Nagata Microtia and Reconstructive Plastic Surgery Clinic

Shimotoda, Japan

Nagata Microtia and Reconstructive Plastic Surgery Clinic

Shimotoda, Japan
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Ko E.C.,University of Tokyo | Ko E.C.,Kaohsiung Medical University | Fujihara Y.,University of Tokyo | Ogasawara T.,University of Tokyo | And 5 more authors.
Tissue Engineering - Part C: Methods | Year: 2012

When the chondrocytes are isolated from the native cartilage and proliferate in vitro, they soon lose their original ability to express glycosaminoglycan (GAG) and type II collagen, which is termed dedifferentiation, or decrease cell viability. We first examined in vitro cartilage regeneration of tissue-engineered pellets that consisted of human auricular chondrocytes and atelocollagen and that were incubated in vitro under stimulation with bone morphogenetic protein-2 (BMP-2), insulin, and T3. We then examined the administration of those growth factors into the scaffold or in the medium and explored the possibility that the atelocollagen, the hydrogel scaffold of the chondrocytes, may function for drug delivery of the factors. BMP-2 in the atelocollagen with the supplement of insulin and T3 in the medium could not only produce a greater GAG matrix in a shorter period but also sustain cell viability with lower mortality. The insulin in the medium could be better administered only for 2 weeks, rather than 3 weeks, which would save time and cost, hence shortening the in vitro culture of chondrocytes. Our protocol of mixing BMP-2 into the atelocollagen with the supplement of insulin and T3 hormone might provide a new insight into the development of tissue engineering in chondrogenesis. © 2012, Mary Ann Liebert, Inc.


Tanaka Y.,University of Tokyo | Saijo Y.,Tohoku University | Fujihara Y.,University of Tokyo | Yamaoka H.,University of Tokyo | And 6 more authors.
Journal of Bioscience and Bioengineering | Year: 2012

The tissue-engineered cartilages after implantation were nonuniform tissues which were mingling with biodegradable polymers, regeneration cartilage and others. It is a hard task to evaluate the biodegradation of polymers or the maturation of regenerated tissues in the transplants by the conventional examination. Otherwise, scanning acoustic microscopy (SAM) system specially developed to measure the tissue acoustic properties at a microscopic level. In this study, we examined acoustic properties of the tissue-engineered cartilage using SAM, and discuss the usefulness of this devise in the field of tissue engineering. We administered chondrocytes/atelocollagen mixture into the scaffolds of various polymers, and transplanted the constructs in the subcutaneous areas of nude mice for 2. months. We harvested them and examined the sound speed and the attenuation in the section of each construct by the SAM. As the results, images mapping the sound speed exhibited homogenous patterns mainly colored in blue, in all the tissue-engineered cartilage constructs. Contrarily, the images of the attenuation by SAM showed the variation of color ranged between blue and red. The low attenuation area colored in red, which meant hard materials, were corresponding to the polymer remnant in the toluidine blue images. The localizations of blue were almost similar with the metachromatic areas in the histology. In conclusion, the SAM is regarded as a useful tool to provide the information on acoustic properties and their localizations in the transplants that consist of heterogeneous tissues with various components. © 2011 The Society for Biotechnology, Japan.


Iwata K.,University of Tokyo | Asawa Y.,University of Tokyo | Fujihara Y.,University of Tokyo | Tanaka Y.,University of Tokyo | And 5 more authors.
Current Aging Science | Year: 2010

In cartilage regenerative medicine, which is highly expected in the face of our aging society, insulin is the potent factor for culture media. To secure the safety of culture media, we attempted to use medical insulin formulations, and compared their effects on human articular or auricular chondrocytes between regular human insulin (R) and neutral protamine hagedorn insulin (N). In monolayer culture with the media containing either R or N, the cell growth reached approximately 15-fold-increase in 6 days, which showed no significant difference between them. These cells showed the equivalent ability to produce cartilage matrices, both in vitro and in vivo. Also, in the 3D culture of the dedifferentiated chondrocytes' either R or N increased gene expression of type II collagen at 3-4 folds in the combination with other growth factors, compared with basal medium, while insulin could similarly enhance both the redifferentiation and cartilage maturation. The in vitro half-life of each insulin in the presence of chondrocytes neither decreased within 3 days, suggesting little degradation in the culture media, unlike in the body. Although both R and N showed similar biological effects on cultured chondrocytes, we may choose the R for clinical practice because of its pure composition. © 2010 Bentham Science Publishers Ltd.


