New Insights in Hydrogels for Periodontal Regeneration

Periodontitis is a destructive inflammatory disease characterized by microbial infection that damages the tissues supporting the tooth (alveolar bone, gingiva, periodontal ligament, and cementum), ultimately resulting in the loss of teeth. The ultimate goal of periodontal therapy is to achieve the regeneration of all of the periodontal tissues. Thus, tissue engineering approaches have been evolving from simple membranes or grafts to more complex constructs. Hydrogels are highly hydrophilic polymeric networks with the ability to simulate the natural microenvironment of cells. In particular, hydrogels offer several advantages when compared to other forms of scaffolds, such as tissue mimicry and sustained drug delivery. Moreover, hydrogels can maintain a moist environment similar to the oral cavity. Hydrogels allow for precise placement and retention of regenerative materials at the defect site, minimizing the potential for off-target effects and ensuring that the treatment is focused on the specific defect site. As a mechanism of action, the sustained release of drugs presented by hydrogels allows for control of the disease by reducing the inflammation and attracting host cells to the defect site. Several therapeutic agents, such as antibiotics, anti-inflammatory and osteogenic drugs, have been loaded into hydrogels, presenting effective benefits in periodontal health and allowing for sustained drug release. This review discusses the causes and consequences of periodontal disease, as well as the advantages and limitations of current treatments applied in clinics. The main components of hydrogels for periodontal regeneration are discussed focusing on their different characteristics, outcomes, and strategies for drug delivery. Novel methods for the fabrication of hydrogels are highlighted, and clinical studies regarding the periodontal applications of hydrogels are reviewed. Finally, limitations in current research are discussed, and potential future directions are proposed.


Introduction
Periodontal disease is a multifactorial disease in which pathogenic bacteria initiate the host immune response, leading to the destruction of the tissues surrounding and supporting the teeth, such as gingiva, periodontal ligament, alveolar bone, and cementum [1].These four tissues are part of the periodontium, a supporting apparatus essential for the restoration and proper functioning of periodontal tissues (Figure 1) [2].Apart from anchoring the teeth in their respective alveolar pockets, the periodontium also stabilizes them by distributing and absorbing masticatory forces, and it serves as a barrier against several pathogens [2].Periodontal disease first manifests as gingivitis, a dental inflammation initiated by dental plaque accumulation and affected by the host response, which dictates the disease progression [3].Although improvements in oral hygiene habits may reverse gingivitis, a lack of treatment can lead to periodontitis, which is characterized by the destruction of collagen fibers, alveolar bone absorption, and the formation of soft tissue

