Bioengineering Tooth and Periodontal Organoids from Stem and Progenitor Cells

Abstract

Tooth and periodontal organoids from stem and progenitor cells represent a significant advancement in regenerative dentistry, offering solutions for tooth loss and periodontal diseases. These organoids, which mimic the architecture and function of real organs, provide a cutting-edge platform for studying dental biology and developing therapies. Recent methodologies have been developed to optimize conditions for organoid production, advancing dental regenerative medicine, disease modeling, and developmental studies. The integration of bioengineering strategies with culture techniques enhances both our understanding and the therapeutic potential of these organoids. Additionally, factors such as the extracellular matrix, growth factors, and culture systems profoundly influence organoid formation and maturation. This review explores various bioengineering approaches for generating organoids, emphasizing the pivotal role of stem and progenitor cells.

1. Introduction

Organoids are three-dimensional, miniaturized, and simplified versions of organs produced in vitro that replicate the complexity of an organ. They are derived from stem- or progenitor cells and can self-organize into structures and functions that resemble their in vivo counterparts [1,2,3]. Currently, these organoids can be constructed to represent oral and maxillofacial tissues, such as tooth germ [4], periodontal tissue [5,6], temporomandibular joint, lingual, and taste bud organoid [7,8,9,10,11,12].

Tooth and periodontal organoids offer a promising avenue for developmental studies, disease modeling, and therapeutic interventions. These organoids, which mimic the complex structures and functions of teeth and periodontal tissues, allow for the exploration of the mechanisms of dental development and the underlying mechanisms of various oral diseases in a controlled environment [13,14]. Additionally, they serve as valuable models for testing potential treatments and interventions [15]. A significant breakthrough in this field is the development of enamel-producing cells within these organoids, addressing the major issue of enamel’s inability to regenerate itself in vivo [16]. This advancement holds the potential to revolutionize dental medicine by providing innovative solutions for tooth decay and other enamel-related problems.

Despite their advantages in translational applications, there are several remaining challenges that still need to be addressed, such as the maturation, morphological properties, and functional physiology, which can hinder the optimum utilization of these organoids. To address this problem, several approaches have been employed, including genetic modification, cell and tissue engineering, modification of the culture environment, and physical/biochemical induction [17].

In this review, we summarized the current challenges and engineering technologies associated with tooth and periodontal organoids. We examine the existing limitations and discuss potential strategies for addressing them to enhance the translational application of these organoids in regenerative dentistry.

2. General Cell Sources

The development of tooth and periodontal organoids involves specific cell sources to replicate the complex structures and functions of these tissues [18] (Figure 1). This process utilizes stem cells and other progenitor cells, each offering unique advantages, challenges, and methodologies.

2.1. Pluripotent Stem Cells (PSCs)

Dental progenitor or tooth germ cells are favorable for generating tooth organoids because of their inherent regenerative potential and accessibility from dental tissues. However, these cells are mostly derived from third molar teeth, limiting their practical application to posterior teeth due to genetic differences with anterior teeth.

PSCs, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), can proliferate indefinitely and differentiate into any cell type, providing a versatile tool for organoid development [19]. They can be genetically manipulated to ensure uniformity and reduce variability among resulting organoids. However, the use of ESCs raises ethical issues, although iPSCs, reprogrammed from adult cells, mitigate some of these concerns. Another advantage of using these cells is their broader range of differentiation into various cell types compared to adult stem cells [20]. However, challenges remain, including difficulties in achieving functional and morphological maturation, ensuring purity, preventing teratoma formation, and addressing a higher chance of immune rejection compared to adult stem [21,22].

Directing hPSCs to differentiate into specific dental and periodontal cell types requires complex and finely tuned protocols. Somatic cells are reprogrammed to become iPSCs using factors like Oct4, Sox2, Klf4, and c-Myc [23]. Specific growth factors and signaling molecules guide iPSCs toward dental epithelial and mesenchymal lineages necessary for tooth and periodontal organoid formation [24]. A recent study successfully derived bioengineered tooth germ organoids from mouse pluripotent stem cells (PSCs) through stepwise differentiation into dental mesenchymal cells (DMCs) and dental epithelial cells (DECs) using a transwell culture method [25]. The organoid structure derived from these PSCs exhibited identical mechanical properties to in vivo native mouse teeth, making it applicable for generating various types of teeth.

2.2. Adult Stem Cells (ASCs)

Adult stem cells (ASCs) offer several advantages for generating organoids due to their inherent regenerative potential, which is beneficial for tissue repair and regeneration. They are particularly useful in creating organoids that closely mimic the native tissue environment [26]. Additionally, ASCs, such as mesenchymal stem cells (MSCs), generally have lower immunogenicity compared to pluripotent stem cells (PSCs), which reduces the risk of immune rejection and makes them suitable for applications where immune compatibility is a concern [22].

However, there are notable challenges associated with using ASCs. One major limitation is their availability; ASCs are only accessible from specific tissues, which can limit their scalability and the ease of obtaining sufficient quantities for large-scale applications [27]. Furthermore, ASCs may not possess the full range of differentiation potential required to generate all necessary cell types for creating fully functional organoids. This can result in incomplete or suboptimal organoid models that may not fully replicate the complexity of the target tissues [26].

