Eun-Kyung Yang

A comparison of lyophilized amniotic membrane with cryopreserved amniotic membrane for the reconstruction of rabbit corneal epithelium

Many researchers have employed cryopreserved amniotic membrane (CAM) in the treatment of a severely damaged cornea, using corneal epithelial cells cultured on an amniotic membrane (AM). In this study, two Teflon rings were made for culturing the cells on the LAM and CAM, and were then used to support the AM, which is referred to in this paper as an Ahn’s AM supporter. The primary corneal epithelial cells were obtained from the limbus, using an explantation method. The corneal epithelium could be reconstructed by culturing the third-passage corneal epithelial cells on the AM. A lyophilized amniotic membrane (LAM) has a higher rate of graft take, a longer shelf life, is easier to store, and safer, due to gamma irradiation, than a CAM. The corneal epithelium reconstructed on the LAM and CAM, supported by the two-Teflon rings, was similar to normal corneal epithelium. However, the advantages of the LAM over that of the CAM make the former more useful. The reconstruction model of the corneal epithelium, using AM, is considered as a goodin vitro model for transplantation of cornel epithelium into patients with a severely damaged cornea.

Assessment of Toxic Potential of Industrial Chemicals Using a Cultured Human Bioartificial Skin Model: Production of Interleukin 1α and Hydroxyeicosatetraenoic Acids

Cytotoxicity assays using artificial skin are proposed as alternative methods for in vitro tests to minimize animals used in ocular and dermal irritation testing. The responses of the artificial skins were studied to a well-characterized chemical irritant, such as toluene, glutaraldehyde and sodium lauryl sulfate (SLS), and a nonirritant, such as polyethylene glycol. The evaluation of irritating and nonirritating test chemicals was also compared with responses seen in human dermal fibroblasts and human epidermal keratinocytes grown in monolayer culture. The responses monitored included the MTT mitochondrial functionality assay. In order to better understand the local mechanisms involved in skin damage and repair, the productions of several mitogenic proinflammatory mediators such as interleukin-1α (IL-1α), 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-HETE were investigated. Dose-dependent increases in the levels of IL-1α and HETEs were observed in the underlying medium of the skin systems exposed to two skin irritants, glutaraldehyde and SLS. The results of the present study show that both human artificial skins can be used as efficient testing models for the evaluation of skin toxicity in vitro and for screening the contact skin irritancy in vitro.

Development of isolation and cultivation method for outer root sheath cells from human hair follicle and construction of bioartificial skin

Obtaining a sufficient amount of healthy keratinocytes from a small tissue is difficult. However, ORS cells can be a good source of epithelium since they are easily obtainable and patients do not have to suffer from scar formation at donor sites. Accordingly, the current study modified the conventional primary culture technique to overcome the low propagation and easy aging of epithelial cells during culturing. In a conventional primary culture, the average yield of human ORS cells is 2.1×103 cells/follicle based on direct incubation in a trypsin (0.1%)/EDTA (0.02%) solution for 15 min at 37°C, however, our modified method was able to obtain about 6.9×103 cells/follicle using a two-step enzyme digestion method involving dispase (1.2 U/mL) and a trypsin (0.1%)/EDTA (0.02%) solution. Thus, the yield of primary cultured ORS cells could be increasd three times higher. Furthermore, a total of 2.0×107 cells was obtained in a serum-free medium, while a modified E-medium with mitomycin C-treated feeder cells produced a total of 6.3×107 cells over 17 days when starting with 7.5×104 cells. Finally, we confirmed the effectiveness of our ORS cell isolation method by presenting their ability for reconstructing the bioartificial skin epitheliumin vitro

Construction of artificial epithelial tissues prepared from human normal fibroblasts and C9 cervical epithelial cancer cells carrying human papillomavirus type 18 genes

