Biofabrication – Revolutionizing the future of regenerative periodontics

Abstract

Periodontium is a compartmentalized and highly specialized tissue responsible for tooth stability. Loss of tooth attachment due to periodontitis and trauma is a complex clinical burden affecting a large parcel of the adult and elderly population worldwide, and regenerative strategies to reestablish the native conditions of the periodontium are paramount. Biofabrication of scaffolds, through various techniques and materials, for regenerative periodontics has significantly evolved in the last decades. From the basics of occlusive membranes and graft materials to the complexity of converging 3D printing and Bioprinting using image-based models, biofabrication opens many possibilities for patient-specific scaffolds that recapitulate the anatomical and physiological conditions of periodontal tissues and interfaces. Thus, this review presents fundamental concepts related to the native characteristics of the periodontal tissues, the key to designing personalized strategies, and the latest trends of biofabrication in regenerative periodontics with a critical overview of how these emerging technologies have the potential to shift the one-size-fits-all paradigm.

1. Introduction

The periodontium is a dynamic and highly specialized tissue anatomically composed of four major structures surrounding the teeth (i.e., gingiva, alveolar bone, periodontal ligament, and cementum) [1,2]. While the gingiva plays a protective role, working as a barrier separating the remaining compartments from the oral environment – cementum, periodontal ligament (PDL), and alveolar bone act as the supporting and hierarchically assembled tissues that maintain the teeth in the socket and allow mechanical load and stress distribution [1].

Periodontitis (gum disease) is a pervasive chronic inflammatory, bacteria-triggered oral disorder affecting two-thirds of Americans over 65 [3–6]. Left untreated, it destroys the periodontal attachment apparatus – hard (alveolar bone and cementum) and soft (gingiva and PDL) tissues, eventually resulting in tooth loss [6]. To date, the management of periodontal tissue destruction has encompassed scaling and root planing, flap surgery, biologics (e.g., enamel matrix derivative), bone grafting, and guided tissue regeneration (GTR) employing a degradable membrane that, while serving as a barrier against soft tissue infiltration, allows resident progenitor cells to promote the regeneration of periodontal tissues [7–12].

Although existing therapeutics in the current standard of care can lead to tissue healing and some degree of tissue regeneration, the low predictability and efficacy in cases of extreme tissue destruction call for improved strategies that can better replicate the three-dimensional (3D) and multi-tissue complexity of periodontal defects [7,11,13,14]. There are currently no approaches to predictably regenerate defects with considerable bone loss and avoid tooth extraction. Thus, there is an emerging quest for personalized (tissue- and defect-specific) solutions that can guide the coordinated growth and development of the periodontal attachment apparatus to prolong the lifetime of natural dentition. Clinically speaking, this represents a disruption in the one-size-fits-all biomaterial paradigm, in which simply using a barrier membrane against soft tissue infiltration into the defect to allow resident progenitor cells to promote neotissue formation was the primary goal.

Biofabrication, a rapidly evolving field that uses additive manufacturing (a layer-by-layer deposition process) to engineer structurally tailored scaffolds, has been successfully deployed in medicine to treat tracheobronchomalacia [15]. Regrettably, data in periodontics using similar technology (selective laser sintering, SLS) showed that, although a personalized scaffold could be made [16], its ability to obtain structures with a degradation rate matching that of bone and PDL neoformation remains intangible. Critically, the invariably bulky nature of those scaffolds and the oversight that soft and hard periodontal tissues regenerate at distinct rates highlight the need for biomaterials with tissue-specific degradation and technologies to create physiologically relevant scaffolds. As recognized by our group and others [7,11,13–20], scaffolds for periodontal regeneration should present well-defined tissue-specific characteristics due to differences in volume, architecture, cell type, and structural properties of its components. Notably, the system works as a bundle that cannot be dissociated, and thus, its soft-to-hard tissue interfaces, microscale organization, and transitions demand accurate enabling technologies [21–27].

Gingival recession (tooth root exposure), a prevalent condition affecting the gums, is a public health issue, particularly in elderly and underserved populations. Besides compromised esthetics, it significantly amplifies the likelihood of dentin sensitivity, root caries, plaque retention, and attachment loss. Regrettably, although collagen membranes and, more recently, living matrices have been exploited for root coverage and soft tissue repair, autogenous grafts (connective tissue and free gingival grafts) from the palate remain the standard of care – despite issues related to donor site morbidity and tissue shortage. Based on the recent progress of biofabrication technologies (3D printing and bioprinting), new opportunities for developing patient-specific gingival grafts have gained attention [28].

Over the past decade, various biofabrication techniques have been used to establish cell-free and cell-based strategies for periodontal tissue regeneration. These tissue engineering strategies utilize scaffolds with a single bulk characteristic, architecture, or material (monophasic) or scaffolds with two or more specific compartments, various architectures, and materials within the same construct (multiphasic). Cell-free monophasic and multiphasic scaffolds are usually three-dimensional (3D) printed to resemble specific anatomical characteristics and provide tissue-specific support for predictable regeneration [18,29–31]. Meanwhile, animal models and clinical studies have investigated the prospects associated with cell-based strategies using periodontal tissue-derived cell sheets [32–35]. Excitingly, the advent of 3D bioprinting has also inspired the design of scaffolds using hydrogel-based “bioinks” that mimic the native conditions of the extracellular matrix (ECM) to house stem cells capable of differentiating toward the targeted tissue(s) from gingival grafts to periodontal tissues.

Computer-aided design and computer-aided manufacturing (CAD/CAM) have become mainstream – thanks to the launch of CEREC in 1987, as the first system to combine digital scanning (of tooth preparation) with a milling unit to fabricate personalized restorations (Fig. 1A).

Fig. 1.

(A) Digital workflow for restorative and prosthetic dentistry using CAD/CAM techniques. (B) Digital workflow for biofabrication of personalized and patientspecific scaffolds for craniofacial regeneration through additive manufacturing.

Adapted with permission from de Carvalho [36].

Inspired by the disruption driven by CAD/CAM technology, the field of biofabrication has the potential to establish and implement personalized solutions to restore the periodontium. Although technologies are still being matured, by focusing on additive manufacturing, one can leverage high-resolution 3D imaging (e.g., cone beam computed tomography, CBCT) to design and then fabricate, using 3D (bio)printing scaffolds/grafts either chairside or at a biomanufacturing facility if patients’ cells are involved. Indeed, establishing a clinically viable workflow based on 3D imaging obtained from computed tomography to help generate personalized (patient-specific) scaffolds can revolutionize the future of regenerative periodontics (Fig. 1B) [36]. Therefore, here we present some fundamental concepts to understand the complexity of the periodontal tissues and explore how cutting-edge biofabrication strategies have the potential to pave the way toward the regeneration of hierarchically organized and fully functional periodontal tissues.