2.2.1. Dental and Orofacial Stem and Progenitor Cells (DSCs)

Dental stem cells are a diverse group of cells with remarkable regenerative potential, categorized based on their origin within dental tissues (Figure 1). The primary types include dental pulp stem cells (DPSCs) [28], which are derived from the dental pulp of adult teeth and are known for their ability to differentiate into various cell types such as odontoblasts, chondrocytes, and adipocytes. Stem cells from human exfoliated deciduous teeth (SHED) are another type, isolated from the pulp of baby teeth, with higher proliferative rates and greater differentiation potential compared to DPSCs [29]. Periodontal ligament stem cells (PDLSCs) are found in the periodontal ligament and are crucial for regenerating periodontal tissues, including cementum, periodontal ligament, and alveolar bone [30]. Additionally, dental follicle progenitor cells (DFSCs) come from the dental follicle surrounding the developing tooth germ and can differentiate into periodontal ligament cells, osteoblasts, and cementoblasts [31]. Stem cells from the apical papilla (SCAP) are located at the tip of the developing tooth root and are vital for root formation and regeneration [32]. Gingival fibroblast stem cells (GFSCs) are derived from the gum tissue and play a role in wound healing and tissue repair [33]. Alveolar bone marrow stem cells (AB-MSCs) originate from the jawbone, which plays a crucial role in bone regeneration, especially in dental implants and repairing bone defects [34]. Each type of dental stem cell holds unique properties that make them valuable for various applications in dental tissue engineering and regenerative medicine.

DPSCs possess a remarkable ability to differentiate into various cell types, including odontoblasts crucial for dentin formation [35]. These cells can be obtained relatively easily from extracted teeth, such as wisdom teeth, making them accessible for research and clinical applications [29]. However, DSCs face challenges such as limited proliferation compared to other stem cell types and significant donor-to-donor variability in their regenerative potential, impacting result consistency. DPSCs are isolated from dental pulp tissue through enzymatic digestion and cultured in specific media to promote their proliferation and differentiation [36]. Various biochemical and physical cues are applied to induce DPSCs to differentiate into odontoblasts, ameloblasts, and other dental cell types.

PDLSCs are well suited for regenerating periodontal tissues due to their origin from the periodontal ligament. These cells have a high capacity for promoting angiogenesis, crucial for tissue regeneration and healing [37]. However, obtaining PDLSCs requires periodontal ligament tissue, which can be more challenging compared to DPSCs. Additionally, PDLSCs can undergo senescence more rapidly than other stem cell types, limiting their long-term usability [30]. PDLSCs are harvested from the periodontal ligament of extracted teeth and cultured in specialized media to maintain their stemness and proliferative capacity [38]. Techniques such as scaffold-based or hydrogel-based systems are employed to support the formation of periodontal organoids that mimic the native periodontal environment [39].

2.2.2. Mesenchymal Stem Cells (MSCs) from Non-Dental and Orofacial Tissue

Non-orofacial MSCs, such as bone marrow-derived MSCs (BM-MSCs) and adipose-derived stem cells (ADSCs), exhibit high potential for osteogenic and adipogenic differentiation [40,41] as well as secrete growth factors and possess immunomodulatory properties, enhancing dental tissue regeneration [42,43,44,45]. This capability allows effective modeling of alveolar bone and periodontal ligaments within periodontal organoids. The use of BM-MSCs has been particularly effective in representing the bone matrix in periodontal organoids, providing valuable insights into osseous tissue recovery and the interactions between tooth-supporting structures [41]. In tooth organoids, these MSCs can also partially represent a component of dental pulp tissue [46]. These widely available non-dental MSCs offer a readily accessible and versatile cell source for tooth and periodontal organoids.

2.2.3. Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs), typically derived from bone marrow or peripheral blood, contribute primarily to studying immune tissue [47]. Incorporating HSCs into these organoids allows for the investigation of immune responses, particularly in inflammatory conditions such as periodontitis, where immune cell infiltration and cytokine production play crucial roles [48]. This integration of HSCs helps to create a more physiologically relevant model for periodontal diseases, enabling the study of immune-mediated tissue destruction and repair processes.

2.3. Somatic Cells

In addition to PSCs and ASCs, somatic cell types can be integrated into tooth and periodontal organoids. Epithelial cells, essential for enamel formation, and endothelial cells, crucial for vascularization, can be derived from various tissues or reprogrammed [33,45]. Fibroblasts provide structural support and produce extracellular matrix components [49,50], while immune cells, including macrophages and T cells, help study immune responses and inflammation [51]. These additional cells contribute to the complexity and functionality of organoids, despite challenges such as maintaining cell phenotype, ensuring stable integration, and managing variability in cell quality [52].

Using somatic cells to generate tooth and periodontal organoids presents several difficulties. Advantages of somatic cells include their ability to more accurately recapitulate the native tissue architecture and function, providing a closer representation of in vivo conditions [53]. This can be crucial for studying tissue-specific phenomena and developing realistic disease models. Despite these advantages, somatic cells, such as primary cells, are often difficult to adapt in vitro due to their lower proliferation rates, limited lifespan, and high variability [54].

3. Tissue Engineering Approach

3.1. Scaffold Free Culture

Scaffold-free systems rely on the self-assembling properties of cells to form three-dimensional structures without the need for exogenous scaffolding materials. Techniques such as cell sheets or promoting cell aggregation using hanging drops and low-adhesion plates are commonly used (Figure 1). Sasai et al. [55] describe how scaffold-free culture methods can be used to generate complex organoid structures, demonstrating the self-organization capabilities of pluripotent stem cells in the absence of scaffolds. These methods offer significant advantages for the development of tooth and periodontal organoids by promoting direct cell-cell interactions, which more closely mimic native tissue microenvironments [56,57]. Direct cellular interaction enhances cell communication, signaling, and tissue organization, leading to improved tissue maturation and functionality [58]. The absence of exogenous scaffolds also reduces the risk of immune responses, inflammation, and scaffold-related complications such as degradation products, thereby enhancing biocompatibility.

