Regenerative Approaches in Gingival Tissue Engineering

Abstract

Gingival tissue engineering aims to regenerate damaged or diseased gingival tissues by applying biomaterials, growth factors, and stem cells. This chapter explores advancements and strategies in gingival tissue engineering. It begins by introducing the goals and anatomy/physiology of the gingiva. Biomaterial selection and design for gingival scaffolds and delivery methods for bioactive molecules to stimulate tissue growth are discussed. Stem cells are highlighted for their role in gingival regeneration - their isolation, characterization, and differentiation. Strategies like cell-based approaches, scaffold-free techniques, and hybrids combining cells, scaffolds, and growth factors are outlined. Preclinical and clinical studies assessing treatment safety/efficacy and methods to evaluate outcomes are reviewed. Challenges around improving cell viability, integration, and function are examined. Future directions focus on addressing these challenges. Ethical considerations and regulatory aspects are addressed to ensure responsible translation into clinical practice. This chapter provides insights into the current state and prospects of regenerative approaches in gingival tissue engineering, including their potential to impact gingival disease treatment and oral health promotion.

1. Introduction

The gingiva is the mucosal tissue surrounding and supporting the teeth. It provides the first defense against plaque and a protective seal around teeth and bone as a keratinized epithelium adhered to highly vascular connective tissue. The gingiva encircles the cervical tooth portion in a collar-like fashion from the gingival margin to the mucogingival junction. It contains oral gingival epithelium, sulcular epithelium lining the gingival crevice, and junctional epithelium attaching to the tooth surface [1]. This epithelium shields the underlying connective tissue, comprising dense collagen bundles, fibroblasts, ground substances, and a rich blood supply. I, III, and V collagen types provide tensile strength and elasticity: cementum and alveolar bone anchor gingival collagen fibers to the tooth [2].

The healthy gingiva maintains vital oral functions. The gingival-tooth seal protects underlying tissues. The junctional epithelium forms a selective barrier, allowing crevicular fluid nutrients but excluding bacterial byproducts. Rich vascularity supports cellular components and the collagen matrix. Angiogenesis enables wound healing. Gingival fibroblasts synthesize matrix molecules, preserving tissue integrity. Host defenses provide microbial resistance, including fluid flow, antimicrobials, and immune cells. These features sustain standard gingival form and function [3].

However, gingiva is constantly at risk of inflammatory damage from plaque accumulation, trauma, and systemic conditions. Gingivitis, the mildest form, causes marginal gingival inflammation without tissue or bone loss. Severe chronic gingivitis can advance to periodontitis, with alveolar bone resorption and eventual tooth loss. Other afflictions include mucositis, gingival hyperplasia, and necrotizing ulcerative gingivitis. Complex cellular and extracellular changes underlie disease progression, including inflammatory cell infiltration, cytokine and protease release, collagen degradation, and disrupted epithelial sealing [4]. These alterations destroy the collagen matrix, compromise the protective niche, and lead to degeneration.

While conventional treatments such as scaling, root planning, and antibiotics can resolve inflammation, they cannot regenerate deficient gingival tissues. There is an unmet need for regenerative approaches to restoring form and function by stimulating cell proliferation, matrix synthesis, and revitalizing physiology. Tissue engineering strategies utilizing biomaterial scaffolds, cellular components, and biological factors offer immense promise for regenerating damaged or inadequate gingiva [5]. Upcoming sections will explore scaffolds, cell sources, bioactive molecules, and treatment modalities being developed to engineer functional gingival substitutes and stimulate endogenous regeneration.

Gingival tissue engineering aims to restore or replace damaged or lost gingival tissue, an integral component of oral health and esthetics. As part of broader regenerative dentistry efforts, substantial focus has targeted engineering gingival tissues, which play a pivotal role in maintaining dental structural integrity. Composed of epithelial and connective tissue, the gingiva functions as a biological barrier protecting the underlying alveolar bone and periodontal ligament. Aberrations in gingival tissues can enable harmful inflammation and eventual tooth loss. Traditional surgical treatments have limitations such as donor site morbidity and inconsistent outcomes, underscoring the need for more effective and reliable alternatives [6, 7].

Stem cells, particularly gingival mesenchymal stem cells (GMSCs), are critical for tissue regeneration owing to their self-renewal and multi-lineage differentiation capabilities. Scaffolds provide structural frameworks supporting cell attachment, proliferation, and differentiation using various natural and synthetic materials. Bioactive molecules such as growth factors and cytokines deliver signals guiding cell migration, proliferation, and differentiation to achieve desired tissue formation [8]. Emerging technologies, including 3D bioprinting and nanotechnology, enhance gingival engineering by enabling precise scaffold fabrication and controlled growth factor delivery for more efficient and effective tissue regeneration.

1.1 Regenerative approaches

Regenerative approaches in gingival tissue engineering can regenerate the entire gingival tissue complex, including the epithelium, connective tissue, and bone. Several different regenerative techniques have been investigated for the regeneration of gingival tissue [9]. These approaches can be broadly categorized into three groups:

• Cell-based approaches involve using cells to regenerate the gingival tissue. The cells can be either autogenous (derived from the patient’s body) or allogeneic (derived from a donor).

• Scaffold-based approaches involve using a scaffold to provide a structure for the growth of new gingival tissue. The scaffold can be made from various materials, including synthetic polymers, natural polymers, and metals.

• Growth factor-based approaches involve using growth factors to stimulate the growth of new gingival tissue. Growth factors are proteins that promote cell growth and differentiation.

2. Cell sources for gingival tissue engineering

Cell-based approaches, mainly stem cells, are among the most promising strategies for regenerating gingival tissue. These techniques have the potential to fully regenerate the gingival tissue complex, encompassing epithelium, connective tissue, and bone. Cells for these methods can either be autogenous, sourced from the patient’s body, or allogeneic, derived from donors [10]. Autogenous cells are often favored as they pose a reduced risk of rejection. Mesenchymal stem cells (MSCs) are the primary cell types employed in this endeavor [11]. Found in various tissues, including bone marrow, adipose tissue, and teeth, MSCs can differentiate into diverse cell types, such as epithelial cells, connective tissue cells, and bone cells. Beyond MSCs, other cells such as epithelial cells, fibroblasts, and endothelial cells have also been researched for their applicability in gingival tissue engineering [12]. Constructing gingival substitutes necessitates viable cell populations that can replicate native tissue structure and function. In this light, gingival fibroblasts, MSCs, and induced pluripotent stem cells are emerging as crucial cell sources [13]. This discussion will delve into these cell types and the crucial aspects surrounding their selection and application in gingival regeneration.

