Introduction

Construction of oral mucosa is a relatively recent field of tissue engineering that aims to treat and fill the tissue defects caused by facial trauma or malignant lesion surgery, as well as to provide a model to study the biology and pathology of oral mucosa. A tissue-engineered oral mucosa may further serve as a vehicle for gene therapy, and as an in vitro system, alternative to in vivo testing of oral care products [1]. It is a potential solution for many patients with congenital defects such as cleft palate or with lost tissues due to diseases such as gingivitis or tumor excision following a craniofacial cancer diagnosis. In case of skin, the “gold standard” for the treatment of wounds has been the use of split-thickness skin grafts [2]. However, for the treatment of wounds in the oral cavity, the small size of the donor oral tissue is a limitation [3]. Besides, the use of skin grafts for the treatment of oral wounds results in negligible assimilation even years after transplantation due to differences between skin and oral mucosa [4]. These led the researchers to employ tissue engineering to develop suitable oral mucosa equivalents designed and constructed according to the needs of the patient to assist in the reconstruction of oral cavity.

3D full-thickness oral mucosa equivalents and oral epithelial cell sheets are the predominant approaches in oral mucosa engineering, which aim to reconstruct oral tissue using cells with or without biodegradable scaffolds, respectively.

Any 3D oral mucosa equivalent should contain a scaffold which, when seeded with fibroblasts, would form a lamina propria equivalent. The choice of the scaffold material is a crucial one since the success of the tissue-engineered implant largely depends on it. The ideal scaffold used in oral mucosa engineering must not induce a toxic or immune response or result in excessive or prolonged inflammation. It should be slowly biodegradable, support the reconstruction of normal tissue and have similar mechanical and physical properties to the oral mucosa it is designed to substitute. In addition, it should be readily available and capable of being prepared and stored with a long shelf life [2]. Porosity is another very important property of the scaffold, because a lamina propria is a highly vascularized tissue and a scaffold should possess optimum pore size and interconnected porosity (>40–60 %) to allow perfusion of the medium, intrinsic vascularization, adequate fibroblast infiltration and proliferation, and cell to cell communication [5]. Polymeric material of the scaffolds used in oral mucosa engineering can be derived from natural sources or obtained by synthesis.

The aim of this review article was to cover the old and new generation biomaterials and scaffold production techniques used in oral mucosa engineering, including the ones used in authors’ laboratories to develop novel oral mucosa models. The review will start with a brief introduction to the similar yet different structures of human oral mucosa and skin, will discuss the strategies developed so far to reconstruct oral mucosal defects, and it will end with future perspectives on oral mucosa engineering.

Structure of Oral Mucosa

Oral mucosa represents the barrier between the mouth and the oral cavity, and serves to protect the underlying tissue from mechanical damage and from entry of microorganisms and toxic materials [6]. Its structure is more similar to skin than to any other mucosa in the body, and just like skin, with which it forms a junction at the lips, it is basically composed of a stratified epithelium and an underlying dense connective tissue, i.e. lamina propria (Fig. 1). The two are attached to each other at the basement membrane region. Epithelium consists mainly of tightly packed epithelial cells. Lamina propria, on the other hand, is composed of fibroblasts, connective tissue, capillaries, inflammatory cells (macrophages), and extracellular matrix (ECM) (Fig. 1). The physiological features that distinguish oral mucosa from skin are its pink color due to extensive vasculature, its moist surface, higher permeability due to its lipid content, absence of appendages (such as hair follicles, sebaceous glands, sweat glands), and its pattern of keratinization [6–8]. Oral mucosa has distinct regions; some require more strength like the hard palate and gingiva, and some others require more elasticity like the cheek, lips, and the floor of the mouth. According to the function of the region in the oral cavity, the epithelium of oral mucosa may be keratinized (masticatory mucosa), nonkeratinized (lining mucosa) or both (specialized mucosa) as in the case of dorsum of the tongue. In contrast, the epithelium of the skin is keratinized regardless of its location in the body [9].

Fig. 1
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General structure of oral mucosa (Image adapted, with permission, from ref. [109])

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Reconstruction of defects in the oral mucosa

Grafts

For the reconstruction of oral mucosal defects, the main sources for the transplants are the inner cheek and the palate [4]. Palatal mucosal grafts are routinely used to cover mucosal defects caused by vestibuloplasty [10]. However, oral tissues are limited in size and quantity, therefore, skin transplants and intestinal mucosa are commonly used to cover extensive defects [4]. This approach, however, has serious disadvantages; one is donor site morbidity and the other is the differences in the properties of the skin, such as hair growth and pattern of keratinization [3]. These result in negligible assimilation even years after transplantation [4]. These issues created the need to find an alternative approach and this was the development of oral mucosa equivalents using regenerative medicine and tissue engineering.

