Could 3D printing be the future for oral soft tissue regeneration ?

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Article

Reference

Could 3D printing be the future for oral soft tissue regeneration ?

NESIC, Dobrila, et al.

Abstract

Oral soft tissue defects are a frequently encountered problem in dental praxis. Tooth loss, tooth root or implant recessions, infections or trauma require soft tissue reconstruction. The autologous graft remains the gold standard for gingiva and oral mucosa augmentation.

However, prolonged pain, limited amount of harvested tissue, and increased risk of infection have prompted the search for off-shelf alternatives. Several acellularized dermal matrices have been studied without satisfactory results. A newly developed collagen-based sponge is currently in clinical studies for long term evaluation. In these approaches however, the matrix needs to be tailored chair-side for each specific defect. 3D printing technology represents a promising solution as it offers precise production of an individualized 3D graft based on a defined shape and inner structure via a specific computer-aided design using a biomaterial of choice. Combined with smart biocompatible polymers (bioinks) that can be co-printed with cells in a specific architectural design, a more natural-like tissues can be engineered. More natural oral mucosa and gingiva [...]

NESIC, Dobrila, et al . Could 3D printing be the future for oral soft tissue regeneration ? Bioprinting , 2020, vol. 20, p. e00100

DOI : 10.1016/j.bprint.2020.e00100

Available at:

http://archive-ouverte.unige.ch/unige:150553

Disclaimer: layout of this document may differ from the published version.

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Review article

Could 3D printing be the future for oral soft tissue regeneration?

Dobrila Nesic

^*

, St ephane Durual, Laurine Marger, Mustapha Mekki, Irena Sailer, Susanne S. Scherrer

Division of Fixed Prosthodontics and Biomaterials, University Clinics of Dental Medicine, University of Geneva, Geneva, Switzerland

A R T I C L E I N F O Keywords:

3D printing Human gingiva Oral soft tissues Biomaterials Tissue engineering

A B S T R A C T

Oral soft tissue defects are a frequently encountered problem in dental praxis. Tooth loss, tooth root or implant recessions, infections or trauma require soft tissue reconstruction. The autologous graft remains the gold standard for gingiva and oral mucosa augmentation. However, prolonged pain, limited amount of harvested tissue, and increased risk of infection have prompted the search for off-shelf alternatives. Several acellularized dermal matrices have been studied without satisfactory results. A newly developed collagen-based sponge is currently in clinical studies for long term evaluation. In these approaches however, the matrix needs to be tailored chair-side for each specific defect. 3D printing technology represents a promising solution as it offers precise production of an individualized 3D graft based on a defined shape and inner structure via a specific computer-aided design using a biomaterial of choice. Combined with smart biocompatible polymers (bioinks) that can be co-printed with cells in a specific architectural design, a more natural-like tissues can be engineered. More natural oral mucosa and gingiva will find application in regenerative dental medicine, offer relevant organotypic cultures for basic research and provide testing platforms for drugs or chemical compounds. Tissue-engineered gingival equivalents comprising epithelial and connective tissues layers have been developed. 3D printing approaches have been applied for skin regeneration and the formation of vascular channels. Combining the gained knowledge from these studies may offer valuable cues on how to choose the best approach to create 3D printed patient-tailored gingival tissue to achieve the functionally and esthetically satisfying solution for the patient.

1. Introduction

Oral soft tissues are complex biologic systems that defend the oral cavity against exogenous substances, pathogens, and mechanical stresses.

Tooth loss, disease, trauma, or congenital disorders cause various oral soft tissue defects and necessitate treatment. When determining the type of treatment, special considerations need to be respected so that mastication, speech, and aesthetics can be appropriately restored and/or maintained. In restorative dentistry, remodeling and resorption of the buccal/oral bone upon tooth extraction represents a major problem [1].

Consequently, placement of implants or implant- or tooth-borne crowns/bridges often necessitate prior bone and/or soft tissue augmentation procedures to ensure satisfyingﬁnal outcomes [1]. With a constant increase of the world population longevity, the number of extracted teeth worldwide and the restorative dentistry interventions will continue to rise, and a need for efﬁcient and predictable tissue augmentation treatments will intensify. Although successful oral mucosa/gingiva

regeneration can be achieved by autologous transplantation, several disadvantages still hinder this type of treatment [2]. Inﬂiction of the second wound, limited autologous tissue amount that can be harvested, and pain have all prompted the development of alternatives. Tissue engineering and regenerative medicine are interdisciplinaryﬁelds aiming to develop procedures and technologies to regenerate and/or replace diseased or missing tissues. Today, advances in biomaterial sciences and production technologies allow fabrication of smart, biocompatible polymers that can be combined with cells and signalling molecules to regenerate a myriad of tissues, including oral mucosa/gingiva.

Three-dimensional (3D) printing, also referred to as additive manufacturing or solid freeform fabrication, is a novel technology that has seen an amazing expansion in recent years and opened new horizons for regenerative medicine [3,4]. 3D printing technology allows the production of an individualized 3D object based on a deﬁned shape, a biomaterial of choice, and a speciﬁc computer-aided design. The possi- bility to co-print living cells within a hydrogel has lifted 3D printing to

* Corresponding author. Division of Fixed Prosthodontics and Biomaterials, University Clinic of Dental Medicine, University of Geneva, Rue Michel-Servet 1, CH- 1211, Geneva 4, Switzerland.

E-mail address:dobrila.nesic@unige.ch(D. Nesic).

Contents lists available atScienceDirect

Bioprinting

journal homepage:www.elsevier.com/locate/bprint

https://doi.org/10.1016/j.bprint.2020.e00100

Received 16 January 2020; Received in revised form 3 September 2020; Accepted 8 September 2020

nc-nd/4.0/).

Bioprinting 20 (2020) e00100

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another level, opening a variety of possibilities to create patient-tailored tissues“a la carte”. The major asset of the 3D printing approaches is the precision of biomaterial and cell deposition to produce a pre-deﬁned shape as well as mimic the complex tissue architecture [4]. The achieved highly natural tissue organization can improve the functional characteristics of tissues afterin vitroculture and/orin vivoapplication and accelerate tissue regeneration. This review aims to outline the recent advancements in the fabrication of oral mucosa/gingiva tissue-engineered constructs and discuss 3D printing procedures developed for skin and blood vessels to provide grounds for the development of 3D printing approaches for oral soft tissues regeneration.