Stem cells, owing to their self-renewal and differentiation capacities, play a central role in addressing challenges in gingival tissue regeneration. These cellular strategies aim to promote new tissue growth and represent a revolution in regenerative therapies. Present studies are delving into the various stem cell types, their roles in tissue regeneration, and the remaining hurdles [14]. The domain offers immense potential for enhancing treatments for gingival tissue-related ailments.

2.1 Types of cells used in cell-based approaches

Various cell types and scaffolding materials facilitate tissue regeneration in gingival tissue engineering. Cells used in these approaches can be categorized as autogenous, derived from the patient’s body, or allogeneic, sourced from a donor [15]. Autogenous cells are generally preferred due to their lower risk of rejection. Mesenchymal stem cells (MSCs) are the most commonly used in tissues such as bone marrow and adipose tissue. These cells are prized for their ability to differentiate into multiple cell types, including epithelial and connective tissue cells [16]. Other cell types such as epithelial cells, fibroblasts, and endothelial cells are also explored for their potential in gingival tissue engineering. In addition to MSCs, dental tissues such as dental pulp and periodontal ligaments serve as sources for dental-derived mesenchymal stem cells (D-MSCs) [17]. Gingival mesenchymal stem cells (GMSCs) are increasingly preferred for their ease of isolation and robust proliferative capacity. Non-dental sources such as bone marrow and adipose tissue are also used, although their isolation is often more invasive [18]. Induced pluripotent stem cells (iPSCs), reprogrammed from adult somatic cells, offer a potentially limitless and ethically sound source of cells for gingival regeneration [19]. Overall, the field leverages a diverse range of cell types and materials, each with its advantages and challenges, to advance the science of gingival tissue engineering.

2.1.1 Gingival fibroblasts

Gingival fibroblasts are highly specialized cells responsible for synthesizing collagenous and elastic extracellular matrix components that provide the structural integrity of connective tissues. They also facilitate wound healing and tissue remodeling through cytokine secretion and matrix metalloproteinase expression [20]. Given their natural role in generating gingival tissues, gingival fibroblasts are logical candidates for cell-based regenerative therapies. Autologous gingival fibroblasts can be isolated from gingival tissue biopsies of the patient and expanded in culture. Allogeneic fibroblasts may also be sourced from tissue donations if autologous cells are limited [21]. Fibroblasts can be seeded into various natural and synthetic scaffold materials where they attach, migrate, proliferate, and deposit extracellular matrix. In vivo, implantation of fibroblast-seeded constructs in animal models has regenerated gingival tissue with reduced inflammation and collagen regeneration. Scaffolds populated with xenogeneic human gingival fibroblasts also integrated well when transplanted in canine models. A key advantage of gingival fibroblasts is their innate ability to recapitulate native tissue morphology and properties. However, their limited proliferative capacity hinders scale-up, which may be addressed by reprogramming to an induced pluripotent state. Obtaining autologous cells also requires additional biopsies [21]. Allogeneic sources can help overcome such constraints but may risk rejection. The highly specialized functional attributes make gingival fibroblasts a promising cell source for gingival tissue engineering [10].

2.1.2 Mesenchymal stem cells

Mesenchymal stem cells (MSCs) derived from bone marrow, adipose tissue, and other sources offer an alternative cell source for gingival regeneration. MSCs can self-renew and differentiate into various mesenchymal lineages, including gingival fibroblasts [16]. Sources such as bone marrow and adipose tissue are more abundant than gingiva and support more significant expansion. MSCs from these tissues have generated new collagen and accelerated wound closure in gingival defect models [18]. Their immunosuppressive properties and limited immunogenicity make allogeneic MSCs less likely to induce adverse immune reactions. However, MSCs still need appropriate cues to commit to the gingival fibroblast lineage and produce tissue-specific ECM. Without directed differentiation, they may generate mineralized bone or fat inadvertently. Genetic reprogramming using transcription factors or miRNA may help preferentially guide MSC fate toward gingival fibroblasts [18]. Overall, the wide availability and differentiation capacity make MSCs promising alternatives to gingival fibroblasts, contingent on providing proper microenvironmental signals.

2.1.3 Induced pluripotent stem cells

The recent advent of induced pluripotent stem cells (iPSCs) created by reprogramming adult somatic cells to an embryonic-like state has expanded cell source options. iPSCs can self-renew indefinitely and give rise to all three germ layers. Researchers have generated iPSCs from gingival fibroblasts and directed their differentiation into gingival mesenchymal progenitors capable of collagen and elastic fiber synthesis [22]. Patient-derived iPSCs can yield autologous cells, thereby minimizing immunogenic concerns. Their pluripotency also offers advantages over finite primary cells for scaling production. However, reprogramming is technically complex and resource intensive. Efficient differentiation and purification protocols are needed to convert iPSCs into gingival lineages while avoiding tumorigenic risks from residual undifferentiated cells [23]. The functional capabilities of iPSC-derived cells also remain to be fully characterized. With further standardization, iPSCs may provide a robust cellular platform for individualized gingival tissue engineering [24].

2.2 Cell source selection considerations

The choice of cell source for gingival tissue engineering warrants careful consideration of several criteria [25]:

• Tissue origin and native function

• Proliferative and differentiation capacity

• Ease of isolation and expansion

• Donor availability and immunogenicity

• Functional properties upon directed differentiation.

• Genetic and epigenetic stability

• Risk of uncontrolled growth

Ideally, the cell source should have an innate ability to form gingival structures and sufficient proliferative potential for scale-up, exhibit stable phenotype, and integrate with host tissues upon implantation without adverse effects [26]. Autologous sources are preferred when feasible to avoid immunological rejection. As each cell type has its advantages and limitations, combining cells at optimal ratios may help balance the desired regenerative attributes. Further research is still needed to characterize and compare the regenerative capacity of different cell populations for gingival tissue engineering. In summary, gingival fibroblasts, MSCs, and iPSCs each offer unique properties that could be harnessed to restore cellularity and tissue function in engineered gingival constructs. Matching project goals to appropriate cell sources and providing inductive scaffold environments will be vital to realizing the full regenerative potential of these cells and achieving optimal clinical outcomes [27].

2.3 Gene and cell-based approaches in gingival regeneration

Gene-based treatments represent an emerging paradigm in healthcare, utilizing genetic manipulation techniques to add, modify, or inhibit specific genes. This allows a patient’s cells to generate therapeutic substances, thereby overcoming the drawbacks of topical applications, such as limited duration of effectiveness [28]. Gene and cell-based therapies have revolutionized gingival regeneration, offering more effective and less invasive treatment alternatives [29]. Gene therapy involves the manipulation of genetic material within a person’s cells to treat or prevent disease. Gingival regeneration typically involves introducing genes that encode growth factors such as Platelet-Derived Growth Factor (PDGF) and Transforming Growth Factor-beta (TGF-β) to stimulate cell proliferation and matrix formation. Efficient gene delivery systems are crucial for the success of gene therapy. These systems are designed to introduce therapeutic genes into target cells, and their choice can significantly impact the treatment’s efficacy, safety, and duration. Various vectors, including viral vectors such as adenoviruses and non-viral vectors such as liposomes, are used for this purpose. Localized delivery methods, such as hydrogels or scaffolds, can also reduce systemic side effects [30].

