Cocktail Formula and Application Prospects for Oral and Maxillofacial Organoids

Abstract

Oral and maxillofacial organoids (OMOs), tiny tissues and organs derived from stem cells cultured through 3-d cell culture models, can fully summarize the cell tissue structure, physiological functions and biological characteristics of the source tissues in the body. OMOs are applied in areas such as disease modelling, developmental and regenerative medicine, drug screening, personalized treatment, etc. Although the construction of organoids in various parts of the oral and maxillofacial (OM) region has achieved considerable success, the existing cocktail formulae (construction strategies) are not widely applicable for tissues of various sources due to factors including the heterogeneity of the source tissues and the dependence on laboratory technology. Most of their formulae are based on growth factor niches containing expensive recombinant proteins with their efficiency remaining to be improved. In view of this, the cocktail formulae of various parts of the OM organs are reviewed with further discussion of the application and prospects for those OMOs to find some affordable cocktail formula with strong operability and high repeatability for various maxillofacial organs. The results may help improve the efficiency of organoid construction in the laboratory and accelerate the pace of the clinical use of organoid technology.

Introduction

An organoid is a model system of tissues and organs with simplified structure and small size. Combined with growth factor niches (simulation of signaling molecules in vivo) on 3-d scaffolds, it is derived and self-organized from embryonic stem cells (EPSCs), induced pluripotent stem cells (IPSCs), adult stem cells (ASCs), specific cell lines, or body cells [1, 2] (Fig. 1), their construction strategies include cells, scaffolds, and growth factor niches. As a 3-d cell culture model, organoid collectively represents the characterization of the source organ’s cell in terms of its structure, function, and molecular. It cannot only summarize the cell lineage of living tissues, but also to a great extent ensure its maintenance and expansion [3, 4]. Moreover, when simulating organ development and disease, or when developing new therapies (especially personalized treatment) [2, 5–7]. an organoid can better mimic the microenvironment of the interaction between cell and extracellular matrix (ECM) and between cells in the body [8], so as to maintain the stable inheritance of the genome in long-term course of culture [9], and restore the genome at both transcription and protein levels [10]. It is noteworthy that studies have shown that the images displayed by organoids at the metabolic level are clearer than those shown by living tissues in terms of resolution [11]. In view of the above advantages, since 2009 when the Hans Clevers’ research team from The Netherlands confirmed that intestinal stem cells cultured in vitro can form organoids [12], organoids have undergone significant development, and they are widely used in preclinical medicine, clinical medicine, and other related scenarios.

Fig. 1.

The formation of an organoid. † RSPO: R-spondin-1; EGF: Epidermal growth factor; FGF: Fibroblast growth factor; B27: Medium supplement. ‡ Organoid formation process: 1. Fresh tissue organ specimens were obtained by biopsy. 2. Digestion into single cells (further screening of ASCs or direct use of immortalized cell lines/reprogrammed IPSCs). 3. Resuspend the cells to the scaffolding system (commonly used Matrigel), and then transfer to the Petri dish with pipette to form a teardrop-shaped dome. 4. Mix growth factor niches (eg. EGF, RSPO, Noggin, FGF, B27, EMDM/F12). 5. Add niches to form organoid culture systems, and form organoids after 6. periods of time (time varies with tissue differences, Oral and maxillofacial organoids are generally 7–10 days)

Oral and maxillofacial (OM) organs are various, including cheeks, tongues, lips, palate, teeth, salivary glands, upper and lower jaws, and their constituent tissues are also distinctive, such as the hardest tissue in the whole body–dentin [13], the most active osseous tissue–alveolar bone, special organs of epithelial differentiation–taste buds [14], and so on. There is currently no universal culture system of organoids that does not take the sources of tissues into account due to the heterogeneity between OM tissues and between cells. Even for organoids of the same tissue, construction strategies vary among laboratories due to different technologies and focuses, and there is no unified measurement standard, so construction strategies are called as “cocktail formulae”. Despite the fact that most oral and maxillofacial organoids (OMOs) have been successfully constructed, they are only used at the proof-of-concept level while barely being applied to clinical practice. However, in this paper, the construction strategies were reviewed in an attempt to find more classic, desirable, and convenient cocktail formulae for various maxillofacial organs; furthermore, the application and prospects of OM organs are further discussed to improve the efficiency of organoid construction in laboratories and accelerate the pace of organoid technology in clinical use.

