Gürkan Raşit BAYAR, Yavuz Sinan AYDINTUĞ, Ömer GÜNHAN, Kamile ÖZTÜRK, Aydın GÜLSES

Ex Vivo Produced Oral Mucosa Equivalent by Using the Direct Explant Cell Culture Technique

Objective: The aim of this study is the histological and immunohistochemical evaluation of ex vivo produced oral mucosal equivalents using keratinocytes cultured by direct explant technique. Material and Methods: Oral mucosa tissue samples were obtained from the keratinized gingival tissues of 14 healthy human subjects. Human oral mucosa keratinocytes from an oral mucosa biopsy specimen were dissociated by the explant technique. Once a sufficient population of keratinocytes was reached, they were seeded onto the type IV collagen coated “AlloDerm” and taken for histological and immunohistochemical examinations at 11 days postseeding of the keratinocytes on the cadaveric human dermal matrix. Results: Histopathologically and immunohistochemically, 12 out of 14 successful ex vivo produced oral mucosa equivalents (EVPOME) that consisted of a stratified epidermis on a dermal matrix have been developed with keratinocytes cultured by the explant technique. Conclusion: The technical handling involved in the direct explant method at the beginning of the process has fewer steps than the enzymatic method and use of the direct explant technique protocol for culturing of human oral mucosa keratinocyte may be more adequate for EVPOME production. Turkish Anahtar Kelimeler: Ağız mukozası, keratinosit, epidermis, dermal matriks Amaç: Bu çalışmanın amacı, direkt eksplant teknik kullanılarak, vücut dışında ağız mukozası keratinositlerinden üretilen ağız mukozası eşdeğerlerinin, histolojik ve immünohistokimyasal olarak incelenmesidir. Gereç ve yöntem: Ağız mukozası örnekleri, 14 sağlıklı insanın keratinize dişeti dokularından elde edilmiştir. İnsan ağız mukozası keratinositleri, ağız mukozası örneklerinden, eksplant teknik ile ayrıştırılmıştır. Yeterli çoğunluğa ulaştıktan sonra keratinositler, tip IV kollajen kaplı “Alloderm” üzerine ekilmiş ve kadavra kaynaklı insan dermal matriksi üzerine ekimlerinden 11 gün sonra histolojik ve immünokimyasal incelemeye tabi tutulmuştur. Bulgular: Histopatolojik ve immünohistokimyasal olarak, 14 örnekten 12sinde, dermal matriks üzerinde, eksplant teknik ile oluşan keratinositlerden elde edilen çok katlı yassı epitelden oluşan vücut dışında üretilmiş ağız mukozası eşdeğeri başarılı bir şekilde oluşturulmuştur. Sonuç: Enzimatik tekniğe göre direct eksplant yöntem, sürecin başında daha az sayıda basamak gerektirmekte ve vücut dışında üretilmiş ağız mukozası eşdeğeri üretimi için ağız mukozası keratinositlerinin elde edilmesinde daha uygun bir yöntem olarak öne çıkmaktadır.

Keywords:

