Tissue-engineered Oral Mucosa

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Tissue-engineered Oral Mucosa K. Moharamzadeh, H. Colley, C. Murdoch, V. Hearnden, W. L. Chai, I. M. Brook, M. H. Thornhill and S. MacNeil J DENT RES published online 19 January 2012 DOI: 10.1177/0022034511435702 The online version of this article can be found at: http://jdr.sagepub.com/content/early/2012/01/19/0022034511435702

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CRITICAL REVIEWS IN ORAL BIOLOGY & MEDICINE

Tissue-engineered Oral Mucosa

K. Moharamzadeh1*, H. Colley1, C. Murdoch1, V. Hearnden1,3, W.L. Chai2, I.M. Brook1, M.H. Thornhill1, and S. MacNeil3 1

School of Clinical Dentistry, University of Sheffield, Claremont Crescent, Sheffield, S10 2TA, UK; 2Department of General Dental Practice and Oral and Maxillofacial Imaging, University of Malaya, Kuala Lumpur, Malaya, Malaysia; and 3 Kroto Research Institute, University of Sheffield, Broad Lane, Sheffield, UK; *corresponding author, k.moharamzadeh @sheffield.ac.uk J Dent Res X(X):xx-xx, XXXX

Abstract Advances in tissue engineering have permitted the three-dimensional (3D) reconstruction of human oral mucosa for various in vivo and in vitro applications. Tissue-engineered oral mucosa have been further optimized in recent years for clinical applications as a suitable graft material for intra-oral and extra-oral repair and treatment of soft-tissue defects. Novel 3D in vitro models of oral diseases such as cancer, Candida, and bacterial invasion have been developed as alternatives to animal models for investigation of disease phenomena, their progression, and treatment, including evaluation of drug delivery systems. The introduction of 3D oral mucosal reconstructs has had a significant impact on the approaches to biocompatibility evaluation of dental materials and oral healthcare products as well as the study of implant-soft tissue interfaces. This review article discusses the recent advances in tissue engineering and applications of tissue-engineered human oral mucosa.

KEY WORDS: fibroblast, keratinocyte, biocompatibility, biomaterial, regeneration, gingiva.

DOI: 10.1177/0022034511435702 Received September 30, 2011; Last revision November 22, 2011; Accepted December 19, 2011 © International & American Associations for Dental Research

Introduction

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n recent years, three-dimensional (3D) tissue-engineered models of human oral mucosa have been developed, optimized, and characterized. Different biomaterials have been used in the past decades as connective tissue scaffolds, involving a variety of cell-culture techniques and tissue-engineering approaches to reconstruct human oral mucosa (Moharamzadeh et al., 2007). Major applications of the engineered oral mucosa include clinical transplantation, in vitro investigations of the interaction of materials with oral mucosa, oral disease modeling, and evaluation of drug delivery systems. In recent years, research has focused on optimization of these models for specific in vivo and in vitro applications, and interesting studies have been published demonstrating the capabilities of the advanced oral mucosal reconstructs to highly resemble the native oral mucosa and simulate the clinical situation, thus reducing the need for animal experiments. This review article aims to discuss the recent advances in tissue engineering of oral mucosa and the latest applications of the oral mucosal models developed in recent years.

Progress on Oral Mucosa Engineering In our previous review (Moharamzadeh et al., 2007), 3D oral mucosal models developed prior to 2006 were thoroughly discussed, including in-depth details of tissue-engineering strategies used for the production of human oral mucosal composites, their relative advantages and drawbacks, as well as systematic classification and critical analysis of different biological and synthetic matrices used as scaffolds for the tissue engineering of skin and oral mucosa and the role of cell source and culture media on these tissue reconstructs. Since 2006, numerous studies have reported the development of 3D oral mucosal models with modified cell sources, scaffolds, and culture media. The suitability of 10 different types of collagen-based and synthetic scaffolds was evaluated in terms of biocompatibility, biostability, porosity, and the ability to mimic normal human oral mucosa morphology in a study comparing normal oral keratinocytes with the TR146 cell line and a variety of cell-seeding techniques (Moharamzadeh et al., 2008b). Cross-linked collagen-/chitosan-based membranes showed that superior biostability compared with non-crosslinked collagen membranes and optimal conditions for encouraging fibroblast infiltration and epithelial stratification, without invasion into the connective tissue layer, could be achieved by laminating a porous scaffold with a collagenlaminin gel on the surface onto which the keratinocytes were seeded. This was