Overview of Oral and maxillofacial organoids (OMOs)

The self-organization and derivation of organoids mostly depend on the understanding and application of signaling molecules during organ development. However, the development of OM organs has a strict time and spatial control process: in the third week of the embryo, the frontal nasal process develops at the lower end of the forebrain; in the fourth week, the mesenchymal cells under both sides of the frontal nasal process rapidly proliferate into six pairs of branchial arches. After that, through epithelial-mesenchymal transition, part of the neural crest cells from the midbrain and the first and second rhombohedral arches proliferates into the first branchial arch. At this point, the frontal nasal process and the first branchial arch have formed the initial oral cavity. After a series of differentiation, association, fusion, and epithelial-mesenchymal interactions, OM organs are formed one after another, which period relies on the regulation of many signaling molecules and genes. For example, the development of salivary glands [15–20] (Fig. 2), First, under the interaction of epithelium and mesenchymal mediated by FGF, SOX, retinoic acid, and ARP protein, the thickened primitive oral epithelium invaginates into the concentrated stroma containing endothelial plexus to form initial buds. Subsequently, driven by signaling molecules such as EFG, SHH, integrin, BMP, MMP, SMEM, WNT, HS, MAPK, collagen, laminin, and fibrin, the initial buds formed pseudo-glands after fissure formation and branch morphogenesis. Under the regulation of EGF, FGF, WNT, SHH, TGF, EDA, ACH, VIP, etc., the midline of the branch of the pseudo-glands expands into catheter, and finally forms the terminal buds of the salivary gland. The functions and regulatory mechanisms of some of the signaling molecules and genes have been revealed, but a detailed understanding is needed. Nevertheless, rapid development has been achieved in the construction of OM organs, which in return, contributes to the deciphering and understanding of the developmental process of OM organs [21].

Fig. 2.

Signal molecules and gene regulatory networks in salivary gland development. † There are four stages of salivary gland development: A Thickened primitive oral epithelium:(from the upper cortex of the primitive mouth). B Initial buds (developed from epithelial invagination to mesenchymal). C Pseudo-glands (From initial buds through branching morphogenesis development). D Terminal buds (From pseudo-glands through duct expansion). ‡ The Tanaka team's organoids give a good overview of the four stages that lead to terminal bud formation. Transplanted into mice with salivary gland defects, the salivary gland function was perfectly played

OMOs are generally developed from ESCs, IPSCs and ASCs. Coming from a wide range of sources, ESCs and IPSCs collectively termed pluripotent stem cells (PSCs) [22]. They can be obtained from embryos or living humans, and even re-edited into cell lines [23–25]. Moreover, they can also differentiate into any type of adult human cells and tissues to simulate the development of organs [26–28]: at first, PSCs are specifically induced to differentiate into the germ layer from which the target organ is derived, such as the endoderm (pancreas, thymus, lung, intestine, stomach, lung, liver, thyroid, etc.), the mesoderm (dermis, bone, heart, blood, etc.), and the ectoderm (epidermis, nervous system, brain, etc.); thereafter, PSCs further develop into target organoids through the control of culture medium conditions. The development of salivary glands organoids from the Tanaka team [29] summarizes this process. However, there are some challenges: first, controlling the differentiation of PSCs into different cell and tissue types requires employing spatio-temporal models of the germ layer and a priori-knowledge of those genes and molecules of organ development, which is still lacking [30]; second, chromosomal and gene mutations may accumulate during the differentiation and culture of PSCs, which would be tumorigenic [31]; thirdly, PSCs from allogeneic sources are immunogenic [31]. Relatively speaking (Table 1), ASCs that are generally taken from the target organ or the tissue of the patient during the biopsy show no rejection reaction [32, 33]. Studies have found that only one synonymous base substitution was found in organoids derived from ASCs after over three months of culture [34], implying that they have strong hereditary stability during long-term proliferation [33]. In addition, having a strong pedigree, ASCs cannot differentiate into tissue types other than the target organ [35]. Having to be identified in the target organs and tissues at the very beginning, ASCs are then isolated and implanted in a scaffold containing niche factors to acquire organoids. Therefore, the following issues must be considered: the accessibility of the anatomical parts of the sources of ASCs [23]; the identification and separation of ASCs groups in some issues remain difficult; the demand for ASC differentiation and maturity on niche has yet to be clarified [36].