One cervical cancer cell line, C9, carrying human papillomavirus type 18 (HPV18) genes that is one of the major etiologic oncoviruses for cervical cancer was characterized. This cell line was further characterized for its capacity related to the epithelial cell proliferation, stratification and differentiation in reconstituted artificial epithelial tissue. Thein vitro construction of three dimensional artificial cervical epithelial tissue has been engineered using C9 epithelial cancer cells, human foreskin fibroblasts and a matrix made of type I collagen by organotypic culture of epithelial cells. The morphology of paraffin embedded artificial tissue was examined by histochemical staining. The artificial epithelial tissues were well developed having multilayer. However, the tissue morphology was similar to the cervical tissue having displasia induced by HPV infection. The characteristics of the artificial tissues were examined by determining the expression of specific marker proteins. In the C9 derived artificial tissues, the expression of EGF receptor, an epithelial proliferation marker proteins for stratum basale was observed up to the stratum spinosum. Another epithelial proliferation marker for stratum spinosum, cytokeratins 5/6/18, were observed well over the stratum spinosum. For the differentiation markers, the expression of involucrin and filaggrin were observed while the terminal differentiation marker, cytokeratins 10/13 were not detected at all. Therefore the reconstituted artificial epithelial tissues expressed the same types of differentiation marker proteins that are expressed in normal human cervical epithelial tissues but lacked the final differentiation capacity representing characteristics of C9 cell line as a cancer tissue derived cell line. Expression of HPV18 E6 oncoprotein was also observed in this artificial cervical epithelial tissue though the intensity of the staining was weak. Thus this artificial cervical epithelial tissue though the intensity of the staining was weak. Thus this artificial epithelial tissue could be used as a useful model system to examine the relationship between HPV-induced cervical oncogenesis and epithelial cell differentiation.

Reconstruction of Rabbit Corneal Layer Composed of Corneal Fibroblasts and Corneal Epithelium on the Lyophilized Amniotic Membrane

Many researchers have employed the cryopreserved amniotic membrane (CAM) and corneal epithelial cells in the treatment of a severely damaged burned cornea, with corneal epithelial cells cultured on an amniotic membrane (AM). The lyophilized amniotic membrane (LAM) has a higher graft take and a longer shelf life; it is easier to store and safer because of gamma irradiation. Two Teflon rings (Ahns supporter) were made for culturing the cells on the LAM, and were then used to support the LAM. To reconstruct a corneal layer composed of corneal fibroblasts and epithelium, the corneal fibroblasts were first cultivated on the stromal side of LAM for five days, followed by epithelial cells culture on the epithelial side, by using the air-liquid interface culture. The reconstructed corneal layer composed of corneal fibroblasts and corneal epithelial cells has a much healthier basal layer of corneal epithelium than the reconstructed corneal epithelium, which was got by using only corneal epithelial cells, and resembles the epithelium of normal corneas, without the horny layer. Thus, the reconstruction of the corneal layer by using a LAM is considered to be a good in vitro model, not only for its application in toxicological test kits, but also for transplantation in patients with a severely damaged cornea.

Tissue-Engineered Skin Substitutes Using Collagen Scaffold with Amniotic Membrane Component

Human skin substitutes are needed for implantation and wound repair based on the new concept of tissue engineering in combination with biomaterials and cell biological technology. However, failure sometimes occurs when the wound healing is delayed in vivo due to acute inflammation resulting from the early degradation of the transplanted biomaterials. Accordingly, the current study modified conventional biomaterials to overcome early degradation and strong inflammation. In a conventional skin substitute, the animal origin collagenous materials have a slight antigenicity as xenogenic materials, however, the modified method was able to obtain a low antigenicity and anti-inflammation effect using atelo-collagen and an amniotic component. The tyrosine content in the developed atelo-collagen, representing the antigenicity, was reduced from 0.590% to 0.046% based on an HPLC analysis. In addition, to reduce the inflammation and foreign material reaction, an amniotic component was applied to the atelo-collagen materials. While, to enhance the wound healing, the modified skin substitute was developed as a composite matrix of an atelo-collagen scaffold with an amniotic membrane component. A quantitative analysis of hEGF in the amniotic membrane was also performed using different processing methods. Finally, a tissueengineered skin substitute was constructed by cultivating skin cells in the collagen scaffold attached to an amniotic membrane.