Despite their benefits, scaffold-free culture methods have notable limitations. The lack of mechanical support inherent in these methods can be a significant drawback, as it is essential for guiding tissue organization and architecture. This limitation can result in less predictable tissue formation and may impede the engineering of larger and more complex tissues [56]. Additionally, scaffold-free approaches may face challenges in scalability and reproducibility, particularly when constructing larger tissue structures or organoids, as controlling the size and shape of tissue constructs without a scaffold is more challenging [59,60]. This condition is also related to limited nutrient transfer, toxic metabolic byproducts, and oxygenation when the structure grows excessively larger than 500 µm in diameter [61]. Moreover, these methods may offer less control over tissue morphology and structure, which can be critical for tissue engineering applications that require precise architectural control. To address this problem, cell sheet technology can be applied mainly when aimed at the transplantation of engineered periodontal tissues [44] (

Table 1. Selected examples of tooth and periodontal organoids.

Organoid Type	Cell Source	Culture Strategy	Application	Ref.
Tooth organoid	Induced pluripotent stem cells (iPSCs)	Scaffold-free (ultra-low attachment U shape well plates)	Tooth development and disease model	Alghadeer, et al. [16]
	Hertwig’s epithelial root sheath (HERS) and dental papilla cells (DPCs)	Scaffold free (cell aggregates)	Dentin tissue regeneration	Duan et al. [62]
	Stem cell population from the apical papilla (SCAP)	Organoid on chip	Model for therapeutic testing and regenerative endodontics	Zhang et al. [63]
	Stem cell population from the apical papilla (SCAP)	Organoid on chip	Biocompatibility testing platform for biomaterials	França et al. [32]
	Dental mesenchymal cells (DMCs) and dental endothelial cells (DECs)	Poly(lactic-co-glycolic acid) (PLGA) scaffold containing a nano-hydroxyapatite	Tooth development model	Van-Manen, et al. [64]
	Dental follicle (DF) tissue from unerupted wisdom teeth	Matrigel embedding	Amelogenesis development model	Hemeryck et al. [65]
	Post-natal porcine dental epithelial (pDE), porcine dental mesenchymal (pDM) progenitor cells, and human umbilical vein endothelial cells (HUVECs)	GelMA encapsulation	Tissue regeneration using bioengineered tooth	Smith et al. [66]
	Dental pulp stem cells (DPSCs) population	Scaffold free (spheroid)	Transplantation and disease model	Itoh et al. [67]
Periodontal organoid	Periodontal ligament (PDLs) and dental follicle cells (DFCs)	Scaffold free (cell sheet)	Engineered periodontal tissue transplantation	Chu, et al. [31]
	Mouse bone marrow mesenchymal stem cells (mBMSCs) and dental endothelial cells (DECs)	GelMA based-bioprinting	Disease model of periodontal tissue defect	Miao et al. [68]
	Periodontal ligament cells (PDLCs), which contain a population of adult stem/progenitor cells	Scaffold free (cell aggregates)	Implantable graft for periodontal regeneration	Basu et al. [5]
	Keratinocytes, dental fibroblasts, and endothelial cells	Organoid on a chip	Disease model of gingival inflammatory	Gard et al. [69]
	Gingival fibroblasts and keratinocytes	Scaffold free (cell sheet)	Disease model of bacterial and viral infection in periodontitis	Golda et al. [70]

).

In their development, the formation of the tooth relies on the interaction between mesenchymal and epithelial cells. This principle was applied by Nakao et al. to develop a three-dimensional bioengineered tooth germ culture by mixing dental mesenchymal cells (DMCs) and dental epithelial cells (DECs). Using this method, they successfully structurally mimic the tooth, both in vitro and after transplantation into a mouse tooth cavity in vivo, demonstrating the integration of blood vessels and nerve fibers [71]. Using a similar approach, another study replicated the results in a larger animal [72,73].

A study conducted by Basu, et al. demonstrates that scaffold-free organoid constructs, engineered using PDLCs containing dental progenitor cell populations, can self-assemble into organized multi-tissue structures resembling natural periodontal tissues. These constructs form a cylindrical structure with a mineralized cementum-like core and a periodontal ligament (PDL)-like periphery, maintaining their organization both in vitro and in vivo [5]. Such engineered tissues could be used for periodontal regeneration or as model systems to study PDLC biology. Similarly, Itoh et al. developed scaffold-free 3D dental pulp stem cell organoid constructs for dental pulp regeneration. These constructs remained viable in vitro and self-organized into pulp-like tissues with rich blood vessels when implanted in human tooth root canals in immunodeficient mice [5].

Kim et al. successfully generated mineralizing dental epithelial organoids (DEOs) from adult dental epithelial stem cells (aDESCs) isolated from mouse incisor tissues [74]. These DEOs expressed ameloblast markers and could be maintained in vitro for over five months with signaling pathway modulators. When transplanted under murine kidney capsules, they produced hydroxyapatite crystallites resembling natural dental tissues, indicating their potential for regenerative enamel therapy.