2. Anatomy of the periodontium and its interfaces

The human periodontium developed as a compartmentalized structure composed of the alveolar bone and the cementum that covers the radicular dentin connected by the periodontal ligament, forming a gomphosis-type fibrous specialized joint. Those structures are protected by the gingival tissue facing the tooth, the soft component of periodontal anatomy [1,37]. The organization of the periodontal tissues and their interfaces create a complex biomechanical system to support the tooth and physiologically distribute occlusal forces. To develop personalized (i.e., defect and tissue-specific) scaffolds for periodontal regeneration, it is necessary to understand these specialized compartments’ natural organization and behavior. Thus, the following sections present relevant information supported by seminal studies related to the periodontium’s anatomy, histology, and physiology [1,37–39].

2.1. Gingiva - the protective soft component

The gingival tissue can be described as part of both the oral mucosa and the periodontium as it displays a unique architecture going from the mucogingival junction (externally) to the limit of the papilla and invaginates facing the tooth until the beginning of the periodontal ligament space (internally) [40]. Histologically, the gingiva presents organized epithelium and connective tissues playing specific roles that help preserve homeostasis in the oral environment, and this is a critical aspect to consider while planning restorative procedures, the placement of grafts, and designing soft tissue regeneration strategies.

Even though buccal topography is extremely relevant from the perspective of tissue esthetics, when the focus is tissue regeneration, the emphasis is on the internal portion of the gingiva. For instance, the gingival volume is determined by the amount of connective tissue underneath the epithelial layer, and the clinician should consider that factor while planning GTR strategies. The gingiva plays a protective role by covering and attaching to the alveolar bone to prevent direct injuries and seal against bacterial penetration. That is only possible due to the organization of two different epithelia – oral epithelia and junctional epithelia – and the densely arranged specialized lamina propria with a dense collagenous network with good blood supply and innervation [40]. While the oral epithelium covers the tissue until the internal margin of the gingival sulcus, the junctional epithelium acts as a sealing component going from the internal margin of the sulcus to the cementum-enamel junction. The junctional epithelium is loosely attached to the enamel surface through desmosomes, and it has an essential role in preserving periodontal tissue homeostasis. Since microorganisms always populate the subgingival space, the junction between the junctional epithelium and the enamel surface works as a mechanical barrier against infection of the periodontium. Besides the physical protection, the junctional epithelium also presents some mechanisms of response against inflammation and regulating the innate response [41] (Fig. 2A).

Fig. 2.

(A) Schematic representation and histological view of the gingival tissue. (B) Schematic representation and histological staining of the cementum-PDL-alveolar bone interfaces with collagen bundles arrangement in circular and radial orientation. (C) Image of immunofluorescence of the periodontal ligament enthesis and fibers orientation along the root surface influenced by the stage of development and mechanical loading. (D) Periodontal ligament enthesis and collagen bundles at the interfacial zones PDL-cementum and PDL-alveolar bone.

(a) Adapted with permission from Bosshardt [41]. (b) Adapted with permission from Ho [42]. (c) Adapted with permission from Lee [38]. (d) Adapted with permission from Lin [39].

Immediately adjacent to the junctional epithelium, the loose connective tissue of the lamina propria is the ultimate layer of soft tissue above the alveolar crest. This layer of connective tissue is predominantly formed by collagen fibers arranged in different orientations according to the area and position of the tooth in the arch. It is vascularized and densely populated by fibroblasts. The bundles of fibers are firmly attached to the tooth and alveolar bone to provide the biomechanical stability of the gingival tissue. Because of these characteristics, the area is called connective attachment [40]. The combined distance of the oral epithelium, the junctional epithelium, and the connective attachment form the “biological space,” usually ~ 3 mm long in healthy tissue, that protects the alveolar crest, the PDL, and radicular cementum from exposure.

Due to the high number of cases of gingival dehiscence and root exposures caused by periodontal disease or occlusal trauma, the management of soft tissue is an integral part of periodontal tissue regeneration. Controlling the progression of the lesions by removing the cause and reestablishing the biological space through proper tissue management is essential to preserve tooth stability. From an esthetic perspective, gaining a specific volume of tissue to adjust the gingival contour and emergence profile is also relevant to promoting more natural results in prosthetic and implant dentistry. Nevertheless, the gold standard method for gingival grafts is still based on plastic surgery collecting connective tissue from a donor site – commonly the palate – to be implanted in the receptor area where the soft tissue is necessary to cover the defect [43]. The procedure creates a second surgical site on the palate, which increases patients’ morbidity and presents associated risks of wound healing complications [44]. In this sense, we further discuss biofabrication-based strategies for regenerating oral soft tissues in Section 4.4.

2.2. Bone, ligament, and cementum - the supportive components and their interfaces

Alveolar bone, periodontal ligament (PDL), and radicular cementum represent the inner compartments of the periodontal tissues. They are hierarchically organized with well-arranged interfacial transitions to support and distribute occlusal and masticatory loading and stabilize the teeth.

Cementum is a mineralized tissue layer deposited on the external surface of the radicular dentin and primarily works as anchorage for the periodontal ligament fibers to attach to the tooth [1]. From a histological perspective, it is composed of 50 % minerals and 50 % organic matrix, and it can be divided into acellular (cervical to middle third) and cellular (apical third and furcation) based on cell absence or presence within the tissue matrix, respectively. The first plays a critical role in PDL attachment, where the principal fibers of the periodontal ligament (Sharpey’s fibers) are anchored, and the latter is responsible for biological responses to compensate for minor resorptions [1,45]. The other mineralized compartment of the periodontium is the alveolar bone, the bone surrounding the alveolus where the tooth is inserted. It comprises an outer layer of cortical bone with trabecular bone close to the middle regions of the alveoli, permeated with Harvesian canals and populated by stem cells. The alveolar bone is responsible for the dissipation of the masticatory stresses through its integration with the PDL. This phenomenon is possible due to the arrangement of the so-called “bundle bone,” the inner layer lining of the alveolar process facing the root surface, intimately connected to PDL, forming an enthesis [1,39,42].