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consistent with previous studies demonstrating that seeding keratinocytes onto a type IV collagen-laminin surface enhanced both keratinocyte attachment and migration (Kim et al., 1994). A porous collagen–glycosaminoglycan (GAG)–chitosan scaffold, along with normal oral keratinocytes and fibroblasts, was used by Kinikoglu et al. (2009) to produce a highly differentiated non-keratinized full-thickness oral mucosa equivalent. In these mucosal models, strong expression of cytokeratin 13 by oral mucosa epithelial cells was independent of fibroblast–epithelial cell interactions. However, the thickness of the epithelium was influenced by fibroblast–epithelial cell communication. Cell lines such as TR146 have been used in oral mucosa engineering in some commercially available 3D models. Compared with normal oral keratinocytes, TR146 cells do not form a fully differentiated oral epithelium (Yadev et al., 2011). However, the need for reproducibility and lack of batch-to-batch variability may make the use of this cell line desirable for in vitro biocompatibility test models. An immortalized human oral keratinocyte cell line (OKF6/TERT-2) has been cultured on fibroblast-populated collagen gels to enhance the reproducibility of the 3D oral mucosal test model without using oral cancer cell lines and to avoid dyskeratotic changes in oral mucosal reconstructs (Dongari-Bagtzoglou and Kashleva, 2006). It is important to realize that the ideal cell source, scaffold, and culture medium for tissue-engineered oral mucosa depend on the intended application. Although for in vitro applications the reproducibility of the model is one of the main priorities, for in vivo and clinical applications, a robust tissue-engineered model based on normal oral keratinocytes seeded on a scaffold with good handling characteristics and optimal biodegradability, and cultured in a growth medium compatible with the receiving host, is desirable. Pena et al. (2010) recently developed and characterized a completely autologous oral mucosa equivalent based on a fibrin glue scaffold obtained from the patient’s own blood plasma. Fibroblast-populated fibrin glue was seeded with the patient’s keratinocytes to produce a host-compatible oral mucosal graft. The advantage of this autologous scaffold included good handling characteristics with no surface retraction as seen in collagen gels and reduced risk of immunorejection. However, the use of this tissue-engineered mucosa remains to be evaluated clinically.

Tissue-Engineered Oral Mucosa for In Vivo Applications Skin Substitutes Several commercially available dermal or skin substitutes have been developed for clinical applications. DermagraftTM, developed by Advanced Tissue Sciences Inc. (La Jolla, CA, USA), is a dermal substitute composed of a biodegradable polymer mesh populated with dermal fibroblasts (Purdue, 1997). ApligrafTM, developed by Organogenesis (Canton, MA, USA), is a composite graft composed of allogenic keratinocytes grown on a fibroblastpopulated bovine collagen gel (Eaglstein et al., 1995). Other living skin substitutes include OrcelTM (Ortec International Inc., New York, NY, USA), PolyactiveTM (H.c. Implants b.v., The Netherlands), and Hyalograf 3DTM (Fidia Advanced Biopolymers,

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Abano Terme, Italy). It has been reported that tissue-engineered skin can affect the host cells and promotes tissue regeneration and remodeling by producing several cytokines and growth factors such as interleukin(IL)-1, IL-3, IL-6, CXCL8, transforming growth factors (TGF)-α and β, and fibroblast growth factor (Lee, 2000). Bilayered cell therapy with these tissueengineered products has been suggested as a potential approach for the treatment of gingival soft-tissue defects as an alternative to autologous free gingival grafts (McGuire et al., 2008; Nevins, 2010).

