Tissue-Engineered Oral Mucosa Constructs for in Vitro Research and Clinical Applications

Monolayer cultures of epithelial or gingival fibroblasts have been used for the study of oral mucosa and the effect of external stimuli, such as different types of dental materials. However, monolayer models lack polarized cell phenotype and systemic components, which affect their function and response to stimuli [1,2]. Several tissue-engineered oral mucosa models have been developed to overcome these limitations with applications in different fields [24]. These 3D cultures provide a higher degree of complexity than monolayer cell cultures being closer to explant cultures, providing an in vitro model resembling the in vivo situation [2]. At least two tissue-engineered oral mucosa models are commercially available, Skin EthicTM Oral Epithelium and Gingival Epithelium constructs from EPISKIN (Lyon France), or the Epi OralTM and Epi GingivalTM constructs from Mat Tek Corporation (Ashland, MA, USA). Other commercial skin tissue models are validated to be used as an alternative to animal testing to proof cosmetics safety [5]. Thus, it could be envisaged that in the near future oral tissue models might be approved to test cosmetic dental raw materials or formulations such as tooth pastes or mouthwashes. However, the existing commercial models lack a fibroblast/collagen matrix component, which is critical in human oral mucosa [2].


Mini Review
Monolayer cultures of epithelial or gingival fibroblasts have been used for the study of oral mucosa and the effect of external stimuli, such as different types of dental materials. However, monolayer models lack polarized cell phenotype and systemic components, which affect their function and response to stimuli [1,2]. Several tissue-engineered oral mucosa models have been developed to overcome these limitations with applications in different fields [2][3][4]. These 3D cultures provide a higher degree of complexity than monolayer cell cultures being closer to explant cultures, providing an in vitro model resembling the in vivo situation [2]. At least two tissue-engineered oral mucosa models are commercially available, Skin Ethic TM Oral Epithelium and Gingival Epithelium constructs from EPISKIN (Lyon France), or the Epi Oral TM and Epi Gingival TM constructs from Mat Tek Corporation (Ashland, MA, USA). Other commercial skin tissue models are validated to be used as an alternative to animal testing to proof cosmetics safety [5]. Thus, it could be envisaged that in the near future oral tissue models might be approved to test cosmetic dental raw materials or formulations such as tooth pastes or mouthwashes. However, the existing commercial models lack a fibroblast/collagen matrix component, which is critical in human oral mucosa [2].
In order to produce a full thickness tissue-engineered mucosa with epithelial and fibroblasts components, several strategies have been followed using different scaffolds or cells from different origin ( Table 1). These differences in scaffold and cell origin are very important depending on the aim of the study or the application of the oral system. In vivo clinical applications include both intraoral graftings (i.e., for use in reconstructions of the oral cavity after tumor resection or the treatment of gingival recession) and extra-oral applications (i.e., for use in urethoplasty, grafting of burn wounds or eyelid reconstruction) [1,6]. For these applications, human primary cells (from the same patient if possible) are used in the fabrication of the oral graft [6]. Besides, in vitro applications include cosmetic testing or study of different dental materials such as titanium implants [7]. These 3D cultures can also be used in oral cancer research to facilitate the study of mechanistic aspects of the disease, early invasion, tumor growth, new treatments or diagnostic tests [8]. For in vitro applications, the use of immortalized cells is preferred, since primary cells are difficult to maintain for a long period of time and results can differ for every cell donor. Buccal carcinoma cell lines may not represent normal epithelial cells; since the results produced using these cells should be interpreted with caution and validated with other cells [2]. Another in vitro application is the use of tissue-engineered oral mucosa as model for the emerging research field that studies soft tissue interaction with dental implant abutments. Human primary oral keratinocytes and human primary oral fibroblasts have been used to produce constructs for this purpose [9,10]. In addition, a three-dimensional tissue engineered bone-oral mucosal model has been produced to asses both soft and hard tissue integration on titanium implants [11].

Conclusion
In conclusion, the use of tissue-engineered oral mucosa constitutes both a therapeutic and a research tool that depending on the given application shall be constructed by a different strategy. Clinical application for intraoral grafting procedures [12,13] In vitro testing [10] Deepidermalized dermis Primary canine oral keratinocytes and Immortalized fibroblasts (3T3) Graft for dog cleft palate repair [14] Keratinocytes derived from human oral palate In vitro preparation of bioartificial mucosa. The authors conclude that a plate scaffold should be used [15] Amniotic membrane Human amniotic membrane Primary rabbit oral epithelial cells Cornea autologous transplantation in rabbits [16] Collagen Collagen type-I Human oral immortalized keratinocytes and immortalized fibroblasts (3T3) In vitro testing [2] immortalized human oral keratinocytes and primary human oral fibroblasts In vitro testing [17] Collagen-chitosan Primary human oral keratinocytes In vitro testing [18] Collagen-elastin

Primary human gingival keratinocytes and primary fibroblasts
Clinical application in periodontal therapy [19] Primary human palatal mucosa keratinocytes and primary human fibroblasts In vitro testing [20] Collagen-GAG-chitosan Primary human palatal mucosa and primary human fibroblasts In vitro testing [20] CollaCote ® (Zimmer Dental) Primary human oral epithelial cells and human gingival fibroblasts In vitro testing [21] Fibrin

Fibrin matrix
Primary human fibroblasts and keratinocytes from oral mucosa Clinical application for ankyloglossia [22,23] Primary human fibroblasts and keratinocytes from oral mucosa Clinical application for hemifacialmicrosomia and ankyloglossia [24] Synthetic Scaffolds Poly (ethylene terephthalate)

Primary human palatal mucosa keratinocytes and primary human fibroblasts
In vitro Testing [20] Polycarbonate membrane Electro-spun poly L-lactic acid Electro-spun polystyrene Polylactic glycolic acid Primary dog epidermal keratinocytes Clinical application for mucosa prosthesis [25]