Stem cells, particularly mesenchymal stem cells (MSCs), have shown promise as carriers for gene delivery and as active agents in cell-based therapies for gingival regeneration. These cells can differentiate into various cell types, including fibroblasts, essential for forming new gingival tissue [30]. Biodegradable scaffolds infused with MSCs can be placed in the area of gingival recession to provide a 3D matrix for cell attachment and growth, eventually leading to tissue regeneration [31]. Gene and cell-based therapies hold significant promise for improving the outcomes of gingival regeneration. Challenges such as optimizing the number of cells that can be effectively modified and potential risks such as viral recombination and immune reactions need to be addressed. However, rigorous clinical trials are needed to establish their efficacy and safety, and ethical considerations also play a crucial role in their widespread adoption [32]. As the field progresses, interdisciplinary collaboration among geneticists, cell biologists, and periodontists will be essential for successfully implementing these advanced therapies [33].

2.4 Delivery methods

Various methods are employed for delivering cells and scaffolds to the defect site in gingival tissue engineering. These include direct implantation, where cells and scaffolds are directly placed into the affected area, and in vitro cell culture, where cells are cultured in a lab setting before being implanted [15]. Another approach is ex vivo cell expansion, in which cells are cultured and expanded in vitro, then combined with a scaffold for implantation. While significant strides have been made in cell-based approaches, several limitations remain. One of the primary concerns is obtaining a sufficient number of cells for transplantation.

Additionally, there is a pressing need to develop scaffolds that meet multiple criteria, including biocompatibility, biodegradability, and mechanical strength. The efficiency and safety of delivery methods also require further refinement to ensure the successful transplantation and integration of cells into the host tissue [34]. Overall, these challenges underscore the complexities involved in advancing gingival tissue engineering.

Cell-based approaches in gingival tissue engineering are still nascent but show promising potential for revolutionizing the treatment of periodontal diseases and other conditions affecting gingival tissue [35]. While considerable progress has been made, challenges persist, including cell sourcing, safety, and ethical considerations. Maintaining cell viability during culture and post-implantation is another hurdle, as is directing stem cell differentiation into specific cell types in a controlled manner. Future research is expected to focus on developing new cell sources, scaffolds, and delivery methods and improving the survival and integration of transplanted cells [36]. Advances in cell biology, scaffold design, and biomaterials are likely to address existing challenges. Standardized protocols for cell handling and application are also anticipated to emerge. Despite these challenges, stem cells have significantly impacted the field with their remarkable regenerative capacity. As our understanding of stem cell biology, gene regulation, and tissue-specific signaling pathways deepens, the scope for refining stem cell-based approaches in gingival regeneration expands [37]. Issues such as optimizing cell sources, cell proliferation, differentiation conditions, and scaling up for clinical applications remain active research areas [36]. The future of gingival tissue engineering is promising, with stem cells offering a revolutionary paradigm. Continued research into the complexities of stem cell biology and their interactions with biomaterials and bioactive molecules are crucial for realizing their full potential in clinical applications.

3. Scaffolds for gingival tissue engineering

In gingival tissue engineering, scaffolds are pivotal, offering structural support and environmental cues to direct functional tissue regeneration. Various biomaterials have been explored, including natural polymers, synthetic polymers, and extracellular matrix (ECM)-derived scaffolds. The choice of scaffold material hinges on several factors, notably its mechanical strength, biocompatibility, and biodegradability [38]. Scaffolds mimic the natural gingival tissue’s extracellular matrix (ECM), providing a three-dimensional framework for cell attachment, proliferation, migration, and differentiation. While cost-effective and easy to fabricate, synthetic polylactic acid (PLA) and polyglycolic acid (PGA) can sometimes raise concerns about biocompatibility. On the other hand, natural polymers derived from biological sources, such as collagen, fibrin, and hyaluronic acid, are more biocompatible, albeit potentially harder and pricier to process. Though highly biocompatible, metals are the most expensive and technically challenging to work with [39, 40].

Ideal scaffold criteria encompass biocompatibility, ensuring they are non-toxic and non-inflammatory; biodegradability, allowing them to be absorbed naturally over time; robust mechanical strength; high porosity for cell infiltration and nutrient diffusion; and functionalized surface chemistry promoting cell adhesion and differentiation [41]. Technological advances, notably electrospinning, and 3D printing, have transformed scaffold design and fabrication. These techniques offer unprecedented control over complex structures, closely replicating the natural ECM. Scaffold success also depends on its tailoring to the defect site’s specifics and the scaffold’s ability to foster a conducive environment for cell growth, differentiation, and potential controlled release of growth factors [42]. Clinical trials have already spotlighted promising scaffold-based strategies. For instance, a collagen and silk-based scaffold has shown its merit in treating periodontal disease. Another promising approach uses growth factor-loaded scaffolds, notably improving gingival tissue regeneration in gingival recession cases [43]. As this field evolves, research is bound to delve deeper into optimizing scaffold materials and designs.

Selecting the proper biomaterials is quintessential in gingival tissue engineering. Biomaterials, as scaffolds, support tissue regeneration by fostering cell attachment, proliferation, and differentiation [44]. Essential for biomaterial selection is replicating the native tissue’s mechanical environment. It should also permit tissue in-growth and nutrient diffusion, with its degradation rate matching tissue regeneration, ensuring the scaffold’s resorption as new tissue forms [45]. Moreover, all biomaterials must conform to regulatory bodies’ rigorous safety and biocompatibility standards. With advances in nanotechnology and a better grasp of cell-material interactions, the future in this field looks promising, aiming to fine-tune these materials for predictable outcomes in gingival tissue engineering [46].

In gingival tissue engineering, scaffold-based strategies are paramount. These scaffolds, essentially frameworks, are designed to support the growth and regeneration of new gingival tissue. The selection of the appropriate scaffold material takes into account various criteria, including but not limited to its mechanical resilience, compatibility with living tissues (biocompatibility), and its ability to degrade over time (biodegradability) [47]. The essence of scaffolds lies in their ability to emulate the extracellular matrix (ECM) of the native gingival tissue. The ECM is crucial as it presents a three-dimensional architecture that dictates cell behavior, ensuring cell adhesion, proliferation, migration, and eventual differentiation. The ultimate goal, driven by this replication, is the development of functional gingival tissue. In terms of materials, natural ones such as collagen, fibrin, and hyaluronic acid are favored for their bioactivity and biocompatibility. However, they need to be more mechanically robust. Conversely, synthetic counterparts such as polylactic acid (PLA) and polyglycolic acid (PGA) can be tailored for particular mechanical attributes. While these are generally non-immunogenic, they might not offer the natural bioactivity seen in organic materials [48].

