In recent years, a number of microfabrication methodologies have been investigated for application in biomedical research (Table 1). One of these approaches is lithography which allows for the development of structured surfaces at the micrometer and nanometer scales (15). Photolithography involves the transfer of a computer-designed pattern to a surface of interest, which is then used as a guide to modify the material by further processes such as photopolymerization or etching (16). One of its advantages is the ability to generate patterns with defined 2D micrometric geometries (17, 18). Electrospinning is another technique used in biomedical sciences where a fiber pattern is generated by ejecting a polymer solution under a high-voltage electric field into a metallic collector (28). Due to their cost-effective fabrication, high surface area, and tailored porosity, electrospun fibers have been used in oral sciences as tissue engineering scaffolds, wound dressings, and drug delivery systems (29). Furthermore, as electrospun scaffolds can be loaded with molecules, growth factors, nanoparticles, and pharmacological agents, their potential as vehicles for drug delivery can open a wide range of novel applications in dentistry in the future (29).

Other microfabrication techniques of interest include three-dimensional (3D) printing via fused deposition modeling or laser stereolithography, methods that allow the creation of biomaterials via additive manufacturing (24, 25). In these approaches, 3D materials are designed in specialized software and divided into a series of 2D layers with an equal thickness (31), which are then printed layer-by-layer until the full material is completed (32). Furthermore, the fabrication of microspheres for the bespoke delivery of drugs and molecules has also been explored with potential uses in bone and tissue regeneration (26, 27).

Microfluidic chip-based models, fabricated via soft lithography and molding, typically consist of polydimethylsiloxane (PDMS) platforms presenting channels and reservoirs that allow fluids to be controlled and manipulated at the microscale (19–21). These chips provide several advantages including precise control of experimental conditions (e.g., flow, the concentration of chemical species, rate of chemical reactions) while allowing inspection of biological samples via microscopy and reducing laboratory costs by decreasing the number of reagents needed for each experiment (17). Perhaps the most promising avenue in microchip-based models is the development of lab-on-a-chip and organ-on-a-chip systems in biomedicine and dentistry. On the one hand, lab-on-a-chip approaches can be used as diagnostic tools for small samples of a bodily fluid such as blood, saliva, or urine, in order to identify proteins, hormones, or pathogens, among others (22). On the other hand, organs-on-a-chip are intended to replicate the environment and physiology of a particular tissue or organ in a complex three-dimensional in vitro model (23). Therefore, these systems are being used to study the physiology of tissues and organs of interest, as well as specific pathologies and potential pharmacological treatment of these conditions. To date, microfabricated chips have been manufactured to replicate the function of organs such as the pulmonary system, cardiovascular system, brain, liver, and kidneys, among others (22).

In addition to the above, there remains an interest in integrating different organs on a chip into one overall system to evaluate the interaction between different organs in the laboratory. This approach, also known as human-on-a-chip or multiorgan-on-a-chip, would have the potential to study not only the therapeutic effect of a drug in a certain target organ but also evaluate its potential toxicity to other relevant organs (33). However, the fabrication of these models is a great challenge due to the difficulty of integrating different systems on a microfabricated chip in an effective and cost-efficient way (34).

When a tooth is subjected to acute damage such as a carious lesion, trauma, or dental fracture, it can cause irreversible inflammation or necrosis of the dental pulp. Given this scenario, one of the most utilized therapeutic options is endodontic treatment; however, several complications may arise including disruption of the mechanical properties of the tooth that significantly increases fracture risk (35). On the other hand, a tooth loses its sensory capacity after endodontic treatment; therefore, any secondary caries lesions on the tooth may not generate symptoms and complicate the detection of these pathologies in a timely manner. Faced with these limitations, pulp regeneration strategies were proposed many years ago as a therapeutic alternative in cases of irreversible pulpitis or pulp necrosis. Nevertheless, there are multiple limitations that hinder its widespread implementation in clinics, such as the limited vascularization of the tooth that hinders pulpal regeneration cell viability post-intervention (36). To overcome these difficulties, a range of microfabrication techniques are being explored as possible alternatives for the study of pulpal regeneration as well as for the development of new clinical approaches (Table 2).