ECM proteins
Bone sialoprotein, collagen, enamel matrix proteins, fibronectin, hyaluron, laminin, osteocalcin, osteopontin, proteoglycans, osteonectin, tenascin In periodontal regenerative therapies, several scaffold forms have been used to support tissue regeneration and healing, including membranes, sponges, fibers, 3D-printed scaffolds, and hydrogels.In particular, hydrogels offer several advantages compared to other forms of scaffolds, such as tissue mimicry and sustained drug delivery.Hydrogels are highly hydrophilic polymeric networks with the ability to simulate the natural microenvironment of cells, and they can be developed from natural or synthetic polymers [15].Moreover, hydrogels can maintain a moist environment that is similar to the oral cavity.Hydrogels allow for the precise placement and retention of regenerative materials at the site of interest [16].This minimizes the potential for off-target effects and ensures that the treatment is focused on the specific defect site.
Hydrogels derived from natural polymers, including alginate, cellulose, chitosan, collagen, fibrin, gelatin, and hyaluronic acid, have been used in several biomedical appli- Periodontitis is a public health problem, and its most severe form is the eleventhmost prevalent human disease [5].Periodontitis affects 20% to 50% of the worldwide population, and its severity can be associated with sociodemographic variables, such as age and income [6].In fact, different age groups are disproportionally affected by the disease, since the prevalence and severity of periodontitis tend to increase with age [5].Furthermore, Nazir and colleagues showed that low-income subjects had significantly higher chances of having severe periodontal disease than high-income subjects [5].In addition, periodontal disease represents a risk factor for surgical infection after mandibular fracture and for mandibular fracture [7].
Because it is caused by bacterial inflammation and plaque accumulation, clinical treatments of periodontitis focus on cause-related, non-surgical, and conservative approaches, such as plaque removal and local inflammation control [8].Thus, the first clinical strategy is usually a debridement treatment involving mechanically cleaning the periodontal pockets to remove bacteria.However, if deep pockets are present, resective surgery might also be needed [9].Although these therapies minimize symptoms and prevent further disease progression, they are not able to restore all the lost tissues, including the periodontal ligament, leaving patients with functional and aesthetic sequelae [10].Aiming to surpass the limitations of the current treatments and to improve the outcomes of standard therapy, tissue engineering strategies have been explored for periodontal regeneration.
The biology of periodontal regeneration involves a complex interplay between various elements, including cells (fibroblasts, osteogenic cells, and immune cells), bioactive molecules (growth factors), and the extracellular matrix (ECM) [11].The interaction between these components is highly orchestrated.Following injury or disease, cells respond to signals from the local environment and bioactive molecules.They then produce the necessary ECM components to create new tissue.Growth factors and other signaling molecules guide the cells' behavior, influencing their differentiation and function [12] (Table 1).The whole process faces several biological and clinical challenges, such as its spatiotemporal healing coordination, the competition between tissues, and the clinically challenging surgical environment [13].In fact, to achieve complete regeneration, it is necessary to reconstruct the whole periodontium, including the alveolar bone and new cementum, with the insertion of the functionally oriented collagen fibers of a newly formed periodontal ligament [2,14].In periodontal regenerative therapies, several scaffold forms have been used to support tissue regeneration and healing, including membranes, sponges, fibers, 3D-printed scaffolds, and hydrogels.In particular, hydrogels offer several advantages compared to other forms of scaffolds, such as tissue mimicry and sustained drug delivery.Hydrogels are highly hydrophilic polymeric networks with the ability to simulate the natural microenvironment of cells, and they can be developed from natural or synthetic polymers [15].Moreover, hydrogels can maintain a moist environment that is similar to the oral cavity.Hydrogels allow for the precise placement and retention of regenerative materials at the site of interest [16].This minimizes the potential for off-target effects and ensures that the treatment is focused on the specific defect site.
Hydrogels derived from natural polymers, including alginate, cellulose, chitosan, collagen, fibrin, gelatin, and hyaluronic acid, have been used in several biomedical applications, such as 3D cell cultures, drug delivery systems, wound healing, and tissue regeneration [17,18].Natural hydrogels are biocompatible, bioactive, they present low cytotoxicity, and their structure resembles native tissues [19,20].However, hydrogels do not have strong mechanical properties and they present batch-to-batch variability in composition, impairing the tuning of the material properties [21].Natural hydrogels often rely on proteins or polysaccharides with batch-specific variations in their characteristics, such as molecular weight, charge, and structural integrity.These variations can affect the overall properties of the hydrogel, including its mechanical strength, swelling capacity, and biocompatibility.On the other hand, synthetic hydrogels, such as polyvinyl alcohol (PVA), polyethylene glycol (PEG) [22], polyacrylic acid (PAA), or polyacrylamide (PAAM), do not present these disadvantages; however, they lack the endogenous factors that are required to promote cell behavior, such as migration, proliferation, and differentiation [21].Different techniques can be used to fabricate hydrogels tailored to specific applications with the required chemical and physical properties [18].This review covers the current available treatments of periodontal disease, describing the use of bone graft materials, guided tissue regeneration, and enamel matrix derivatives.Alternative tissue engineering strategies are explored, in particular the use of 3D hydrogels.The recent research on 3D hydrogels for periodontal regeneration is summarized and discussed.Novel methods for the fabrication of hydrogels are highlighted, and clinical studies regarding the application of hydrogels for periodontal applications are reviewed.Finally, the shortcomings and future perspectives of using 3D hydrogels for treatment of periodontitis are discussed.

Current Treatments
Periodontal therapy aims to regenerate all the tissues damaged due to periodontal disease.The regeneration of these tissues that compose the periodontium involves distinct cell types, including periodontal ligament stem/stromal cells (PDLSC), alveolar bone cells, cementoblasts, and epithelial cells [23,24].Among the current treatments, guided tissue regeneration (GTR) membranes and bone grafts are the most used in clinics [11].Importantly, the clinical image of the defect site in clinical settings plays a critical role in optimizing the probability of successful regeneration.However, these approaches present a lack of compartmentalization between the periodontal defect and the surrounding soft tissue, leading to poor regenerative outcomes.Hence, several strategies have been introduced to regenerate the whole periodontium, such as the use of enamel matrix derivatives (EMD) and other growth factors, as well as tissue engineering strategies, including 3D hydrogels.