3.2. Matrix Embedded Culture

Matrigel, a gel-like protein mixture sourced from Engelbreth–Holm–Swarm (EHS) mouse sarcoma cells, offers a supportive extracellular matrix (ECM) environment crucial for the growth and differentiation of organoids. Matrigel embedding is commonly employed for culturing diverse types of organoids, including those derived from oral and maxillofacial tissues (Table 1). This ECM provides advantages such as closely mimicking the in vivo environment, supporting complex structures, and being suitable for long-term culture [52,75,76,77,78]. However, challenges associated with Matrigel use include variability in matrix composition, high cost, and ethical concerns regarding animal-derived products that remain a concern [79,80]. Alternatives to Matrigel, a commonly used basement membrane matrix for cell culture, can be derived from natural and synthetic sources. Natural alternatives include extracellular matrix (ECM) proteins such as collagen [81], laminin [81,82], and fibronectin [83], which offer biocompatibility and support cell adhesion and growth. Synthetic alternatives include hydrogels like polyethylene glycol (PEG) and alginate, which offer tunable mechanical properties and degradation rates. PEG hydrogels can be functionalized with cell-adhesive peptides to enhance cell attachment and growth [84,85], while alginate provides a 3D environment for encapsulating cells and promoting their proliferation and differentiation [79,86].

Hemeryck et al. developed a novel epithelial organoid model derived from human teeth. By isolating dental follicle (DF) tissue from unerupted wisdom teeth, they successfully generated long-term expandable Matrigel-embedded dental epithelial organoids [65]. These organoids exhibited characteristics of tooth epithelial stemness, similar to the epithelial cell rests of Malassez (ERM) found in the DF, which houses dental epithelial stem cells. When exposed to epidermal growth factor (EGF), the organoids underwent transient proliferation followed by epithelial–mesenchymal transition (EMT), closely replicating the processes observed in the ERM in vivo. Additionally, these ERM stemness organoids demonstrated the ability to initiate an ameloblast differentiation process [65].

3.3. Scaffold Based Culture

Scaffold-based culture methods play a crucial role in the advancement of engineered tooth and periodontal tissue or organoid models, offering a platform to replicate the intricate architecture and function of these tissues [87]. In these approaches, cells are seeded onto a supportive scaffold that provides structural integrity and cues for cell attachment, proliferation, and differentiation [41] (Figure 1). A variety of scaffolds, including synthetic polymers, natural polymers, and decellularized tissues, are employed to mimic the extracellular matrix (ECM) environment of native tissues [88].

Studies have explored the use of bioactive scaffold materials such as hydrogels, which can mimic the ECM and provide cues for cell differentiation and tissue regeneration [89] (Table 1). Additionally, advances in 3D bioprinting technology have enabled the fabrication of complex scaffold structures with precise control over spatial organization and composition, facilitating the development of biomimetic tooth and periodontal tissue constructs [90]. Several attempts have been made to improve the generation of tooth organoids by modifying scaffolds. For example, to spatially control tooth development, porcine DMCs and DECs were seeded in a poly(lactic-co-glycolic acid) (PLGA) scaffold containing nano-hydroxyapatite to further promote differentiation [64]. Fiber alignment by electrospinning influenced cell orientation during the early weeks after seeding, but it had no long-term impact on cell alignment or the deposition of organized calcified matrix once the cells reached confluency.

Smith et al. developed a biomimetic tooth bud model using post-natal porcine dental epithelial (pDE), porcine dental mesenchymal (pDM) progenitor cells, and human umbilical vein endothelial cells (HUVECs) encapsulated within GelMA hydrogels. Their study demonstrated that GelMA constructs supported predictable mineralized dental tissue formation of specified size and shape, overcoming previous limitations of small tooth structures formed within scaffold pores. Encapsulated pDE-HUVEC constructs in 3% GelMA and pDM-HUVEC constructs in 5% GelMA supported dental cell differentiation and vascular mineralized tissue formation, presenting a promising approach for bioengineered tooth replacement in humans [91]. Another attempt, developed by Bektas et al., aimed to generate tooth germ organoids using modified hydrogel microparticles (HMPs) derived from gelatin methacrylate (GelMA) as scaffolds for human dental pulp stem cells (hDPSCs) and porcine dental epithelial cells (pDE) [66]. These co-cultured structures exhibited polarized, differentiated dental cells, suggesting potential in tooth regeneration strategies.

Decellularized organ scaffolds can provide an extracellular matrix to control the shape of tissue formation and guide the specific differentiation of the tooth bud [92]. Using decellularized tooth buds (dTBs), Zhang et al. seeded human dental pulp cells (DPCs) and porcine DECs together with human umbilical vein endothelial cells (HUVECSs) to enhance the vascularization of the pulp-like tissue. This recellularized tissue construct showed significant production of organized dentin and enamel-like tissues [53]. Another investigation was conducted to improve the incorporation of growth factors, bioactive molecules, and stem cells into scaffold-based cultures to enhance tissue regeneration and promote functional integration with the host environment [93,94,95,96]. For instance, to mimic the tissue environment, the tooth germ organoid can be formed in polycaprolactone (PCL) together with neural growth factors embedded inside to further induce tooth germ differentiation and self-tissue rearrangements with functional tissue innervation [65].

While offering a tissue-like environment, there is a need to further enhance several aspects. These improvements entail optimizing scaffold properties such as biocompatibility, mechanical strength, and degradation kinetics to better support tissue development [97,98]. Moreover, ensuring the sustained stability and functionality of engineered tissues remains a prominent research focus [99].