Meanwhile, the PDL is a specialized fibrous connective tissue with bundles of fibers distributed through the whole tooth socket, attaching the tooth to the alveolar bone, with the primary function of stabilizing the tooth into the alveolus and distributing the occlusal and masticatory stimuli to the adjacent bone. The thickness of the ligament varies according to the area, but its overall range goes from 150 to 380 μm [1]. PDL space has a heterogeneous cell population predominantly composed of fibroblasts, mostly cited as periodontal ligament cells, some epithelial cells close to the cementum, and a population of mesenchymal stem cells (periodontal ligament stem cells, PDLSCs). While the PDL fibroblasts are closely attached to the bundle spaces and constantly working on remodeling PDL fibers, PDLSCs have demonstrated the ability to differentiate towards PDL, cementum, and bone lineages, which makes this cell population of particular relevance in cell-based regenerative strategies [46]. The organization of the PDL fibers varies according to the region of the root. It can be divided into six major groups: 1) Gingival fibers, 2) Alveolar crest, 3) Horizontal, 4) Oblique, 5) Apical, and 6) Interradicular for multirooted teeth. Adjacent teeth also present transeptal fibers connecting one tooth to another going over the alveolar crest [37,40,42]. Altogether, these bundles of fibers are responsible for tooth stability, distribution of mechanical forces, proprioception, and gingival attachment. The arrangement of these fibers forming periodontal ligament enthesis is primarily dictated by mechanical loading as it occurs for all entheseal systems (Fig. 2C) [38,47].

While planning regenerative strategies, the transition zones and interfaces PDL-bone and PDL-cementum are crucial. PDL is distributed in both radial and circumferential planes of each tooth (Fig. 2B), and the attachment zones present collagen bundles intricately embedded in bone and cementum called Sharpey’s fibers, forming functionally graded interfaces (Fig. 2D) [1,37,39,42]. The fibers are mineralized at these interfaces, and they are constantly remodeled in health and disease to adapt to variations in load, slight disturbances in occlusal forces, and therapeutic stimuli such as orthodontic treatment [48]. Moreover, there are significant changes in the elastic modulus of the PDL from the non-mineralized body of the bundles to the mineralized interfacial zones [42], and recapitulating this hierarchical organization with proper biological, biochemical, and mechanical features is challenging [38].

Another important factor that differentiates the reconstruction/regeneration of the periodontal enthesis from other bone-ligament attachments is that in periodontal disease, the reason for the injury is not overuse or excess of load but an infection. In this sense, besides all the microscale aspects of insertion and variables related to size, tooth, load distribution, and region of the defect, infection eradication is paramount to designing clinically relevant therapeutic strategies in periodontal regeneration.

3. Traditional regenerative strategies to minimize the burden of periodontal defects

Periodontal regeneration strategies have been clinically performed for over 40 years to manage hard and soft tissue defects with a considerable success rate [8–10]. To date, the management of periodontal tissue destruction has encompassed scaling and root planing, flap surgery, biologics (e.g., enamel matrix derivative, growth factors, etc.), bone grafting, and guided tissue regeneration employing a degradable membrane that, while serving as a barrier against soft tissue infiltration, allows resident progenitor cells to promote the regeneration of periodontal tissues [7,12,43,49,50]. Guided Tissue Regeneration (GTR) and Guided Bone Regeneration (GBR) procedures have been performed to gain bone volume, manage soft tissue repair, and improve the prognosis of teeth compromised by periodontal defects. Combining the basic cleaning and scaling procedures with root conditioning, bone grafts, biologics, and membranes to cover the defects has been widely used to treat intra-bony and furcation defects in favorable clinical scenarios [51]. Although existing therapeutics can lead to some degree of tissue regeneration, the low predictability and efficacy in cases of extreme tissue destruction call for improved strategies. These concepts have been extensively reviewed before [7,11,12,51,52], and here we present a brief description of distinct approaches related to bone grafts and membranes for periodontal regeneration.

3.1. Graft materials for alveolar bone regeneration

For years, bone grafts and substitutes to fill alveolar defects have been used in clinical periodontology and implant dentistry [50,53]. The purposes for using bone grafts and substitute materials include filling the defect’s space, stimulating new bone formation, and providing conditions for reestablishing stability in the compromised area [54]. However, the different characteristics in the structure and function of the maxillary and mandibular bone make the strategies for oral bone regeneration unique and challenging [50]. In fact, the reconstruction of small and large bone defects in the oral cavity has been recently reviewed elsewhere [50,55], and here we bring a succinct and general description of the conventional strategies and materials for GBR.

Depending on the site, the desired outcome, and the size of the defect, the graft can be placed as blocks, granules, or cement obtained from different sources [36,50,56]. Cortical and cancellous autogenous bone grafts harvested from other sites in the oral cavity (e.g., symphysis, anterior ramus) are still considered the gold standard for bone augmentation even though the procedure demands the exposure of a second surgical site and increase patient morbidity [50,57,58]. Other alternatives include the use of allografts (bone from a different donor), which significantly increases the risk of immunogenicity and rejection of the graft, xenografts (usually bovine-derived particulate bone), and alloplastic bone substitutes (e.g., hydroxyapatite, β-tricalcium phosphate, bioglass) [50,54]. Various xenograft (Bio-Oss^®, Gen-Os^®) and alloplastic (Bone Ceramic^®, Cytrans^® Granules) materials have been launched for clinical use.

Despite the demonstrated clinical outcomes, based on the established concept of the triad for regeneration using scaffolds, biomolecules, and a source of progenitor cells to favor tissue response, none of the materials present all the necessary conditions to provide complete regeneration of alveolar bone defects and, in most cases, combining different grafts and substitutes has been a more successful strategy. Moreover, conventional bone grafts still present limitations in recapitulating the anatomical features of the area to be repaired. In this sense, we further discuss the current trends in 3D printing of bioceramic scaffolds for alveolar bone regeneration in Section 4.5.

3.2. Functionalized periodontal membranes

GTR/GBR strategies have significantly impacted the prognosis of compromised periodontal tissues toward positive outcomes, which is advantageous in mitigating the complications of periodontal disease worldwide. Bone grafts and substitutes are covered with occlusive membranes to protect the site and allow efficient healing and new tissue formation (Fig. 3A) [53,59]. Periodontal membranes are used to prevent soft tissue invagination, reduce the risk of infection, and provide enough spatiotemporal conditions to support bone regeneration. An ideal membrane for periodontal regeneration should present the following: biocompatibility, bioactivity, porosity/occlusive properties, mechanical stability, integration with the tissue, exposure tolerance, and biodegradability [60].

Fig. 3.

(A) Schematic illustration of the protective and occlusive function of a periodontal membrane covering the bone defect and cross-section SEM images of a functionally graded membrane with three integrated layers. (B) Representative images of induced periodontal disease in rat model and antibiotic-laden electrospun scaffolds for periodontal regeneration (top). Micro-CT reconstruction of the periodontal defects 2 and 6 weeks after different treatments indicating the healing potential of the periodontal membrane; histological sections and quantification of the inflammatory response according to each proposed treatment (bottom). (C) Micro-CT reconstruction of calvaria defects filled with functionalized membranes containing TCP and morphometric quantification illustrating the amount of bone formed as a result of the treatment.

(a) Adapted with permission from Bottino [17]. (b) Adapted with permission from Ferreira [61]. (c) Adapted with permission from Mahmoud [62].