2. The structure and function of oral mucosa and gingiva

Oral mucosa is the mucous membrane lining the inside of the oral cavity. It comprises 1) the masticatory mucosa (gingiva and a cover of the hard palate), 2) the specialized mucosa (dorsal cover of the tongue), and 3) the lining mucosa, encompassing all other parts of the buccal cavity [5]. Gingiva belongs to masticatory mucosa, covering the alveolar bone and surrounding the teeth. Marginal (free) gingiva forms a “collar”

around the tooth (Fig. 1). The attached gingiva extends from the marginal gingiva and is tightly attached to the periosteum of the alveolar bone. Oral mucosa extends from the attached gingiva from which it is separated by the mucogingival line.

Structurally, gingiva consists of oral epithelium and underlying

connective tissue, lamina propria, which provides support and nourish- ment [6]. The marginal gingiva comprises a thin, non-keratinized epithelium (towards the tooth) and keratinized epithelium (towards the oral cavity), and lamina propria containing loosely connected collagenﬁbres [7]. Attached gingiva consists of the thick, keratinized epithelium and lamina propria with well-organized and dense collagen ﬁbres (Fig. 2). Attached gingiva is indispensable for the maintenance of teeth, periodontal tissues, alveolar bone as well as dental implants. It forms a protective barrier against harmful environmental agents such as pathogens, chemicals, and constant abrasion.

The main cell type in the gingival epithelium are keratinocytes.

Through a process of differentiation through several layers from lamina propria towards the surface (stratum corneum, granulosum, spinosum, and basale), keratinocytes form a defending stratified squamous epithelium (Fig. 2). Other cell types, namely Langerhans and Merkel cells, as well as melanocytes, are also found in the gingival epithelium. The gingival connective tissue consists of a papillary layer, withfinger-like projections of connective tissue intermingled with the overlying epithelium - rete pegs, and the reticular layer, located further underneath towards the tooth-root surface or the alveolar bone. The components of the lamina propria include cells, extracellular matrix, blood and lymphatic vessels, and nerves [5,7]. Fibroblasts interconnect to form a network and produce collagenfibres (organized according to mechanical demands), hyaluronic acid, and proteoglycans. Other cell types comprise macro- phages, mast cells, and a variety of inflammatory and immune cell types.

Fig. 1. Illustration of microscopic structures of human soft oral tissues in relation to the clinical image.

Fig. 2. Illustration of microscopic features of the attached gingiva.

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Oxygen and nutrients are supplied by gingival blood vessels originating from the periodontal ligament, the marrow spaces of the alveolar process, and the supraperiosteal blood vessels [5,6]. These vessels in turn supply major capillary plexuses that are located in the connective tissue adjacent to the oral epithelium and the junctional epithelium. The gingival tissues are supplied with lymphatic vessels that drain principally to submaxillary lymph nodes. Branches of the trigeminal nerve provide sensory and proprioceptive functions. The overall complexity of the biological systems allows for the necessary and diverse oral soft tissue functions.

3. Oral soft tissue defects: current treatments and limitations

Oral soft tissues play a vital role in the structure and function of the oral cavity. The insufficiency of oral mucosa due to tooth loss, gingival recessions, infections, or trauma require tissue reconstruction [2]. Soft tissue augmentation is frequently used to regain reduced or lost tissue in edentulous patients, cover an exposed root or implant, increase buccal mucosal soft tissue thickness, or coronal soft tissue height [2]. The treatment of choice has to comply with functional mastication, speech, and aesthetics. Depending on the location and the need, different techniques are used. Today, the gold standard for soft tissue augmentation is an autologous connective tissue graft, which can be either a free gingival graft (FGG) or a subepithelial connective tissue graft (SCTG) [2,8]. The use of an autologous tissue graft, however, presents several disadvantages and limitations 1) the amount of tissue that can be harvested is restricted as the height, length, and thickness of the palate depend on the anatomical position and vary among patients; 2) harvesting technique can be surgically challenging; 3) limited amount of tissue can be obtained per intervention, and 4) patients complain about prolonged postsurgical pain and numbness [9,10]. To reduce the morbidity caused by graft harvesting, soft tissue substitutes have been developed [11]. The requirements for an ideal non-autologous graft for soft tissue augmentation comprise biocompatibility, volume, and mechanical stability, biodegradability and tissue integration, secure handling, and low cost without compromised efficacy [11]. Freeze-dried skin allografts were among the first products introduced in mucogingival surgery. They were initially used as a replacement for FGG in combination with an apically positioned flap for the augmentation of keratinized tissue [12]. Subsequently, allogenic dermal substitutes initially developed for covering full-thickness burn wounds were introduced to increase keratinized tissue, cover exposed roots, deepen the vestibular fornix, and augment localized alveolar defects [13–15]. Unfortunately, the outcomes were associated with high shrinkage rates of the grafted areas and delicate clinical handling. Moreover, histology analysis indicated a significant structural difference in comparison to the gingival tissue [16]. To reduce scar retraction and enhance the healing process, xenogeneic (porcine) collagen matrix was developed and evaluated as a replacement for autogenous tissue to increase the width of keratinized tissue and cover gingival recessions [17–19]. The clinical evaluation indicated a sub- stantial enhancement of the keratinized tissue width with similar outcomes in comparison to the FGG [20–22]. Another porcine dermis-derived acellular matrix was also used for extensive keratinized tissue augmentation, resulting in some tissue contraction [23]. Recently, a novel, porous yet volume-stable matrix, consisting of slightly cross-linked reconstituted collagen fibres, has been introduced and shown to increase soft tissue volume similarly to the SCTG [24,25]. These biological scaffolds hold promise for reduction of morbidity, surgical time, and cost for future oral soft tissue regeneration treatments. How- ever, they are delivered in standardised shapes and dimensions and have to be tailored chair-side for each soft tissue defect, they do not reproduce precise inner tissue architecture of a particular oral site, and remain surgically demanding. A functionally and aesthetically satisfying solution for oral soft tissue augmentation thus remains a challenge. 3D printing technology could provide three solutions for a better adapted, customized graft: 1) a 3D printed graft based on the individual defect shape with the tissue-specific inner structure produced on demand in a centralized

facility; 2) a graft 3D printed chair-side based on the individual defect shape with the tissue-speciﬁc inner structure 3) a graft 3D printed chair-side directly into the defect (in situ), based on the individual defect shape with the tissue-speciﬁc inner structure.