In vivo Animal Studies In vivo animal studies have been carried out to investigate the role of grafted tissue-engineered oral mucosa in wound healing. Pena et al. (2011) evaluated an autologous fibrin glue-based engineered oral mucosa by subcutaneous implantation in athymic mice. Although the results of their study showed that the epithelium remained viable and expressed a cytokeratin profile similar to that of native oral mucosa, it may be difficult to extrapolate the data to the clinical situation, where wounds are left open and the grafted material is air-exposed. In a similar study, Rouabhia and Allaire (2010) grafted engineered oral mucosa based on human gingival keratinocytes cultured on a fibroblast-populated biodegradable collagen membrane on the dorsum of athymic mice. Using silicone transplantation chambers, they were able to protect the graft material. Histology on day 15 post-grafting revealed a well-organized epithelium with no sign of necrosis. Ophof et al. (2008) studied the effects of implantation of autologous canine oral keratinocytes, cultured on acellular dermis, on intra-oral wound healing. Cultured keratinocytes were lost on week 1 after grafting, and the substitutes did not improve the healing of palatal wounds, leading the authors to suggest that the revascularization of the wound area was too slow to allow for survival and integration of the substitutes. However, Yoshizawa et al. (2011) grafted an engineered oral mucosal equivalent consisting of human oral keratinocytes cultured on Alloderm into a buccal mucosal defect in athymic mice. Although histological evaluation of the grafted engineered mucosa after 1 week showed that the cultured epithelium had peeled away and was lost, epithelial migration and complete coverage of the membrane with epithelium were observed on day 14 post-grafting. In their control group, which had Alloderm membrane with no keratinocytes, no epithelial coverage was observed. They suggested that the cultured keratinocytes function as an in situ bioreactor, releasing numerous cytokines and growth factors such as vascular endothelial growth factor (VEGF), transforming growth factor, amphiregulin [a protein of the epidermal growth factor (EGF) family], and heparin-binding EGF-like growth factor, which promote not only vascularization but also the migration of keratinocytes from the wound edge after grafting.

Intra-oral Clinical Applications Treatment of patients with cleft palate is often complicated due to the shortage of oral soft tissue and the presence of scarred

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tissue from previous surgeries. Tissue-engineered palatal mucosa based on a biodegradable collagen membrane has been developed for potential use in treatment of patients with cleft palate (Luitaud et al., 2007). Cryopreservation of labial mucosa harvested during initial lip surgery has been reported to be a useful approach to preserve keratinocytes for subsequent reconstruction of the mucosa (Xiong et al., 2010). Tissue-engineered oral mucosa has also been used in the field of periodontal plastic surgery for the treatment of patients with inadequate attached gingival. In a randomized clinical trial, cultured gingival grafts on a biodegradable collagen scaffold have been shown to be safe and capable of generating keratinized attached gingival tissue (Mohammadi et al., 2007). The same group reported successful application of tissue-engineered gingival graft for regeneration of peri-implant gingival tissue at a lower premolar implant with insufficient attached keratinized gingiva (Mohammadi et al., 2011). It has been shown that the presence of elastin in the connective tissue layer of the engineered oral mucosa modulates the keratinization of the overlying epithelium (Hsieh et al., 2010).

Extra-oral Applications Autologous free grafts from buccal mucosa are currently the first-choice treatment for substitution urethroplasty (Watkin, 2008). However, in patients with inadequate buccal mucosa, the use of tissue-engineered mucosal grafts can be extremely valuable and reduces the likelihood of oral complications following the harvest of extensive areas of buccal mucosa. There is a consensus among surgeons that tissue-engineered graft material will become a standard treatment in the future (Barbagli and Lazzeri, 2007). In our group, tissue-engineered buccal mucosa was evaluated clinically in recent years for urethral reconstruction. Bhargava et al. reported the outcome in a 3-year follow up of five patients who received substitution urethroplasty with tissue-engineered oral mucosa (Bhargava et al., 2008). While one patient suffered fibrosis and contraction after several months, requiring graft excision, and another showed fibrosis of half of the graft, the remaining patients showed good ‘take’ and survival of grafts over 3 years. The study indicated that graft contraction can be a complication of the use of engineered oral mucosa for some patients; hence this was then investigated in vitro. Glutaraldehyde pre-treatment of the de-epidermized dermis scaffold and restraint of the tissue-engineered graft during culture were both identified as useful approaches for future clinical exploration (Patterson et al., 2011). Biodegradable polymer-based electrospun scaffolds have also been used to produce engineered oral mucosa for urethra reconstruction but are not yet used clinically (Selim et al., 2011). Esophageal stenosis is one of the major complications of aggressive endoscopic resection. In animal experimental models, tissue-engineered oral mucosal grafts were effective in promoting re-epithelialization and suppressing inflammation which causes esophageal scarring and stenosis after endoscopic submucosal dissection (Yang and Soetikno, 2007; Takagi et al., 2010).