Bone Graft Materials
Bone graft materials have been used to fill in periodontal defects, promoting alveolar bone regeneration (Figure 2).To achieve bone regeneration, the residual bony walls of the periodontal defect need to be able to provide mechanical support and blood supply to the bone graft [25].Bone grafts have a supportive function and should present osteogenic, osteoconductive, and osteoinductive properties to allow for new bone formation as well as bioactive sites for cells to proliferate while recruiting host stem cells into the defect site [25,26].Different types of grafts have been used, such as autologous, allogeneic, and xenogeneic grafts [27].Furthermore, considering the material, grafts can be further divided into five distinct categories: natural, synthetic, composite, growth factor-based bone substitutes, and bone substitutes with infused living cells [26].Natural bone grafts represent the majority of bone grafts used worldwide, and include autografts, allografts (such as demineralized bone matrix and freeze-dried bone matrix), xenografts (such as chitosan or silk from other species), and plant-based materials (such as algae-and coral-based grafts) [26,28] (Table 2).Nevertheless, natural bone grafts present several disadvantages.In fact, although autografts are considered the gold standard for bone graft materials due to their osteogenic properties and low risk of immunogenicity, the harvest procedure is associated with pain and higher morbidity at the donor site.On the other hand, allografts may trigger Different types of grafts have been used, such as autologous, allogeneic, and xenogeneic grafts [27].Furthermore, considering the material, grafts can be further divided into five distinct categories: natural, synthetic, composite, growth factor-based bone substitutes, and bone substitutes with infused living cells [26].Natural bone grafts represent the majority of bone grafts used worldwide, and include autografts, allografts (such as demineralized bone matrix and freeze-dried bone matrix), xenografts (such as chitosan or silk from other species), and plant-based materials (such as algae-and coral-based grafts) [26,28] (Table 2).Nevertheless, natural bone grafts present several disadvantages.In fact, although autografts are considered the gold standard for bone graft materials due to their osteogenic properties and low risk of immunogenicity, the harvest procedure is associated with pain and higher morbidity at the donor site.On the other hand, allografts may trigger an immune response due to graft rejection by the recipient [28][29][30].Therefore, synthetic bone substitutes aim to mimic the properties of natural bone while tackling the aforementioned limitations [26].Calcium phosphate ceramics, calcium phosphate cements, metals, and polymers are examples of the synthetic bone substitutes used [26].Different materials can also be combined in order to achieve an optimal set of mechanical properties, forming composites.Examples of commercialized composites are NanoBone TM (76% w/w nanocrystalline hydroxyapatite and 24% w/w silicon) [31] and Fortoss Vital TM (β-tricalcium phosphate (β-TCP) in a calcium sulphate matrix) [32].Aiming to improve the osteoinductive properties of the grafts, growth factor-based bone substitutes have been used.Platelet-derived growth factor (PDGF), bone morphogenetic proteins (BMP) such as BMP-2 and BMP-7, fibroblast growth factor 2 (FGF-2), parathyroid hormone (PTH), insulinlike growth factors (IGF), and platelet concentrates such as platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) have been used for alveolar bone grafts and substitutes [26,29].
Table 2. Bone grafts and substitute materials used for periodontal applications, categorized according to their source.
Infuse TM bone graft is an example of a commercialized product for periodontal regeneration.Infuse TM bone graft is approved for use in sinus augmentation and localized alveolar ridge augmentation [40].It contains recombinant human BMP-2, inducing new bone formation [26].
Finally, bone substitutes with infused living cells have been explored to treat periodontal defects, aiming to increase the osteogenic and osteoconductive properties of the material [26].Mesenchymal stem/stromal cells (MSC) have been widely used in the dentistry field, since they are able to differentiate towards an osteogenic lineage and are able to regenerate large bone defects when combined with a scaffold [26,41].
Although they are able to regenerate bone tissue, current bone grafts alone are not able to prevent epithelium downgrowth and have also been used in combination with other approaches, such as GTR [42].In fact, bone grafting procedures have demonstrated the formation of a long junctional epithelium rather than a new connective tissue attachment [27].

Guided Tissue Regeneration
GTR uses membranes to act as physical barriers, avoiding connective and epithelial tissue downgrowth into the defect [43,44] (Figure 3).Besides excluding epithelial cells from the defect, these membranes also provide space for cells, such as PDLSC, osteoblasts, and cementoblasts, to repopulate the wound area while increasing wound stabilization [29,45].