Epithelial cell sheets

Driven by the problems associated with oral mucosal grafts and by the success of epidermal cell sheets used in the treatment of skin defects caused by severe burns, ulcers, etc., researchers recently started to grow autologous oral epithelial cell sheets in the laboratory starting with small oral biopsies (Fig. 2). Cell sheet engineering makes use of several techniques such as culturing on temperature responsive culture dishes [11], on human amniotic membranes [12] and on collagen membranes [13]. The former is the oldest and the most established method, where temperature-responsive culture dishes are created by the covalent grafting of the temperature responsive polymer poly(N-isopropylacrylamide) (PNIPAM) on ordinary polystyrene-based tissue culture dishes [14]. Under normal culture conditions at 37 °C, the dish surfaces are relatively hydrophobic and cells attach, spread, and proliferate as on typical tissue culture dishes. However, upon decrease of the temperature below the polymer’s lower critical solution temperature (LCST) of 32 °C, the PNIPAM graft becomes hydrophilic and swells, forming a hydration layer between the dish surface and the cultured cells, causing their spontaneous detachment without enzymatic treatments such as trypsinization. By avoiding the proteolytic treatment, critical cell surface proteins such as ion channels, growth factor receptors, cell to cell junctions and their deposited ECM remain undamaged, and cells can be non-invasively harvested in the form of sheets [15].

Fig. 2
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A transparent oral mucosal epithelial cell sheet prepared for ocular surface reconstruction

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The advantage of oral mucosal epithelium over the skin is its high regenerative capacity allowing the harvest site to heal very rapidly and without scar formation [16]. In addition, oral mucosal epithelial cell sheets were proven to be superior to epidermal cell sheets in some other respects. For example, it took 12 days to form an epithelial sheet from small epithelial segments as compared to 14 days in the case of a skin epithelial sheet [17]. Furthermore, in vitro, mucosal epithelial sheets could maintain their viability for 30 days as opposed to 22 days for skin epithelial sheets [17]. Clinical results were good, with accelerated healing, smooth and keratinized graft site, and with no infection or scar contraction [3, 10, 18, 19]. Oral mucosal epithelial cell sheets were also used for ocular surface reconstruction in bilateral cases of limbal stem cell deficiency [20–24], for the treatment of esophageal ulcerations [25, 26], and for bladder reconstruction [27].

Full-thickness Oral Mucosa Equivalents

At the beginning, there was one major problem with the oral epithelial cell sheets used in the reconstruction of the oral mucosa: they were fragile, difficult to handle, and also had low engraftment rates [1]. It was reported that the presence of a mesenchymal tissue assisted in the adherence and maturation of the epithelial graft, and minimized wound contraction while encouraging the formation of a basement membrane [28]. This led the scientists to construct 3D, full-thickness oral mucosa equivalents consisting of an epithelium and an underlying lamina propria to support the former (Figs. 3, 4).

Fig. 3
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Schematic representation of the construction of a full-thickness oral mucosal equivalent

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Fig. 4
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Histological analysis of a full-thickness oral mucosal equivalent (OME) composed of an epithelium and a lamina propria (a) HPS staining of the OME. Cell nuclei were stained by hematoxylin (blue), cytoplasm by phloxine (pink), and extracellular matrix of connective tissue by saffron (orange/yellow) (b) Immunofluorescence staining of the OME. Epithelium was stained by anti-keratin 13 (green), cell nuclei by propidium iodide (red) Bar 50 µm (color figure online)

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The advantages of these 3D models were that they were easier to handle and able to fill deep defects; they resembled the native tissue more due to their more complex structure with both an epithelium and a lamina propria separated by a basement membrane. They also allowed the incorporation of different cell types, had high degree of differentiation and the strength for histological assessment. Using these 3D models, it might be possible to monitor tissue growth or damage, together with the expression of tissue proteins or mRNA in situ [29].