Development and application of bioartificial skin.

formation of living tissues for cell biology research, wound repair, test systems for therapeutics, and drug delivery (1). Intensive investigations have resulted in developing technologies for a variety of organs and applications. General strategy of development of bioartificial organ by tissue engineering technology is composed of following steps: (i) a mass of primary cell should be prepared by isolation from specific tissue and pure proliferation; (ii) a fabricated biocompatible polymer must be manufactured Into tissue-scaffold with suitable structure and shape; (iii) the primary cells should be cultured into the biocompatible tissue-scaffold and constructed into bioartificial tissue or organ, and then that was implanted into human body. The most advanced development in tissue engineering has been achieved for the skin replacement. The engineering of skin tissue has been studied from a variety of approaches. Typical wound dressing, which are mainly natural or synthetic polymers alone, are used only to enclose damaged area and care for it without prevention of external infection and internal water loss. In addition to these roles, introducing the viable cells into biocompatible polymers develops bioartificial skins by tissue engineering assist wound repair and regeneration. In addition, serious damage in depth or burn of wide area to reach to 30-40% area, the bioartificial skin should be required besides the synthetic wound dressing and autologous grafting. Skin is composed of two tissues, a connective tissue or dermis and a covering epidermis. During connective tissue repair, fibroblasts exhibit several different activities. At first, they migrate from adjacent tissues into the wound region and then proliferate and synthesize a collagen-rich extracellular matrix which effectively fills the wound. The extracellular matrix of the dermis provides the structural and biological support for epidermis. The fibroblasts also contribute to the remodeling of newly synthesized extracellular matrix (2, 3). Collagenous tissue as a biomaterial possesses many favorable characteristics and advantages over synthetic materials. The resemblance to human tissue suggests that it has a performance advantage over alternative materials (4). Several types of collagen based artificial skin have been developed so far. Bell et al produced a tissue-like structure by contraction of collagen gel by human fibroblast (5). There has been an increasing number of laboratories, both academic and industrial, involved in developing various models of skin for clinical use. Advanced Tissue Sciences, Inc. (La Jolla, CA, USA) has characterized an in vitro skin model consisting of keratinocytes and fibroblasts grown on a nylon mesh (6). Organogenesis Inc. (Canton, MA, USA) has examined the effect and behavior of ApligrafTM in a broad range of venous ulcer conditions (7).

Reinforced Bioartificial Skin in the Form of Collagen Sponge and Threads

Bioartificial skin requires high mechanical strength-scaffold to overcome the problem of easily being torn during handling and suturing. In addition, the scaffold should not even have potential toxicity to cell culture and host tissue. Therefore, in this study, we suggest how to make a stronger sponge without using any cross-linking treatments, which will show the latent cytotoxicity on the implant material. We made sponge type of bioartificial skin using its three major components, collagen, dermal fibroblasts, and epidermal keratinocytes. The sponge-type collagen scaffold for skin cell culture was prepared by freeze-drying of 7.5 mg/ml of type I rat tail collagen solution. We reinforced collagen sponge by incorporation of collagen mesh which was made by stacking five layers of 9×9 collagen threads. By this method we could increase tensile strength by as much as three times higher compare to the collagen sponge cross-linked using glutaraldehyde. The ultimate tensile strength of collagen sponges reinforced with collagen threads, uncross-linked collagen sponges, and collagen sponges cross-linked by glutaraldehyde were 0.086, 0.0038, and 0.029 MPa respectively.

Development of Artificial Skin Using Raft Culture Technique and its Application to Study the Mechanism of Oncogenesis

Artificial skin has been developed in vitro using tissue culture technique for application to artificial organ transplantation. We have been also trying to apply this technique to set up a system for elucidating a mechanism of oncogenesis. As a model system for studying oncogenesis, we have been syudying papillomavirus-associated cervical cancer. For epidermal cells squamous carcinoma cell lines, cervical cancer cell lines, and normal human keratinocytes were used. These cells were cultured in raft system to much artificial tissues and these artificial tissues were analyzed histochemically and immunologically.