Table 1.

Comparison of multiple factors in the application of PSCs and ASCs to organoids

Comparison	Pluripotent stem cells (PSCs)	Adult stem cells (ASCs)
1. Source	From a wide range of sources and derived from the proliferation of ESCs and the reorganization of adult cells [23, 24]	From relatively limited sources, with poor accessibility of the source tissue cells [35]
2. The degree of cell enrichment	High with the self-renewal and functional reorganization of the entire group [25]	Low with the side of group with stem cell-like properties difficult to identify and separate [36]
3. Tumorigenicity	High with genes accumulating and mutating during differentiation [31]	Low with strong hereditary stability [33]
4. Immunogenicity	High as a result of the expansion of allogeneic cells [31]	Low as a result of the expansion of autologous cells [32, 33]
5. Pedigree	Weak with the capability of differentiating various organs [28]	Strong with the sole capability of differentiating the source tissues and organs [35]
6. Prior knowledge and growth factor niche	Highly and widely demanded [30]	Seldom demanded [30]
7. Ethical issues	ESCs is Serious, IPSCs is Minor	Minor
8. Extent of differentiation	Low	High

PSCs Pluripotent Stem Cells; ASCs Adult Stem Cells; ESCs Embryonic Stem Cells

Prior knowledge and growth factor niches: molecular and genetic networks that regulate development

(ECM) is an extracellular network structure composed of proteins and polysaccharides, which transmit physical and biological signals to influence cellular behavior. The 3-d organoid scaffold, a simulation of the structure and function of ECM, can be divided into natural scaffolds and synthetic scaffolds according to source (Table 2). As a representative of natural scaffolds, Matrigel is derived from the basement membrane matrix extracted from an Englebreth-Holm sarcoma of mouse [37]. Therefore, with good biocompatibility, it is often used in the construction of organoids, however, there are still obvious limitations of Matrigel: first, it is a heterogeneous source material, which may be subject to immune and ethical restrictions when used in human bodies; second, it contains nearly 2000 proteins, mainly laminin and Nidogen and 14,000 peptides [38], which may cause unexpected cellular responses and affect the analysis of organoid indicators; in addition, Matrigel from different mice will lead to the non-repeatability of experiment; finally, coming from benign tumors, Matrigel may be tumorigenic. In view of these problems, some natural scaffolds with good reproducibility, easy separation and controllable components have been developed and widely used, such as collagen, fibrinogen, hyaluronic acid and alginate. However, ‘natural’ material cannot avoid dissimilar species and ethical concerns. Synthetic scaffold usually made of polymers such as polyethylene glycol (PEG) and polycaprolactone (PLC) are also used as alternatives, which come into being. Polyethylene glycol (PEG), which is non-toxic to cells and minimally stimulating tissues, is a representative of this type of scaffold and supports the expansion of cells and the formation of organoids [39]. In addition, synthetic scaffolds that assembles bioactive molecules, can also control their biochemical properties. For example, Murakami et al. enhanced tongue cancer cell proliferation and invasion ability after coating collagen on silicate fiber scaffolds. These studies also partly confirm that organoids have relatively low biochemical requirements for scaffolds, making the physical properties of the latter more important, including: hardness, compliance, geometric structure, viscosity, elasticity, and stress relaxation. Studies have shown that elasticity is a decisive parameter that affects the proliferation and differentiation of stem cells [40, 41]. In the salivary glands, a tissue scaffold with an elastic modulus of 48 kPa can promote the morphogenesis and differentiation of the salivary glands and save the damaged salivary glands [42]. In summary, a suitable organoid scaffold system requires both physical and biochemical conditions. Besides, the aforementioned research is enlightening: first, we can find various nutrients that are small in quantities, shared by organisms and non-reactive to each other or inert substances shared by organisms, so as to form a scaffold with certain physical properties, which, at this point, has a clear physical definition with no chemical properties to activate cells. Then, certain active factors are employed to define its biochemical properties through modification.