Diving deeper into the materials, we notice a spectrum of choices, each with unique advantages and challenges. Synthetic polymers, for instance, are often the go-to materials due to their affordability and ease of production. Yet, there might be concerns over their biocompatibility. On the other hand, natural polymers are obtained from biological entities such as collagen, silk, and alginate. While they score high on the biocompatibility scale, their production may be intricate and pricier. Despite their unmatched biocompatibility, metals come with a hefty price tag and demand advanced technical know-how. When zeroing in on the perfect material for gingival tissue regeneration, considerations span various domains, including cost implications, fabrication simplicity, and biocompatibility, ensuring a holistic approach to tissue engineering [49].

3.1 Natural biomaterials

Derived from natural sources, these polymers are notable for their inherent bioactivity and biocompatibility attributes primarily due to their composition, which often mirrors the native ECM components [50]. The vast array of natural biomaterials, collagen, chitosan, hyaluronic acid, alginate, silk, and cellulose has been rigorously studied as potential scaffolds for gingival tissue engineering. Such materials offer the dual advantages of biocompatibility and biodegradability and closely resemble the natural extracellular matrix (ECM), which fosters enhanced cellular interactions [51]. Yet, they are not without their challenges. The variability from one batch to another can be a significant concern, potentially compromising reproducibility. Moreover, these biomaterials might still elicit immune responses in specific scenarios despite their natural origin. Such considerations have inevitably driven research toward synthetic alternatives, offering more consistency and controlled properties [52].

Collagen: Collagen is the predominant ECM protein in gingival connective tissues, making it a logical scaffold choice. Type I and III collagens are the major isoforms, providing tensile strength and elasticity. Collagen scaffolds can be fabricated by freezing reconstituted collagen suspensions into porous sponges, which support gingival fibroblast attachment, proliferation, and matrix deposition [53]. Injectable collagen hydrogels that gel in situ have also been applied for gingival augmentation. Chemically crosslinked collagen sponges exhibited slower degradation and supported superior tissue regeneration compared to unmodified collagen in preclinical models [54]. Composite collagen-chitosan scaffolds also enhanced structural integrity. The excellent biocompatibility, degradability, and ability to promote cell migration and matrix remodeling make collagen a versatile scaffold material for gingival engineering.

Chitosan: Chitosan is a natural polysaccharide derived from chitin with structural similarity to glycosaminoglycans in ECM. It contains reactive amine groups amenable to chemical modification and crosslinking into porous scaffolds using freezing/lyophilization or particulate leaching techniques. Chitosan supports gingival fibroblast adhesion and matrix synthesis while having mucoadhesive and hemostatic properties beneficial for wound healing [55]. Composite chitosan-collagen scaffolds promoted gingival tissue regeneration better than either component alone in vivo. Ultrafine chitosan fibers produced by electrospinning also facilitated gingival cell proliferation. The combination of tunable mechanical properties, biocompatibility, and bioactivity make chitosan a promising biomaterial for gingival scaffolds [55].

Hyaluronic Acid: Hyaluronic acid (HA) is a major ECM component in connective tissues, including the gingiva. Crosslinked HA hydrogels support fibroblast encapsulation and migration while reducing inflammation and scarring. Injectable HA hydrogels containing gingival fibroblasts regenerated oral mucosal defects better than collagen controls. Thiol-modified HA hydrogels also enhanced gingival fibroblast adhesion, migration, and matrix deposition. Composite HA-gelatin hydrogels exhibited shear-thinning behavior favorable for injection [56]. Tuning the stability and mechanics of HA-based hydrogels holds promise for gingival scaffold development.

Alginate: Alginate is a linear polysaccharide derived from seaweed that forms hydrogels upon reacting with divalent cations like calcium. Injectable alginate gels containing RGD peptides facilitated gingival fibroblast adhesion and proliferation. Reinforcing alginate scaffolds with collagen nanofibers enhanced their stiffness and elasticity. Alginate microbeads have also been applied as cell carriers for gingival tissue engineering. The mild gelation conditions, tunable porosity, and hydrophilicity make alginate attractive as an injectable scaffold material [57].

Silk Fibroin: Silk fibroin protein extracted from Bombyx mori silkworm cocoons can be fabricated into scaffolds using solvent casting/particulate leaching, electrospinning, or 3D printing techniques [58]. Silk hydrogels and porous sponges supported gingival fibroblast attachment, proliferation, and collagen synthesis. Patterned silk films with micro-grooved surface topography enhanced gingival cell alignment and matrix deposition along the groove direction. The robust mechanical properties, adjustable degradation rates, flexibility in processing, and excellent biocompatibility make silk fibroin a promising scaffolding material for gingival tissue engineering.

Bacterial Cellulose: Bacterial cellulose produced by Acetobacter species has emerged as an appealing scaffolding material given its nanofibrous structure similar to native ECM. It supported superior proliferation of gingival fibroblasts compared to plant-derived cellulose [59]. The highly hydrated porous network promoted cell ingrowth while the mechanical integrity maintained space for tissue regeneration. Bacterial cellulose scaffolds implanted in gingival defects accelerated wound healing compared to traditional dressings. The combination of porosity, hydrophilicity, and bioactivity highlights the potential of bacterial cellulose for engineering gingival substitutes.

3.2 Synthetic biomaterials

Due to their distinct advantages, synthetic polymers have emerged as attractive alternatives in gingival tissue engineering. Their tunable physical properties, ability to control degradation kinetics, and reproducibility in their fabrication make them stand out. Among the gamut of synthetic polymers, polyesters such as polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA), and their co-polymer poly(lactide-co-glycolide) (PLGA) have been prominently spotlighted [60]. These materials, particularly PGA, PLA, and PLGA, have seen considerable adoption because of their precision in tailoring mechanical properties and degradation rates. This control provides a customization capability that natural biomaterials often cannot match [61]. However, one caveat with synthetic materials is their inherent inertness. This can necessitate further surface modifications to bolster cellular interactions and promote optimal tissue integration [62].

PLGA: PLGA is FDA-approved for various drug delivery and medical device applications. Highly porous PLGA sponges fabricated by solvent casting/particulate leaching supported gingival fibroblast adhesion, proliferation, and infiltration [63]. PLGA microspheres encapsulating bioactive factors have been incorporated in gingival scaffolds to achieve controlled growth factor delivery. The acidic degradation byproducts of PLGA can lower local pH and cause tissue irritation. Copolymerizing with basic monomers like polyethylene glycol (PEG) to form PLGA-PEG-PLGA triblock polymers helps mitigate this issue. Overall, the tunable degradation, processability, and FDA approval make PLGA a popular synthetic scaffold material [64].