GTR membranes are conventionally divided into non-resorbable (PTFE, titanium-reinforced PTFE) and resorbable (natural and synthetic polymers) [7,52]. Despite the biocompatibility and good clinical outcomes evidenced using non-resorbable membranes, they behave only as physical barriers to prevent soft tissue invagination and do not exert biological activity. Moreover, these membranes demand a second surgical intervention for their removal, which increases the risk of infection and damage to the newly formed periodontal tissues [59,60]. Meanwhile, resorbable membranes have in their favor easy handling, the possibility of using various polymers from natural or synthetic origins, and degradability [7,52,60]. Resorbable GTR membranes can also be functionalized with various biomolecules (antibiotics, growth factors, peptides, metallic oxides, bioceramics) to induce tissue response and local regeneration [7,52]. Besides commercially available materials, the field has evolved towards functionalized resorbable membranes, predominantly synthesized through electrospinning-based techniques.

Mono- and multilayered membranes have been prepared using solution electrospinning targeting infection control, anti-inflammatory response, and osteogenic properties to optimize periodontal regeneration. It is well understood that controlling the infection is paramount for the success of GBR/GTR due to the naturally biofilm-populated sites in the periodontal region. Thus, to reduce the risk of bacterial infection, antimicrobial-loaded electrospun membranes have been synthesized for local and sustained release of antibiotics or metallic ions that have activity against periodontal pathogens (Fig. 3B) [63–67,61,68]. Besides, electrospun membranes functionalized with biomolecules that induce mineralization have been tested to accelerate new bone tissue formation. Those models included loading the polymeric solution with hydroxyapatite, β-TCP, Bioglasses, and other inorganic materials with osteogenic potential (Fig. 3C) [69–72,62,73]. Apart from inorganic compounds, biomolecules such as proteins, peptides, growth factors, corticosteroids, and naturally derived agents are also commonly incorporated into polymeric solutions for the synthesis of electrospun membranes for antibacterial, anti-inflammatory, and osteogenic purposes [74–79]. However, some of these biomolecules are sensitive to the strong solvents that dissolve the polymers used to prepare the membranes, and a more complex apparatus of coaxial electrospinning is necessary to create core/shell fibrous membranes and guarantee the stability, proper delivery, and activity of the loaded molecule [7].

The use of core/shell fibrous membranes synthesized through coaxial electrospinning has other significant applications for preparing bifunctional materials with more than one expected response like antimicrobial/osteogenic or anti-inflammatory/osteogenic in a controlled fashion [75,80]. Bifunctional resorbable membranes can also be obtained by synthesizing multilayered scaffolds where the layer facing the bone has osteogenic/osteoinductive properties, and the layer facing the gingiva has antimicrobial potential [17,81,82]. For those functionally graded membranes, it is necessary to design stable interfaces between layers to prevent membrane delamination (Fig. 3A). Unfortunately, regardless of the adopted strategy, the mat-like aspect of these membranes and the limited control over fiber deposition in conventional electrospun membranes [83,84] limit their ability to mimic the hierarchical organization of the periodontium and its interfaces. Thus, biofabrication-based strategies such as 3D printing and bioprinting have offered new possibilities regarding the development of defect- and tissue-specific scaffolds/grafts for periodontal regeneration [52]. The following section of this article presents the current trends in biofabrication platforms for regenerative periodontics.

4. Patient-specific scaffolds: the next level of biofabrication for periodontal regeneration

The contributions GBR/GTR strategies have made to ease the burden of periodontal defects are remarkable and have significantly changed the clinical prognosis in periodontal therapy. However, as mentioned in the previous sections, those therapies do not fully repair the complex architecture and hierarchical organization of the periodontal tissues and interfaces. Moreover, the need for more personalized therapeutics, the integration of 3D reconstruction from CT scans, and the constant evolution of biofabrication technologies, including many 3D printing and Bioprinting methods, have shifted how we can address periodontal regeneration toward patient-specific scaffolds.

4.1. Building complex architectures for periodontal regeneration

4.1.1. Natural and synthetic materials

The success of the regenerative strategy is also related to the nature of the scaffold and its interaction with the host tissue; thus, selecting the material with the best properties can be challenging. Various natural and synthetic materials are available for 3D printing scaffolds/constructs for periodontal tissue regeneration, and fundamental knowledge about their properties and best indications will lead to more predictable outcomes.

Natural polymers are frequently used to recapitulate the native conditions of the extracellular matrix (ECM) in biofabrication strategies with the most relevance for bioprinting in the form of hydrogels. Collagen is the most predominant ECM protein with several applications in regenerative dental medicine, including bioprinted cell-laden scaffolds for periodontal regeneration [85] and commercially available GTR membranes [7,60]. Commercially available decellularized ECM and other alternatives of natural polymers for biofabrication, like hyaluronic acid, gelatin, chitosan, and alginate, have been tested individually or as blends to optimize their properties and general applications [18,86–88]. However, natural polymers present low mechanical properties, limiting their applications in load-bearing areas and sometimes making printing tick scaffolds with multiple layers challenging. In this sense, reinforced materials and additional methods of crosslinking could be used in a layer-by-layer fashion. At the same time, FRESH Bioprinting provides stability for printing these materials through sacrificial baths [89]. The most common additional crosslinking method is functionalizing these polymers with a methacrylate group for subsequent light curing. For instance, gelatin methacryloyl (GelMA) has been successfully used as a core material, either functionalized or not, to synthesize electrospun membranes, 3D printed, and Bioprinted scaffolds for periodontal regeneration [18,62,79,90].

Synthetic polymers are also used in biofabrication strategies for periodontal regeneration. Thermoplastic polymers such as polycaprolactone (PCL), polylactic acid (PLA), and polylactic co-glycolic acid (PLGA) are predominantly used due to their biodegradability, mechanical stability, and biocompatible properties [52]. The versatility of these polymers relates to relatively low melting temperatures (70 °C – 90 °C) and fast cooling for use in extrusion-based printing where solvents are not necessary. For instance, PCL is FDA-approved and has been tested for periodontal regeneration through casting [19,91], Fused deposition modeling (FDM) [32], and melt electrowriting (MEW) [18,30,31]. Nevertheless, ensuring these polymers’ degradability rates are compatible with the spatiotemporal tissue neoformation is necessary.

Besides using natural and synthetic polymers, bioceramics such as hydroxyapatite, beta-tricalcium phosphate (β-TCP), amorphous magnesium phosphate, and bioglasses have been used to obtain 3D printed scaffolds for alveolar bone and periodontal regeneration [92–96]. Calcium-phosphate and silicate-based materials or their precursors found in the inorganic phase of mineralized tissues provide cues to osteogenesis, osteoinduction, and/or osteoconduction [78–80] and have been used as graft materials for alveolar bone reconstruction.