Within the literature, the manufacture of scaffolds made of both natural and synthetic polymers in which dental pulp stem cells can be cultured has been reported (37). The microfabrication techniques used to manufacture these scaffolds include electrospinning, 3D printing, self-assembly, and micro/nanosphere systems (39). Amongst these, Li et al. designed a microsphere-based injectable system that acts as a scaffold for dental stem cell proliferation (40). Here, researchers manufactured a scaffold system made of biodegradable polylactic acid (PLA) microspheres which contained heparin-conjugated gelatin nanospheres containing vascular endothelial growth factor (VEGF). This system acted as both a transport and scaffolding medium for dental pulp stem cells, while at the same time acting as a sustained release system for VEGF over time (40). Furthermore, the fibrillar structure and high porosity of these materials were comparable to the extracellular matrix of collagen. Finally, the authors noted that in this system, the degradation and absorption of microspheres produced a sustained and controlled release of VEGF when injected into the root canal of extracted human teeth and subsequently implanted into an animal model. After 9 weeks, the proliferation and differentiation of dental pulp stem cells toward odontoblasts were observed, accompanied by a large number of blood vessels throughout the pulp (40). Furthermore, Albuquerque et al. have developed an antibiotic-containing PDS fiber scaffold using electrospinning. Metronidazole, minocycline, and ciprofloxacin were incorporated into the scaffold and tested against endodontic pathogens such as Enterococcus faecalis and Actinomyces naeslundii. The scaffold had the ability to sustainably release the antibiotic mixture causing bacterial death without affecting dental pulp stem cell proliferation and migration (38). However, studies regarding the use of microfabrication in dental pulp regeneration are mostly restricted to in vivo or in vitro experiments, so the extrapolation of these results into the clinical setting remains quite limited. In the future, researchers hope to use these and other microfabrication approaches—such as vasculature engineering—to generate scaffolds that promote dental pulp revascularization and regeneration after injury or restorative treatment with predictable outcomes (58).

In dentistry, the interaction between biomaterials and oral tissues is a crucial component for biocompatibility and the long-term success of dental restorations. However, many difficulties remain in order to correctly replicate the in vivo tissue-biomaterial interface in the in vitro setting. To solve this problem, França et al. have been pioneering the implementation of microfluidic-based systems to replicate the physiology of dental pulp and its interaction with different biomaterials (41). For this, they designed a device called “tooth-on-a-chip” by using a combination of PDMS and polymethylmethacrylate (PMMA) by employing 3D printing and microfabrication. By designing two parallel channels separated by a block of human dentin, stem cells from the apical papilla were co-cultured with different dental biomaterials to replicate the pulp-dentin-biomaterial interface and evaluate the pulp response in real time. In an initial publication, they evaluated the cytotoxicity of materials used in restorative dentistry such as hydroxyethyl methacrylate, resin-based adhesive systems, and phosphoric acid by employing their tooth-on-a-chip system (41). Subsequently, in a second study, the authors used the same device to evaluate the effect of different calcium silicate-based cements and their ability to induce cell proliferation in pulp cells (42). Authors observed differences in cell proliferation, viability, morphology, transforming growth factor-β expression, and antimicrobial activity according to the employed biomaterials (42). Overall, these microfluidic systems are highly reproducible in vitro platforms for the high-throughput evaluation of biomaterials for restorative dentistry; however, clinical studies are still needed to validate the in vivo effectiveness of these approaches. Nevertheless, the further development of tooth-on-a-chip devices shows great promise for the real-time evaluation of biomaterials, molecules, and drug therapies against cells and biofilms of clinical interest (Table 2).

Furthermore, microfabrication techniques are gaining traction as a tool for the development of dental materials such as fiber-reinforced composites, which have shown improved mechanical properties compared to regular-filled composites (43). In Tian et al., the incorporation of nylon 6 fibers into a resin matrix was achieved with electrospinning, resulting in spun nylon 6 nanofibers with an average of 250 nm diameter. The fiber was then milled and incorporated into a resin matrix in various mass fractions (1%, 2%, 4%, and 8%) to observe their effect on the mechanical properties of the resulting biomaterial. Results demonstrated an increase in flexural strength, elastic modulus, and work of fracture for the small mass fraction groups (1% and 2%) (30). Similar to the previously discussed literature, this work is also mostly focused on in vitro formulations and testing, and further efforts must be made to validate the use of these microfabricated biomaterials in the clinical setting.