PCL: PCL is more hydrophobic and slower degrading than PLGA due to its higher crystallinity. Highly porous PCL scaffolds fabricated via electrospinning or fused deposition modeling/3D printing facilitated gingival fibroblast ingrowth. Composite PCL-gelatin and PCL-chitosan scaffolds improved hydrophilicity for better cell interaction compared to pure PCL. PCL’s slower degradation provides longer-term scaffolding but can delay tissue remodeling [65]. Strategies to increase hydrophilicity and introduce degradation via copolymerization continue to expand PCL scaffold design options.

PLA and PGA: PLA and PGA degrade via bulk hydrolysis to non-toxic lactic and glycolic acid metabolites. Pure PGA degrades more rapidly than PLA and is mechanically brittle. While PGA alone had limited tissue regenerative capacity, composite PGA-chitosan fibrous scaffolds improved gingival cell infiltration and matrix deposition. Reinforcing PLA with collagen nanofibers enhanced scaffold stiffness, elasticity, and cell compatibility for gingival applications [66]. Though less commonly applied than PLGA, PLA and PGA warrant further exploration for gingival scaffold engineering. The tunable mechanical properties, degradation kinetics, and processability make synthetic polyesters versatile scaffolding materials. However, their lack of bioactivity necessitates modification with ECM components or bioactive signals to improve gingival cell interactions [45].

3.3 ECM-derived scaffolds

Decellularized ECM retains composition and architecture resembling native tissues while removing cellular antigens. Standard decellularization methods include physical disruption, chemical detergents, and enzymatic digestion [67]. Decellularized dermal matrices supported gingival fibroblast repopulation and new matrix deposition in vitro. Porcine small intestinal submucosa ECM scaffolds also clinically facilitated gingival soft tissue regeneration with reduced pain compared to free gingival grafts [68]. Xenogeneic ECM from animal tissues carries the risk of immune rejection and disease transmission. Allogenic human gingival ECM has been explored as an alternative, but sourcing sufficient donor tissue is challenging. While ECM scaffolds recapitulate the native microenvironment, standardized decellularization and sterilization protocols are needed to ensure the safety and retention of structural integrity.

3.4 Design considerations for gingival scaffolds

Various natural, synthetic, and ECM-derived biomaterials have shown promise for gingival tissue engineering. However, rational scaffold design requires considering critical anatomical and functional requirements [69]:

Biocompatibility and Bioactivity: The scaffold must support gingival cell adhesion, migration, proliferation, differentiation, and matrix deposition without eliciting inflammation or toxicity. Natural polymers generally exhibit intrinsic bioactivity, while synthetic scaffolds require ECM-mimetic modifications to enable cell interactions.

Mechanical Properties: The scaffold should provide adequate stiffness and elasticity to maintain space for gingival tissue regeneration under masticatory forces. Yet, excessive rigidity impedes functional integration, necessitating careful modulation of stiffness [70]. Reinforcing weak hydrogels with stiffer nanofibers or blending synthetic and natural polymers helps achieve optimal mechanics.

Degradation Rate: Scaffold degradation must match the rate of tissue regeneration to provide temporary support without hindering long-term remodeling. Synthetic polyesters allow tuning degradation from weeks to months by varying molecular weight and crystallinity. Composite scaffolds with natural polymers help balance degradation.

Porosity and Interconnectivity: The scaffold architecture should facilitate cell infiltration and matrix deposition throughout the construct thickness while retaining space for vascular integration. Highly porous scaffolds with interconnected channels support better cell penetration and tissue in-growth.

Injectability and Gelation: For minimally invasive delivery, scaffolds that gel in situ upon injection are ideal for filling irregular defect geometries. Shear-thinning hydrogels that liquefy under shear stress but re-gel at rest are promising injectable scaffold candidates [71]. Scaffold design involves navigating tradeoffs between competing parameters to meet functional requirements for gingival regeneration. Blending synthetic and natural polymers at optimal ratios, advanced processing methods to generate porous interconnected networks and strategic biofunctionalization offer promising directions to engineer tailored gingival tissue scaffolds for regenerative applications [72].

4. Bioactive molecules for gingival regeneration

In gingival tissue engineering, scaffolds are frequently modified to include bioactive molecules such as growth factors, peptides, or cytokines to enhance their regenerative capabilities. These modifications provide not only a mechanical framework but also a biochemically active environment that is conducive to tissue regeneration. Strategies for incorporating these bioactive molecules often involve encapsulating micro- or nanoparticles, allowing for sustained release over time. Bioactive molecules serve a pivotal role in the orchestration of tissue regeneration. They act as critical communicators and coordinators, directing essential cellular processes such as proliferation, migration, and differentiation [73]. These molecules are indispensable in controlling the complex interplay of cellular activities required for the successful regeneration of gingival tissue. Bioactive molecules are integral components in gingival tissue engineering, serving as regulators that guide cell behavior and tissue development. Their inclusion in scaffold designs and other delivery systems enhances the potential for successful tissue regeneration, although challenges in their optimal application and sustained release remain to be addressed [43].

Types of Bioactive Molecules: Growth factors and cytokines are the most commonly used bioactive molecules in gingival tissue regeneration. Growth factors such as epidermal growth factor (EGF), fibroblast growth factors (FGFs), and platelet-derived growth factor (PDGF) have a range of beneficial effects. They promote cell proliferation and migration, stimulate the formation of new blood vessels (angiogenesis), and can even influence the differentiation of cells into specialized types [74]. For instance, FGF-2 is known to enhance the proliferation of gingival fibroblasts and keratinocytes, which are essential cells for the regeneration of gingival tissue. On the other hand, cytokines such as interleukins and tumor necrosis factors primarily serve to modulate immune responses but also have a role in influencing cell behavior and tissue regeneration. They are critical in the intricate cellular interactions that control inflammation and wound healing. Both growth factors and cytokines are vital bioactive molecules in gingival tissue engineering. While growth factors are more directly involved in stimulating tissue growth and development, cytokines have a broader role, including the modulation of immune responses and facilitating wound healing [75]. Both molecules contribute to the complex orchestration of cellular activities required for successful tissue regeneration.