Oral Disease Modeling Oral Cancer In vitro models of oral carcinogenesis at various stages of malignant transformation have been developed drawing on the ease of isolation of normal oral epithelial cells and the use of wellestablished immortalized dysplastic and neoplastic cell lines cultured on collagen scaffolds (Costea et al., 2005). Recently, Gaballah et al. (2008), using clinically isolated keratinocytes and fibroblasts, developed models of mild, moderate, and severe dysplasia on collagen scaffolds. Models produced with mortal dysplastic keratinocytes failed to match the original dysplastic nature of the clinical lesion, and the severity of the dysplasia also increased with passage number, suggesting a phenotypic drift (Gaballah et al., 2008). Marsh et al. (2011) showed increased invasion of OSCC cells into a collagen/Matrigel-based extracellular matrix in the presence of myofibroblasts compared with normal oral fibroblasts, with no invasion in models absent in fibroblasts. To assist with invasion studies, investigators have devised digital image analysis methods to quantify tumor invasion in 3D cultures (Gaggioli et al., 2007). Despite being more physiologically relevant than monolayers, the basic structure of the connective tissue component and the reconstituted basement membrane in organotypic models allows for only a somewhat simplistic representation of the native stromal microenvironment (Wolf et al., 2009). Recent studies have suggested that the extracellular matrix has a dual role, acting not only as a barrier that invading malignant cells need to transverse, but also as an anchorage point, allowing invading cells to attach (Gaggioli et al., 2007). Furthermore, contrary to the well-established view that focal breaks in the basement membrane correlate to malignant progression, recent findings from the collagen matrix organotypic model suggest that expression of basement membrane proteins (laminin-332, type IV collagen, and fibronectin) is enhanced with cancer progression, and that the basement membrane is both degraded and synthesized to support the invading front (Kulasekara et al., 2009). Mucosal models that utilize a more complex, native, connective tissue containing a defined basement membrane to investigate oral dysplasia and invasive carcinoma may provide a greater insight into the molecular mechanisms controlling pre-malignant dysplasia and invasion in vivo. We have recently developed different models to replicate the different stages of carcinogenesis: dysplasia, carcinoma in situ, and early invasive carcinoma (Fig. 1) (Colley et al., 2011). Essentially, wellcharacterized oral squamous cell lines were seeded as cell suspensions or as multi-cellular tumor spheroids with oral fibroblasts onto a de-epidermized acellular dermis, since this is more representative of native dermis (Wolf et al., 2009). These in vitro models gave the morphological appearance and histological characteristics of dysplasia and tumor cell invasion seen in vivo. We suggest that such models could facilitate study of the molecular processes involved in malignant transformation, invasion, and tumor growth, as well as in vitro testing of new treatments, diagnostic tests, and drug delivery systems for OSCC.

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Figure 1. Tissue-engineered models of (A) normal oral mucosa, (B) dysplastic epithelium, and (C) early invasive oral squamous cell carcinoma. To produce the engineered normal oral mucosa model, we seeded normal oral fibroblasts and keratinocytes onto a DED scaffold. After 72 hrs, the composites were raised to the air/liquid interface and cultured for a further 14 to 21 days. To reconstruct an in vitro model of dysplastic oral mucosa and early invasive oral carcinoma, we replaced the normal oral keratinocytes with either DOK or Cal27 cells, respectively. (Scale bar = 100 µm.)