The use of microfabrication has also potentiated cutting-edge research on periodontal regeneration (Table 2). Periodontal tissues such as the alveolar bone, periodontal ligament (PDL), and oral mucosa are highly complex, and as such, there is a need to develop small-scale biomimetic biomaterials to effectively regenerate areas with extended tissue destruction following periodontal disease (59). To do so, some groups have utilized 3D-printed microfabrication to generate surfaces with micropillar cues to promote the deposition of aligned collagen fibers for PDL regeneration (44). After seeding with PDL cells, authors observed cellular alignment and the deposition of orientated collagen amongst the micropillars in an animal model, showing promise as an approach for the guided reconstruction of soft tissues (44). Furthermore, Suzuki et al. fabricated a collagen membrane to be used as scaffolding for both extraoral and intraoral epithelial regeneration by mimicking the shape and distribution of dermal papillae (46). Firstly, the authors employed lithography to generate microfabricated surface topographies in order to mimic the tissue pattern of the epidermis and dermis. Subsequently, a tilapia collagen mixture was poured into the molds and further crosslinked in order to improve the mechanical properties of the membrane (46). Overall, the authors observed epithelial regeneration directly associated with the membrane structure and suggest that this technique may have the potential to function as a membrane that allows cell regeneration in a biomimetic way.

In other work, Lee et al. have focused on creating microstructured materials with region-specific properties to simulate the cementum-PDL-alveolar bone complex for optimized periodontal regeneration (45). For this, authors engineered specific microarchitectures for each region and selectively loaded the scaffold with either amelogenin, connective tissue growth factor, or bone morphogenic protein to promote cementum, PDL, and alveolar bone regeneration, respectively. These micro-channeled substrates were generated via 3D bioprinting and were found to promote distinct cell differentiation, collagen deposition, and mineralization within the construct in both in vitro and in vivo experiments (45). On the other hand, Vurat et al. designed a microfabricated model to replicate the interface between the PDL and alveolar bone, similar to a tooth-on-a-chip system. The authors used 3D bioprinting to fabricate hydrogel blocks containing human periodontal ligament fibroblasts and osteoblasts. Subsequently, both hydrogels were united and the resulting microtissue was included in a PDMS chip containing a circuit that allowed the diffusion of culture media to monitor cell viability and migration over time (47). The authors used tetracycline as a model drug for the antibiotic treatment of periodontal disease and were able to measure its uptake by the scaffold-encapsulated cells. Overall, the use of these models to study the response of periodontal and alveolar bone cells to drug loading or bioactive molecules is of great interest for future studies on the treatment of periodontal diseases. However, more clinical research is still needed in order to translate these systems into clinics and patient care.

Furthermore, oral bone regeneration is also an area that has gained much interest due to the increased use of dental implants for tooth replacement. Currently, the treatment for bone defects is mainly based on the use of bone grafts that provide an osteoconductive media for regeneration. Therefore, the complementation of bone regeneration with microfabricated osteoinductors such as growth factors is currently being explored. In this context, Ma et al. developed an injectable system of poly (lactic acid) (PLA) microspheres carrying gelatin nanospheres bound to bone morphogenetic protein (BMP-2). This system allowed both improved cell adhesion and a sustained release of BMP-2 from the microspheres over time (48). There is also a growing interest in the use of scaffold-free approaches for regenerative periodontics and endodontics, including the use of extracellular vesicles, spheroids, and tissue strands that aim to promote the development of a biomimetic extracellular matrix for regeneration (60). The microfabrication of tissue-specific stem cell niches that promote local tissue regeneration is also of great interest (8). Nevertheless, most of these investigations remain strictly in vitro and challenges remain in order to translate these microfabricated technologies and biomaterials into standardized and reproducible clinical approaches, particularly as the microarchitecture of the periodontal region is highly complex compared to other tissues.

A crucial element for the success of titanium dental implant treatments in dentistry is the correct integration of the implant into the periodontium (including the alveolar bone) and the surrounding soft tissues. To achieve this, it is known that the surface topography and roughness of the implant are important factors to achieve intimate contact between the alveolar bone and the titanium surface (61). Therefore, it is no surprise that the use of microfabrication techniques has been implemented to attain biologically active titanium surfaces (Table 2) (62).