Table 2.

Comparison of various factors in the applications of natural and synthetic scaffolds to organoids

Comparison	Matrigel scaffolds	Synthetic scaffolds represented by PEG
1. Source	Coming from the basement membrane matrix extracted from sarcoma of mouse	Coming from manually developed polymeric material
2. Components	Not defined: including hundreds of thousands of peptides and proteins with potential tumorigenicity	Defined: pure polymeric materials
3. Repeatability of experiment	Low for its non-modifiable biochemical properties	Great
4. Biochemical properties	Difficult to modify	Able to be modified through the assembling of active molecule
5. Physical properties	Difficult to change	Easy to change
6. Ethical issues	Serious	Minor

PEG polyethylene glycol

Growth factor niches which are used to simulate the complex composition of signaling molecules in the body play an important role in the regulation of cell growth, differentiation, proliferation, and organ development and maturation. However, these growth factors are usually selected from certain active cytokines, hormones, vitamins, and small-molecule inhibitors, including Wnt-3A, R-spondin-1, Noggin, Sox, Y-27632, EGF, B27, gastrin, etc., and can support the organoid culture of most oral tissues in F12 medium [43]. The study of Driehuis (Table 3, Fig. 3) covered these matters and successfully derived organoids from various tissues of the head and neck [44]. In the living body, the development of different tissues and organs is stimulated by the combination of different signaling molecules, and these stimuli are precisely controlled in terms of dosage, time, space, etc. Therefore, it is difficult to simulate it through the existing organoids with evenly distributed ecological factors. Recently, more mature single-cell sequencing will contribute by studying the molecular representation of tissues and organs to facilitate the a priori development and screening of signaling molecules; finding the optimized culture conditions by high-throughput sequencing comparing the single-cell profiles of organoids under different culture conditions and those of target tissues and organs [45].

Table 3.

The Driehuis team’s construction strategy (cocktail formula) may be the possible general model for OMOs

The general characteristics of construction strategies	Primary cells	Extracellular matrix (ECM) scaffolds	Growth factors	hormones	Recombinant proteins	Small molecule inhibitors/agonists	Culture medium
Driehuis team’s construction strategy	Healthy or cancerous oral primary cells (mucous membrane, mouth floor, tongue and gingival/alveolar process)	BME2 type with reduced Cultrex growth factor	B27, N-acetyl-L-cysteine, nicotinamide	Prostaglandin E2, Forskolin	RSPO, Noggin, EGF, FGF10, FGF2	CHIR99021, A83-01	DMEM/F12 medium

OMOs Oral and maxillofacial organoids; ECM Extracellular matrix; RSPO R-spondin-1; EGF Epidermal growth factor; FGF Fibroblast growth factor; B27 Medium supplement

Fig. 3.

Tongue cancer organoid. †Note: Our previous study based on the Driehuis team’s cocktail formula and Cell line CAL-27

Strategies for the construction of various oral and maxillofacial organoids (cocktail formulae)

