Alternatives to animal experiments for eye irritation

2. Reconstructed tissue models

2.1 Reconstruction of tissues

Several in vitro models with reconstructed tissues with cells from human skin have been introduced. These systems include ocular models, which are composed of a combined epithelium and stroma or just serve as models of the corneal epithelium. Several skin models are constructed in a similar way, but they also include a stratum corneum. The similarities between the reconstructed tissue models and the normal human tissues makes many practical applications in toxicology and pharmacology possible (Rasmussen, 1996). Experiments with reconstructed tissue models have so far given good concordances to results from animal experiments on many types of individual chemicals and finished products for eye and skin irritation, corrosivity and photorirritation. The reconstructed tissue models can also be used to evaluate efficacy issues, e.g. sun protection, wound healing and anti-aging.

2.1 Reconstruction of tissues

Skin equivalents were first developed for treatment of large burns, where engineered grafts are needed. Both chemical systems with foam pads obtained by co-polymerizing dermal macromolecules, and biological systems with viable skin cells have been used. Culturing of keratinocytes directly on the burn wound bed sometimes results in wound contraction, and preliminary use of a dermal equivalent has been shown to improve the wound healing. Epidermal cell cultured sheets are used to replace the missing epidermis. Hormones and other growth factors are supplied from the patients body, and the keratinocytes will reconstitute to a fully differentiated epithelium with a stratum corneum.

Reconstructed skin models are composed either of pure keratinocyte cultures or of combinations of fibroblasts and keratinocytes. For this reason, they present a very simplified system compared to the in vivo situation. The large challenge in the construction of the tissue models has been to produce a multilayered epidermis with a stratum corneum without the assistance of a live organism. Keratinocytes are seeded to filters or other substrates, and they first form a coherent layer of basal cells, and then several epithelial cell layers. If the process is stopped at this stage, an ocular model has been produced. When the epidermal tissues afterwards are exposed to air, the multilayered epidermis may be covered by a coherent layer of corneocytes, a thin stratum corneum. The artificial epidermis is composed of layers of keratinocytes similar to the natural epidermis, and it has been shown to mimic the ultra structure well by light and electron microscopy.

Reconstructed tissue models can be used to study the differentiation of keratinocytes, including changes in proteins and lipids. Biochemically, reconstructed skin models have been shown to mimic normal skin well, but the models also differ significantly structually and biochemically from the in vivo situationen. Large differences in the relative composition of various lipids and their structural organization have been demonstrated. In addition, high rates of diffusion through the not fully cornified cells of the stratum corneum in vitro have been found.

Several tissue models are solely composed of an epidermal tissue cultured on non-viable dermal replacements, such as a deepidermized dead dermis, microporous filters or a collagen matrix. The presence of a viable dermis may, however, have a significant impact on the functions of the tissue. During wound healing in vitro, the growth of the keratinocytes is significantly increased by the presence of epidermal growth factors and other substances that are produced by the basal membrane and the dermal fibroblasts. Additionally, the presence of an epithelium, a basal membrane and a stroma in the in vitro models may be used in evaluations of recovery from lesions to the tissue. When the outer parts of an epithelium solely have been damaged, the consequences in general are small, because the tissue is able to regenerate. If the basal membrane and the stroma also are damaged, the risk that permanent tissue lesions are introduced is dramatically increased.

A live dermis can be constructed in several ways. Fibroblasts may, for example, be embedded in a collagen gel. The cells quickly adhere to the collagen fibers, the gel contracts, and the tissue culture medium is expelled. After 2-3 days in culture, a plate of white, condensed tissue, which is a suitable basis of culturing epidermal cells, has been created. A viable dermal tissue can also be constructed by culturing fibroblasts on a nylon net. The fibroblasts adheres to the net, secretes various dermal macromolecules, and the tissue gradually covers the meshes. A three-dimensional stroma, which can serve as a basis for culturing other cell types, has then been constructed.

A series of commercial tissue models with fibroblast tissue grown on nylon net as a basis have been available. The SKIN² ZK 1100 model was solely composed of fibroblast tissue. In the SKIN² ZK1200 ocular model, the fibroblast stroma was supplemented with 3-4 layers of keratinocytes. SKIN² ZK1200 was a model of the cornea, which has a layer of epidermal cells of corresponding thickness. SKIN² ZK 1300 systems with a stratum corneum have been used as skin models. A model with human oral epithelial cells has been introduced for test of dental materials. Additionally, artificial skin to be used in transplantations has also been constructed using nets as a basis.

The tissue models are metabolically active, and they can also be used to study the interaction between different cell types. Experiments with melanocytes cultures in multilayered keratinocyte tissues have shown that several characteristics from the in vivo situation can be reestablished. The melanocytes establish in the basal layer of keratinocytes, and start to produce the pigment melanin. The pigmentation of the tissue increases after exposure to UV light. This model has been evaluated to be well suited for test of chemicals and finished products for phototoxic or photoprotective effects.

Exposure of tissues to local irritants or UV light may change the enzyme activity and the growth pattern of the cells, and increase the levels of irritation markers such as prostaglandines, leukotrienes and cytokines. Measurements of such parameters in monolayer cell cultures have been evaluated as possible alternatives to animal experiments for test for local irritancy. In general, results obtained from this type of systems have been in good agreement with data on in vivo eye irritation for water soluble substances. In several large blind studies of mixed chemicals and products, such methods have, however, been shown to be inadequate in predicting the in vivo response. Experiments with ocular models composed of reconstructed tissue are now among the most promising alternatives to animal experiments. Several protocols with tissue models have been developed to test for corrosivity, skin irritation, and phototoxicity, and such methods are subject to further validation.