Enamel Matrix Derivatives
Recently, EMD have been used to treat periodontal defects.EMD are mainly composed of enamel matrix proteins, 90% of which are amelogenins, and the remaining 10% are prolin-rich non-amelogenins, tuftelin, and other serum proteins [47].Several studies have reported that EMD can mimic the biological processes that occur during periodontal tissue formation; however, the mechanism of action remains unclear.In fact, EMD are involved in the formation of acellular cementum; the most important tissue for the insertion of collagen fibers [47].Furthermore, this mixture of proteins plays a significant role in the development of the periodontium [48], since EMD upregulate Runt-related transcription factor 2 (RUNX2) and Osterix (OSX) transcription factors [48] and increase the production of transforming growth factor-β (TGF-β), BMP, vascular endothelial growth factors (VEGF), and FGF-2 [49].In addition to the stimulatory effects of growth and transcription factors during periodontal wound healing, EMD can retard epithelial downgrowth [49].Emdogain ® (Straumann, Basel, Switzerland) is a commercially available product composed of EMD derived from the developing teeth germs of six-month old piglets combined with a vehicle solution of propylene glycol alginate [8].Emdogain ® is an injectable hydrogel.It is minimally invasive and possesses antimicrobial effects, eliminating the need for antibiotic coverage [9].However, being a porcine-derived product, Emdogain ® might present ethical concerns and trigger an immune response once applied in humans.Even so, Emdogain ® has been one of the most used approaches in clinics, in part due to EMD being quite similar among mammalian species, which translates in a smaller antigenic potential [9,30].Regarding expected outcomes, in specific cases, this treatment is effective for the regeneration of alveolar bone, cementum, and periodontal ligament [50].Although several studies have reported the capacity of EMD to promote the regeneration of periodontal tissues as well as to improve the clinical attachment levels and reduce probing pocket depth [9,49,51], a high degree of heterogeneity among the results has also been shown [9].Moreover, due to its gel-like consistency, Emdogain ® has been used in combination with other biomaterials, such as bone grafts and membranes [51].

Three-Dimensional Hydrogels as a Novel Treatment
The ultimate goal of periodontal therapy is to achieve the regeneration of the alveolar bone, cementum, gingiva, and periodontal ligament.Thus, tissue engineering approaches have been evolving from simple membranes or grafts to more complex constructs.Hydrogels have shown interesting results for periodontal regeneration [29], since they can address one of the major limitations of current treatments: the inability to exert spatiotem- Although periodontal regeneration using GTR strategies has shown quite satisfactory results in animal models, the same was not observed in clinical settings.The clinical outcomes varied according to the nature of the periodontal defect as well as the skills and experience of the clinician [13].These poor clinical outcomes are associated with the inability of progenitor cells to repopulate the defects in a certain spatial or temporal order [13]; a very important requirement to achieve multiple tissue regeneration and functional restoration [42].
These membranes should exhibit four critical parameters: biocompatibility, an adequate degradation time matching the rate of new tissue formation, proper mechanical/physical properties, and sufficient sustained strength to avoid membrane collapse [46].Polytetrafluoroethylene (PTFE) membranes either with or without titanium reinforcement were the gold standard of non-resorbable membranes [23,29]; however, this type of membrane presents several drawbacks.Among them, there is a need for a second surgery for their removal, representing additional costs, as well as pain and discomfort for the patients, negatively affecting regenerative outcomes [23,29,43].To address these shortcomings, biodegradable membranes were introduced.Indeed, resorbable membranes are used to reduce patient discomfort and to accelerate tissue healing through their bioactive properties [23].Although natural membranes present good biocompatible and bioactive properties, they also present some disadvantages, such as poor mechanical properties and fast and unpredictable degradation rates [23].Synthetic resorbable membranes have also been used, since their degradation and mechanical properties can be easily tailored [23].Polylactic acid (PLA), polyglycolic acid (PGA), and their copolymers are the most used for these purposes [11].
Compared to the use of hydrogels, GTR membranes involve a surgical procedure with potential complications.Furthermore, maintaining the stability and integrity of the barrier membrane during the healing period is challenging.Membrane displacement can compromise the success of the procedure.

Enamel Matrix Derivatives
Recently, EMD have been used to treat periodontal defects.EMD are mainly composed of enamel matrix proteins, 90% of which are amelogenins, and the remaining 10% are prolin-rich non-amelogenins, tuftelin, and other serum proteins [47].Several studies have reported that EMD can mimic the biological processes that occur during periodontal tissue formation; however, the mechanism of action remains unclear.In fact, EMD are involved in the formation of acellular cementum; the most important tissue for the insertion of collagen fibers [47].Furthermore, this mixture of proteins plays a significant role in the development of the periodontium [48], since EMD upregulate Runt-related transcription factor 2 (RUNX2) and Osterix (OSX) transcription factors [48] and increase the production of transforming growth factor-β (TGF-β), BMP, vascular endothelial growth factors (VEGF), and FGF-2 [49].In addition to the stimulatory effects of growth and transcription factors during periodontal wound healing, EMD can retard epithelial downgrowth [49].Emdogain ® (Straumann, Basel, Switzerland) is a commercially available product composed of EMD derived from the developing teeth germs of six-month old piglets combined with a vehicle solution of propylene glycol alginate [8].Emdogain ® is an injectable hydrogel.It is minimally invasive and possesses antimicrobial effects, eliminating the need for antibiotic coverage [9].However, being a porcine-derived product, Emdogain ® might present ethical concerns and trigger an immune response once applied in humans.Even so, Emdogain ® has been one of the most used approaches in clinics, in part due to EMD being quite similar among mammalian species, which translates in a smaller antigenic potential [9,30].Regarding expected outcomes, in specific cases, this treatment is effective for the regeneration of alveolar bone, cementum, and periodontal ligament [50].Although several studies have reported the capacity of EMD to promote the regeneration of periodontal tissues as well as to improve the clinical attachment levels and reduce probing pocket depth [9,49,51], a high degree of heterogeneity among the results has also been shown [9].Moreover, due to its gel-like consistency, Emdogain ® has been used in combination with other biomaterials, such as bone grafts and membranes [51].