Tongue epithelial organoid cocktail formula

The tongue is an important muscular organ for taste, digestion, and pronunciation with its back covered with stratified squamous epithelium. These epithelial cells form four special-shaped papillae with the axis formed by the lamina propria: filamentous, circumvallate, and fungiform and foliate papillae. The rate of renewal of epithelial cells on the back of the tongue is very fast: once every 6 to 7 days, for which tongue epithelial stem cells located in the basal layer are believed to be responsible [46]. Through immunohistochemistry and in-situ RNA hybridization, Tanaka et al. [46] found that mouse Bmi1 + , keratin 14 + , and keratin 5 + cells exist on the second and third layers of the base of the interpapillary fossa, and then through multi-chromatography system tracking found that Bmi1 + cell can differentiate into keratinocytes of filamentous papillae but cannot differentiate into taste bud cells. Xiaoyan and his colleagues [47] proved the stem-cell like properties of mouse keratin (Krt) 5 + cells and cultured them on the gas-solution interface of collagen gel that contains feeder cells to obtain tongue epithelial organoids. However, keratin (Krt) 5 + cells only differentiated into stratified squamous epithelium of Trp63 + , keratin 14 + , and Foxa2 + without papillary structure. As mentioned, all-round stem cells (Stem cells that can differentiate into all the lineages in an organ) have not been identified, and whether it is caused by inherent genetic memory of the cells or by insufficient culture components remains unclear. However, Hisha and Tanak’s teams [48, 49] used the mouse entire tongue epithelial cell population containing stem cells to establish three tongue epithelial organoids under the new organoid culture system: concentric round organoids with cutin, solid nodular organoids, and circular organoids arranged in a grid (Table 4). Therefore, the cocktail formula in this study can be used as a representative formulation of tongue organoids at present. However, their research did not fully summarize the morphological heterogeneity of the epithelium of the back of the tongue; these entire cell-derived organoids can mature after implantation, indicating that the defect is not attributed to lack of stem cells, but may be related to insufficient culture components. This requires us to make more efforts in the screening of niche factors and the identification of tongue stem cells to develop a better culture system.

Table 4.

Representative strategies for the construction of various oral and maxillofacial organoids (cocktail formula)

Formulae	Cells	Scaffold systems	Growth factor niches	Results	References
1	Unsorted lingual epithelial cell	Matrigel	N2, B27, N-acethyl cysteine, Glutamax, EGF, noggin, spondin1-hFc, Y-27632, DMEM/F12 medium	Forming three tongue epithelial organoids: concentric round organoids with cutin, solid nodular organoids, and circular organoids arranged in a grid	Hisha and Tanaka [48]
2	The primary ASCs sorted byLgr5 + or Lgr6 +	Matrigel	RSPO, Noggin, Jagged-1, Y27632, N-acetylcysteine, EGF, N2, B27, DMEM/F12 medium	Producing the ever-expanding taste bud organoids that well summarized the types and functions of differentiated and mature taste cells	Ren et al. [51]
3	Pluripotent stem cells (PSCs)	Matrigel with reduced adhesion factor	SB, BMP4, LDN, FGF2, adenovirus introduced Foxc1 and Sox9 genes, in N2, FGF7, FGF10, DMEM/F12 medium	Differentiating into immature ducts and acinar organoids, which can perform all the functions of saliva secretion after being implanted in the body	Tanaka et al. [29]
4	Epithelial and mesenchymal cell populations of lower deciduous molars	Collagen gel	FCS, L-ascorbic acid, L-glutamine, DMEM medium	Generating tooth germ organoids in cap stage, which were implanted into the renal sacs of mice to form the correct tooth structure	Fu Wang et al. [60]
5	Primary cancer cells of the oral cavity (floor of mouth, tongue and gingivae/alveolar process)	BME2 type with reduced Cultrex growth factor	B27, N-acetyl-L-cysteine, nicotinamide, Forskolin, EGF, A83-01, FGF10, human FGF2, prostaglandin E2, CHIR99021, RSPO, Noggin, DMEM/F12 medium	Oral cancer organoids were formed with an efficiency of 60%, and the molecular and morphological characteristics of the original tumor were summarized	Driehuis et al. [44]

OMOs Oral and maxillofacial organoids; ECM Extracellular matrix; RSPO R-spondin-1; EGF Epidermal growth factor; FGF Fibroblast growth factor; B27 Medium supplement; SB Inhibitor of TGF-β; LDN Inhibitor of BMP; ASCs Adult Stem Cells; FCS Fetal calf serum