4. 3D printing technology

3D printing allows the production of an individualized 3D object based on a material of choice and a speciﬁc computer-aided design (CAD). The process begins with the design of a 3D model, created by CAD software. The model is then converted into cross-sectional slices and sent to the 3D printer, which deposits layer after layer of the chosen material to produce the desired object. In the last decade, 3D printing technology has been broadly used in different medicalﬁelds including regenerative medicine, dentistry, production of anatomical models and surgical guides as well as drug formulations [3,4,26,27]. 3D printable scaffolds are used to build tissue models without or with cells allowing studying the pro- cesses of complex cellular interactions during tissue formation, matura- tion, and disease. Moreover, such tissue models are employed for drug screening and testing [28,29].

Two types of printing approaches have been established in the medical and dental fields: 1) indirect, where scaffolds with the desired structure are printed to be subsequently populated with cells, and 2) direct, where cells are co-printed within the scaffold. The latter has been named“bioprinting”. Hydrogels, in which cells reside for the printing purpose are named“bioinks”[30,31]. Hydrogels are hydrophilic polymers cross-linked to form porous matrices with high water content, high biocompatibility, and low toxicity [32]. To be suitable for 3D bioprinting, a hydrogel must be viscous enough for the printing process, yet not mechanically harm the cells. On the other hand, it has to retain the 3D shape and structure upon printing. Four leading 3D printing technologies employing hydrogels for soft tissues are extrusion, laser-assisted, inkjet bioprinting and stereolithography [33–35]. In extrusion bioprinting, pneumatic (pressure) or mechanical (plunger) force extrudesfilaments, which have to undergo fast gelation to retain the desired shape and structure. Laser-assisted bioprinting is based on a laser pulse that pro- duces local heating of a cell-containing solution causing the dropping of cells in an orderly manner on the other side of a platform/substrate. In inkjet bioprinting, a defined volume offluid (with or without cells) is jetted onto a platform to obtain a precise pattern. Droplets are deposited using either thermal or piezoelectric energy. Stereolithography printing is based on photocuring of light-sensitive polymers by precise light beaming. The major advantage of extrusion and inkjet techniques is the building of complex cell-laden biomimicking tissue equivalents using a multi-head approach with different cell types and biomaterials. The main disadvantage is that cells or bioactive molecules have to be in a liquid/semi-liquid state to allow deposition and subsequently solidify into the required structure [36]. The main advantages of stereolithography are high accuracy and construct complexity, while the major disadvantage remains a light source causing cell damage [33]. Due to the accurate positioning of different materials and/or cell types, 3D printing approach will offer distinct advantages: the production of precise geometries to perfectly fit any defect and faithful reproduction of the complex tissue architecture [4].

To mimic the dynamic nature of tissues, a novel approach, named 4D bioprinting has emerged, where time has been added to 3D bioprinting as the fourth dimension [37]. Based on either intrinsic signals or in response to external stimuli 3D printed construct would mature in time, resulting in a change of shape, structure and function. Similar to 3D printing, 4D printing also encompasses the approach of printing the biomaterial only or the biomaterial combined with cells [38]. 4D bioprinting relies on smart, responsive biomaterials capable of change when exposed to temperature, pH, humidity, electrical or magneticﬁelds, light, sound, or a combination thereof [38,39]. Additionally, changes at the cell level, such as response to coating/encapsulation, self-organization and/or new matrix deposition with concurrent biomaterial degradation can trigger

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changes leading to the production of a more dynamic tissue [37]. An important aspect, particular for 4D printing, is the necessary mathe- matical modelling of the anticipated biomaterial transformation or cellular responses [40]. With the future development of smart biomaterials, 4D bioprinting will add to the possibilities of reconstructing complex, heterogenous tissues, including oral mucosa.

5. Biomaterials used for 3D printing of soft tissues

Scaffolds or biomaterials provide the initial mechanical support to the tissue defect and allow for cell migration, adhesion, and differentiation to foster guided tissue regeneration. Hydrogels offer modifiable chemical composition, and adjustable mechanical and biodegradation properties [41,42]. Hydrogels biomaterials can be classified into naturally derived or synthetic. Natural biomaterials comprise agarose, alginate, collagen, gelatine, hyaluronic acid, chitosan, fibrin, cellulose and silk [41].

Biocompatibility, biodegradability, hydrophilicity, and resemblance to the native ECM are the signiﬁcant assets of natural materials, while feeble mechanical properties represent their main weakness [43]. Syn- thetic biomaterials comprise polylactide acid (PLA), polyglycolic acid (PGA), poly-lactic-co-glycolic acid (PLGA), polycaprolactone (PCL), methacrylated gelatine, Pluronic®-127 or polyethylene glycol (PEG), and have been used in soft tissue 3D printing approaches [44,45]. The main advantages of synthetic polymers are tailoring the mechanical properties and degradation kinetics by altering polymer structure. However, due to the lack of bioactive molecules, they are poor substrates for cell adhesion or migration [43]. To overcome the limitations of each group, different composite biopolymers have been developed and extensively studied [41,46]. Bioprintability of hydrogels used for soft tissue 3D printing is governed by their rheological properties and the targeted bioprinting technique [45]. A set of bioink requirements for extrusion-based, dro- plet-based or laser-based bioprinting techniques have been thoroughly explained and described [45].

To maintain the desired graft shape, hydrogels need to solidify. Cells can be either seeded after gelation to avoid harsh printing/curing conditions, or co-printed within the hydrogel (bioprinting). Bioink gelation can be achieved via physical cross-linking (ionic, hydrophobic initiated with temperature changes or “self-assembly”), chemical cross-linking providing better mechanical stability (glutaraldehyde, genipin, irradiation-induced photopolymerization), or enzymatic (thrombin) cross-linking [45]. Different material solidiﬁcation methods, namely optical polymerizing agents, UV, LED, and lasers are already present in dental clinics for hardening of restorative composites as well as bonding materials. These methods would allow smooth clinical translation for the chair-side soft-tissue solidiﬁcation providing that they are compatible with chosen hydrogels or cell-containing bioinks.