Oral Infection Models In investigators’ attempts to understand the mechanisms of pathogenesis, tissue-engineered oral mucosal models have been extensively used to investigate the interaction of oral microbes with the oral epithelium. Most studies to date have examined the interaction of the fungus Candida albicans with tissue-engineered oral mucosa as a model of oral candidiasis. Much of the information generated on the interaction of C. albicans and other Candida species with the oral epithelium has come from the use of reconstituted human oral epithelial (RHOE) models in which the buccal squamous cell carcinoma cell line (TR146) is cultured on a polycarbonate transwell insert at an air-liquid interface to form a multilayered epithelium (Schaller et al., 2006). This simple in vitro model has produced a plethora of data on host-Candida interactions, such as the mode of C. albicans hyphal invasion into oral tissue, host signal transduction mechanisms activated, and cytokines released upon stimulation with C. albicans (Moyes et al., 2010); but recently, questions have been raised about the accuracy of this model in replicating the histological features of the oral mucosa, and thus its usefulness as an in vitro model. Mostefaoui and colleagues were the first to use 3D tissue-engineered oral mucosa (normal oral keratinocytes seeded onto a fibroblast-populated bovine collagen matrix) to investigate oral candidiasis in vitro (Mostefaoui et al., 2004), and the use of this in vitro model is becoming increasingly popular (Fig. 2A). These more complex models are based on normal or immortalized oral keratinocytes cultured on top of an oral fibroblast-containing collagen gel or human de-epithelialized dermis at an air-to-liquid interface, and show remarkably similar levels of keratinocyte differentiation, basal cell proliferation, and cytokeratin expression to normal oral mucosa (DongariBagtzoglou and Kashleva, 2006; Yadev et al., 2011). Moreover, we have recently shown that C. albicans-infected tissue-engineered oral mucosal models display increased cytokine (CXCL8, IL-1β) release and β-defensin 2 expression compared with RHOE models, making them more representative models of in vivo oral candidiasis (Yadev et al., 2011). These tissue-engineered oral mucosal models have been used to show that C. albicans-secreted aspartyl proteases are required for E-cadherin degradation at epithelial cell junctions during hyphal invasion of

oral tissue (Villar et al., 2007), and that several C. albicans genes (Hwp1, Hyr1, Ssa1, IPT1) and their products are crucial for adhesion to and invasion of the oral epithelium during oral candidiasis (Sun et al., 2010; Dwivedi et al., 2011; Rouabhia et al., 2011). Apart from their use in investigating mechanisms of fungal pathogenesis, tissue-engineered oral mucosal models have also been used to examine bacterial-derived oral infections. Most work to date has focused on the invasiveness and tissue destruction capacity of the Gram-negative anaerobic bacterium Porphyromonas gingivalis, which is an important etiological agent in chronic periodontitis. Not only were strains of P. gingivalis and F. nucleatum found to be internalized by the upper layers of oral keratinocytes in collagen matrix infection models (Fig. 2B), but also they were observed in the deeper layers and in the connective tissue, suggesting that these bacteria can penetrate the epithelium during infection (Gursoy et al., 2010).

Drug Delivery Studies The oral mucosa also has potential as a site for the systemic delivery of drugs which could provide an alternative to parenteral delivery (Hearnden et al., 2011). An effective test model is required for the development of delivery systems capable of transmucosal or intra-mucosal delivery in an optimal manner with minimal local tissue damage, drug delivery to the desired location with ideal speed, and minimal drug degradation. We have shown (Hearnden et al., 2009) that fluorescently labeled polymersomes, a nano-scale polymer-based delivery system, can be tracked through tissue-engineered oral mucosa by laser scanning confocal microscopy, enabling progression of polymersomes to be tracked over time. The polymersomes were able to reach the basal keratinocytes of the tissue-engineered epithelium, and there was evidence of polymersomes crossing into the de-epithelialized dermis, something which is often obstructed by the permeability barrier. The use of tissue-engineered models allows for the evaluation of epithelial drug delivery systems without the need for animal models and facilitates initial toxicity testing prior to in vivo trials. The potential for these kinds of experiments is a clearly advantageous application of tissueengineered oral mucosa.