Oral mucosa, keratinocyte, epidermis, dermal matrix,

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Alcalde J, Garcia-Tapia R, Espinosa JM, Perez N. Clinical applica- tion of free microvascular flaps in reconstructive surgery of the oral cavity. Acta Otorrinolaringol Esp 1994;45:457-60.
Leong DJ, Wang HL. A decision tree for soft tissue grafting. Int J Periodontics Restorative Dent 2011;31:307-13.
Kao SY, Lui MT, Fong J, Wu DC, Wu CH, Tu HF, et al. A method us- ing vestibulo-sulcoplasty combining a split-thickness skin graft and a palatal keratinized mucosa graft for peri-implant tissue secondary to oral cancer surgery. J Oral Implantol 2005;31:186-91. [CrossRef]
Jhaveri HM, Chavan MS, Tomar GB, Deshmukh VL, Wani MR, Miller PD Jr. Acellular dermal matrix seeded with autologous gin- gival fibroblasts for the treatment of gingival recession: a proof- of-concept study. J Periodontol 2010;81:616-25. [CrossRef]
Kaner D, Friedmann A. Soft tissue expansion with self-filling os- motic tissue expanders before vertical ridge augmentation: a proof of principle study. J Clin Periodontol 2011;38:95-101. [CrossRef]
Kedjarune U, Pongprerachok S, Arpornmeaklong P, Ungkuson- mongkhon K. Culturing primary human gingival epithelial cells: comparison of two iso lation techniques. J Craniomaxillofac Surg 2001;29:224-31. [CrossRef]
Klingbeil MFG, Herson MR, Cristo EB, Pinto JrDS, Yosshito D, Mathor MB. Comparison of two cellular harvesting methods for primary human oral culture of keratinocytes. Cell Tissue Bank 2009;10:197-204. [CrossRef]
Billingham RE, Reynolds J. Transplantation studies on sheets of pure epidermal epithelium and on epidermal cell suspensions. Br J Plast Surg 1952;23:25-36. [CrossRef]
Izumi K, Takacs G, Terashi H, Feinberg SE. Ex vivo development of a composite human oral mucosal equivalent. J Oral Maxillofac Surg 1999;57:571-7. [CrossRef]
Hotta T, Yokoo S, Terashi H, Komori T. Clinical and histopatho- logical analysis of healing process of intraoral reconstruction with ex vivo produced oral mucosa equivalent. Kobe J Med Sci 2007;53:1-14.
Izumi K, Feinberg SE, Lida A, Yoshizawa M. Intraoral grating of an ex vivo produced oral mucosa equivalent: a preliminary report. Int J Oral Maxillofac Surg 2003;32:188-97. [CrossRef]
Izumi K, Song J, Feinberg SE. Development of a tissue engi- neered human oral mucosa: from the bench to the bed side. Cells Tissues Organs 2004;176:134-52. [CrossRef]
Horch RE, Debus M, Wagner G, Stark GB. Cultured human kera- tinocytes on type I collagen membranes to reconstitute the epi- dermis. Tissue Eng 2000;6:53-67. [CrossRef]
Schultze-Mosgau S, Lee BK, Ries J, Amann K, Wiltfang J. In vitro cultured autologous pre-confluent oral keratinocytes for experi- mental prefabrication of oral mucosa. Int J Oral Maxillofac Surg 2004;33:476-85. [CrossRef]
Lauer G, Schimming R. Tissue-engineered mucosa graft for re- construction of the intraoral lining after freeing of the tongue: a clinical and immunohistologic study. J Oral and Maxillofacial Surg 2001;59:169-75. [CrossRef]
Flaxman BA, Lutzner MA, Van Scott EJ. Cell maturation and tis- sues organization in epithelial out-growths from skin and buccal mucosa in vitro. J Invest Dermatol 1967;49:322-32. [CrossRef]
Lauer G. Autografting of feeder-cell free cultured gingival epi- thelium. Method and clinical application. J Cranio Maxillofacial Surg 1994;22:18-22. [CrossRef]
Daniels JT, Kearney JN, Ingham E. Human keratinocyte isolation and cell culture: a survey of current practices in the UK. Burns 1996:22:35-9. [CrossRef]
Bayar GR, Aydintug YS, Gulses A, Elci P, Sarper M. A pilot study of the primary culture of the oral mucosa keratinocytes by the direct explant technique. OHDMBSC 2011;10:88-92.
Wanichpakorn S, Kedjarune-Laggat U. Primary cell culture from human oral tissue: gingival keratinocytes, gingival fibroblasts and periodontal ligament fibroblasts. Songklanakarin Journal of Sci- ence and Technology 2010;32:327-31.
Kinikoglu B, Auxenfans C, Pierrillas P, Justin V, Breton P, Burillon C, et al. Reconstruction of a full-thickness collagen- based human oral mucosa equivalent. Biomaterials 2009;30:6418-25. [CrossRef]
Freshney RI. Primary Culture. In: Willey-Liss Culture of animal cells: a manual of basic techniques, 5th ed.Hoboken NJ: Wiley- Liss, 2005: 176-97. [CrossRef]
Reid CBA, Cloos J, Snow GB, Braakhuis BJM. A simple and re- liable technique for culturing of human oral keratinocytes and fibroblasts. Acta Otolaryngol 1997;117:628-33. [CrossRef]
Lauer G, Otten JE, Von Specht BU, Schilli W. Cultured gingival epithelium. A possible suitable material for pre-prosthetic sur- gery. J Craniomaxillofac Surg 1991;19:21-6. [CrossRef]
Izumi K, Feinberg SE, Terashi H, Marcelo CL. Evaluation of trans- planted tissue-engineered oral mucosa equivalents in severe com- bined immunodeicient mice. Tissue Eng 2003;9:163-74. [CrossRef]
Nakamura T, Endo K, Cooper LJ, Fullwood NJ, Tanifuji N, Tsuzuki M, et al. The successful culture and autologous transplantation of rabbit oral mucosal epithelial cells on amniotic membrane. Invest Ophthalmol Vis Sci 2003;44:106-16. [CrossRef]