3.4. Microphysiological System/Organoid on Chip

Microphysiological devices, also known as organ-on-a-chip systems, integrate microfluidic technology to create small-scale, physiologically relevant models of human organs (Figure 1). These systems can replicate the complex microenvironment of tissues, including mechanical and biochemical cues, making them ideal for studying organoid development and function [100] (Table 1). This technology enables precise control over culture conditions, the ability to study dynamic processes, and the potential for cultivating organoid models to study inter-organ interactions [69,101,102].However, this culture system exhibits several challenges related to technical complexity, high cost, and scalability issues.

The study by Zhang, et al. explored how cell density and GelMA concentrations influence microfluidic chip-based tissue constructs. Optimal conditions were identified with 2 × 10⁴ cells/μL density and 5% GelMA concentration promoting cell growth and uniform distribution, enhancing osteogenic or odontogenic differentiation of stem cells from the apical papilla (SCAP) [63]. This suggests the potential for simplified dentin-on-a-chip models using GelMA with SCAP at the recommended seeding density. França et al. developed a similar tooth-on-a-chip model to mimic the dentin pulp interface. This model allows for live-cell imaging of dental pulp cell responses to biomaterials. Stem cells from the apical papilla (SCAPs) were cultured on a dentin matrix-coated wall within a microfluidic device, and standard dental materials were tested for cytotoxicity and metabolic activity. Real-time tracking revealed dental pulp cells’ potential contribution to proteolytic activity in the model hybrid layer, highlighting the tooth-on-a-chip’s utility for studying dental pulp cell responses to biomaterials [32].

Investigations by Niu et al. utilized a microfluidic chip to mimic dentin tubule structures, facilitating odontoblast culture. By varying microchannel sizes, they found that 2 μm channels induce odontoblast process growth, mirroring in vivo conditions. This chip offers a valuable tool for studying odontoblast physiology and developing dental disease treatments, such as dentin hypersensitivity [103].

Gard et al. introduced a high-throughput microfluidic organ-on-chip model of human periodontal organoid, including keratinocytes, dental fibroblasts, and endothelial cells. This triculture model accurately replicates physiological tissue structure, mucosal barrier formation, and protein biomarker expression and secretion, enabling the study of inflammation over several weeks. By administering specific small molecule inhibitors, they mitigated the inflammatory response, demonstrating the potential for identifying new therapeutic targets for gum disease and facilitating preclinical drug efficacy testing [69].

3.5. 3D Bioprinting Technologies

Three-dimensional (3D) bioprinting technology has emerged as a promising tool in regenerative dentistry, enabling the precise fabrication of three-dimensional tissue constructs by layering bioinks (cells, matrix, growth factors, or scaffold) [104,105]. In oral and maxillofacial organoid research, 3D bioprinting can recreate complex structures like teeth and periodontal tissue with high precision, which is crucial for studying tissue development and regenerative medicine [106,107] (Figure 1). This technology also enables customizable designs for patient-specific organoids, enhancing the potential for personalized medicine [108,109] (Table 1).

Murphy and Atala emphasized the advantage of 3D bioprinting in creating patient-specific implants and tissue models [110]. The technology supports scalable production, enabling large-scale 3D bioprinting for various potential clinical applications [111]. However, current bioprinting techniques often face challenges in achieving the necessary resolution to recreate the fine details of natural tissues, which can affect tissue viability and functionality [112,113]. Additionally, the high costs of bioprinting equipment and materials may limit accessibility in some research and clinical settings [114,115]. Current bioinks may lack the necessary mechanical properties and biocompatibility required for certain oral and maxillofacial tissues [113]. Despite these challenges, Daly et al. discussed the applications of 3D bioprinting in tissue engineering, highlighting its ability to fabricate anatomically accurate and functional tissue constructs, including those for craniofacial regeneration [116].

A novel multi-component hydrogel, comprising GelMA, sodium alginate, and bioactive glass microspheres, was developed for 3D bioprinting to regenerate periodontal tissues. This hydrogel exhibited excellent printability and biocompatibility, along with enhanced osteogenic differentiation in vitro. Furthermore, Miao et al. revealed that this system performs better when loaded with mouse bone marrow mesenchymal stem cells and growth factors (BMP2 and PDGF), which effectively promoted the regeneration of gingival tissue, periodontal ligament, and alveolar bone in vivo to address periodontal defects [68].

4. Gene Profiling, Genetic Engineering, and Adjustment of Mechanical and Biochemical Conditions

Gene profiling and genetic engineering are powerful tools revolutionizing our understanding and manipulation of biological systems, even in complex structures like tooth and periodontal organoids [117]. Gene profiling analyzes gene expression patterns within cells, offering insights into their behavior and function [118]. By unraveling the genetic signatures of these organoids, scientists can identify key genes involved in tooth and periodontal development, homeostasis, and disease processes [119]. Genetic engineering enables precise manipulation of these genes, offering potential avenues for therapeutic interventions or regenerative medicine approaches. This combination of techniques holds promise for addressing dental and periodontal disorders, paving the way for personalized treatments tailored to individual genetic profiles [56,57].