Bioceramics can be either printed as the main component of the scaffold, primarily through direct ink writing (DIW) [97], mixed in polymeric blends [98,99], or used as coatings to functionalize polymeric materials through other extrusion-based biofabrication methods [92,100]. The similarities of bioceramic materials with the inorganic component of the bone are a favorable aspect for using them in personalized strategies of craniofacial bone regeneration and developing appropriate formulations to control brittleness, degradation ratio, and integration with the organic phase of the tissue are essential for the success and predictability of treatment outcomes. Importantly, from a clinical standpoint, periodontal tissues are under constant and cyclic loading. A suitable material for periodontal regeneration should support the mechanical stimuli, but relying only on the mechanical properties might result in detrimental biological properties. At the same time, using highly biocompatible materials with excellent antibacterial and/or osteogenic properties might impact the scaffold’s mechanical properties and long-term stability. One should know that balancing materials’ mechanical, chemical, and biological properties to address periodontal regeneration is paramount. Moreover, the printability, accuracy, and fidelity to reproduce the exact shape of the design are essential to guarantee the use of anatomically relevant scaffolds with the area’s features to be repaired. Therefore, designing optimized hybrid materials and converging multiple biofabrication techniques in a single platform to build multiphasic scaffolds from 3D defect reconstructions are alternatives to replicating the characteristics of the periodontium and interfacial zones.

4.1.2. Techniques of biofabrication for regenerative periodontics

A myriad of biofabrication technologies is available to devise scaffolds with complex architectures for dental and craniofacial tissue regeneration [52,101]. The decision for one or more methods (technology convergence) of biofabrication should be made considering the materials’ properties and the scaffold’s ability to recapitulate anatomical and physiological features, optimize tissue response, and improve clinical outcomes. In that sense, image-based CT scan designs have helped develop anatomically relevant scaffolds for regenerative periodontics through various strategies with good anatomical fidelity and printing accuracy. All these techniques have been extensively reviewed before [52,89,97,101,102], and here we share some brief details about their specificities while building personalized scaffolds for regenerative periodontics.

Among the various biofabrication technologies, Fused Deposition Modeling (FDM) is the most common method of printing thermoplastic polymers like PCL and PLA by extruding filaments of molten polymers through a nozzle to form 3D shapes [103]. The filaments are deposited according to a computer-aid design (CAD), and the fast-cooling properties of the polymers at room temperature allow the stacking of layers without collapsing after calibration of the printing parameters like speed, pressure, and layer thickness [103]. On the one hand, FDM allows control over the polymer’s deposition, reduces the toxicity risk for the cells compared to solution electrospinning, and omits the postprocessing step of solvent evaporation. Nevertheless, the thickness of FDM filaments (160 μm – 700 μm) is a limitation when considering the submicron to nanoscale details in the interfacial transitions of the periodontium [52].

One of the most recent alternatives used to circumvent the limitations faced with FDM while building personalized scaffolds for periodontal regeneration is Melt Electrowriting (MEW) [102]. This technology combines the fundamental concepts of filament deposition from FDM with the high voltage applied to the extruded polymer used in solution electrospinning to form submicron to microscale fibers with precise alignment and organization [83,102]. The moving collector going at speeds slightly above the speed of the molten polymeric jet (critical translation speed, CTS) results in straight filaments to build complex and highly structured scaffolds with a wide range of geometries [83,102]. Of note, curved filaments printed with speeds below the CTS could also approximate the fibers to the biomechanical behavior of tendons and ligaments [104,105]. The polymer’s major properties, the collector’s speed, the applied voltages and pressure, and the distance from the nozzle to the collector influence the characteristics of melt electrowritten scaffolds. Moreover, playing with the critical translation speed and G-Codes to compensate for inconsistencies in the printing path makes it possible to build a wide range of shapes in a single-step platform [106], which has a significant impact while replicating the periodontal tissues and interfaces. Preparing scaffolds with irregular architectures using only MEW still requires advanced knowledge in computational models with sophisticated systems and in-house-made apparatus. Nevertheless, MEW allows convergence with other biofabrication platforms toward developing compartmentalized and patient-specific scaffolds for periodontal regeneration.

Direct Ink Writing (DIW) or robocasting is another relevant extrusion-based system with applications in craniofacial regeneration [97]. DIW is used to print bioceramic pastes using a robotic arm to control the deposition of filaments at room temperature layer-by-layer [107,108]. The ceramic paste/slurry is usually formed by mixing the bioceramic powder with organic binders (e.g., polyvinyl alcohol copolymers). Caution must be taken to find the correct concentrations of the materials to prevent the nozzles from clogging during the printing process [109]. DIW is a relatively low-cost technique with fast printability and good resolution ranging from 5 μm to 200 μm. The easy preparation of the slurries and the possibility of combining different materials make DIW a versatile and valuable technique to print scaffolds/grafts with osteogenic/osteoinductive properties for alveolar bone augmentation and regeneration. The method’s limitations include the need for post-processing to remove the binding material [97,107] in minor structural changes that could impact the micromorphology of the complex architectures needed in periodontal regeneration strategies.

The abovementioned biofabrication methods are usually applied to obtain materials/scaffolds that do not carry cells during the printing process. In the meantime, bioprinting consists of 3D printing scaffolds where the material already carries the cells to target the specific tissue. Bioprinting protocols use highly biocompatible materials (bioinks) that mimic the ECM characteristics to allow the proper cell type to survive and differentiate in the targeted tissue [52,89]. Bioprinted scaffolds can be achieved through stereolithography, extrusion, and inkjet-based platforms. Bioprinting through stereolithography (SLA) or vat polymerization demands a light source to cure a bioink functionalized with photoinitiators like GelMA. SLA has excellent resolution and printing accuracy, making it a suitable method in regenerative dental medicine [110]. Extrusion-based bioprinting dispenses cell-laden bioinks through a nozzle using pneumatic or mechanical pressure [111]. On the other hand, the inkjet system of bioprinting produces droplets in a programmable and controlled manner [112]. Bioprinting has gained more relevance in the last few years in cell-based therapies due to their easy and controllable manner of building 3D scaffolds and delivering cells to the tissue to be regenerated following the anatomical features compared to other alternatives, such as cell sheets or injectable models. However, due to the nature of the bioinks, usually based on natural polymers, bioprinted scaffolds lack mechanical stability. Therefore, when combined with other relevant 3D printing strategies, bioprinting holds excellent potential in regenerative periodontics.