Mechanisms of Action and Delivery of Bioactive Molecules: Bioactive molecules bind to specific receptors on target cells, initiating a series of intracellular signaling cascades that influence various aspects of cell behavior, such as proliferation, migration, and differentiation. These molecules also play a vital role in angiogenesis, forming new blood vessels essential for delivering nutrients and oxygen to newly forming tissues [76]. Additionally, bioactive molecules can modulate immune responses, aiding inflammation control and minimizing excessive scar formation. Delivering these bioactive molecules to the site requiring gingival tissue regeneration is critical to their effectiveness. Various strategies have been devised for this purpose, most commonly involving their incorporation into biocompatible scaffolds. These scaffolds can be engineered to release the bioactive molecules in a controlled and sustained manner, ensuring their continuous presence at the site of tissue regeneration [77]. This approach allows optimal use of these molecules in stimulating and guiding the regenerative process.

While bioactive molecules play a pivotal role in gingival tissue regeneration, their application comes with challenges. One of the primary concerns is the stability of these molecules, as they often have a short half-life and can degrade quickly. Another issue is potential side effects, mainly when administered in high doses. Controlling the release of these molecules in a targeted and sustained manner is also a complex task [78]. The financial burden of producing these molecules, especially growth factors, can significantly hinder their widespread use. Despite these challenges, the future of bioactive molecules in gingival tissue engineering looks promising. Research will likely focus on finding more stable and potent molecules, developing advanced delivery systems, and investigating ways to boost the body’s production of these crucial agents. As our understanding of bioactive molecules continues to grow, bolstered by technological advancements, their role in guiding and enhancing the regeneration of complex tissues like the gingiva is expected to expand [78]. Their ability to control cellular behavior makes them indispensable in the ongoing quest for effective gingival tissue regeneration.

4.1 Growth factors

Growth factors represent a promising avenue for gingival tissue regeneration. Essential proteins such as platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), and fibroblast growth factor (FGF) are instrumental in cell growth and differentiation [79]. As essential mediators, these growth factors regulate cellular processes, including cell migration, proliferation, and extracellular matrix deposition [80]. However, challenges arise, such as dose optimization, temporal release kinetics, and potential unintended effects. The stability of these molecules, particularly when integrated into biomaterial scaffolds, warrants further investigation. Advanced tools, like CRISPR for gene editing and innovative drug delivery systems, hint at more accurate applications of these bioactive agents. Despite the potential of growth factor-based methods in gingival tissue engineering, rigorous research and verification are indispensable. Pursuing knowledge in this domain is vital to bringing these bioactive agents from the laboratory to the clinic, setting the stage for the future of gingival regenerative therapies. As polypeptide signaling molecules, growth factors influence cellular functions such as proliferation, migration, differentiation, and extracellular matrix synthesis. They are pivotal during embryonic development, wound healing, and tissue maintenance [81]. Their powerful impact on cellular activities has driven extensive research on their application in regenerative treatments, particularly in gingival and periodontal regions.

Epidermal Growth Factor (EGF): EGF was one of the first growth factors discovered with its isolation from mouse submaxillary glands in 1962. It binds to the EGF receptor (EGFR) on cell surfaces to activate intracellular signaling cascades that stimulate epithelial cell proliferation, migration, and differentiation. EGF has been shown to enhance gingival fibroblast proliferation and migration in vitro and support re-epithelialization in gingival wound healing models. Controlled delivery of EGF using collagen sponges or hydrogels demonstrated significant gingival tissue regeneration and pocket-depth reduction in beagle dogs with chronic periodontitis [82]. EGF’s mitogenic and mitogenic effects on gingival keratinocytes and fibroblasts make it a promising agent for gingival tissue engineering approaches. However, the potent stimulatory effects of EGF also raise concerns about potential excessive tissue growth, requiring careful dose optimization [83].

Fibroblast Growth Factors (FGFs): The FGF family encompasses 22 members, each binding to one of the four FGF tyrosine kinase receptors. Their primary roles include regulating angiogenesis, fibroblast activation, extracellular matrix synthesis, and stem cell renewal. FGF-2, FGF-4, FGF-7, and FGF-10 have been critically assessed for their potential in periodontal regeneration. FGF-2, in particular, has been highlighted in preclinical gingival wound healing models for its ability to promote granulation tissue formation, angiogenesis, and collagen synthesis. When FGF-2 was released using gelatin hydrogels, there was a marked enhancement in gingival tissue regeneration in beagle dogs with chronic periodontitis, as Ward [84] reported. Additionally, Phase I/II human trials have pointed to the potential benefits of FGF-2 therapy in reducing gingival recession. It is noteworthy, however, that FGF-2’s pleiotropic effects can be double-edged; aberrant signaling might trigger uncontrolled angiogenesis or hyperproliferation. This underscores the importance of carefully optimizing both the delivery system and dosage. FGF-2 stands out in its promise to promote cell proliferation and angiogenesis. Its interaction with receptor tyrosine kinases sets off a series of intracellular events leading to cellular activation [85, 86]. Given its potential, it has been thoroughly explored in both in vitro and animal studies, where it has been shown to boost fibroblast proliferation, playing a pivotal role in wound closure angiogenesis and positioning itself as a prime candidate for gingival regeneration.

Transforming Growth Factor-beta (TGF-β): The TGF-β superfamily in mammals consists of three isoforms: TGF-β1, TGF-β2, and TGF-β3. These factors operate via transmembrane serine/threonine kinase receptors, orchestrating cellular processes such as proliferation, differentiation, migration, and apoptosis. TGF-β has an indispensable role in wound healing. It galvanizes fibroblast activation, angiogenesis, extracellular matrix deposition, and fine-tunes inflammatory responses. Research suggests that exogenous TGF-β can expedite gingival wound closure, amplify collagen production, and curtail inflammation. Yet, its potential drawbacks, such as hypertrophic scarring and fibrosis, emphasize the necessity of precise, localized delivery in optimal doses [87]. The engineering of controlled release systems tailored for TGF-β is a burgeoning field in gingival tissue engineering. Marconi et al. [88] underscored that this multifunctional cytokine, specifically in the gingival tissue milieu, stimulates fibroblast differentiation and boosts extracellular matrix synthesis, with collagen being a pivotal byproduct.

Platelet-Derived Growth Factor (PDGF): Activated platelets release PDGF during the clotting process and play a pivotal role in tissue repair and wound healing. It encourages the proliferation and sustenance of fibroblasts, smooth muscle cells, and progenitor cells. Clinical studies and trials, including a notable Phase I/II trial, have evidenced the efficacy of locally applied PDGF in gingival regeneration. In this trial, the controlled release of PDGF from a collagen wound dressing notably enhanced gingival augmentation in patients with marked gingival recession, outperforming the surgery results alone. Moreover, PDGF has been integrated into bone graft materials to boost periodontal tissue regeneration [89]. Its ability to stimulate cell proliferation and angiogenesis underpins its utilization in gingival tissue regeneration. The interaction of PDGF with its receptor activates a series of intracellular pathways, driving the mobilization and activation of essential cells for tissue repair. Further research is essential to explore the potential synergistic effects of PDGF with other growth factors or cytokines.