Three-Dimensional Hydrogels as a Novel Treatment
The ultimate goal of periodontal therapy is to achieve the regeneration of the alveolar bone, cementum, gingiva, and periodontal ligament.Thus, tissue engineering approaches have been evolving from simple membranes or grafts to more complex constructs.Hydrogels have shown interesting results for periodontal regeneration [29], since they can address one of the major limitations of current treatments: the inability to exert spatiotemporal control over the wound healing process [52].In fact, hydrogels possess great advantages from a clinical perspective, such as injectability, easy accessibility, and their potential to deliver the necessary cues to induce the migration of the host cells and to accelerate periodontal tissue formation (Figure 4).vantages from a clinical perspective, such as injectability, easy accessibility, and their potential to deliver the necessary cues to induce the migration of the host cells and to accelerate periodontal tissue formation (Figure 4).In addition to biochemical cues, the mechanical properties and biodegradability of hydrogels are also important.In fact, the stiffness of the material needs to be similar to the tissue's stiffness in order to promote successful adaption of the material to the root sur- In addition to biochemical cues, the mechanical properties and biodegradability of hydrogels are also important.In fact, the stiffness of the material needs to be similar to the tissue's stiffness in order to promote successful adaption of the material to the root surface.Additionally, hydrogels must degrade within a period of time similar to the growth of new tissue [53].

Hydrogel Composition
Hydrogels can be composed of several materials and combined with bioactive molecules to induce new tissue formation [53] (Tables 3 and 4).Hydrogels are mainly classified into natural and synthetic polymers.

Natural Polymers
Hydrogels composed of natural polymers are derived from natural sources.Natural polymers exhibit remarkable biocompatibility and biodegradability.Their hydrophilic nature promotes cell adhesion, proliferation, and differentiation.However, the mechanical strength and stability of natural polymers are not as high as those of synthetic hydrogels, which can limit their applications [19,20].

Collagen
Collagen hydrogels have also been extensively studied for periodontal regeneration.Although collagen hydrogels have been mostly used as membranes for GTR, studies have evaluated their influence on periodontal regeneration when applied directly to the defect.In fact, Sato and colleagues have demonstrated that the application of a collagen gel loaded with FGF-2 in a dog defect model yielded promising results, with the formation of new collagen fibers and cementum [66].

Gelatin Methacrylate
Gelatin methacrylate (GelMA) hydrogels are also frequently used for periodontal regeneration.Pan and colleagues evaluated the effect of a GelMA hydrogel with embedded human PDLSC both in vivo and in vitro and observed enhanced proliferation and differentiation of PDLSC within the hydrogel, as well as newly bone formation when the hydrogels were placed on rat alveolar defects [67].GelMA hydrogels have also been enriched with nanohydroxyapatite [68].These constructs enhanced osteogenic differentiation of human PDLSC, and in vivo studies in a mouse model showed increased formation of mineralized tissue [68].Apart from being used alone, GelMA has also been combined with other materials, such as polyethylene glycol diacrylate (PEGDA) [69].

Hyaluronic Acid
Being part of the natural ECM, hyaluronic acid has also been used for periodontal regeneration [70].Fawzy El-Sayed and colleagues studied the effect of a hyaluronic acid hydrogel loaded with interleukin 1 receptor antagonist (IL-1ra) and gingival MSC in a swine periodontal defect model [71].Both IL-1ra-loaded and unloaded constructs proved its potential for periodontal regeneration, yielding a higher clinical attachment level, probing depth, periodontal attachment level, cementum regeneration, and bone regeneration in addition to a lower junctional epithelium [71].

Self-Assembling Peptides
Recently, self-assembling peptide (SAP) hydrogels have also been investigated for periodontal repair [72][73][74].Overall, SAP used in rat periodontal defects showed greater organization of periodontal fibers in the defect, as well as decreased epithelial downgrowth [72] and enhanced new bone formation [74].