Tooth decay poses a significant challenge due to the enamel’s inability to naturally regenerate. Recent research has made significant strides in presenting enamel-producing cells using organoids, potentially revolutionizing dental medicine. Alghadeer et al. developed enamel-producing organoids by replicating the genetic program guiding fetal stem cells to become ameloblasts. Using single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) and the Monocle program, they mapped gene expression patterns driving this differentiation. This approach successfully created organoids capable of secreting enamel proteins [16], representing a 3D organoid system that models amelogenesis imperfecta, thus advancing enamel therapies.

The generation of organoids can be enhanced using CRISPR-Cas9 gene editing technology to modify cellular responses and promote maturation. By targeting specific genes, this method triggers the expression of substances that aid in cell differentiation [120]. For example, a CRISPR-Cas9-based technique can activate transcription factors, adjust tissue-regulated networks, and improve the maturation of PSC-derived organoids [121]. While this approach shows promise, the effectiveness of using genetic editing tools to control organoid self-organization is limited by incomplete information on cell behavioral complexity and regulatory networks, highlighting the need for further research in this area.

Irfan et al. investigated the role of the C5L2 receptor in inflammation-induced odontoblastic differentiation of dental pulp stem cells (DPSCs) by using CRISPR knockout (KO) and a TrkB antagonist. They found that C5L2 CRISPR KO enhances mineralization and expression of dentin proteins DSPP and DMP-1 in TNFα-stimulated DPSCs, with TrkB playing a critical regulatory role in this process [122].

Mechanical stimuli, such as compression, tension, and shear stress, can enhance the proliferation, differentiation, and mineralization of cellular components in tooth and periodontal organoids [123,124]. The scoping review by Rad et al. evaluated the impact of mechanical forces on the behavior of various dental stem cells (DSCs) through a comprehensive analysis of 18 in vitro studies published between 2008 and 2019. The study examined the effects of tension, hydrostatic pressure, compression, simulated microgravity, and vibration on osteogenic/odontogenic differentiation, proliferation, adhesion, and migration of DSCs, including periodontal ligament stem cells (PDLSCs), dental pulp stem cells (DPSCs), and stem cells from apical papilla (SCAP). The review found that various mechanical forces, except uniaxial tension, generally promoted the osteogenic/odontogenic differentiation of DSCs, with specific forces like tension and simulated microgravity enhancing PDLSC and DPSC proliferation both in tooth and periodontal organoids. Additionally, carrier features and the duration of tension application influenced the extent of osteogenic differentiation in DSCs [123].

The application of mechanical forces can induce the differentiation of DPSCs into odontoblast-like cells, which are responsible for forming dentin, a key component of tooth structure. This differentiation is mediated through various signaling pathways and the expression of specific genes related to odontogenesis. Moreover, mechanical stimuli can modulate the extracellular matrix production and the secretion of growth factors by DPSCs, further promoting tissue regeneration and repair. The study emphasizes the potential of using mechanical forces in combination with other biochemical factors to optimize the regenerative capabilities of DPSCs in clinical applications [125].

Research by Jia et al. investigated the effects of different cyclic stress loadings on inflammatory human periodontal ligament cells (hPDLCs). They found that excessive stress loading (150 kPa) significantly increased the expression of inflammatory and osteoclastic markers, while moderate cyclic pressure (0–90 kPa) promoted osteogenic differentiation. The study suggests that appropriate mechanical stimulation is crucial for maintaining bone homeostasis in periodontal organoids and highlights the differential impact of stress levels on hPDLC function [126].

The study by Wang et al. investigated the effects of shear stress on the endothelial differentiation of stem cells from human exfoliated deciduous teeth (SHEDs). The researchers found that shear stress significantly upregulated the expression of arterial markers and angiogenic proteins, enhancing the formation of vessel-like structures in vitro. Additionally, shear stress increased VEGF protein levels, and the process involved VEGF-DLL4/Notch-EphrinB2 signaling, demonstrating shear stress’s role in inducing arterial endothelial differentiation of SHEDs [127].

Research by Shi et al. demonstrates that flow shear stress (FSS) promotes the proliferation of periodontal ligament (PDL) cells, essential for periodontal regeneration. Their study reveals that FSS remodels the cytoskeleton and focal adhesion, activates the p38 pathway, and facilitates YAP nuclear translocation, providing mechanistic insights and potential applications for tooth and periodontal regenerative medicine [128].

Honda et al. investigated the effect of shear stress on odontogenic cells, focusing on its role in tissue-engineered odontogenesis. Cells from the porcine third molar at the early crown formation stage were seeded on a biodegradable polyglycolic acid fiber mesh, with and without exposure to shear stress. In vitro, shear stress significantly enhanced the expression of odontogenic-related mRNAs and proteins, as well as alkaline phosphatase activity, without affecting cell proliferation. In vivo, constructs exposed to shear stress for 12 h formed enamel and dentin tissues after 15 weeks, unlike controls. This study concludes that shear stress facilitates odontogenic cell differentiation and tooth tissue engineering [129].

A study by Zhao et al. highlights the role of dental pulp stem cells (DPSCs), a type of mesenchymal stem cells from dental pulp, in biological regeneration. DPSCs can differentiate into multiple cell types and promote osteogenesis, odontogenesis, chondrogenesis, and angiogenesis, regulated by numerous intra- and extracellular factors. The review focuses on the impact of biomechanical cues, such as substrate stiffness, physical stress, and cell spreading, on DPSC function, discussing related signaling components, current findings, and potential applications in regenerative medicine and tissue engineering [28].