Bioinks for 3D printing can be also produced from decellularized matrix components [41]. Decellularized extracellular matrices have a major advantage: they contain all tissue components preserved in the correct proportions and the tissue-specific signalling factors which together provide an instructive environment for cell migration, proliferation and differentiation [47]. An extensive analysis of decellularized porcine skin-derived bioink, including concentration, viscosity, cross-linking degree, and cell viability lead to the production of precise, mouse fibroblasts-laden 3D printed skin-like constructs [48]. Bioink derived from decellularized porcine skin was bioprinted with human dermalfibroblasts for the production of skin-like grafts [49]. Decellu- larized porcine small intestinal submucosa slurry was developed and cryo-printed with rat dermal fibroblasts for skin regeneration [50].

Decellularization of gingiva tissue has already been achieved [51].

However, the production a printable bioink will require modiﬁcations and adjustments.

Hydrogels permit incorporation of instructive, bioactive agents [52].

The presence of signalling molecules can guide residing, host-tissue cells or provide delivered cells with the necessary instructions towards tissue regeneration. Biphasic PCL/hyaluronic acid-based hydrogel

incorporating bone morphogenic protein 2 (BMP2) enabled bone for- mationin vivo[53]. A heparin-collagen gel containing BMP2 supported by 3D printed bioceramic scaffold induced osteogenesis of dental pulp MSCin vitroand ectopic bone formation in a rat model [54]. A combination of BMP2 and vascular endothelial growth factor (VEGF) in a PLGA-PEG-PLGA hydrogel led to osteogenesisin vitroand bone regeneration in a rabbit model [55]. 3D printing of osteogenic-peptide laden tricalcium phosphate/PGA topped with PLA, and a collagen gel incorporating transforming growth factor beta 1 (TGFβ-1) allowed cartilage and bone-speciﬁc differentiation of bone marrow MSC for the regeneration of osteochondral defects [56]. The presence of VEGF in a collagen gel induced endothelial cells to form capillary-like structures [57].

Alginate hydrogel incorporating PRP, known to contain a cocktail of growth factors, has demonstrated improved endothelial and MSC proliferation and migration [58]. In another study, a gelatin-sulfonated silk composite scaffold with incorporatedﬁbroblast growth factor 2 (FGF-2) demonstrated improved host cell migration and superior wound healing, and dermal vascularization upon insertion in a full-thickness skin defect [59]. Development of such functionalized (instructive) biomaterials would facilitate regeneration based on patient residing cells and could prove particularly effective for oral soft tissue given the high prolifera- tive, migrating and healing capacities of gingivalﬁbroblasts [60].

6. Tissue engineering of oral mucosa and gingiva equivalents 6.1. Tissue engineering (TE) of gingiva, biomaterials and cell sources

Tissue engineering approach aims at rebuilding a functional tissue that could either replace or facilitate regeneration of the missing tissue.

The three pillars of TE combine biomimetic scaffolds as the initial structural support, cells as tissue masons, and bioactive molecules as the signal instructors [61]. Scaffolds allow the production of the new tissue by maintaining space while enticing host cell migration, proliferation, and differentiation. Bioactive scaffolds incorporate growth factors to direct cell fate. Cell-based treatments can be autologous, or allogeneic and can be delivered in suspension or seeded on a scaffold [62]. TE concept has been applied to establish 3D organotypic cultures resembling the natural gingiva for clinical as well as for research purposes. Due to many similarities in structure and function, the initial concepts heavily relied and overlapped with approaches employed for TE of skin [63]. An ideal full-thickness TE gingiva should contain 1) a supporting connective tissue, i.e. lamina propria containingfibroblasts within an ECM, mainly consisting of collagenfibers; 2) a continuous basement membrane which separates lamina propria from the epithelium, and 3) a stratified squamous epithelium containing densely packed keratinocytes that undergo differentiation as they move towards the surface. Oral mucosa/gingival TE construct can consist of either a monolayer keratinocyte sheet (one cell layer), or ideally, a bi-layer construct containing epithelial and dermal tissues to allow the formation of a continuous basement membrane. Initially, to obtain the epithelial layer, keratinocytes were cultured in cell sheets with a cell feeder layer [64]. Despite subsequent im- provements, the clinical application of epithelial layers demonstrated long production time, delicate handling, inconsistent engraftment, and wound retraction. The incorporation of dermal substitutes and fibroblasts provided the necessary support of lamina propria and led to the fabrication of TE gingiva/oral mucosa equivalents [63].

Biomaterials developed and used in gingiva TE during the past de- cades can be classiﬁed into 1) naturally derived such as acellular human dermis or amniotic membrane, 2) collagen-based, including combinations with chitosan, elastin or glycosaminoglycans, 3)ﬁbrin-based, 4) gelatine-based, including combinations with chitosan and hyaluronic acid, 5) synthetic such as polycaprolactone or polyglycolic acid, and included different combinations [65].

The main primary cell source used to construct oral mucosa for clinical application as well as for thein vitrostudies were primary keratinocytes andﬁbroblasts isolated from autologous biopsies. Thein vitro

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studies often used transformed cell lines for the sake of availability, reproducibility, and standardization. These cell lines, however, contain compromised genetic material, not offering proper physiological responses.

In clinics, TE gingiva based on patientﬁbroblasts and keratinocytes incorporated in aﬁbrin-base scaffold was successfully used to augment keratinized tissue around teeth [66]. Another approach combined acellular dermal matrix with patient keratinocytes to treat keratinized tissue defects [67]. The method has been subsequently upscaled for large (over 15 cm²) tissue defects [68]. Besides clinical applications, gingiva organotypic models represent invaluable tools to study oral mucosa biology and often replace animal studies for drug targeting, vaccination development, and testing of new therapeutics. In research, they are used to understand the physiological role of human oral mucosa barrier properties as well as different pathologies, including oral cancer, bacterial, and fungal infections. Finally, oral mucosa models are used for cytotox- icity and biocompatibility testing of oral health care products [65].