Synthetic Polymers
In addition to natural hydrogels, synthetic hydrogels, such as PEG and PEGDA hydrogels have also been explored [75].Liu et al. developed a PEGDA hydrogel combined with stromal cell-derived factor 1 (SDF-1) and showed that this hydrogel promoted proliferation, migration, and osteogenic differentiation of PDLSC, and it promoted osteogenesis when used in a rat periodontitis model [76].Tanongpitchayes and colleagues have developed a combination of a polyacrylamide-based hydrogel and nanohydroxyapatite [77].This hydrogel effectively enhanced pocket regeneration in dogs with periodontitis.Chitin, poly(lactic-co-glycolic acid) (PLGA), nanobioglass ceramic (nBGC), cementum protein 1 (CP-1), PRP, FGF-2

Human dental follicle stem cells
The incorporation of the additives nBGC, CP-1, PRP, and FGF-2 resulted in improved cementogenic, osteogenic, and fibrogenic differentiation of human dental follicle stem cells, similar to hydrogels without additives in induction media.Pan, J. et al., 2020 [67] GelMA, PDLSC Cell culture surface Human PDLSC PDLSC proliferated at a similar rate in the hydrogels and in 2D culture.Hydrogels with DA at a higher concentration showed lower cell viability.Both cell types proliferated and migrated in the hydrogels.
Hydrogels with a DA concentration of 1/20 showed vertical cell penetration from the top of the hydrogel in the depth, with PDLSC having a slightly higher migration potential.

Table 4.
In vivo studies on hydrogels for periodontal regeneration.

Immature dog teeth with apical periodontitis
The incorporation of chitosan hydrogels in dogs did not improve the formation of new mineralized tissues along the root canal walls or the histologic evidence of the regeneration of a pulp-dentin complex.Moreover, they demonstrated optimal properties for bone tissue engineering applications.

Methods of Fabrication
Hydrogels can be fabricated by crosslinking networks and can be classified as physically or chemically crosslinked hydrogels [89,90].Physical crosslinking is typically accomplished through physical mechanisms, including crystallite formation, polymer chain complexion, hydrophobic interaction, and the establishment of hydrogen bonds [91].Chemical or covalent crosslinking results from covalent bond junctions.Physically crosslinked hydrogels exhibit reversibility due to conformational changes that prevent dissolution in aqueous media, while chemically crosslinked hydrogels are permanent and irreversible owing to configurational changes.The macroscopic properties of hydrogels, such as the degree of swelling, mechanical characteristics, and the transport of molecules through the hydrogel meshes, are impacted by both the type and degree of crosslinking [92].
In the field of tissue engineering, hydrogels have traditionally presented limited mechanical strength and structural complexity.However, due to their expanding applications, there is a growing need for advanced engineering methods that allow for precise control of both the physical and chemical properties of hydrogels, enabling the creation of more well-defined structures.Recent advanced engineering methods, including 3D bioprinting and in situ gel formation, have been developed for fabricating hydrogels for periodontal applications.

Three-Dimensional Bioprinting
Three-dimensional bioprinters can create customized scaffolds that closely mimic the natural architecture of periodontal tissues [93].Bioprinters can deposit cells, biomaterials, and bioactive molecules with high precision, allowing for the creation of complex periodontal structures and ensuring that the right cell types are in the right locations.Furthermore, 3D bioprinting enables the incorporation of PDLSC into the scaffold, promoting tissue regeneration by differentiating into the necessary cell types.Regarding drug and growth factors delivery, bioprinters can precisely control the release of growth factors or antimicro-bial agents, which is essential for managing inflammation, promoting tissue regeneration, and preventing infections in periodontal applications.
A recent study from Miao and colleagues developed a 3D bioprinted multi-component hydrogel for cell delivery in periodontal tissue regeneration [93].The hydrogel consisted of GelMA, sodium alginate, and a bioactive glass microsphere.Furthermore, this hydrogel was used as a bioink to load mouse bone marrow MSC and growth factors (BMP-2 and PDGF) to develop scaffolds for periodontal applications.The cells loaded in the hydrogel maintained good cellular viability after 3D bioprinting and presented enhanced osteogenic differentiation in BMP-2-and PDGF-loaded hydrogels.Moreover, when the hydrogels were transplanted in beagle dog periodontal defects, a significant regeneration of gingival tissue, periodontal ligament, and alveolar bone was observed [93].
In a different study from Yan et al., a 3D bioprinted periodontal construct was developed with high architectural integrity using a GelMA/decellularized ECM cell-laden bioink [94].Dental follicle cells were encapsulated into the bioink.After incorporation of the 3D bioprinted constructs into a critical-size periodontal defect model, enhancement of the regeneration of periodontal tissues in beagles was observed.In particular, anchoring structures of the bone-ligament interface, well-aligned periodontal fibers, and highly mineralized alveolar bone were observed [94].