Formula 5 may be the possible general model for OMOs. Because they successfully derived organoids from Oral and maxillofacial various tissues

Cocktail recipe for taste bud organoids

As special organs for epithelial differentiation, taste buds are composed of three types of slender cells, which are widely distributed in the epithelium of the anterior/posterior tongue, soft palate, and epiglottis. Studies have shown that Lgr5 or Lgr6 label the adult taste bud stem/progenitor cells of the anterior/posterior tongue [50, 51]. Ren et al. [51] embedded mouse single Lgr5 + or Lgr6 + taste stem cells in Matrigel containing R-spondin-1, Noggin, Jagged-1, Y27632, N-acetylcysteine, EGF, N2, and B27, producing the ever-expanding taste bud organoids that summarized the types and functions of differentiated and mature taste cells (Table 4, Fig. 4). Therefore, the Cocktail Formula can be used as a representative formulation of bud organoids at present. It is worth noting that the epithelium of posterior tongue is derived from the endoderm, while the anterior tongue and palatal epithelium are derived from the ectoderm. Lgr5 is known to mark stem cells in endoderm-derived tissues such as the small intestine, colon, and stomach, but not suitable for the anterior tongue, suggesting that taste buds of different origins may have different stem cell subsets. When screening candidate markers of anterior tongue and palate stem cells, the idea can move closer to the proven ectoderm epidermal stem cell markers (such as intergrinβ1, K15, etc.). In addition, in the experiment of Aihara et al. [52] produced multi-layered spherical taste bud organoids through the 3-d culture of mouse circumvallate papillae containing stem cells, but there is no formation of taste pore structure in body tissues, which may be related to the lack of neuron innervation. As early as 2011, Miyako et al. [53] embedded mouse TBD-a5, a cell line derived from taste buds, in collagen gel and cultured. They found that the TBD-a5 line developed into cell clusters with a natural taste bud structure: well differentiated epithelial cells formed a cavity surrounded by cells with basement membrane structures, and on the inner surface the microvilli and tightly jointed structures of taste pores were found. However, the organoids alluded to in this research did not summarize all types of taste cell. In fact, the existing niche factors have met the conditions of the culture of taste bud organoids. However, a co-culture with other cells (such as tongue epithelium, neurons, mesenchymal cells) to simulate innervation and cell-epithelial/mesenchymal interaction may promote the generalization and structure formation of taste bud organoid cells.

Fig. 4.

Structure of taste bud and tooth. † A Taste bud: The Ren team’s taste bud organoids reconstructed the structure of taste buds and summarized the types of taste cells that differentiated and matured, and even some of those cells responded to taste enhancers. ‡ B Tooth: The Yun Jeong team’s dentin organoids reconstructed elongated odontoblasts and mineralized dentin on cell surfaces

