Senos venosos durales

Cytokeratin 10 expression was not detected in any of the sections of cultured oral keratinocytes (Figure 2G, J, M) or in the normal palatal mucosa (Figure 2D). In contrast, cytokeratin 10 expression was found in the supra-basal layer of the normal epidermis (Figure 2A) and in the sections of epidermal keratinocytes on top of both types o f skin-derived substrates after 21 days of culture (not shown).

Cytokeratin 16 was expressed in the oral palatal mucosa o f the beagle dog except in the basal layer (Figure 2E), and throughout the cultures composed of oral keratinocytes from day seven on (Figure 2H, K, N). In the epidermis of the skin, cytokeratin 16 was not expressed (Figure 2B). However, the supra-basal layers of the cultures with epidermal keratinocytes were locally stained for cytokeratin 16 (not shown).

The basement membrane component heparan sulphate was not detected in the DED preparations before culture or in the collagen-based substrates. In contrast, it was still present on the uncultured AlloDerm®.

Continuous staining for heparan sulphate was observed at the epithelial­ dermal border o f the normal epidermis (Figure 2C) and the oral mucosa (Figure 2F), and in all cultures composed o f oral keratinocytes and AlloDerm®. In the DED specimen, however, expression o f heparin sulphate was only detected on days 14 and 21 (Figure 2L and O).

E___

C

Figure 2 Immunohistochemical staining fo r cytokeratin 10 on sections o f skin (A), oral mucosa (D) and oral keratinocytes cultured on DED fo r 7 (G), 14 (J), and 21 days (M), staining fo r cytokeratin 16 on skin (B), oral mucosa (E) and oral keratinocytes cultured on DED fo r 7 (H), 14 (K), and 21 days (N), staining fo r heparan sulphate on skin (C), oral mucosa (F) and oral keratinocytes cultured on DED fo r 7 (I), 14 (L), and 21 days (O). Bars represent 50 ¡ m.

2.4 Discussion

The general aim o f these studies is to develop a substitute for oral mucosa, which can limit the iatrogenic effects of cleft palate surgery. In this in vitro study, we evaluated substitutes composed of oral and epidermal keratinocytes o f a beagle dog cultured on several dermal substrates. The morphology and differentiation o f the cultured substitutes were compared to normal palatal mucosa and skin.

The results show that canine oral keratinocytes cultured on a skin- derived substrate at the air-liquid interface form an approximately ten cell layers thick parakeratinized epithelium closely resembling normal palatal epithelium of the beagle dog. We have further shown that oral keratinocytes and epidermal keratinocytes in culture are strikingly different. Epidermal keratinocytes cultured on topof the skin-derived substrates form only upto three cell layers and an orthokeratinized stratum corneum. However, this is similar to the histological characteristics o f normal canine epidermis. This study clearly demonstrates that canine keratinocytes from palatal mucosa and skin are capable to form an epithelium in culture. In both cases, the histological characteristics o f the original tissue were reproduced. This is in agreement with the result of others using human keratinocytes.38,39 Therefore, keratinocytes o f oral origin appear to be suitable for culturing a mucosa-like tissue.

Oral keratinocytes cultured on skin-derived substrates undergo stratification and keratinization from day 7 on. This is in agreement with

-5 C

the results o f others using human keratinocytes. However, all cultures showed a large increase o f stratum corneum thickness and only a limited increase in the number o f suprabasal cell layers with increasing culture

time. The increase in thickness o f the stratum corneum might be explained by the absence o f shedding in culture that normally occurs in vivo due to mechanical abrasion. However, others suggest that this might be dependent on the presence of EGF in the culture medium.40

Regeneration o f rete pegs was observed from day 14 on in the dermal substitute containing a skin-derived substrate and oral keratinocytes. Medalie41 suggested that a skin-derived substrate retains the undulating papillary surface o f normal dermis. The invaginations of this skin-derived surface will be filled by keratinocytes to form rete pegs. However, our skin-derived substrates originally do not posses a papillary surface. An alternative, more suitable, explanation might be that keratinocytes are able to contract the substrate forming a papillary surface. It has been shown before that keratinocytes are able to contract skin-derived substrates and collagen gels in vitro.42,43 The exact nature of this mechanism is still unclear.

None of the collagen-based substrates seeded with oral or epidermal keratinocytes induced the formation o f a stratified epithelium. These cultures exhibited only cell ingrowth into the substrate. This is not in agreement with others, who observed the formation o f a stratified and partially keratinized epithelium upon a collagen gel.44 This difference might be explained by the presence o f fibroblasts in the collagen gel.41,45 In addition, the large pore size of the collagen substrates could explain the ingrowth of keratinocytes. All substrates used in the present study had a pore size o f over 100 ^m. In the future, adding a surface layer o f a smaller pore size or incorporating fibroblasts into the matrix may improve the collagen-based substrates.

The skin-derived substrates containing oral keratinocytes were characterized by the presence of cytokeratin 16 and the absence of cytokeratin 10. Cytokeratin 16 expression was found in the suprabasal layers. This cytokeratin is specifically found in mucosal epithelia, and in hyperproliferative skin.46 This indicates that the cytokeratin expression of oral keratinocytes in these cultures is similar to that of oral mucosa. However, EGF, a component o f the culture medium, can also induce the

47

expression of cytokeratin 16. This might also explain the local staining o f cytokeratin 16 in our cultures with epidermal keratinocytes.

Cytokeratin 10 expression was only observed in the cultures containing epidermal keratinocytes at day 21. Cytokeratin 10 expression in keratinocytes on a collagen-based substrate or a nylon mesh was also observed by others.48,49

A difference between the two skin-derived substrates was the presence of the basement membrane component heparan sulphate on the human donor dermis. Heparan sulphate was not present in the DED since we used trypsin treatment to prepare the substrate. However, heparan sulphate was expressed in the cultures composed o f oral keratinocytes and DED from day 14 on. This is in agreement with others who also show that a structural basement membrane is not a prerequisite for epidermal morphogenesis and differentiation.50

In conclusion, we have shown that the oral mucosal substitutes composed of oral keratinocytes cultured on skin-derived substrates (DED or AlloDerm®) showed histological and immunohistochemical characteristics close to normal oral mucosa. In addition, epidermal keratinocytes cultured on a skin-derived substrate revealed a more skin­ like tissue. Therefore, we hypothesize that oral keratinocytes cultured on a skin-derived substrate are suitable to prevent the iatrogenic effects of cleft palate repair. In future studies, such a mucosal substitute will be implanted on the palate o f a beagle dog in a simulated cleft palate repair. This may ultimately lead to the improvement of cleft palate surgery in humans.

2.5 Acknowledgem ents

The technical assistance of M.P.A.C. Helmich was highly appreciated. We are grateful to Prof. Dr. J. Berden for providing the anti-heparan sulphate JM 403 antibody, to Dr. L.E. Boerboom for providing A llo­ Derm®, to Dr. A. van Kuppevelt for providing the collagen I matrix, and to Dr. E. Middelkoop for providing collagen I-elastin matrix. This work was supported by a grant from the American Association of Orthodontist Foundation and the European Orthodontic Society.

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