In Situ Gel Formation
Regarding periodontal applications, in situ gel formation involves the use of a gel-like material that can be administered directly at the defect site and transformed into a solid state once it comes into contact with oral tissues.The formation of in situ gels is generally a time-dependent process and can vary depending on the specific formulation [95].The gel formation process may involve factors including temperature, pH, or the presence of specific ions or polymers [95].
In situ gel-forming systems have the ability of sustained drug release and have attracted attention due to their easy administration (injectable) and high drug retention (localization) in periodontal defects.
Recently, Gopalakrishna and colleagues developed a piperine-loaded in situ gel [96].Different gel formulations were tested by varying the concentration of deacylated gellan gum crosslinked with sodium tripolyphosphate and poloxamer-407.The optimized formula was implanted into human patients for 14 days, and its anti-inflammatory effectiveness was evaluated.At physiological conditions, the hydrogel was able to form, allowing an efficient residence time of the hydrogel within the defect.Furthermore, it was possible to observe a significant reduction in the mean plaque score, gingival index and pocket depth, and anti-inflammatory potential compared to the control group [96].
In a different work, Swain et al. developed a moxifloxacin hydrochloride-loaded in situ gel for the treatment of periodontitis [97].Different formulations were tested by varying temperature sensitive (poloxamer 407), ion sensitive (gellan gum), and pH sensitive (carbopolol 934P) polymers.The optimized formulation contained 19.072% w/v poloxamer 407 and 0.245% w/v gellan gum, which has a desired gel temperature of 36 • C and gelling time of 102 s, and 98% of the drug released after 9 h [97].
Ranch and colleagues developed a doxycycline hyclate-laden in situ gel composed of poloxamer 407, chitosan, and polytethylene glycol 600 [98].After testing, the gelation temperature of the optimized in situ gel was 34 ± 1 • C with a sufficient strength and texture profile for periodontal applications.Furthermore, the in vitro dug release assays demonstrated a sustained release from the developed gels (24 h) compared to commercially available gels (7 h).Interestingly, doxycycline hyclate retained its antimicrobial efficacy when formulated as an in situ gelling system [98].

Clinical Studies on the Application of Hydrogels for Periodontal Repair
Clinical studies on hydrogels for periodontal applications have been investigated [99].Hyaluronic acid hydrogels loaded with human fibroblast growth factor 2 were used to treat periodontal defects [20].After 1 year of treatment, the clinical parameters of periodontal wound healing were significantly improved in a total of 30 patients [20].
Olszewska-Czyz and colleagues have shown that the use of hyaluronic acid hydrogels as an adjunctive to non-surgical periodontal therapy demonstrated more favorable clinical results after 3 months [100].A reduction in inflammation was observed, measured by bleeding on probing (−6% compared to the control group) and gain in periodontal attachment (1 mm more than control group), while it had no effect on the probing depth reduction.Furthermore, no side effects were reported [100].
In a different study, Tamura et al. reported the clinical effects of the sustained release of basic fibroblast growth factor (bFGF) from gelatin hydrogels in patients with periodontal disease presenting bone defects [101].A total of 23 patients were treated with a mucoperiosteal flap operation.At the time of surgery, each bone defect was filled with a bFGF-gelatin hydrogel.One year after the treatment, there were significant improvements in clinical parameters, such as probing pocket depth reduction, clinical attachment gain, and radiographic bone fill.Furthermore, no adverse effects were observed [101].
Gad and colleagues formulated solid lipid microparticles gels encapsulating doxycycline hydrochloride and metronidazole and proved the clinical efficacy of these gels in periodontal patients [102].