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Figure 2. Tissue engineered models of oral mucosal infection.(A) Hyphal invasion of a wild-type strain of Candida albicans (CAF2-1) into the epithelium of a tissue-engineered oral mucosa model after 48 hrs in culture. Hyphae (stained purple with periodic acid-Schiff) can be seen penetrating through the oral epithelium (stained brown with immunohistochemistry for the pan-cytokeratin marker MNF16) and into the fibroblastcontaining native connective tissue (bar = 200 µM). (B) P. gingivalis-infected tissue-engineered oral mucosa. P. gingivalis (brown dots) within the upper layers of the oral mucosa were visualized after infection by immunohistochemistry with a monoclonal antibody raised against P. gingivalis (bar = 20 µM).

Imaging and Spectroscopic Diagnostic Technologies The diagnosis of many oral mucosa conditions requires surgical biopsies and subsequent histological and immunohistochemical analyses. A non-invasive point-of-care diagnostic imaging system could dramatically improve patient outcomes, especially in conditions such as OSCC, where early detection, monitoring of disease progression, and definition of surgical boundaries are highly sought after. Tissue-engineered oral mucosa has great value during the development stages of novel imaging systems, since new imaging technologies can be tested without the need for patients or technology specially adapted and tested for in vivo use. For example, the potential for optical coherence tomography OCT as a method to detect oral squamous cell carcinoma has been recently evaluated (Smith et al., 2011). In this study, OCT was shown to differentiate successfully between the connective tissue (fibroblast-populated de-epithelialized dermis) and epithelium generated from normal oral keratinocytes (Fig. 3). When tissue-engineered models of OSCC and dysplasia were imaged by OCT, severe epithelial disruption could be detected. Other work has focused on the use of scanning acoustic microscopy to characterize tissue-engineered models generated from oral keratinocytes cultured on fibroblast-free Alloderm, particularly for epithelial surface irregularities (Winterroth et al., 2011), which can help with quality control and monitoring of tissue-engineered oral mucosa production.

3d Oral Mucosal Biocompatibility Testing Models Biocompatibility of Dental Materials and Oral Healthcare Products Conventional cytotoxicity assays use monolayer cultures of cells, either monocultures or co-cultures. It has also been shown

Figure 3.  Tissue-engineered oral mucosa imaged by optical coherence tomography. Differences in the optical properties of different portions of tissue-engineered oral mucosa visible by different brightness intensities in gray-scale image.

that when experiments are moved into three dimensions, there is often a cytoprotective effect observed, with TC50 values higher for 3D models than for the traditional two-dimensional (2D) models (Sun et al., 2006). In addition, cytokine and growth factor release from the 3D oral mucosal models in which both viable and non-viable cells are present differs from those of the monolayer cell culture systems in which the majority of cells are viable (Xu et al., 2009). Commercially available 3D oral mucosal biocompatibility test models have been used to assess the biological effects of different types of dental materials, including bonding adhesives (Vande Vannet and Hanssens, 2007), orthodontic wires (Vande Vannet et al., 2007), and other metals used in dentistry, such as nickel (Trombetta et al., 2005). MatTek’s split-thickness 3D buccal mucosal model (EpiOralTM, MatTek Corp., Ashland, MA, USA) has been recently used to investigate the influence of ethanol and ethanol-containing mouthrinses on the permeability of oral mucosa in vitro (Koschier et al., 2011). In our previous studies, a full-thickness

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Figure 4.  Histological sections of (A) freshly excised normal oral mucosa exposed to PBS; (B) excised mucosa exposed to a high concentration (2%) of SLS; (C) excised mucosa exposed to a high concentration (45%) of ethanol; (D) 3D oral mucosal model exposed to PBS; (E) 3D oral mucosal model exposed to 2% SLS; and (F) 3D oral mucosal model exposed to 45% ethanol. Hematoxylin and eosin, original magnification ((A × 10, B × 10, C × 10, D × 10, E × 10, F × 10).