Ko E.C.,University of Tokyo | Fujihara Y.,University of Tokyo | Ogasawara T.,University of Tokyo | Asawa Y.,University of Tokyo | And 4 more authors.
Journal of Biomedical Materials Research - Part A | Year: 2011

Three-dimensional culture of the tissue-engineered cartilage constructs may increase the matrix production, but central necrosis must occur if the construct becomes large. To increase the cell viability in the middle part of constructs and to enhance the in vivo cartilage regeneration, we attempted to administer the insulin into the scaffold. Insulin is known to strongly enhance the matrix production in the chondrocytes. The pellets of human auricular chondrocytes with atelocollagen hydrogel were 3D-cultured in the medium. The comparison among three groups (insulin mixed in the atelocollagen, insulin added to the medium, and control group, i.e.; insulin in neither atelocollagen nor medium) revealed that both insulin mixed in the atelocollagen and that in the medium could effectively promoted the cell viability and matrix synthesis of the chondrocytes. The daily assay also showed the gradual release of insulin from the atelocollagen hydrogel, suggesting that this material may work as a control release of insulin. We actually transplanted the poly-L-lactide porous scaffolds carrying the chondrocytes and the atelocollagen mixed with or without insulin, into the nude mice, showing that glycosaminoglycan accumulation was evident in the group with insulin although less without insulin. We thus showed the possibility to enhance the in vivo cartilage regeneration, when administered insulin into the atelocollagen hydrogel. © 2011 Wiley Periodicals, Inc.


Yamaoka H.,University of Tokyo | Yamaoka H.,Teikyo University | Nishizawa S.,University of Tokyo | Nishizawa S.,Teikyo University | And 7 more authors.
Cell Proliferation | Year: 2010

Objective: Chondrocytes inevitably decrease production of cartilaginous matrices during long-term cultures with repeated passaging; this is termed dedifferentiation. To learn more concerning prevention of dedifferentiation, we have focused here on the fibroblast growth factor (FGF) family that influences chondrocyte proliferation or differentiation. Materials and methods: We have compared gene expression between differentiated cells in passage 3 (P3) and dedifferentiated ones in P8 of human cultured chondrocytes. We also performed ligand administration of the responsive factor or its gene silencing, using small interfering RNA (siRNA). Results: FGFs 1, 5, 10, 13 and 18 were higher at P8 compared to P3, while FGFs 9 and 14 were lower. Especially, FGF18 showed a 10-fold increase by P8. Ligand administration of FGF18 in the P3 cells, or its gene silencing using siRNA in the P8 cells, revealed dose-dependent increase and decrease respectively in type II collagen/type I collagen ratio. Exogenous FGF18 also upregulated expression of transforming growth factor beta (TGF-β), the anabolic factor of chondrocytes, in P3 chondrocytes, but P8 cells maintained a low level of TGF-β expression, suggesting a decrease in responsiveness of TGF-β to FGF18 stimulation in the dedifferentiated chondrocytes. Conclusion: FGF18 seems to play a role in maintenance of chondrocyte properties, although its expression was rather high in dedifferentiated chondrocytes. Upregulation of FGF18 in dedifferentiated chondrocytes implied that it may be a marker of dedifferentiation. © 2009 Blackwell Publishing Ltd.


Iwata K.,University of Tokyo | Asawa Y.,University of Tokyo | Nishizawa S.,University of Tokyo | Mori Y.,University of Tokyo | And 3 more authors.
Biomaterials | Year: 2012

To promote clinical application of cartilage tissue engineering, we should establish a serum-free chondrocyte growth medium. The serum-free medium would increase the cell numbers by more than 20-fold within one week, which proliferation ability almost matches that of serum-based one. For that, we examined the combinations of growth factors and the methods to enhance their effects by making use of the interaction with biomaterials. From various growth factors that are contained within the serum, we made the cocktail of FGF-2 (100 ng/mL), insulin (5 μg/mL), EGF (10 pg/mL), PDGF (625 pg/mL) and TGF-β (5 pg/mL), which increased the chondrocyte numbers by approximately 3-fold for 7 days. Moreover, we used the biomaterials including albumin and hyaluronan as the carrier of those factors. By direct mixing of those factors with biomaterials before the administration to the medium, the medium containing those mixture showed the chondrocyte growth of approximately a 25-fold increase by day 10. In this medium, the FGF-2 or insulin concentration hardly decreased, and rather enhanced the activation of ERK. Due to the optimal usage of biomaterials, this serum-free medium will realize a constant harvest of chondrocytes and could contribute to the safety and quality in regenerative medicine. © 2011 Elsevier Ltd.


Yonenaga K.,University of Tokyo | Nishizawa S.,University of Tokyo | Fujihara Y.,University of Tokyo | Asawa Y.,University of Tokyo | And 4 more authors.
Tissue Engineering - Part C: Methods | Year: 2010

To optimize the chondrocyte numbers obtained after collagenase digestion for cartilage tissue engineering, we examined the enzyme concentration and incubation time for collagenase digestion. The appropriate cell density in the chondrocyte primary culture was also verified. The collagenase digestion conditions that maximized the viable cell numbers were 24h in 0.15% and 0.3% collagenase, 6h in 0.6%, and 4h in 1.2%, leading to ∼5×105 cells from 0.05g. When seeded at 10,000 cells/cm2, all of these cells became almost confluent within 1 week. Cells digested in 0.3% for 24h or 0.6% for 6h and seeded at 3000 cells/cm2 may also be acceptable, and similarly reached confluence within 1 week. Results regarding expression of the p53, tumor necrosis factor-α, and interleukin-1β genes, as well as apoptosis enzyme-linked immunosorbent assay results, show that excessive collagenase exposure may decrease chondrocyte viability or activity. We recommend a 24-h incubation in 0.3% collagenase or 6h in 0.6% collagenase, and a cell-seeding density of 3000-10,000 cells/cm2. These conditions maximize the harvest of isolated chondrocytes from a small amount of biopsied tissue and significantly aid in obtaining a large quantity of cultured cells in a short period. © Copyright 2010, Mary Ann Liebert, Inc.