Vascular Endothelial Growth Factor (VEGF): VEGF plays an instrumental role in angiogenesis, forming new blood vessels from pre-existing vasculature. In gingival regeneration, adequate blood supply is crucial for providing essential nutrients and facilitating the removal of waste products. VEGF, therefore, emerges as a key factor in enhancing the success rate of tissue-engineered constructs [90].

Bone Morphogenetic Proteins (BMPs): While primarily associated with bone regeneration, some BMPs, like BMP-7, have positively affected soft tissue healing, including gingival tissue [82]. They promote cellular differentiation and are often used with other growth factors for synergistic effects.

4.1.1 Delivery methods for growth factors

Various methods are available to deliver growth factors to gingival tissue engineering defect sites. These include direct injection, where growth factors are directly administered into the affected area; scaffold-based delivery, where growth factors are loaded onto a biocompatible scaffold that is then implanted; and gene therapy, which involves delivering genes encoding for growth factors, allowing for their sustained release over time [79, 91]. However, growth factor-based approaches come with their own set of challenges. One significant issue is the short half-life of growth factors in the body, necessitating frequent administration or higher doses for effective treatment. Additionally, there is the risk of side effects such as inflammation or other complications, which must be carefully managed. The field is still in its infancy but shows promising potential for revolutionizing the treatment of periodontal diseases and other conditions affecting gingival tissue. Future research should focus on developing new growth factors and delivery methods. There is also a need for a deeper understanding of the mechanisms by which growth factors promote tissue regeneration. As research progresses, growth factor-based approaches could offer a new and effective treatment avenue for patients with damaged or lost gingival tissue.

4.2 Anti-inflammatory cytokines

The host immune/inflammatory response is a double-edged sword in tissue regeneration. While an acute inflammatory phase is needed to initiate tissue remodeling, chronic inflammation impairs healing and causes tissue destruction [92]. Locally delivered anti-inflammatory cytokines can potentially suppress gingival inflammation and promote a pro-regenerative microenvironment.

Interleukin-4 (IL-4): IL-4 is a seminal anti-inflammatory cytokine produced by T helper 2 (Th2) cells, mast cells, and basophils. It strongly inhibits pro-inflammatory cytokines such as TNF-alpha, IL-1beta, and IL-6 and upregulates anti-inflammatory factors like IL-1 receptor antagonists. IL-4 stimulates the proliferation and migration of fibroblasts and keratinocytes to facilitate tissue regeneration [93]. IL-4 delivery promoted re-epithelialization and granulation tissue formation in gingival wound healing models. IL-4-containing scaffolds also reduced neutrophil infiltration and inflammatory markers in periodontal lesions. The multifaceted anti-inflammatory and pro-regenerative effects make IL-4 an attractive candidate for gingival tissue engineering applications [94].

Interleukin-10 (IL-10): IL-10 is an immunoregulatory cytokine secreted by macrophages, Th2 cells, and regulatory T cells. It suppresses Th1 cell differentiation and inhibits the synthesis of pro-inflammatory cytokines such as IFN-gamma, IL-1, TNF-alpha, and MMPs. IL-10 knockout mice show impaired gingival wound closure, indicating its importance for tissue repair [95]. Delivery of IL-10 in collagen scaffolds reduced inflammatory cell infiltration and alveolar bone loss in ligature-induced periodontitis models in rats. Recombinant human IL-10 has been tested clinically and shown to limit periodontal tissue destruction. Optimization of IL-10 dosage and release kinetics is necessary to harness its therapeutic potential while avoiding immunosuppression [96].

Interleukin-13 (IL-13): IL-13 is an anti-inflammatory cytokine secreted by activated T cells that shares many functional similarities with IL-4. It suppresses inflammatory cytokine production by macrophages and neutrophils [97]. IL-13 delivery in a poly(lactic-co-glycolic acid) (PLGA) gel facilitated gingival wound healing in dogs, with increased epithelium formation, collagen content, and angiogenesis. It also reduced osteoclast numbers to limit alveolar bone loss [98]. The broad anti-inflammatory effects make IL-13 well-suited to mitigate inflammation during gingival regeneration.

4.3 Matrix remodeling enzymes

The destruction and remodeling of the extracellular matrix (ECM) via proteolytic enzymes mediate the tissue breakdown and healing phases of periodontal disease progression. Therefore, regulating the balance between ECM synthesis and degradation biochemically is an essential regenerative strategy. Critical matrix remodeling enzyme families include:

Matrix Metalloproteinases (MMPs): MMPs are zinc-dependent endopeptidases that can cleave all ECM components. They play complex dual roles in wound healing—debriding damaged tissues in early inflammation while enabling cell migration and tissue remodeling in later phases. Multiple MMPs, including MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, and MMP-13, are upregulated in gingival crevicular fluid during periodontitis [99]. While inhibition of MMPs using tetracyclines and their chemically modified variants (doxycycline, minocycline) limits periodontal tissue destruction, some MMP activity is needed for organized matrix deposition [100]. Optimizing the timing and dosage of MMP inhibitors is critical for balancing their protective and regenerative effects.

Tissue Inhibitors of Metalloproteinases (TIMPs): TIMPs are endogenous inhibitors of MMPs that regulate their proteolytic effects. The four mammalian TIMPs (TIMP 1–4) have varying specificity toward different MMPs [101]. The TIMP/MMP balance is disrupted in chronic periodontitis with excessive MMP activity. Locally elevating TIMP levels using gene therapy vectors or recombinant protein delivery inhibited MMPs and reduced bone loss in experimental periodontitis [7]. However, excess TIMP delivery over long periods may likewise impair tissue turnover and healing. Transient TIMP upregulation to curb acute MMP spikes during gingival regeneration is prudent.

Lysyl Oxidase (LOX) Family: LOX enzymes catalyze collagen crosslinking to strengthen and stabilize the ECM. Reduced LOX activity leads to gingival tissue deterioration and collagen breakdown in periodontitis. Locally administered LOX preserved collagen content and attachment levels in experimental periodontitis. LOX also stimulated fibroblast proliferation and osteogenic differentiation, indicating therapeutic potential beyond crosslinking ECM proteins [102]. However, high LOX activity can also make collagen fibers rigid and brittle. Therefore, fine-tuning the timing and extent of LOX-mediated crosslinking will be essential to balance ECM flexibility and strength for gingival regeneration.