Cocktail recipe for salivary gland organoids

Salivary glands are composed of acini and ducts with their development mainly depending on the interaction between epithelium and mesenchyme. As the basic development model of salivary glands, branching morphogenesis is also the key to the construction of salivary gland organoids, which is a process regulated by a complex network of ecological niche factors. Among the research on oral organoids, salivary glands are the most mature, with the previous studies having successfully derived fully functional salivary gland organoids from cell lines, ASCs, and embryonic stem cells. CD24hi/CD29hi cells have been confirmed to have stem cell-like properties. Lalitha et al. [54] expanded them in MM medium with Y-27632, a strategy that may be conducive to solving the rarity of ASCs. Sarah et al. [55] utilized the same strategy to conduct organoid culture on human salivary gland cells (unsorted) for the first time and verified that c-Kit + cells are a subgroup with stem-cell properties, believing that expanding CD24hi/CD29hi cells in MM medium with Y-27632 is not the best for long-term expansion of an in vitro strategy, as cell apoptosis is found to increase when cell renewal potential decreases. However, this phenomenon does not exclude the inherent growth restriction or species differences of cells. For example, Srinivasan [56] reported a human stem cell population with multiple markers (KRT5, KRT14, MYC, KIT, and ETV5), which can extend for a long time in hyaluronic acid water medium modified by YIGSR, IKVAV, and PlnDIV peptide and only supplemented with B27, EGF, and FGF2. In addition to the maintenance and expansion of stem cell-like properties, differentiation and induction are also crucial for organoids, to which Tanaka et al. [29] have made a significant contribution: with SB, BMP4, LDN, and FGF2, they induced mouse PSCs in a step-wise manner, which then formed in the oral ectoderm, into which Foxc1 and Sox9 genes were introduced by adenovirus and it was induced to differentiate into salivary gland organs in DMEM/F12 Matrigel of N2, FGF7, and FGF10. After implantation in the body, it can exert all salivary secretion functions (Table 4, Fig. 2). Therefore, the proposed cocktail formula can be used as a representative formulation of salivary gland organoids at present. Moreover, based on these, Athwal et al. [57] has developed a differentiation method, which derived organoids from mouse saliva SIMS cell lines. The co-culture system further enables us to restore the epithelial and mesenchymal dialogue to ensure the formation of branched structures. Ogawa et al. [58] injected the dissociated mouse mesenchymal cells and salivary epithelial cells into two adjacent Matrigel, respectively, and co-cultured them in DMEM/F-12 supplemented with fetal bovine serum and penicillin/streptomycin, which differentiated into branches and ducts that achieved the recovery of the full function of salivary gland organs. In addition, like cell sources and ecological molecules, scaffolding systems are also important. Based on the genetic memory of salivary gland stem cells, Shin et al. [59] differentiated and assembled human salivary organoids in a niche-free PCL nano-porous scaffold.

Cocktail recipe for dental organoids

Tooth germ organoids have been successfully constructed in pigs and dogs: after the self-organization of single epithelial cells of tooth germ or mesenchymal cells of the pig in the ultra-low adhesion plate suspension culture, they were mixed and packed into the collagen scaffold and the tooth germ organoids in the cap stage were found to be generated, which were implanted in mouse renal sacs, forming the correct structure of teeth [60] (Table 4). Therefore, the proposed cocktail formula can be used as a representative formulation of saliva, however, in terms of autogenous tooth germs that are often difficult to obtain with most of them coming from third molars limited in numbers, it may be difficult to use them to regenerate and restore anterior teeth because of their genetic lineage. iPSCs can avoid these shortcomings. So far, several studies have successfully derived tooth germ organoids from iPSCs [61, 62]. In one study, mouse iPSCs were differentiated into embryoid bodies (EB) through hanging drop culture, which then were respectively placed in SF2 medium or DMEM medium to induce dental epithelial stem cells or mesenchymal stem cells, and both types of cells were then co-cultured to produce tooth germ organoids. In addition, some easy-to-obtain ASCs were identified and isolated with organoids successfully derived therefrom, including periodontal ligament stem cells, dental pulp and dental follicle stem cells, etc., thus achieving partial tooth tissue restoration. Jeong et al. [62] reported for the first time a plasma-like dentin organoid (Fig. 4). They condensed a mixture of human dental pulp stem cells and Matrigel into organoids in a differentiation medium and observed calcified matrix and odontoblast differentiation around them. Then, by adding Biodentine to the medium, they found that it can further stimulate differentiation. irootBP, MTA, Biodentine, and calcium hydroxide were found to greatly contribute to the revascularization as commonly used clinical drugs, which cannot only promote the proliferation, migration, and adhesion of stem cells, but also help odontoblasts to produce dentin bridges and reactive dentin to promote mineralization with their main component Ca ions and a weakly alkaline pH. To sum up, we suspect that Ca ions and a weakly alkaline environment may play an important role in the differentiation of dental tissue stem cells and the formation of organoids, which, of course remains to be researched.