For example, Doll et al. have published different strategies to design and fabricate surface patterns on titanium frameworks (49). They initially proposed a subtractive method by acid etching using photolithography. However, as the substrates in which lithography techniques are used are usually flat, a flexible photomask was manufactured to apply this technique onto a dental implant surface by using a mechanism allowing rotation of the implants while they are exposed to ultraviolet light (49). By employing this method, they obtained microscale features up to 1.5 µm sizes with the hope of improving fibroblast and epithelial cell adhesion to dental implants, in order to increase soft tissue interactions with dental implant biomaterials in the future.

On the other hand, the same group of researchers published a proposal for an additive method using a photolithography method. However, instead of using chemical etching, they proposed the use of anodic oxidation in which an oxide layer is formed on the titanium surface (50). Furthermore, in a recent investigation, Moreira et al. developed micropatterned silica coatings consisting of either lines or micropillars to increase osteointegration in zirconia dental implants (51). The resulting surfaces displayed increased hydrophilicity compared to controls and therefore showed great promise to promote cellular attachment and long-term osseointegration.

Microfabrication has also been used to generate real-time sensors for monitoring inflammation surrounding dental implants. In this context, Kim et al. developed a temperature-based polymer microsensor to monitor implant survival and predict potential failures (52). By using photolithography, authors fabricated polymer microfilms that were wrapped around dental implant abutments and were able to sense temperature changes in the surrounding environment. The microsensor sensitivity paired with its adequate mechanical and chemical resistance suggests its potential use in clinics to develop personalized diagnoses and long-term follow-up for dental implant treatments.

The formation of multispecies biofilms in the oral cavity is an important problem in dentistry as they play a key role in the development of diseases such as dental caries, periodontitis, and peri-implantitis (63–65). More specifically, the dysregulation of the oral microbiota (dysbiosis) due to the over-proliferation of certain pathological strains is associated with site-specific ecological changes (66). Until now, the incubation of microorganisms in closed systems under static conditions where nutrients are depleted, and waste products accumulate has been the most utilized approach. Nowadays, however, thanks to microfabrication it is possible to assess biofilm formation on surfaces using microfluidic devices that simulate different hydrodynamic flow conditions (67). Furthermore, microfabricated systems can be used to study the effect of antibiotics and antimicrobial molecules on bacteria, as well as the resulting changes that occur at the bacterial and biofilm levels (Table 2) (68).

Within this context, there are several approaches to characterize bacterial adhesion and biofilm formation using microfabrication-derived approaches. For example, Straub et al. monitored bacterial adhesion in real-time using a microfluidic system coupled with optical microscopy, observing how medium composition can impact biofilm formation (53), and Tang et al. utilized a microfluidic chip to explore the biofilm dynamics of antibiotic-resistant Escherichia coli (54). More specifically in dentistry, Alvarez-Escobar et al. have recently employed lithography techniques to study intraoral bacterial adhesion to substrates (55). Authors designed PDMS plates with different surface patterns that were subsequently incorporated into intraoral retainers for 24-hour in vivo bacterial adhesion and biofilm formation. Although no significant differences in biofilm formation were observed among substrates, this pilot study proposes a method to study bacterial adhesion directly onto different dental biomaterials or dental tissue specimens (55).

Finally, microfabrication and microfluidic techniques are also being developed as novel low-cost screening strategies for oral cancers, particularly with the objective of early detection and treatment (Table 2). Proof of concept and early designs for lab-on-a-chip-based microfluidic systems looked to automatize the detection of certain saliva-based markers for oral squamous cell carcinoma (56). However, more recently, Zoupanou et al. designed a microfabricated chip with the ability to immobilize circulating tumor cells (57). To do this, a PMMA microfabricated chip containing a microfluidics circuit was constructed using laser ablation techniques and further treated to bind epithelial cellular adhesion molecule (EpCAM), an important membrane biomarker expressed by tumor cells of epithelial origin. By using this system, researchers were able to effectively immobilize circulating tumor cells with the aid of anti-EpCAM antibodies and demonstrated the potential of this system as a plug-and-play device for rapid and easy oral cancer screening (57).