4.4 Antimicrobial peptides (AMPs)

AMPs are essential innate immune system components with broad-spectrum antimicrobial efficacy against bacteria, fungi, and viruses. They provide a first line of defense against pathogens by disrupting microbial cell membranes. AMPs such as human beta-defensins (hBD) and LL37 are expressed in the oral epithelium and protect against periodontal bacteria [103]. However, chronic inflammation in periodontitis reduces AMP levels. Locally restoring AMP concentrations by exogenous delivery or gene therapy can help control pathogenic biofilms. For instance, a lentiviral vector expressing hBD3 inhibited the growth of Porphyromonas gingivalis and protected against bone loss in a mouse periodontitis model. AMPs exert direct antimicrobial effects while modulating host immune responses [104]. Harnessing these multifaceted actions can help establish a balanced regenerative microenvironment in the gingiva.

5. In vitro models for gingival tissue engineering

In vitro model systems are critical for preliminary testing of gingival tissue engineering strategies before committing to complex animal studies or clinical trials. Two-dimensional (2D) cultures, three-dimensional (3D) tissue constructs, and bioreactor platforms each offer distinct advantages to evaluate gingival cell interactions, matrix production, and mechanical properties prior to in vivo implantation. This section will discuss key considerations in designing and utilizing these in vitro models to accelerate the development of functional gingival substitutes.

5.1 2D gingival cell cultures

Standard 2D cultures involve seeding gingival fibroblasts or epithelial cells as monolayers onto flat tissue culture plastic or surfaces coated with ECM proteins such as collagen and laminin. While lacking native 3D tissue structure, 2D models allow straightforward analysis of fundamental cell behaviors such as adhesion, spreading [105], migration, proliferation, and matrix deposition in response to biomaterial substrates and bioactive factors. 2D cultures are readily adapted to high throughput screening to quickly test arrays of scaffolding materials, surface chemistries, protein coatings, and drug formulations. Parameters such as scaffold porosity, pore size, stiffness, and ligand density can be modulated to determine optimal conditions for cell attachment and matrix synthesis. Labeling intracellular cytoskeletal proteins helps visualize cell morphologies reflecting biocompatibility. Proliferation assays quantify growth rates over time. Immunofluorescence, PCR, and ELISA detect expression of ECM proteins, genes, and soluble factors at the protein and mRNA level [106]. These techniques provide quantitative endpoints to compare outcomes across scaffold variants. However, 2D cultures lack physiologic cell-cell and cell-matrix interactions in 3D microenvironments. The polarity, mechanical forces, and transport gradients experienced by cells in 3D tissues cannot be recapitulated. The simplicity of 2D models provides valid starting data to guide scaffold design, but 3D cultures and bioreactor systems are ultimately needed to evaluate actual tissue formation.

5.2 3D gingival tissue constructs

3D cultures aim to mimic native gingival tissue architecture by culturing cells within porous scaffolds or hydrogel matrices. Embedding gingival fibroblasts and epithelial cells within collagen gels has enabled the generation of bilayered constructs with epithelial-connective tissue polarity [107]. Perfusion bioreactor culture of such constructs enhanced cell viability and matrix deposition compared to static culture. Co-culturing gingival fibroblasts with endothelial cells in porous chitosan scaffolds also promoted pre-vascularization in vitro prior to implantation. 3D printing has enabled precise positioning of multiple cell types within engineered gingival constructs. Gingival fibroblasts printed within photocrosslinkable GelMA hydrogels exhibited higher viability and cell density than other bio-inks. Co-printing epithelial cells and fibroblasts recapitulated epithelial-connective tissue interfaces. Multi-layered constructs with continuous microchannels fabricated by 3D bioprinting enhanced nutrient diffusion to inner cell layers [108]. Such controllable 3D architectures allow the modeling of physiologic cell distributions and interactions within gingival tissue. 3D cultures further permit analysis of matrix accumulation, remodeling, and mechanical properties over long-term culture using techniques such as histology, immunohistochemistry, PCR, western blotting, and rheometry. Testing degradation, swelling, stiffness, stress relaxation, and fractal properties provide functional endpoints to compare scaffold performance under physiologic 3D conditions [109]. Overall, mimicking native gingival tissue architecture in 3D cultures enables more predictable screening of scaffolds before in vivo evaluation.

6. Future directions and conclusions

Gingival tissue engineering is rapidly evolving, amalgamating multiple disciplines to offer revolutionary regenerative medicine solutions. This transformative field endeavors to transcend the constraints of conventional periodontal therapies, like gingival grafts, marked by donor site morbidity and inconsistent results. It embraces innovations in biomaterials, stem cell research, and bioactive molecules to effectively present promising pathways for treating gingival and periodontal diseases. Advanced technologies like 3D bioprinting and computational modeling unlock unparalleled prospects for precision and individualization in treatment methodologies. However, these technologies also introduce technical, ethical, and regulatory challenges. Addressing these necessitates a harmonious integration of scientific, ethical, and regulatory considerations, focusing on a multifaceted and personalized approach to medicine and addressing individual patient needs to enhance the probability of successful outcomes. The paradigm is shifting from merely palliative solutions to genuinely regenerative therapies, emphasizing rigorous scientific investigation, adherence to ethical standards, and dedication to clinical excellence. With the burgeoning progress in regenerative strategies, the journey toward clinical translation is laden with challenges and unexplored opportunities. Continuing research endeavors focus on optimizing various aspects such as scaffold engineering, cell source optimization, and biologic modulation, aiming to inaugurate advanced gingival regenerative therapies.

Emerging Research Avenues:

• Development of Advanced Biomaterials: Crafting innovative biomaterials attuned to direct cellular behavior and modify tissue microenvironment based on biological signals.

• Cell Differentiation and Reprogramming Protocols: Refining protocols to develop stable gingival lineages from iPSCs or MSCs, ensuring robust cell sourcing.

• Exploration of Gingival Biology and Healing Mechanisms: Diving deeper into the developmental biology and healing mechanisms to comprehend genetic and epigenetic regulators for targeted modulation.

• High Throughput Platforms and Advanced Bioprinting: Implementing platforms for rapid screening of regenerative combinations and leveraging advanced printing methods to emulate the intricate gingival architecture.

• Enhanced Integration of Technologies: Merging computer-aided design with AI for elevated scaffold design, testing, and customization.

• Bioreactor Preconditioning and Pre-Vascularization Regimes: Developing regimes to enhance in vivo integration and perfusion of engineered gingival constructs.

• Scale-up of GMP Compliant Production: Streamlining production and application of gingival tissue products for widespread clinical use.

The progression in gingival tissue engineering signals a transformative phase in reconstructing damaged gingival tissues, with concerted interdisciplinary efforts and synergistic academia-industry collaborations being pivotal. These advancements are steering the field toward meeting clinical needs effectively and improving patient outcomes by translating laboratory innovations into practical, regenerative therapies, promising restoration of form, function, and esthetics.

Conflict of interest

The authors declare no conflict of interest.