3D models of full-thickness oral mucosa were reconstructed using acellular cadaver or animal dermis [30–33] and polymer-based scaffolds [34–36]. The constructs containing a de-epithelialized dermis from a cadaver or animal lacked fibroblasts. Fibroblasts are the most important cells for a lamina propria equivalent. They promote the growth and differentiation of epithelial cells and the formation of a basement membrane [28, 29]. The first non-keratinized 3D oral mucosa equivalent was constructed using epithelial cells isolated from the non-keratinized cheek region of the oral cavity [37]. Equivalents reconstructed using epithelial cells isolated from elsewhere result in keratinized structures. The full-thickness oral mucosal equivalents have also been successfully used in urethral reconstruction [38, 41, 42]. Besides tissue reconstruction purposes, 3D oral mucosa models have also served as an alternative to in vivo cytotoxicity testing of oral care products, such as tooth whitening agents [40] and antiseptic mouthwashes [41]. Another area is using them as models which closely mimic the native tissue in the study of the biology and pathology of native oral mucosa. These 3D models shed light into epithelium-mesenchymal tissue interactions [42], efficacy of administered drugs [43], studies of soft tissue-implant interfaces [44], and also diseases such as oral dysplasia, early invasive oral squamous cell carcinoma [45], oral candidiasis [46], and oral mucositis [47].

Scaffolds used in oral mucosa engineering

Natural scaffolds

Natural scaffolds are either cadaver- or animal-derived de-epithelialized acellular matrices or are mostly constructed using natural polymers extracted from animals. Porcine skin [32], de-epithelialized human cadaver dermis [30, 31, 33, 48–51], AlloDerm™ (also a human cadaveric dermis) [8, 49, 52–54], human amniotic membrane [12], and de-epithelialized bovine tongue [55] have been used as acellular matrices for oral mucosa engineering. Natural polymers have the advantage of responding to the environment via degradation and remodeling through the action of the enzymes. They are also generally non-toxic, even at high concentrations [56], which must be of significant importance in oral mucosa engineering. Among natural polymers, collagen is the most commonly used one due to its high biocompatibility and biodegradability. In addition, it is adhesive and cohesive, fibrous, and can be used in combination with other materials. It was the first material used to construct a dermal equivalent in vitro [57, 58]. It might, however, be antigenic through telopeptides, though it is possible to remove these small telopeptides by proteolysis before use [59]. The lamina propria of native oral mucosa itself is mainly composed of collagen, type I along with some collagen type III in the deeper layers [60]. Collagen scaffolds gave promising results in oral mucosa engineering [35–37, 49, 61–65], but other natural materials such as fibrin [34, 66, 67], elastin [49, 68], and glycosaminoglycans [49] were also used, alone or in combination with collagen (Fig. 5).

Fig. 5
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Collagen Type I sponge impregnated with chondroitin sulfate and crosslinked with EDC/NHS at RT

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Synthetic Scaffolds

Synthetic polymers such as poly(glycolic acid) (PGA), poly(lactic acid) (PLA) and their copolymers (PLGA), poly(p-dioxanone) (PDO), and copolymers of trimethylene carbonate (TMC) are popular in tissue engineering because they allow the researcher to tailor the mechanical properties and the degradation kinetics to suit various applications, and they can be fabricated into various shapes with desired morphologic features and chemical moieties [69]. They, however, have not been used in oral mucosa engineering yet. Currently, only two synthetic scaffolds, namely Dermagraft®, which is made of PLGA, and TransCyte®, which is made of silicone, are available for skin tissue engineering.

Recombinant polymers

Recombinant polymers are polypeptides designed using recombinant DNA technology and contain the desired amino acid sequences for advanced applications in biotechnology. Recently researchers started to use them as another category of materials for tissue engineering.

These macromolecules are generically named as “recombinamers” [70, 71]. Using this technology, it is possible to bioengineer protein-based polymers (PBPs) of more complex and well-defined structure. Elastin-like recombinant polymers (ELRs) form a class of these biocompatible PBPs. They are composed of the pentapeptide repeat Val-Pro-Gly-Xaa-Gly (VPGXG), which is derived from the hydrophobic domain of tropoelastin and where X represents any natural or modified amino acid, except proline [72]. ELRs can be designed to respond to physical stimuli such as temperature, redox, pH, light, etc., by incorporation of suitable guest residues in the polypeptide chain at the fourth position [72]. After the finding of the high biocompatibility of the (VPGVG)-based ELRs, their in vitro potential for tissue engineering was tested [71]. Later, short peptide sequences having a specific bioactivity were inserted into the polymer sequence of these polypeptides to improve their properties (Fig. 6). The first active peptides inserted in the polymer chain were the well-known, general purpose cell adhesion tripeptide RGD (R = l-arginine, G = glycine and D = l-aspartic acid) and the REDV (E = l-glutamic acid and V = l-valine), which is specific to endothelial cells. The resulting bioactivated (VPGVG) derivatives, especially those carrying RGD, showed a high capacity to promote cell attachment [70].