Research by Yang et al. investigated the effects of compressive stress on the proliferation and differentiation of human dental pulp cells (hDPCs). Using a four-point bending strain system, they applied low-density cyclic uniaxial compressive stress to hDPCs and found that cell cycle progression and differentiation-related gene expression were significantly promoted. The study concluded that hDPCs are sensitive to compressive stress, enhancing proliferation and odontogenic differentiation in vitro [130]. Another study by Klimcumhom et al. investigated the role of Yes-associated protein (YAP) in human periodontal ligament (PDL) cells under intermittent compressive force (ICF). They found that ICF-induced YAP promotes osteogenesis and inhibits adipogenesis in PDL cells. The study suggests that YAP is a crucial mechanosensitive transcriptional activator in periodontal organoid, highlighting its potential to facilitate periodontal tissue regeneration through manipulation of the Hippo-YAP signaling pathway [131].

Kim et al. investigated periodontal ligament (PDL) regeneration strategies, developing fibrous scaffolds mimicking the PDL matrix. The scaffold-seeded PDL fibroblasts exhibited proliferative and osteogenic potential, with scaffold topography influencing cellular behaviors mediated by yes-associated protein (YAP) signaling. Their findings suggest that fiber–hydrogel complexes could offer a promising approach for guided periodontal ligament regeneration using organoid technology [132].

Biochemical induction plays a crucial role in the development and regeneration of dental tissues, including tooth and periodontal organoids, as well as in the differentiation and proliferation of dental stem cells. This process often involves the application of small molecules or growth factors that can precisely guide cellular behavior and tissue formation [4]. Small molecules, such as retinoic acid and various signaling pathway inhibitors, are employed to modulate specific biochemical pathways, thus promoting the differentiation of stem cells into odontoblasts, ameloblasts, and other specialized dental cells. Growth factors like BMPs (bone morphogenetic proteins) [31], FGFs (fibroblast growth factors) [133], and TGFs (transforming growth factors) [4] are essential in mimicking the natural developmental signals, enhancing cellular communication, and orchestrating the complex processes of tissue morphogenesis and repair [134]. The strategic use of these biochemical agents in vitro allows the differentiation and maturation of functional tooth and periodontal organoids that closely resemble their in vivo counterparts.

The study by Algadheer et al. introduced a new method to create human enamel teeth using human-induced pluripotent stem cells (hiPSCs). The researchers developed a three-step process with specific signaling pathways and molecules. First, hiPSCs were turned into non-neural ectoderm using SU5402, which inhibits the FGF signaling pathway. Next, this ectoderm was converted into oral ectoderm using LDN193189 (a BMP inhibitor) and purmorphamine (an SHH activator). Finally, the oral ectoderm cells were made into enamel-producing ameloblasts using Purmorphamine, XAV939 (a WNT inhibitor), and BMP4 [16].

Zhu et al. developed a fast, three-step method to generate dental epithelial (DE) cells from human induced pluripotent stem cells (hiPSCs) in just 8 days. They used specific small molecules for each step: SU5402 (an FGF signaling inhibitor) to form non-neural ectoderm, LDN193189 (a BMP inhibitor) and purmorphamine (an SHH activator) to form oral ectoderm, and then Purmorphamine, XAV939 (a WNT inhibitor), and BMP4 to turn the oral ectoderm into DE cells [135]. Proangigenic protein SEMA4D and its receptor Plexin B1 are expressed in dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth (SHED), and human dental pulp tissues. Recombinant human SEMA4D (rhSEMA4D) at 25–100 ng/mL significantly induces the expression of endothelial markers VEGFR-2, CD31, and Tie-2 in DPSCs and promotes capillary-like sprouting in vitro [136].

5. Limitation and Possible Improvement by Bioengineering Approaches

5.1. Morphology Mimicry of In Vivo Like-Tissue Complexity

Replicating the identical structure of natural tissues in engineered teeth and periodontal tissues is crucial. For disease modeling, it ensures accurate simulations and a better understanding of dental and periodontal diseases, leading to more targeted treatments. In therapeutic testing and transplantation, structurally and functionally identical tissues integrate better, restoring function, supporting healing, and reducing rejection risks [137]. This similarity enhances the evaluation of drugs, biomaterials, and regenerative therapies in a realistic context, leading to more predictive and safer outcomes before clinical trials.

Despite this importance, reproducing the full complexity and heterogeneity of teeth and periodontal tissues is challenging. Organoids often lack the precise structural organization and the diverse cell types present in natural tissues. Advanced bioengineering techniques, such as 3D bioprinting, can improve the arrangement of those different cell types and biomaterials to mimic the intricate architecture of the actual tissue. Utilizing scaffold materials that provide specific cues for cell differentiation and organization can also improve tissue complexity. For instance, Daly et al. (2021) highlighted the potential of 3D bioprinting to recreate the complex structures of craniofacial tissues, demonstrating improved structural and functional outcomes in tissue constructs [116].

Another significant challenge in tooth and periodontal organoids is the lack of vascularization. Organoids often remain limited in size and complexity due to insufficient nutrient and oxygen supply, which is a direct consequence of inadequate vascular networks within the organoids. Wörsdörfer et al. demonstrated that incorporating endothelial cells into cerebral organoids leads to the formation of a primitive vascular network, suggesting a similar strategy could be effective for oral and maxillofacial organoids [138]. Bioengineering approaches can address this issue by integrating endothelial cells into the organoid culture to promote vascular network formation. However, creating a microvascular tube with a neovascularization capability remains challenging. Replicating this complexity is still beyond the capabilities of current bioprinting technologies [113].