Despite the advantages evidenced by available biofabrication technologies (3D printing and bioprinting), it remains challenging to replicate the compartmentalized arrangement and hierarchical organization of the periodontal tissues when using only one biofabrication method. To date, no individual technology presents all the necessary conditions to generate scaffolds replicating all the mechanical and biological conditions of the periodontal tissues and their interfaces. Therefore, converging multiple technologies and materials into a single biofabrication platform to build multiphasic scaffolds has gained the scientific community’s attention [21]. Some studies have already focused on combining multiple strategies to generate multiphasic scaffolds to improve tissue response and clinical outcomes [18,22,113,114]. Moreover, generating 3D CAD models from patients’ CT scans facilitates the convergence of techniques and materials to build personalized and patient-specific scaffolds with precise fit and desirable mechanical and biological properties to guide hierarchical and spatiotemporal periodontal tissue regeneration. Current trends in the design and application of multiphasic and compartmentalized scaffolds with specific characteristics of the periodontal tissues are, therefore, described in the following sections, focusing on the possible strategies for clinical implementation.

4.2. Compartmentalized scaffolds for periodontal regeneration

Biofabrication of compartmentalized scaffolds for periodontal regeneration has been a field in expansion for almost two decades, with significant progress and some bottlenecks still to be solved. Complex and hierarchically organized architectures in living tissues are challenging to replicate, and personalized strategies for periodontal regeneration demand multiphasic scaffolds with micron-to-nanoscale structures and transition zones [52]. As we previously discussed, engineering all the compartments of the periodontium with all the anatomical, biomechanical, and biological features is challenging, and most of the alternatives use multiple platforms and materials to achieve the expected response.

Compartmentalized scaffolds can be designed either as biphasic (bone-ligament) or triphasic (bone-ligament-cementum) using cell-free or cell-based approaches. Nevertheless, fundamental knowledge about the biology and functionality of each compartment of the periodontium is essential to design relevant models. Due to its unique characteristics, the PDL compartment is of utmost importance. In fact, it is a concern that the main reason for unsuccessful outcomes in periodontal regeneration is poorly arranged PDL fibers that result in nonfunctional tissue [13]. Therefore, numerous studies have focused on biofabrication strategies to create specific microtopography and properly guide PDL regeneration. For instance, micropatterned scaffolds with different orientations have demonstrated the ability to guide PDL cell orientation [19,20,91,115], which is relevant while planning the scaffold design according to the site and extension of the defect to maintain PDL orientation and functionality. It is important to note that narrow grooves in micropatterned systems provide more favorable conditions to guide cell orientation and alignment than wide designs (Fig. 4A–B) [19,20,115]. Even though these methods used casting to build the micropatterned scaffolds, the templates with topographical characteristics were 3D printed from CAD designs to mimic bone and PDL compartments [19,91].

Fig. 4.

(A) Representative images of micro-grooved scaffold designs with various angles for guiding specific PDL organization with fluorescence microscopies illustrating cell alignment following the designed pattern. (B) SEM images and histological staining for the PDL tissue formed on different micropatterned scaffolds. (C) Schematic representation and SEM images of the compartmentalized and functionally graded melt electrowritten scaffolds illustrate the designed bone, PDL, and interfacial zones. In vivo model of implantation in rat mandibular defects with the obtained μCT scans of the regenerative outcomes. (D) SEM images of melt electrowritten scaffolds with different strand spaces for the PDL compartment and histological staining of the oriented newly formed tissue following the pattern of the scaffold.

(a) Adapted with permission from Park [20]. (b) Adapted with permission from Pilipchuk [19]. (c) Adapted with permission from Golafshan [30]. (d) Adapted with permission from Staples [116].

Micropatterning also impacts the other compartments of the periodontium and the interfacial zones. Other studies have more recently used MEW as a single platform to build bone and PDL compartments and the interfaces of the periodontium [30,31,116]. For instance, personalized PCL-based scaffolds with small and large strand spacing for bone (500 μm) and PDL (250 μm) compartments were investigated with significant osteogenic potential, macrophage activity regulation, and successful periodontal tissue arrangement and regeneration controlled by the scaffold design and coating step [31]. Also, multiphasic melt electrowritten scaffolds composed of PCL containing magnesium-phosphate, with 500 μm strand spacing for the bone compartment, and a PDL zone with highly aligned PCL fibers with 50 μm strand space were designed. Two different architectures were prepared at the interfacial zones, where the authors used either a box-shape design with 250 μm strand spacing or randomly deposited fibers of PCL containing magnesium-phosphate (Fig. 4C) [30]. Composite-graded interfacial zones significantly improved the quality of the new tissue formed compared to the scaffolds with no interfacial transition and clear distinction between bone and PDL compartments in vivo. The randomly deposited fibers at the interfacial zone demonstrated better response in terms of recapitulating the enthesis of the periodontal tissue, probably related to the small spacing between fibers when compared to the 250 μm box-shaped design [30]. It has been reported that even smaller architectures and channels seem more critical in guiding PDL orientation. Melt electrowritten pristine PCL-based biphasic scaffolds with microchannels below 100 μm have led to optimized PDL fibers alignment in vivo [116]. The distribution of collagen bundles on the dentin surface followed the pattern of the microchannels in an organized way [116], thus reinforcing the claim that scaffold architecture is paramount for regenerating complex tissues such as the periodontium (Fig. 4D).

Other investigations have focused on multiphasic compartmentalized 3D-printed scaffolds with larger patterns. For instance, triphasic PCL/Hydroxyapatite scaffolds were printed with different pore sizes for cementum (100 μm), PDL (600 μm), and bone (300 μm) compartments, subsequently loaded with dental pulp stem cells (DPSCs) and implanted in vivo to study new tissue formation in an ectopic model [117]. On the other hand, to mimic the three compartments of the periodontal tissues using technology convergence, a PCL/β-TCP biphasic scaffold made with FDM (bone) and electrospinning (periodontal ligament) was designed and combined with periodontal ligament cell sheets, attached to the electrospun membrane side, to achieve multi-tissue periodontal regeneration [32]. The same group later synthesized PCL/β-TCP (bone) scaffolds integrated with melt electrospun meshes (PDL). Osteoblast cells were dispersed on the bone compartment, and PDL cell sheets were attached to the ligament-cementum compartment [92]. The coating with a bioceramic material and the morphological characteristics of the scaffolds significantly improved the response in vitro and in vivo. Moreover, the presence of specific cell lineages for each compartment yielded positive responses. Even though the in vivo model was ectopic, there was evidence of new periodontal fiber alignment and attachment to the root surface with considerable new bone formation at the bone compartment interface [92]. Considering the dynamic evolution of biofabrication technologies, replacing the traditional approach of cell-sheets for Bioprinted scaffolds in cell-based therapies might offer new possibilities in terms of controlling cell distribution in the specific compartments and reducing the time for scaffold preparation.