Fig. 6
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Schematics of the elastin-like recombinant polymer H-RGD6, which contains 6 monomers of the cell adhesion peptide RGD

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ELRs have been used as coatings [73–76], as fibers [77], and films [78, 79] to improve cell attachment, as hydrogels to promote chondrogenesis [80–83], as injectable solutions [84, 85], or as 3D matrices [86]. A scaffold containing an ELR with substrate amino acids for mTGase, recognition sequences for endothelial cell adhesion (REDV), elastic mechanical behavior (VPGIG), and for targeting of specific elastases for proteolytic reabsorption (VGVAPG), was found to be suitable for vascular tissue engineering [80]. In 2011, the first oral mucosa equivalent based on a scaffold made of an ELR functionalized with cell adhesion peptide RGD was reported [87, 88]. The authors found that the presence of the ELR increased the proliferation of epithelial cells, fibroblasts, and the epithelial stem cells in the engineered tissue (Fig. 7). This study, along with others, strongly supports the potential of elastin-like recombinant polymers as scaffold materials for tissue engineering applications.

Fig. 7
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Influence of the ELR on the thickness of the reconstructed epithelium (a) and (b) show that the oral mucosa equivalent constructed of the ELR-collagen scaffold had a thick epithelium with a large number of proliferative basal cells that were Ki67 positive (c) and (d) show that the epithelium was thin in the control collagen scaffold; it was composed of a few cell layers and a few proliferative basal cells that were Ki67 positive Bars 50 µm (Image used, with permission, from ref. 88)

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Scaffold production techniques used in oral mucosa engineering

Several techniques have been developed to fabricate scaffolds for tissue engineering such as solvent casting and particulate leaching, gas foaming, fiber mats and fiber bonding, phase separation, melt molding, emulsion freeze drying, solution casting, electrospinning, and freeze drying [89]. For the porous scaffolds of oral mucosa engineering, in most cases, freeze drying has been employed as the fabrication method.

Freeze drying

Scaffolds for tissue engineering may be produced by a multitude of different and novel techniques which aim to mimic the natural ECM. As a result, the spectrum of scaffold types available with very different properties has expanded [90]. Freeze drying (or lyophilization) of aqueous solutions of polymers such as collagen has been reported for the production of well-defined porous matrices, where the pore sizes and orientation are achieved by the controlled growth of ice crystals during the freezing process [91]. In this process, the solution to be frozen contains the polymer such as collagen and the solvent, freezing traps the polymer in the spaces between the growing ice crystals and forms a continuous interpenetrating network of ice and the polymer (Fig. 8). A reduction in the chamber pressure causes the ice to sublimate, leaving behind the polymer as highly porous foam [92].

Fig. 8
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a and b Collagen fibers obtained by electrospinning from an HFIP solution (c) Collagen foam obtained by freeze drying of a collagen solution in dilute acetic acid

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Freezing temperature, solute and polymer concentration were shown to strongly influence the porous structure of the scaffold obtained by freeze drying. For example, freezing of a collagen solution at −20 °C was reported to result in larger pore sizes than freezing at -80 °C, and the most rapid freezing procedure, −196 °C (used liquid nitrogen), led to the smallest pores [93]. Solute concentration was also shown to influence the pore size in scaffolds produced by freeze drying; an inverse relationship was found between collagen concentration and pore size [94].

Electrospinning

Cells cultured in 3D environments behave differently than those cultured in a 2D environment, in that they adopt more in vivo like morphologies. The environment affects the cell-receptor ligation, intercellular signaling, cellular migration, and also the diffusion and adhesion of proteins, growth factors, and enzymes needed for cell survival and function [95]. The 3D fibrous scaffolds composed of nanoscale multifibrils prepared by electrospinning with the aim of mimicking the supramolecular architecture and the biological functions of the natural ECM as much as possible, have attracted a great deal of attention in the field of tissue engineering [90]. Studies involving electrospinning of collagen type I indicated the possibility to reproducibly electrospin nanostructured scaffolds that preserve the biological and structural properties of collagen [96].