Tanaka Y.,University of Tokyo | Yamaoka H.,University of Tokyo | Nishizawa S.,University of Tokyo | Nagata S.,Nagata Microtia and Reconstructive Plastic Surgery Clinic | And 5 more authors.
Biomaterials | Year: 2010

To broaden the clinical application of cartilage regenerative medicine, we should develop an implant-type tissue-engineered cartilage with firmness and 3-D structure. For that, we attempted to use a porous biodegradable polymer scaffold in the combination with atelocollagen hydrogel, and optimized the structure and composition of porous scaffold. We administered chondrocytes/atelocollagen mixture into the scaffolds with various kinds of porosities (80-95%) and pore sizes (0.3-2.0 mm), consisting of PLLA or related polymers (PDLA, PLA/CL and PLGA), and transplanted the constructs in the subcutaneous areas of nude mice. The constructs using scaffolds of excessively large pore sizes (>1 mm) broke out on the skin and impaired the host tissue. The scaffold with the porosity of 95% and pore size of 0.3 mm could effectively retain the cells/gel mixture and indicated a fair cartilage regeneration. Regarding the composition, the tissue-engineered cartilage was superior in PLGA and PLLA to that in PLA/CA and PDLA. The latter two showed the dense accumulation of macrophages, which may deteriorate the cartilage regeneration. Although PLGA or PLLA has been currently recommended for the scaffold of cartilage, the polymer for which biodegradation was exactly synchronized to the cartilage regeneration would improve the quality of the tissue-engineered cartilage. © 2010 Elsevier Ltd.


Kanazawa S.,University of Tokyo | Fujihara Y.,University of Tokyo | Sakamoto T.,University of Tokyo | Asawa Y.,University of Tokyo | And 4 more authors.
Journal of Tissue Engineering and Regenerative Medicine | Year: 2013

To disclose the influence of foreign body responses raised against a non-absorbable hydrogel consisting of tissue-engineered cartilage, we embedded human/canine chondrocytes within agarose and transplanted them into subcutaneous pockets in nude mice and donor beagles. One month after transplantation, cartilage formation was observed in the experiments using human chondrocytes in nude mice. No significant invasion of blood cells was noted in the areas where the cartilage was newly formed. Around the tissue-engineered cartilage, agarose fragments, a dense fibrous connective tissue and many macrophages were observed. On the other hand, no cartilage tissue was detected in the autologous transplantation of canine chondrocytes. Few surviving chondrocytes were observed in the agarose and no accumulation of blood cells was observed in the inner parts of the transplants. Localizations of IgG and complements were noted in areas of agarose, and also in the devitalized cells embedded within the agarose. Even if we had inhibited the proximity of the blood cells to the transplanted cells, the survival of the cells could not be secured. We suggest that these cytotoxic mechanisms seem to be associated not only with macrophages but also with soluble factors, including antibodies and complements. © 2011 John Wiley & Sons, Ltd..


Nagata S.,Nagata Microtia and Reconstructive Plastic Surgery Clinic
Japanese Journal of Plastic Surgery | Year: 2011

The number of cases of secondary auricular reconstruction for unfavorable primary auricular reconstruction according to conventional methods has been increasing in the past few years. Secondary auricular reconstruction is needed due to incorrect anatomical location, postoperative hair growth, contracture of full-thickness skin (FTS) grafts utilized to cover the insufficient skin surface area of the conchal vault and for ear elevation, both resulting in an increase of tension, vascular compromise, etc. All these factors are capable of causing postoperative complications such as necrosis, resorption (deformation) and/or protrusion of the cartilage framework. During the first stage operation, the scar tissue and mismatched colored grafted skin are excised and the framework is removed. The subcutaneous pedicle flap is constructed in the posterior surface of the lobule and mastoid surface, and passed to the anterior surface of the 3-dimensional costal cartilage frame (3-D frame) to line the incisura intertragica and cavum concha. The newly fabricated 3-D frame is fixed to its proper anatomical location, covered with the temporoparietal fascia flap (TPF) followed by ultra-delicate split-thickness scalp skin (UDSTS). In the second stage operation, a cartilage block is fabricated for auricular projection and covered with the deep temporal fascia (DTF) followed by UDSTS cover. Elevation of the reconstructed auricle with the skin graft only must be absolutely avoided since it will lead to postoperative complications and/or problems in long-term follow-ups and the results will always be a flat and floppy reconstructed auricle. The tissue expander method must also be avoided, for the same reasons. The scientific and systematic auricular reconstruction method for secondary auricular reconstruction for unfavorable primary results with conventional methods is discussed in detail. What must be avoided?

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