Based on the solid evidence shown by compartmentalized scaffolds on providing conditions for the regeneration of the periodontal tissues and interfaces and the knowledge that the scaffold architecture plays a fundamental role in guiding PDL orientation and interfacial transitions, one might ponder that moving the field toward personalized constructs will lead to more predictable regenerative strategies in the future. Selecting the best strategy to print these multiphasic and tissue-specific scaffolds still depends on the choice of material, the defect’s area, and the patient’s conditions. The translation of these models to clinical practice is a constant discussion primarily because of safety/regulatory protocols to establish clinical trials.

4.3. Image-based models for patient-specific regeneration

The high demand for individualized therapeutics and precision oral health treatments is moving the field of biofabrication toward tailoring the specific anatomical and physiological conditions of oral and craniofacial tissues. Currently, the combined evolution of digital processing methods to create CAD designs from a patient’s computed tomography image indicates a favorable scenario for developing personalized scaffolds. The workflow for computational models and image-assisted planning and manufacturing has been proposed elsewhere [118], and, in summary, the process is conducted in four steps: 1) The clinician obtains the 3D image of the defect using CT scans; 2) The CAD model of the scaffold to fit the defect is designed; 3) Biofabrication of the 3D printed/bioprinted patient-specific scaffold and; 4) Surgical implantation of the scaffold/graft in the patient.

Most models that develop personalized image-based devices for the craniofacial area focus on prosthetic devices and maxillofacial surgery to correct large defects from congenital conditions, trauma, or tumor resection [101]. However, the field of periodontal regeneration has been exploring using such technologies to build personalized scaffolds to correct periodontal defects and promote alveolar ridge augmentation. For instance, computational models using reverse engineering strategies digitally validated the design and adaptation of site-specific scaffolds from periodontal defects in swine [91]. Subsequently, the model was optimized and tested for a fenestration defect in rats using PCL-based fibrous-guiding scaffolds with specific anatomical features and a more than 95% adaptation rate [119,120]. At the time, the CAD models were 3D printed in wax molds that served as templates to cast PCL and form the scaffolds with desired anatomical features. The image-based site-specific scaffold provided conditions for PDL regeneration and integration with the bone and cementum compartments, making the newly formed tissue hierarchically organized and functional [119,120].

The high accuracy of the CAD models and precise fit in preclinical tests led to an image-based model for periodontal regeneration that was tested on a human subject [16]. A patient-specific PCL-based scaffold was 3D-printed using selective laser sintering to regenerate a periodontal defect in a lower canine (Fig. 5A). The personalized scaffold was embedded in a platelet-derived growth factor (PDGF)-containing solution before implantation to optimize the local response. The authors performed clinical follow-up for over a year with a significant improvement in tissue insertion, and the scaffold remained stable for up to 12 months [16]. However, the scaffold was exposed and had to be retrieved after 14 months. The slow degradation rate of PCL probably caused the failure of the strategy since after retrieval of the scaffold, about 75 % of the original construct was still intact [16]. Nevertheless, as a proof-of-concept, the case report showed that the workflow to build image-based personalized scaffolds is effective and clinically viable.

Fig. 5.

(A) Representative images of a case report of an image-based patient-specific scaffold for periodontal regeneration with the clinical view of the periodontal defect, CT scan, and image-based CAD models. (B) 3D reconstruction of a mandibular defect in rat and the 3D CAD design and printing accuracy for defect-specific regeneration using melt electrowriting. (C) Clinical and radiographical images, and 3D CAD designs of patient-specific ceramic scaffolds for alveolar bone preservation. (D) Schematic representation with 3D CAD designs and printing accuracy of clinical scale patient-specific ceramic scaffolds for alveolar bone and ridge augmentation.

(a) Adapted with permission from Rasperini [16]. (b) Adapted with permission from Daghrery [31]. (c) Adapted with permission from Kim [121]. (d) Adapted with permission from Anderson [122].

The optimization of many CAD software and the integration and affordability of multiple platforms in the last years have resulted in other anatomically relevant models for alveolar bone and periodontal regeneration. For example, patient-specific bioceramic scaffolds were 3D printed based on CBCT scans for proof-of-concept following a digital workflow with a high printing fidelity while replicating the anatomical features of the defect (Fig. 5D) [122]. Also, the printing fidelity and accuracy of personalized image-based melt electrowritten scaffolds were verified [31]. The CAD design was based on μCT scans of a rat fenestration defect. The anatomical features were planned in the CAD model (Fig. 5B). Even though the CAD model was successfully generated and the external surface of the scaffold presented good fidelity, the limitations of internal adaptation (due to the size and complexity of the defect) can be certainly improved over the coming years [31].

Moreover, a randomized clinical trial was performed using a commercially available bioceramic material to 3D print image-based scaffolds for alveolar bone preservation and later installation of implants (Fig. 5C) [121]. Patient-specific 3D printed scaffolds from CBCT fit into the space of the defects and did not differ from conventional bone grafts in the histomorphometric parameters [121]. Minor concerns related to the presence of residual granulation tissue in the case of alveolar preservation and the need for a specific type of anchorage for these personalized scaffolds [121]. Despite the inherent limitations, the application of the digital workflow for a good number of patients in a randomized trial reinforces that biofabrication strategies are bringing more benefits in terms of patient-specific therapies and reduced clinical/surgical time in bone and periodontal tissue regeneration.

Critically speaking, it is important to reinforce that many factors are involved in the success of regenerative strategies. First, using goodquality CT scans to plan the appropriate architecture of the periodontal tissues and interfaces is critical. Second, the nature and properties of the resorbable material, the biofabrication platform, the biomolecules used for functionalizing the scaffold, and the decision for a cell-free or cell-based approach will dictate the clinical effectiveness of the image-based personalized scaffold. Third, medical conditions and habits of the patient also influence tissue response and prognosis, and the clinical team should be aware of all these factors while planning and executing the appropriate treatment.

4.4. Biofabrication of oral soft tissue grafts

Although the three compartments of the supportive side of the periodontium (bone, PDL, and cementum) have drawn more attention from a regenerative perspective, the gingival tissues are also impacted by periodontal disease, and therapeutic strategies for soft periodontal tissue regeneration (gingiva) are also needed. The established protocols for treating gingival recession are still based on autografts or flap repositioning [43], new opportunities based on biofabrication-based (3D printing and Bioprinting) approaches focusing on mimicking the complexity of the gingiva have been recently reviewed [28].