3D human oral mucosal model was developed and characterized for biocompatibility assessment of dental materials (Moharamzadeh et al., 2008b). The use of this model permitted biocompatibility testing of experimental dental composite resins in direct contact with the surface of the engineered oral mucosa (Moharamzadeh et al., 2008a). This test set-up highly resembled the clinical situation and provided useful and relevant information on the interaction of the oral mucosa with resin-based dental materials with different monomer compositions. A similar model was used for biological evaluation of alcohol-containing antiseptic mouthwashes (Moharamzadeh et al., 2009). Although these 3D models seem more promising than monolayer cell culture systems in the biological assessment of dental materials and healthcare products, the question still remains: How do the responses of these 3D models compare with those of normal oral mucosa in a clinical situation? In an attempt to address this question, we have carried out experiments comparing the response of freshly excised human oral mucosa to various concentrations of ethanol and sodium lauryl sulphate with those of 3D tissue-engineered oral mucosa (Fig. 4). The results suggested that the response of the 3D oral mucosal model to ethanol and SLS exposure had some features similar to the responses of the fresh clinical biopsies of oral mucosa.

Evaluation of Implant-Soft Tissue Interaction To date, most studies of the peri-implant tissue response to different implant systems have been carried out on either in vivo models, such as humans or animals, or on in vitro 2D cell culture

models (Pendegrass et al., 2008). Recently, a 3D tissue-engineered oral mucosal model was developed for the purpose of investigation of the implant-soft tissue interface (Chai et al., 2010). This model consisted of both epithelium and connective tissue layer based on a de-epithelialized acellular dermis scaffold (Fig. 5A). Under light microscopy examination, the epithelium layer of the oral mucosal model formed a peri-implant-like epithelium on the implant surface tested (Fig. 5B), as seen in in vivo models. Further experiments (Chai et al., 2011) also showed evidence of hemidesmosome-like structures formed at the implant-oral mucosa interface under transmission electron microscopy examination (Figs. 5C, 5D). Besides qualitative analysis of the implant-soft tissue interface, this oral mucosal model also allowed for quantitative analysis of the biological seal of the Ti-oral mucosa interface, based on permeability and attachment tests. This newly developed model provides more useful information than the monolayer cell culture systems for the investigation of the implant-soft tissue interface. Nevertheless, it must be appreciated that the oral mucosal model is not yet able to fully substitute for the in vivo situation.

Future Developments Developments in oral mucosa tissue engineering seem to follow advances in skin technology, since composite skin equivalents were developed many years ago for the treatment of burn patients (MacNeil, 2007). The use of stem cells combined with new synthetic electrospun nanofibrous scaffolds has been

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Figure 5. A tissue-engineered oral mucosal model for the investigation of implant-soft tissue interface.  (A) A hematoxylin-and-eosin-stained tissueengineered oral mucosal model. Note a well-formed stratified squamous epithelial layer and some fibroblasts in the connective tissue layer (black arrows). Scale bar = 100 µm. (B) Ground section (Richardson stain) shows the epithelium of the tissue-engineered oral mucosa forming a peri-implantlike epithelium (white arrow) next to the tested titanium surface. Scale bar = 100 µm. (C, D) Transmission electron micrographs showing hemidesmosome-like structures (white arrows) at the implant-tissue-engineered oral mucosal interface. Black triangle depicts the residual titanium oxide layer after the electropolishing procedure to dissolve the bulk of the titanium metal for ultrathin section preparation. Scale bar = 0.2 µm.

introduced for skin tissue engineering (Jin et al., 2011). Genemodified keratinocytes and fibroblasts have recently been shown to enhance skin regeneration in a full-thickness tissueengineered model (Lohmeyer et al., 2011). Other types of cells, such as Langerhans cells, have been incorporated into engineered skin equivalents to develop models capable of identifying potential chemical sensitizers (Ouwehand et al., 2011). Translation of these latest developments in skin engineering to the oral mucosa is under way. New aspects of oral biology of the engineered mucosal reconstructs have been the focus of research in recent years, including angiogenesis (Perez-Amodio et al., 2011) and radiation-induced oral mucositis (Tobita et al., 2010; Lambros et al., 2011). Most researchers in the field of oral biology are

moving on from the traditional monolayer cell culture systems to the newly developed well-characterized and reproducible tissue-engineered oral mucosal models that highly resemble the native human oral mucosa and are more clinically relevant and more informative than the monolayer cell culture systems.

Acknowledgments The authors are grateful to Nish Yadev and Abigail Rice for providing the images of fungal and bacterial invasion of tissueengineered oral mucosa. The author(s) received no financial support and declared no potential conflicts of interest with respect to the authorship and/or publication of this article.

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