Conclusions and Future Perspectives
Current clinical approaches for periodontal regeneration are focused on the use of membranes and bone grafts.Recently, these materials have been used for the delivery of growth factors and bioactive molecules to promote and enhance the wound healing process and further periodontal tissue formation.However, these strategies fail to yield the expected clinical outcomes since they do not promote the regeneration of all the periodontal tissues, including hard tissues (cementum and alveolar bone) and soft tissues (gingiva, periodontal ligament).Thus, hydrogels have been used in several applications in the field of tissue engineering, such as space fillers, vehicles for delivery of bioactive molecules, and as 3D structures to promote new tissue formation [29,52,53].
Periodontal tissue regeneration is a very complex process.Hydrogels have been explored as scaffolds and/or drug delivery systems due to their capacity to incorporate cells into their structures and their ability to degrade on the same timeline as new tissue formation.Additionally, hydrogels can be combined with several bioactive molecules to induce cellular processes, such as migration, proliferation, differentiation, vascularization, and mineralization [96][97][98].Furthermore, hydrogels have excellent biocompatibility, water retention, and controlled release, and they provide support for cellular interaction during periodontal regeneration [15,16].Regarding periodontal applications, since hydrogels are structurally similar to the ECM of several tissues and may be delivered in a minimally invasive manner, they can reduce the inflammatory response to remodel the structure and function of periodontal tissues.In fact, as a mechanism of action, the sustained release of drugs presented by hydrogels allows for the control of disease by reducing inflammation and attracting host cells to the defect site.Several therapeutic agents, such as antibiotics, anti-inflammatory and osteogenic drugs, have been loaded into hydrogels, presenting effective benefits in periodontal health and allowing for sustained drug release [103].In fact, antimicrobial hydrogels are an attractive solution to control infectious diseases and to address the challenges associated with antibiotic resistance due to their unique physicochemical and biological properties and drug delivery capacity.Moreover, combining complementary therapeutic approaches with these antimicrobial hydrogels will improve their effectiveness.These hydrogels can have inherent antimicrobial activities or can be loaded with antimicrobial agents [104,105].
Although there are only a few clinical studies related to the effectiveness of hydrogels for periodontal therapy, some clinical studies have already shown promising results on the application of hydrogels for periodontal regeneration [20,[100][101][102].Several hydrogels loaded with different bioactive agents have shown significant improvements in clinical parameters, such as probing pocket depth reduction, clinical attachment gain, and radiographic bone fill.Although remarkable progress has been made in the application of hydrogels in periodontal regeneration, challenges remain to providing sufficient mechanical strength and more biological properties to these hydrogels to achieve successful regenerative outcomes.In fact, the composition and structure of hydrogels have a significant impact on periodontal tissue regeneration.ECM-derived scaffolds prepared from decellularized tissues or cell cultures have been developed to promote functional tissue remodeling in several clinical applications.Indeed, the decellularized ECM can be manipulated to form hydrogels that can be used as injectable materials to fill irregularly shaped defects.By having ECM structural and biological cues, these hydrogels direct cell behavior and influence new tissue formation.Furthermore, the development of intelligent and multifunctional hydrogels for periodontal tissue regeneration is required for future research.While 3D bioprinting holds significant promise for periodontal applications, it is important to note that the technology is still evolving, and clinical translation is ongoing.Researchers and clinicians are actively working to optimize bioprinting techniques for better outcomes in the treatment of periodontal disease.Additionally, regulatory and safety considerations are crucial when moving from lab research to clinical practice.Further investigation is needed to determine which combinations of biomolecules, cells, and hydrogels can improve clinical results.This research will also require extensive teamwork between clinicians and researchers.This paper reviews the current treatments for periodontal disease and new insights on hydrogels for periodontal regeneration, providing discussions about their different characteristics and outcomes and aiming to contribute to successful periodontal regeneration.

Figure 1 .
Figure 1.The structure of periodontium and the different stages of periodontal disease.It first manifests as gingivitis and then progresses to a more serious infection affecting the soft tissue and the alveolar bone that support the teeth.If left untreated, it can result in tooth loss.Figure created using Biorender.com.

Figure 1 .
Figure 1.The structure of periodontium and the different stages of periodontal disease.It first manifests as gingivitis and then progresses to a more serious infection affecting the soft tissue and the alveolar bone that support the teeth.If left untreated, it can result in tooth loss.Figure created using Biorender.com.

Figure 2 .
Figure 2. Bone loss triggered by periodontal disease.Bone grafts can be used to enhance the alveolar bone to accommodate an implant or to preserve the natural teeth in that defect, promoting new bone formation.Figure created using Biorender.com.

Figure 2 .
Figure 2. Bone loss triggered by periodontal disease.Bone grafts can be used to enhance the alveolar bone to accommodate an implant or to preserve the natural teeth in that defect, promoting new bone formation.Figure created using Biorender.com.

Figure 3 .
Figure 3. Illustration of the guided tissue regeneration technique used for periodontal therapy.Figure created using Biorender.com.

Figure 3 .
Figure 3. Illustration of the guided tissue regeneration technique used for periodontal therapy.Figure created using Biorender.com.

Figure 4 .
Figure 4. Injectable hydrogels for periodontal regeneration.Bioactive cues, such as cells, can be incorporated into the hydrogel to stimulate new tissue formation.Figure created using Biorender.com.

Figure 4 .
Figure 4. Injectable hydrogels for periodontal regeneration.Bioactive cues, such as cells, can be incorporated into the hydrogel to stimulate new tissue formation.Figure created using Biorender.com.

Table 1 .
Cell types and molecules responsible for periodontal regeneration.

Table 1 .
Cell types and molecules responsible for periodontal regeneration.

Table 3 .
In vitro studies using hydrogels for periodontal regeneration.