Most tissue engineering strategies for oral mucosa regeneration use commercially available acellular dermal matrix (Alloderm^®). Even though ex vivo-produced oral mucosa equivalents (EVPOME) [123,124] and oral keratinocytes-based cell sheets [125] have been studied for oral mucosa wound healing, the concepts applied to oral mucosa and gingival regeneration are predominantly translated from wound healing strategies to treat skin injuries. For instance, an integrated dermis and epidermis system made of an alginate/gelatin/fibrinogen blend was successfully bioprinted and presented relevant histological characteristics of native skin [126]. Noteworthy, the bioprinted tissue evidenced desmosomes and hemidesmosomes and pertinent characteristics of cell-to-cell interactions (Fig. 6C) [126]. The organization of the desmosomes and hemidesmosomes in the tissue is a finding that could be translated to relevant models for gingival regeneration and junctional epithelium integration to protect the subjacent alveolar bone.

Fig. 6.

(A) Schematic representation, full size and microscopical views of the designed melt electrowritten mesh infused with GelMA hydrogel for epithelial healing. (B) Cell viability and proliferation on the melt electrowritten mesh with the bioprinted GelMA hydrogels in different conditions. (C) Transmission Electron Micrographic images of the desmosomes, hemidesmosomes, and mature collagen fibrils formed in the multilayered 3D Bioprinted skin tissue. (D) Macroscopic photographs and histological and immunofluorescence images of vascularized bioprinted skin grafts illustrate the optimized healing process with the use of vascularized scaffolds.

(a) Adapted with permission from Dubey [127]. (b) Adapted with permission from Dubey [127]. (c) Adapted with permission from Pourchet [126]. (d) Adapted with permission from Baltazar [128].

A recent model converging melt electrowritten PCL scaffolds with keratinocytes-laden GelMA hydrogels printed via droplet system has been designed for optimized mechanical stability and improved cell viability for skin wound dressings (Fig. 6A–B) [127]. The hydrogel was also functionalized with periostin [127], an important protein found in inflamed sites and mineralized compartments of the periodontal tissues that also play a role in wound healing. Additionally, promoting vascularization and tissue nutrition is essential to prevent hypoxia and failure of the regenerative strategy. In this way, vascularized skin grafts were bioprinted, mixing keratinocytes and endothelial cells with efficiency in preserving the tissue layers and forming microvasculature in vivo (Fig. 6D) [128].

Despite the progress in soft tissue regeneration applied to skin reconstruction, more is needed regarding biofabrication-based approaches toward generating gingival grafts. Since the oral environment is a mucosa, and the gingiva has some specific characteristics, designing strategies exploring the latest advances in biofabrication to recapitulate the native attributes of gingival tissue represents a tremendous opportunity in regenerative periodontics.

4.5. Patient-specific bioceramic and composite scaffolds for alveolar bone and ridge augmentation

The field of periodontal regeneration is closely related to implant dentistry. Thus, GBR strategies are often used for alveolar preservation or ridge augmentation before dental implant installation [49,129]. Despite the absence of cementum or PDL surrounding dental implants, the quality and stability of the alveolar bone around the implant are highly relevant for the device’s longevity. Biofabrication strategies (e.g., 3D printing) have been used to build personalized scaffolds/grafts and tackle alveolar preservation and ridge augmentation mainly through the use of bioceramic materials due to their known biocompatible, osteogenic, and osteoinductive potential. The major aspects regarding the processing of scaffolds for craniofacial bone regeneration have been extensively reviewed [36,52,97,129,130]; however, here we bring some of the anatomically relevant concepts and designs applied to alveolar bone and ridge augmentation.

PCL/β-TCP blends were printed via DIW with two layers that differ in pore interconnectivity to replicate cortical and cancellous bone [99]. The personalized bioceramic scaffolds were printed and implanted on mandibular defects in dogs, and optimized bone regeneration was achieved when the scaffolds were functionalized with bone morphogenetic protein [99]. Also, 3D-printed HAP/PLGA scaffolds with controlled microporosities were implanted in critical-size intrabony mandibular defects in rabbits and evidenced significant osteogenic potential and stability 4 weeks after implantation [131]. Another personalized model for alveolar ridge augmentation tested a “core-cover” scaffold designed from generic CT scans for proof of concept. The core layer was a TCP/HAP porous scaffold, while the cover was printed with PCL for support [132]. Lastly, for alveolar ridge regeneration, patient-specific scaffolds have been printed with a commercially available bioceramic (Osteoink^™, HAP/α-TCP) [122].

Personalized bioceramic scaffolds printed using DIW were recently characterized and implanted in rat mandibular defects for alveolar bone regeneration [94]. From the various bioceramic compositions tested in the study, calcium-silicate-magnesium scaffolds evidenced the overall best mechanical stability and bone regeneration properties after 16 weeks [94]. Notably, the presence of magnesium in bioceramic materials has demonstrated an excellent biological response, and an ECM-amorphous magnesium phosphate bioink was synthesized for maxillofacial bone regeneration [95]. In addition, other bioceramic compounds, such as bioglasses, have also been tested for craniofacial bone regeneration. Boron-containing bioglass scaffolds were 3D printed, implanted in mandibular fenestration defects, and compared to HAP-based scaffolds [133]. Boron-containing bioglass scaffolds significantly increased bone formation and showed better integration to the host tissue than HAP-based scaffolds [133].

Even though personalized bioceramic scaffolds have been tested in several models over the years, additional research is still needed to offer additional pre-clinical and clinical findings related to alveolar bone and vertical ridge regeneration. Targeting these specific bone reconstructions using 3D printing techniques still has room to be explored, mainly regarding complex geometries in large ridge defects in edentulous areas. Notably, the biofabrication of bioceramic scaffolds/grafts involves assessing the need for material postprocessing (e.g., sintering), controlling the material’s brittleness, and synchronizing the degradation rate of the scaffolds in a spatiotemporal fashion with the kinetics of regeneration [36].

5. Remarks and future perspectives

Biofabrication provides conditions to build complex and anatomically relevant scaffolds that can revolutionize how clinicians and surgeons address periodontal tissue regeneration. Building multiphasic scaffolds replicating the characteristics of the periodontal compartments and interfacial transitions using state-of-the-art 3D printing and bioprinting technologies presents great potential in regenerative periodontics. The feasibility and accuracy of image-based scaffolds/grafts to regenerate periodontal tissues following digital workflows have been tested and confirmed by different research groups using various biofabrication platforms. Some challenges remain in the decision-making process for the correct biofabrication strategy, and the convergence of technologies and tissue-specific biomaterials (e.g., ceramics, polymers, composites, and hydrogels) to build multiphasic personalized scaffolds seems more appropriate for recapitulating all the anatomical and biomechanical features of the periodontal tissues. From a clinical perspective, some regulatory and safety concerns remain to be surpassed. Besides, more extensive preclinical and clinical validation of the precise scalability of the anatomical features, as well as the performance and long-term stability of materials for patient-specific scaffolds in regenerative periodontics, is necessary. Therefore, developing standardized methods for testing the potentially successful strategies should involve a task force of researchers, clinicians, and regulatory bodies from various institutions to accelerate the translation from the bench to the clinics.