6.2. New advances in tissue engineering of oral mucosa and gingiva equivalents

Recent years have seen several advances regarding the TE fabrication and application of gingiva/oral mucosa models (Table 1). One aspect is addressing the necessity for vascularization. Using de-epithelized dermis as a scaffold, human gingival keratinocytes and fibroblasts were combined with human dermal microvascular endothelial cells to generate oral mucosa and evaluate the effects of irradiation-induced oral mucositis [69]. The same combination of cells was grown on a collagen membrane to produce a pre-vascularized oral mucosa equivalent with dense capillary structures able to develop into blood vessels [70]. Recently, a layer-by-layer technology to deposit nanofilms on the cell surface was developed and employed to generate vascularized oral mucosa equivalents [71]. Primary human gingivalfibroblasts were coated with gelatine andfibronectin and combined with: 1) gingiva-derived keratinocytes for keratinized oral mucosa, 2) oral mucosa keratinocytes for non-keratinized oral mucosa tissue, and 3) incorporated human umbilical vein endothelial cells (HUVEC) for capillary formation. Vascularized constructs containing lamina propria and the epithelial tissue with a functional basal membrane barrier function were successfully fabricated.

Another aspect is the need for data reproducibility, acquisition of necessary cell amounts, and elimination of individual variation. The

establishment of immortalized human oral epithelial andfibroblast cell lines answered those needs. Primary human keratinocytes transformed with E6/E7 human papillomavirus (HPV) genes were combined with collagen gel to successfully fabricated gingiva equivalents [72]. Spontane- ously immortalized human gingival keratinocytes were employed with primary gingivalfibroblasts in a collagen gel to produce an oral mucosa model for the assessment of bacterial infections and disease progression [73]. A step forward towards a more natural gingival equivalent was the use of gingival cells immortalized through the expression of Telomerase Reverse Transcriptase (TERT) and combined with a collagen gel [74]. This model was then employed to demonstrate how multibacterial oral biofilm promotes epithelial stratification and improves epithelial barrier function [75]. Thefinal aspect was to fabricate an organotypic model that comprised several tissues. Hence, a model comprising bone and oral mucosa has been produced, combining hydroxyapatite/tri-calcium-phosphate with rat osteosarcoma cell line for the bone part, and collagen gel with TERT-immortalized keratinocytes and human oral fibroblasts for the gingiva part [76,77].

While these gingival models allow biological and pathological research, as well as drug and health care products testing, they still fail to meet the clinical need, i.e., the perfectﬁt necessary for consistent functional and esthetical results. 3D printing approach could meet that challenge by allowing the production of various geometries to match any defect while mimicking tissue complexity via the precise positioning of different materials and/or cell types. Given the similarity between the gingiva and skin tissues, 3D printing approaches investigated for skin regeneration represent a good starting point in attempting 3D printing of gingiva constructs.

7. 3D printing approaches for skin regeneration 7.1. 3D printing of skin equivalents

Skin is the largest organ of the body with functions similar to gingiva:

protection against environmental challenges, infections, and mechanical stress [78]. The need for skin replacement/regeneration due to injuries, extensive burns, and different skin conditions prompted TE approaches for skin regeneration to rapidly adopt 3D printing technology. Due to the higher resemblance to the original skin architecture, bioprinted skin constructs are better suited for basic research, drug screening, chemical, and cosmetic testing. The skin consists of an epithelial layer (epidermis Table 1

Tissue engineered gingiva equivalents.

Tissue type Biomaterial Cell types Application Reference

Oral mucosa de-epithelized dermis (Euroskin) human oral keratinocytes, model for oral mucositis [69]

human oralﬁbroblasts,

human dermal microvascular endothelial cells

Oral mucosa collagen membrane Bio-Gide® human oral keratinocytes, TE mucosal equivalents [70]

human oralﬁbroblasts,

human dermal microvascular endothelial cells

Oral mucosa ﬁbronectin and gelatin cell coating human oral keratinocytes, TE mucosal and gingival

equivalents

[71]

human oralﬁbroblasts, human endothelial cells (HUVEC)

Gingival tissue collagen gel on inserts HPV immortalized human oral keratinocytes, organotypic gingival model [72]

HPV immortalized human oralﬁbroblasts Gingiva

equivalent

collagen gel spontaneously immortalized

human oral keratinocytes and

model for host-pathogen interactions

[73]

human oralﬁbroblasts Gingiva

equivalent

collagen gel TERT-immortalized human oral keratinocytes, organotypic gingival model [74]

TERT-immortalized human oralﬁbroblasts, HPV immortalized human oral keratinocytes, HPV immortalized human oralﬁbroblasts Gingiva

equivalent

collagen gel TERT immortalized human oral keratinocytes, model for host-microbiome [75]

TERT immortalized human oralﬁbroblasts interactions for epithelial barrier function

Gingiva&bone collagen gel hydroxyapatite/tri-calcium- phosphate

TERT immortalized keratinocytes human oralﬁbroblasts and rat osteosarcoma cell line

bone-oral mucosa model [76,77]

Abbreviations: HUVEC–human umbilical vein endothelial cells, HPV–human papilloma virus, TERT–telomerase reverse transcriptase

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made of keratinized stratiﬁed squamous epithelium) separated from the underlying connective tissue layer (dermis) by a basement membrane and the subcutaneous insulation (hypodermis) tissue [79].

Skin 3D printing most commonly employed approaches comprise bi- layer bioprinting of ﬁbroblasts and keratinocytes in various types of cross-linkable hydrogels [80–82]. Extrusion, inkjet, and laser-assisted bioprinting techniques were all employed in different studies [83].

Collagen type I, the main component of the skin ECM, was the predom- inantly used hydrogel, followed by alginate, gelatine, and blood de- rivatives such as plasma-derivedfibrin or blood plasma combined with collagen or alginate [82]. Cell types used were either keratinocyte and fibroblast cell lines (HaCaT and NIH3T3) or primary human skin cells. A skin construct of 100 cm² was printed in less than 35 min with an extrusion printer, a bioink consisting of human plasma, humanfibro- blasts, and keratinocytes obtained from skin biopsies [84]. Moreover, this skin construct efficiently grafted onto immunodeficient mice skin.

Excellent comprehensive reviews on skin bioprinting technologies are available in recent articles [78,82,83,85–88].