Fiber-based scaffolds can have advantages over foams such as greater homogeneity, higher porosity, higher interconnectivity, and reproducibility [97].

The studies on skin equivalents based on electrospun scaffolds yielded promising results indicating their potential for oral mucosa engineering. A study comparing collagen-based skin substitutes fabricated using scaffolds produced by either freeze drying or electrospinning concluded that that electrospun scaffolds can be used to fabricate skin substitutes with optimal cellular organization and have more potential to reduce wound contraction than freeze dried scaffolds [98]. These properties are expected to lead to reduced morbidity in patients treated with such skin substitutes [98]. Another study found that collagen nanofibrous matrices effectively accelerated wound healing in early stage wound repair [99]. The authors reported that crosslinked collagen nanofibers coated with ECM proteins, particularly type I collagen, may be a good candidate for biomedical applications, such as wound dressings and scaffolds for tissue engineering [99]. Other non-collagenic electrospun nanofibrous materials were also shown to be effective as skin substitutes. Indeed, high cell attachment and spreading of human oral mucosal keratinocytes and fibroblasts were observed on nanofibrous chitin scaffolds [100]. PLGA-PLLA electrospun scaffolds were able to support growth of keratinocyte, fibroblast, and endothelial cells and production of extracellular matrix [101]. Other nanofibrous scaffolds from collagen/silk fibroin, carboxyethyl chitosan/poly(vinyl alcohol), gelatin, PLGA/chitosan, and poly(ε-caprolactone) [98, 102–105] were also found to promote keratinocyte and/or fibroblast attachment and proliferation, indicating the potential of nanofibrous mats as substrates for oral mucosa regeneration. Various kinds of scaffolds used in the reconstruction of 3D oral mucosa equivalents during the past 10 years are listed in Table 1.

Table 1 Various kinds of scaffolds used in the reconstruction of 3D oral mucosa equivalents during the past 10 years
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Future perspectives

A tissue-engineered oral mucosa offers hope for many patients with congenital defects or tissue loss due to diseases, facial trauma or malignant lesion surgery. In the laboratory, it also provides insight into the behavior of the oral mucosal cells in healthy and pathological tissues. Tissue-engineered skin is now a reality and commercial models such as Integra® and Apligraf® are being successfully used in the clinics. Though skin tissue engineering has a history of 30 years, oral mucosa engineering may benefit from the excellent research done and lessons learned from the former. Currently, in vivo studies using oral mucosal equivalents conducted mostly in mice are yielding promising results. Clinical studies are scarce but they report better healing using tissue-engineered oral mucosa compared to controls. However, long-term clinical outcomes are needed to demonstrate the success of these models.

Oral mucosa is a highly vascularized tissue, much more than skin. As such, like in most fields of tissue engineering, vascularization is a big challenge for the post-transplantation survival of the tissue-engineered product. In skin tissue engineering, microvascularized 3D models were developed in vitro by the incorporation of human umbilical vein endothelial cells (HUVECs), which formed tubular capillaries in the presence of dermal fibroblasts and vascular endothelial growth factor (VEGF) [106]. When grafted, these models were shown to result in faster vascularization leading to better implant tissue survival, due to an early inosculation between the host capillaries and the ones in the skin equivalent. Several scaffolds are also being developed to enhance angiogenesis, such as scaffolds with large pore sizes to promote faster vascularization [107] and injectable 3D scaffolds to release potent angiogenic factors such as basic fibroblast growth factor (bFGF) [108]. Although these models are not in clinical use yet, oral mucosa engineering may benefit from similar approaches to help tissue-engineered oral mucosa survive after grafting.

Stem cells are an attractive source for use in tissue engineering applications due to their high flexibility and self-renewal capacity. Indeed, adult stem cells with their versatility and regeneration potential have been successfully used in tissue engineering of various organs such as skin, cornea, bladder, stomach, heart, and cartilage. Despite advances in the field of stem cell biology, relatively little attention has been paid so far to stem cells present in the oral mucosa. Human oral mucosa stem cells (hOMSC) are a recently described neural crest derived stem cell population. They are clinically important for the treatment of oral mucosa/skin defects and their potential to regenerate epithelial and mesenchymal tissues should be explored by their incorporation into 3D oral mucosa equivalents. These tissue-engineered structures containing hOMSC might further serve as models to investigate oral diseases including carcinogenesis.