5.2. Scaling up and Reproducibility

Scaling up the production of organoids for clinical applications and ensuring reproducibility between batches remains challenging due to variability in organoid size, shape, and functionality. This variability can hinder clinical translation, but implementing standardized protocols and automated culture systems can improve scalability and reproducibility. High-throughput screening and quality control measures are essential for consistent organoid quality. Yin et al. emphasized the importance of standardizing culture conditions and using automated bioreactor systems to enhance both the reproducibility and scalability of organoid production [139].

Another possible cost-effective method is the utilization of growth factor accumulation in conditioned media using a dialysis-based strategy. This accumulation is derived from recycling remaining exogenous growth factors altogether with accumulated endogenous growth factors secreted by the organoid itself [140,141].

5.3. Functional Maturation

Organoids often lack the full functional maturity of adult tissues, limiting their utility in disease modeling and regenerative medicine. Achieving the mature phenotype and function of oral and maxillofacial tissues remains a significant challenge. Providing mechanical and biochemical cues that mimic the in vivo environment can promote the functional maturation of organoids. Co-culturing with other cellular components in related tissues, as well as applying mechanical forces (e.g., shear stress, tension), can enhance tissue maturation. Sano et al. explored the regenerative potential of co-cultured spheroids of human periodontal ligament mesenchymal stem cells (hPDLMSCs) and human umbilical vein endothelial cells (HUVECs) in vitro and in vivo. Co-cultured spheroids showed upregulation of stemness markers, VEGF, and osteogenesis-related genes, along with increased nodule formation compared to monolayer and hPDLMSC spheroid cultures [142].

Another approach to induce maturation in dental pulp cells is by using a maturation inducer molecule. Nie et al. investigated the effects of TGF-beta1 on dental pulp cells in different culture patterns. TGF-beta1 significantly increased cell proliferation, ALPase activity, and the formation of mineralization nodules in cell cultures. In tissue and three-dimensional cultures, TGF-beta1 induced dental pulp cells to differentiate into odontoblast-like cells, forming a pulp–dentinal complex and upregulating dentine-related proteins DSPP and DMP-1. This study highlights the crucial role of TGF-beta1 in odontoblast differentiation and pulp repair [143].

5.4. Integration with the Host Tissue

The integration of tooth and periodontal organoids or engineered tissues with the host tissue environment presents significant challenges that need to be addressed for successful regenerative therapies. Ensuring proper integration of the printed tissue with the host tissue in vivo is another major hurdle. One of the primary issues is the immune response elicited by the host tissue [144]. When foreign cells or tissues are introduced into the body, the immune system may recognize them as threats and initiate an inflammatory response, leading to rejection or impaired function of the implanted organoids [145].

The use of DPSCs and mesenchymal stem cells (MSCs) from various sources as a primary cell source for generating organoids or engineered tissues offers the potential to reduce immune rejection, largely due to their low immunogenicity compared to pluripotent stem cells [146]. Unlike pluripotent cells, DPSCs and MSCs are less likely to trigger a strong immune response, making them more suitable for clinical applications. Moreover, these cells secrete several immunomodulatory cytokines, such as prostaglandin E2 (PGE2), interleukin-6 (IL-6), and transforming growth factor-beta (TGF-β), which possess anti-inflammatory properties [147]. These cytokines help create a more tolerogenic environment by dampening the immune response and reducing inflammation. As a result, the use of DPSCs and MSCs not only minimizes the risk of immune rejection but also mitigates the inflammatory response, making them ideal candidates for tissue engineering and regenerative medicine applications [146].

One promising alternative approach is the use of immunomodulatory treatments to dampen the host immune response and promote tolerance to the implanted tissues. This can be achieved through local delivery of anti-inflammatory agents or systemic immunosuppressive therapies [148]. Additionally, advancements in tissue engineering techniques, such as the use of biomimetic scaffolds and 3D bioprinting, have improved the ability to replicate the native periodontal architecture [148]. These scaffolds can be designed to provide the necessary biochemical cues and mechanical support to facilitate the integration and function of the implanted tissues.

Incorporating stem cells that can differentiate into various periodontal cell types within the scaffolds enhances the regenerative potential of the engineered tissues, promoting better integration and repair of the damaged tooth and periodontal structures. For instance, the complexity of replicating the native tooth and periodontal environment, which includes a diverse array of cell types, extracellular matrix components, and vascularization, further complicates the integration process, making it difficult to achieve functional and structural integration [137]. Ensuring the engineered tissues mimic the native biological and mechanical properties is crucial for their long-term survival and functionality within the host environment [144].

6. Conclusions

In summary, the development of tooth and periodontal organoids from stem and progenitor cells marks a significant advancement in regenerative dentistry, offering promising solutions for tooth loss and periodontal diseases. These organoids, designed to replicate native organ architecture and function, provide a sophisticated platform for studying oral and maxillofacial biology and advancing therapeutic interventions. Recent methodological advancements have improved organoid production conditions, driving forward the field of dental regenerative medicine and enabling disease modeling and developmental studies.

Moreover, critical factors such as the extracellular matrix, growth factors, and culture systems influence dental organoid formation and maturation, impacting their functionality. Optimizing these parameters can enhance organoid efficiency and functionality, bringing this technology closer to clinical applications. Exploring various bioengineering approaches underscores the importance of stem and progenitor cells in tooth and periodontal organoid development, offering potential for innovative regenerative therapies and personalized dental treatments.