7.2. New advances in skin 3D printing

Steps towards a more natural skin bioprinted constructs were achieved with the fabrication of biomimetic skin which incorporated melanocytes or sweat glands. In one study, human dermalfibroblasts were first inkjet printed with cross-linkable collagen hydrogel, followed by printing human melanocytes andfinally keratinocytes. Air-liquid expo- sure resulted in keratinocyte stratification, yielding a freckle-like pig- mented skin constructs [89]. In a similar approach, the 3D printed skin construct showed a better-developed epidermis stratification and a continuous layer of the basement membrane when compared to the manually casted construct [90]. For sweat glands, which have low regenerative potential, 3D printing approaches could provide precise cues to trigger epidermal progenitor to differentiate towards sweat gland cell lineage. Extrusion bioprinting of mouse embryonic cells within alginate-gelatine hydrogel laden with mouse plantar dermis homoge- nates containing BMP4 resulted in glandular morphogenesis with a well-defined architectural design [91]. In another study, a similar approach was undertaken with epithelial progenitors isolated from mouse dorsal skin, and the constructs were transplanted into the burned mice paws. Only constructs that incorporated epidermal growth factor in addition to mouse plantar dermis developed sweat glands and most successfully regenerated burnt mouse skin [92]. In a recent study, a skin equivalent which not only reproduced skin morphology but also demonstrated barrier function and minimal retraction was successfully bioprinted [93]. To fabricate dermis, neonatal human dermalfibroblasts were printed within a hydrogel consisting of gelatin,fibrinogen, collagen type I and elastin. A thin layer of laminin and entactin was used for the basal layer and human keratinocytes were printed as a cell layer on top.

The barrier function was validated through penetration assays and electrical impedance.

The introduction of vascularization took 3D printed skin equivalents to another level. A combination of human dermal ﬁbroblasts, human endothelial cells and human pericytes in collagen type I hydrogel printed as dermis with human keratinocytes in collagen type I hydrogel for epidermis resulted in the formation of microvessels in the dermis and improved epidermal differentiationin vitro, and anastomosis with host vasculature leading to perfusion in a mouse skin wound model [94].

Bioink obtained from decellularized porcine skin was combined with human dermalﬁbroblasts, human epidermal keratinocytes, human adipose tissue-derived MSC, and endothelial progenitor cells, and employed with extrusion and inkjet printing techniques to fabricate a pre-vascularized skin patch [95]. Vasculogenesis was faster and more enhanced with the decellularized skin bioink compared to a standard collagen hydrogel. Moreover, the skin patch efﬁciently closed the wound via re-epithelialization, neovascularization, and anastomosis with the host blood network. Bioprinting of a three-layered skin construct

comprising epidermis, dermis and hypodermis usingfibrin, gelatin and hyaluronic acid as a bioink resulted in successful closure of the full-thickness skin wound in athymic mice [96]. Combination of human keratinocytes and melanocytes for epidermis, fibroblasts, dermal microvascular endothelial cells and follicle dermal papilla cells for dermis and adipocytes for hypodermis allowed regeneration of a dermis harbouring normal basket weave collagen organization, stratification of the epidermis and blood vessel formation.

The ideal clinical treatment of skin wounds would be a directly bioprinted skin patch. Two handheld printers have indeed been developed [97,98]. Skin Printer contains one syringe with human dermalfibroblasts withinfibrin/hyaluronic acid/collagen hydrogel for the dermis, keratinocytes withinfibrinogen/hyaluronic acid hydrogel for the epidermis and another syringe contains thrombin as a cross-linker. Controlled velocity/movement of the printer resulted in an exactflow rate of both solutions, creating sheetsin vitroas well as over the skin wound in mouse and pig skin wound healing models [98]. The second skin printing device contains a 3D wound scanner for collection of wound topography and subsequent guiding of print-heads. Using inkjet printing, the cartridge containing human fibroblasts within a fibrinogen/collagen hydrogel printed dermis immediately crosslinked with subsequent deposition of thrombin. Keratinocytes were next printed in the same way, on top. The approach was validated in a mouse and pig skin full-thickness wound healing models [97]. Furthermore, when compared to the currently employed cell-spraying technologies [99], bioprinted approach showed an accelerated wound healing with re-epithelialization and epidermal thickening.

To translate the knowledge gained with skin 3D printing for the bioprinting of gingiva, several differences between the two tissues have to be considered. The skin contains melanocytes and appendages such as hair follicles and sweat and sebaceous glands [83]. Additionally, while gingiva baths in saliva laden with lytic enzymes and microorganisms, the skin is exposed to air. Consequently, gingiva epithelium, although being stratified, does not contain a clearly defined stratum granulosum, and typically does not undergo cornification [100]. Significantly more living cell layers are found in gingiva epithelium compared to skin epidermis [79]. A critical difference between skin and oral mucosa is wound healing: oral mucosa wounds heal faster, present fewer infections and rarely form scars [101]. Intrinsic properties of the cells [60], their interactions with the surrounding tissue environment and the immune system together with better blood microcirculation could explain the superior healing properties of the oral mucosa [101]. Taken together, 3D printing of gingiva should present fewer hurdles due to the lack of additional cell types (e.g. melanocytes) and structures, namely different glands. On the other hand, thicker epithelium and denser microvascu- lature would need to be envisioned.

8. 3D printing approaches for vascular tissue regeneration 8.1. Gingiva vasculature

The primary role of vasculature is to supply tissues with oxygen and nutrients, simultaneously disposing of waste and carbon dioxide. Blood vessel formation, i.e., vasculogenesis, is a process where endothelial cells produce de novohollow capillaries [102]. Subsequent recruitment of perivascular mural cells (pericytes) and remodeling of the existing blood vessel networks result in a dense vascular plexus. Vascularization (angiogenesis) is a formation of new blood vessels, a complex process requiring interaction between various cells, the ECM and growth factors [102,103]. Vascularization is indispensable for proper regeneration as well as the integration of any TE construct [104]. Gingiva blood supply originates from supraperiosteal blood vessels, which represent terminal branches of several arteries [5]. These blood vessels form numerous branches to yield subepithelial plexus, which in turn forms thin capillary loops to supply connective tissue papillae of the oral epithelium. In the free gingiva, the supra-periosteal vessels connect with the vessels from

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the periodontal ligament and bone [7]. Thus thick, thin as well as branching and loop forming vessels are necessary for the reconstruction of gingiva vasculature. To obtain nutrient and oxygen supply and dispose of waste, cells within tissues have to be at a distance of 100–200μm from blood vessels and capillaries [103]. Hence, depending on the size of the construct and the time required for neo-vascularization, the implanted graft will survive or not. The presence of pre-formed blood vessels could thus enable improved tissue survival and integration upon implantation.

Although blood vessels range in diameter from 3 cm to a few microme- ters, microvessels (capillaries) are vital to maintaining tissue viability.

