominal aortas via the femoral artery of three other beagles. After 1 month of implantation, aortography revealed no stenosis or aneurysmal changes. The luminal surface of the BSGs was completely covered with neointimal tissue, including endothelialization, without any thrombus formation. The cover tissue could fuse the luminal surface of the native aorta with tight conjunctions even at both ends of the stents, resulting in complete impregnation of the strut into the reconstructed vascular wall, which is expected to prevent endoleaks and migration in clinical applications.


Assuntos
Aorta Abdominal/cirurgia , Implante de Prótese Vascular/métodos , Prótese Vascular , Stents , Engenharia Tecidual , Animais , Cães
17.
Biomaterials ; 34(36): 9096-102, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24008038

RESUMO

Surface characteristics of biomaterials such as wettability, rigidity, roughness, and electrical charge affect the fate of transplanted cells such as progenitor cells or stem cells for use in regenerative medicine. Of these, the effects of surface electrical charges on cellular behaviour such as adhesion, proliferation, and differentiation are not well understood. We prepared precisely charged culture surfaces ranging from -28 mV to +21 mV, simply by surface deposition of polyion complex nanoparticles prepared by mixing a positively charged thermoresponsive homopolymer, poly(N,N-dimethylaminoethyl methacrylate), with negatively charged plasmid DNA at various charge ratios. Drastic morphological changes of adipose-derived vascular progenitor cells were generated on the positively charged surface of organized forms at +19 mV. Capillary-like networks or single aggregates of these cells were selectively created depending on cell seeding density. Our findings offer new insights that may aid develop stem cell-processing techniques for use in regenerative medicine.


Assuntos
Tecido Adiposo/citologia , Eletricidade , Íons/farmacologia , Nanopartículas/química , Células-Tronco/citologia , Animais , Contagem de Células , Forma Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Microscopia de Força Atômica , Microscopia de Fluorescência , Tamanho da Partícula , Ratos , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Propriedades de Superfície , Fator A de Crescimento do Endotélio Vascular/metabolismo
18.
Bioconjug Chem ; 24(2): 159-66, 2013 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-23360504

RESUMO

We have developed a substrate-mediated transfection method called "deposition transfection technology" using a poly(N,N-dimethylaminoethylmethacrylate) (PDMAEMA) homopolymer with both thermoresponsive and cationic characteristics. In this study, we enhanced deposition transfection efficiency by using tris(hydroxymethyl)aminomethane (Tris buffer) as a pH adjuster for transfection solution composed of PDMAEMA and plasmid DNA (pDNA). PDMAEMA with a molecular weight of 9.7 × 10(4) g mol(-1) was synthesized by photoinduced radical polymerization. The pH of PDMAEMA solution was increased gradually in the range from 8 to 11 by the addition of Tris, and then the solubility of PDMAEMA was significantly decreased and the dissolution time was extended from 15 to 40 min at Tris/PDMAEMA ratio of 1 and higher. On the other hand, while the polyion complexes (polyplexes) were formed by mixing PDMAEMA with luciferase-encoding plasmid DNA even under an excess amount of Tris at Tris/PDMAEMA ratio of 8, the binding affinity between PDMAEMA and pDNA was decreased with increasing Tris at Tris/PDMAEMA ratio of 2 and higher. When HeLa cells, smooth muscle cells, and cardiac fibroblasts were transfected by the deposition method using polyplex solution containing various amounts of Tris, the transgene expression dramatically increased at a Tris/PDMAEMA ratio of 2 in all cell types, which were more than 150-fold in HeLa cells, 40-fold in smooth muscle cells, and 30-fold in cardiac fibroblasts compared to those in the Tris-free condition. In addition, the enhanced transgene expression by Tris was sustained for over 10 days post-transfection as well as that observed in Tris-free condition. Thus, deposition transfection efficiency can be dramatically enhanced by using Tris buffer as a pH adjuster for polyplex solution.


Assuntos
DNA/administração & dosagem , Metacrilatos/metabolismo , Nylons/metabolismo , Plasmídeos/administração & dosagem , Transfecção/métodos , Trometamina/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Cães , Células HeLa , Humanos , Metacrilatos/química , Nylons/química , Trometamina/química
19.
Bioconjug Chem ; 23(4): 751-7, 2012 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-22435888

RESUMO

A poly(N,N-dimethylaminoethylmethacrylate) (PDMAEMA) homopolymer with both thermoresponsive and cationic characteristics was applied to a vector for use in deposition transfection. PDMAEMA with a molecular weight of 2.5 × 10(5) g mol(-1) was synthesized by photoinduced radical polymerization. Polyplexes approximately 750 nm in size were formed by mixing PDMAEMA with luciferase-encoding plasmid DNA. The polyplexes had a lower critical solution temperature (LCST) of approximately 30 °C. In addition, they exhibited excellent adsorption and durability on a polystyrene surface, as confirmed by a surface chemical compositional analysis. When HeLa cells and primary cells were cultured on a substrate coated with the polyplexes, high transgene expression and cell viability of more than 90% were obtained at low charge ratios (PDMAEMA/plasmid DNA ratio) ranging from 2 to 8. In addition, transgene expression was sustained for over 2 weeks post-transfection whereas decreased expression was observed 5 days post-transfection when the conventional solution-mediated transfection method was used. Thus, high and sustained transgene expression as well as high cell viability can be realized by using small amounts of PDMAEMA as a deposition transfection material.


Assuntos
DNA/química , DNA/genética , Portadores de Fármacos/química , Metacrilatos/química , Nylons/química , Tensoativos/química , Temperatura , Transfecção/métodos , Animais , Sobrevivência Celular/efeitos dos fármacos , Cães , Portadores de Fármacos/toxicidade , Células HeLa , Humanos , Metacrilatos/toxicidade , Nylons/toxicidade , Tensoativos/toxicidade
20.
J Biosci Bioeng ; 111(3): 357-64, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21126909

RESUMO

An ex vivo cartilage defect model for the evaluation of cartilage regeneration using mesenchymal stem cells (MSCs) was developed. Porcine chondrocytes and human MSCs were transplanted into cartilage defects created on the porcine osteochondral and chondral discs and cultivated for 3 weeks. Although the regeneration of cartilage-like tissues was observed after the transplantation of chondrocytes to defects on both of the osteochondral and chondral discs, the transplanted MSCs formed cartilage-like tissues only in the defect on the chondral disc, in which an in vivo cartilage-like structure was partly observed, and a degraded tissue was observed in the defect on the osteochondral disc. The effects of medium additives such as serum, transforming growth factor-ß3 (TGF-ß3), and fibroblast growth factor-2, and the transfection of TGF-ß3 gene to MSCs could be clearly estimated using the cartilage defect model. In conclusion, a chondral disc with defects is useful for evaluating cartilage regeneration using MSCs.


Assuntos
Cartilagem/citologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Regeneração , Engenharia Tecidual , Adulto , Animais , Células Cultivadas , Condrócitos/citologia , Meios de Cultura , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Suínos , Transfecção , Fator de Crescimento Transformador beta3/genética , Fator de Crescimento Transformador beta3/metabolismo
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