Moreover, their network organization is precisely oriented in the direc- tion of cells and ECM of a particular tissue [105].

8.2. 3D printing of vascularized structures

Recent advances in 3D bioprinting technology have enabled the production of complex vascularized structures. Bioprinting extrusion- inkjet- and laser-based and stereolithography techniques were combined with different biomaterials to print tubular structures. The ﬁrst approaches comprised the use of dissolvable/removable (sacriﬁcial) materials such as carbohydrate glass, Pluronic F127, gelatine, and agarose [106]. Later approaches developed procedures to print tubular structures directly. Recent advances in stereolithography approach also allowed biofabrication of tubular structures laden with viable cells [107] and of tissue constructs [108]. Some examples of studies where 3D printing was employed to produce vascularized tissue constructs are presented in Table 2. More extensive reviews can be found in recent publications [106,109].

Employing customized inkjet 3D printing technique, gelatine as a sacrificial material, and collagen as a bioink, afluidic channel network of different designs was obtained [110]. The viability of dermalfibroblasts, printed within collagen, proved dependent on the cell distance from the channels perfused with cell culture medium. Employing the same 3D printed collagen/gelatine approach with human umbilical vein endothelial cells (HUVEC) cultured under static and dynamic conditions, the same group demonstrated that fluidic vascular channel improved cell viability, while static culture allowed for angiogenic sprouting and branching [111]. In another study, bioprinting of cross-linked methacrylated gelatine (GelMa) blended with type I collagen resulted in a

capillary-like network with optimal rheological and shearing properties, allowing high viability of human bone marrow mesenchymal stem cells (MSC) and HUVEC [112]. Agarose, as a sacrificial material and GelMa laden with different cell types was employed to print channels mimicking vascular structures in shape (linear, branching and lattice) and diameter (ranging from 150 to 1000μm), leading to improved cell proliferation as well as osteogenic and endothelial differentiation [113]. To achieve the next level of tissue complexity, PDMS was printed as a structure to sur- round the tissue construct, Pluronic F127 as the sacrificial material, and GelMa was laden with dermalfibroblasts. Upon removal of Pluronic F127,fibroblasts populated GelMa, and the subsequently seeded HUVEC efficiently lined the microchannels [114]. In the same study, a four-layer construct was also produced by co-printing four hydrogels: PDMS, Plur- onic F127, and GelMa laden with human dermalfibroblasts or mouse 10T1/2sfibroblast cell line. HUVEC were again added upon Pluronic F127 removal, and the tissue construct demonstrated good cell distri- bution and high viability. To produce thicker constructs (>1 cm) that would sustain cell growth over more extended periods (>6 weeks), the same group fabricated a perfusion chip [115]. Pluronic F127 and thrombin were printed as cast for perfusable channels, while gelatine, fibrinogen, and MSC or dermalfibroblasts were cast over the printed network. After polymerization, Pluronic 127 was removed, and HUVEC seeded by perfusing the channels. The resulting construct allowed high cell viability over several weeks. This well-perfused network also allowed consistent delivery of differentiation factors, successfully inducing osteogenic differentiation of printed MSC. Another group achieved the fabrication of a thick tissue construct with a branching channel network and a dense cell population that successfully proliferated beyond two weeks [116]. Synthetic biomaterials, PLA and PVA, were used as sacrificial materials, and hepatocytes, printed within a cross-linked gelatine hydrogel, showed improved survival and proliferation in perfused constructs. As a step toward personalized medicine, human decellularized omental tissue was processed to behave as a thermoresponsive hydrogel, laden with omental derived iPSC differentiated into cardiomyocytes and co-printed (2 nozzles) with gelatine-laden iPSC differentiated into endothelial cells [117]. The obtained cardiac patches contractedin vitro and showed striation upon transplantation. To produce larger constructs, the same strategy was used to print in an alginate-based support material that could be subsequently easily removed. Based on digital design, a Table 2

3D printing of vascular tissues.

3D method

Biomaterial Cell types Tests performed Reference

InkJet collagen hydrogel gelatin (sacriﬁcial) human dermalﬁbroblasts hydrostatic pressure, cell viability [110]

InkJet collagen hydrogel gelatin (sacriﬁcial) HUVEC diffusional permeability barrier function, cell viability [111]

InkJet GelMa-collagen blend human bone marrow MSC HUVEC shear strain, viscosity cell viability [112]

Extrusion GelMa agarose (sacriﬁcial) mouse pre-osteoblasts (MC3T3), human endothelial cells (HUVEC)

elastic modulus, swelling mass transport, cell viability, proliferation and differentiation

[113]

Extrusion PDMS, Pluronic F127 (sacrificial), GelMa human dermalfibroblasts, HUVEC, 10T1/2 mousefibroblast cell line

shear strain, cell viability [114]

Extrusion Pluronic F127, thrombin (sacriﬁcial);

gelatin,ﬁbrinogen

human dermalﬁbroblasts, human bone marrow MSC, HUVEC

shear strain, perfusion, barrier function, cell viability and differentiation

[115]

Extrusion PLA, PVA (sacriﬁcial), crosslinked gelatin hepatocellular carcinoma HepG2 cells

elastic modulus

cell viabilitycontinuous perfusion

[116]

Extrusion gelatin (sacriﬁcial), personalized hydrogel from decellularized omentum

human iPSC-cardiomyocytes, human iPSC-endothelial cells

compression, cell viability, cell differentiation [117]

Extrusion alginate, GelMA, PEGTA HUVEC and human MSC shear strain, compressive strength,

cell viability and proliferation

[118]

Extrusion alginate, GelMA HUVEC and neonatal rat cardiomyocytes, human iPSC-cardiomyocytes

elastic modulus, perfusion, cell viability and differentiation [119]

DLP- μCOB

GM-HA, GelMA HUVEC, C3H/10T1/2 and HepG2 compressive strength, hydrogel degradation, cell viability, subcutaneous implantation anastomosis

[121]

Abbreviations: GelMA–gelatin methacrylate: a denatured collagen modiﬁed with photopolymerizable methacrylate groups, allowing covalent cross-linking with UV light, Pluronic F127-a hydrophobic poly(propylene oxide) PPO segment and two hydrophilic poly(ethylene oxide) PEO segments arranged in PEO-PPO-PEO conﬁg- uration, PDMS–poly(dimethyl siloxane), PLA–polylactic acid, PVA–polyvinyl alcohol, iPSC–induced pluripotent stem cells, PEGTA - poly(ethylene glycol)-tetra- acrylate, DLP -μ^COB–digital light processing based on microscale continuous optical bioprinting, GM-HA - glycidal methacrylate-hyaluronic acid, HUVEC–human umbilical vein endothelial cells

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small scale cellularized human heart with major blood vessels was successfully fabricated.

Several studies ventured into the direct fabrication of vascularized constructs by deposition of highly arranged perfusable vascular structures in a single step, thereby bypassing the necessity for a sacriﬁcial material. A 3-layer coaxial nozzle device was used to print the hydrogel consisting of GelMa, alginate, and polyethylene glycol-tetra-acrylate (PEGTA), creating perfusable vascular structures [118]. This biomaterial combination allowed for optimal rheological and mechanical properties and supported adhesion and proliferation of an MSC-HUVEC cell mixture. Bioink consisting of alginate and GelMA was extruded with HUVEC cells to produce multilayer microﬁbrous scaffolds onto which the rat neonatal or human iPSC-induced cardiomyocytes were seeded to generate the endothelial myocardial constructs [119]. Another study employed a different type of bioprinting technique, namely a rapid digital light processing (DLP) bioprinting method, which is based on microscale continuous optical bioprinting (μCOB) and relies on computer-aided photopolymerization [120]. DLP-μCOB, a fast and high-resolution technology, was used to print glycidal methacrylate-hyaluronic acid (GM-HA) or GelMa laden with either 10T1/2, HepG2 and HUVEC to fabricate a construct harbouring uniform or gradient channels [121]. The subcutaneous implantation of pre-vascularized constructs revealed not only the formation of a dense endothelial network but also anastomosis with the host blood vessels.

The advances in fabricating sophisticated vascular channels via 3D bioprinting, particularly those achieved for skin-like constructs, will un- doubtedly provide solid ground in the creation of pre-vascularized gingiva tissue.

9. The outlook: 3D printed gingiva for oral soft tissue augmentation

3D printing represents an appealing biofabrication technique for gingival soft tissue regeneration for several reasons. First, it offers fabrication of a predetermined shape of the graft that couldfit the defect with better accuracy and meet aesthetic demands. Second, due to its ability to precisely pattern a combination of appropriate biomaterials and living cells in pre-defined spatial locations, it can mimic the specific microarchitecture and structural organization of gingival tissue with more fidelity. This may prove particularly advantageous for proper positioning of differentially oriented collagen fibers within lamina propria, thereby offering optimal mechanical function. Third, a

combination of different cells types and the corresponding biomaterials with correct physiological cues could result in a more nature-like gingiva tissue (Fig. 3). Such models would also offer research tools to understand the physiology and pathology of oral soft tissues as well as more relevant means for screening drugs and chemical products.

Despite the immense progress achieved in recent years, uncovering ideal printable biomaterials capable of supporting and stimulating printed cells for gingiva development remains a major challenge. Current bioinks mainly rely on homogeneous biomaterials with several short- comings, such as insufficient mechanical properties and questionable recapitulation of the tissue-specific inner architecture. A gingiva-derived decellularized matrix may be considered and developed as a more appropriate bioink. The main advantage of decellularized natural ECM is the retention of the specific matrix composition in the correct proportions, thus providing an optimal environment for cell attachment, growth, and long-term function. This approach has been successfully demonstrated for adipose, cartilage, and heart tissues [122]. Moreover, tissue-specific response of human MSC to 3D printed decellularized cornea, heart, liver and skin has been recently shown [47].

For small defects, printing the correctly shaped graft with the appropriate tissue architecture may sufﬁce for patient cells ingrowth upon implantation. For larger defects, co-printing cells, ideally autologous, would prove indispensable. In both cases, vascularization remains of utmost importance. In bioprinting pre-vascularized gingiva grafts, the hierarchical order of the vascular tree, containing arterioles, venules, and capillaries should be respected. The bioprinting approaches recently developed for pre-vascularized skin constructs offer various possibilities that could be translated into the fabrication of pre-vascularized gingiva.

Theﬁnely tuned mechanical properties allowing the development of intact and perfusable microvessels would need to be combined with biodegradation rates concurred to the new matrix deposition. Consid- ering all the recent advances made in different ﬁelds: engineering, biomaterial sciences, cellular and developmental biology, successful printing of oral soft tissues may not be that far in the future.

10. Conclusion

Gingiva/oral mucosa augmentation is a daily challenge in dental clinics. Although autogenous tissue grafts give desired outcomes, their availability is limited. Moreover, the surgical procedure is painful and not without risks. The currently available graft substitutes do not offer long term satisfactory functional and esthetical results. Development of Fig. 3. 3D printing approach for fabrication of a more nature-like gingiva tissue. A combination of smart bioinks, ideally derived from decellularized tissues for the preservation of the tissue-specific matrix components, gingiva-specific cell types - gingival keratinocytes, gingival fibroblasts, and endothelial cells - together with the signalling molecules (epidermal growth factor - EGF,fibroblast growth factor-2 - FGF2, tumor growth factor beta - TGFβ, platelet derived growth factor - PDGF, vascular endothelial growth factor - VEGF) could be precisely patterned via extrusion, laser-assisted, and inkjet 3D or stereolithography bioprinting to mimic the complex gingiva tissue architecture.

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3D bioprinting approaches would offer fabrication of a perfectlyﬁtting patient-tailored shape of the graft, and a more nature-like gingiva/oral mucosa inner structure. This review explores recent advancements in the fabrication of oral mucosa/gingiva tissue-engineered constructs and outlines already developed 3D printing procedures for skin and blood vessels that combined could pave the way towards the development of 3D printing approaches for oral soft tissues regeneration.

Authors’contribution

D.N., S.S.S. and S.D. conceptualization. D.N. writing and editing.

D.N., S.D., L.M., M.M., I.S. and S.S.S. critical reviewing of the manuscript.

All authors approved the manuscript.

Funding

This research did not receive any speciﬁc grant from funding agencies in the public, commercial, or not-for-proﬁt sectors.

Declaration of competing interest

The authors declare that they have no known competing ﬁnancial interests or personal relationships that could have appeared to inﬂuence the work reported in this paper.

Acknowledgements

The authors are very grateful to Elena Brioschi (www.2spark.ch) for creating the illustrations.

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