Cocktail recipe for mouth neoplasm

Oral cancer is the sixth most malignant tumor in the world with an increasing incidence [63], and generally poor prognosis. At present, clinical treatment mostly relies on surgery, radiotherapy, and chemotherapy, which are slightly single compared with other those of malignant tumors, for the basic research of oral cancer has been limited. However, the organoid model may provide a more reliable platform for oral cancer overtaking in corners: compared with two-dimensional and xenotransplantation, it can be used to explain the molecular mechanism of oral cancer in detail, test potential drugs, and formulate personalized precision treatment plans. Created by Zhao et al. [64] and others in 2017, the first oral cancer organoid was formed by the human tongue cancer cell line CAL27 on the cytoskeleton of the tongue (Only retained extracellular matrix and proteins tongue tissue after cell removal). In addition, Tam and others [65] used the cancer cell line PCI-13 to establish oral cancer organoids in mouse tail collagen containing FBS and IFNγ. However, these two organoids with simple structure (They form only round clusters of cancer cells) and low cost may benefit mechanism research and preliminary drug screening, but with the difficulty to generalize the heterogeneity of body cells, it is not suitable for precision medical treatment. In view of this, researchers set out to develop a primary cell organoid system for oral cancer. Driehuis et al. [44] described a long-term mucosal organoid system derived from human oral keratinocytes/primary oral cancer cells, with its culture supplemented with B27, N-acetyl-L-cysteine, and niacinamide, human EGF, A83-01, human FGF10, human FGF2, prostaglandin E2, CHIR99021, Forskolin, RSPO, and Noggin. These organoids established from biopsy retain the morphological, genetic and molecular characteristics of the original tissues, and show similar characteristics with the parental tumor after transplantation into mice (Table 4). Therefore, the proposed Cocktail Formula can be used as a representative formulation of salivary gland organoids at present, which may be the possible extending to the entire OMOs. The importance of the stent system has been emphasized above. Some studies [11, 66] have developed a silica fiber scaffold called Cellbed, whose structure is similar to the tissue skeleton of the loose connective tissue of living body in which cells can flow in these gaps without restriction. Compared with Matrigel, its biggest advantage is that it is very simple and has no undefined proteins, with the ability to extract pure cancer cell molecular substances in research and can also be used to develop niche factors. Noi et al. [66] used this system, which accurately simulates the morphology of cancer cells in the human body, to prove that ERK promotes the progression of tongue cancer through corticosteroid activation. In tumor tissues, there are tumor stem cells (tumor stem cells, TSC) with specific cell surface markers, which exhibit self-renewal and are strongly tumorigenic, playing a decisive role in tumor initiation and growth. In previous studies [67], it is often difficult for these cancer stem cells to maintain their stem cell-like properties in vitro for enrichment, expansion, and passage. Fortunately, Zhao et al. [68] have achieved long-term expansion of primary stem CD44 + cells of human oral cancer in organoid systems supplemented with N2, B27, EGF, gastrin, and A83-01, which indicates that such organs may be a suitable model for cancer stem cell research. Besides, Shimokawa et al. [69] knocked-down LGR5-iCaspase9 in organoids to eliminate human LGR5 + cancer stem cells, which led to tumor regression. However, after long-term culture, LGR5 + cancer stem cells reappeared, indicating that long-term organoid culture may induce spontaneous reshape of genetic information of stem cell, which, combined with the proliferation of stem cells by organoids and the elimination of terminally differentiated cells in the process of culturing organoids, may help in the enrichment of cancer stem cells.

Application of OMOs

Compared with other models, organoids have received extensive attention due to their unique advantages. However, OMOs have broad applications and prospects in disease modelling and mechanism research, developmental and regenerative medicine, drug screening and personalized treatment, and toxicology, for which many excellent models have been developed.

Disease modelling and mechanism research

Organoids are a suitable model for studying complex OM diseases. First, for oral cancer, it has been described above; second, for infections and inflammatory diseases, organoids are often added with various cytokines and microbial products or co-cultured with microorganisms to rebuild the environment. For example, there are studies [70] reporting the use of carbachol and forskolin to stimulate the formation of swelling in organoids so as to simulate the inflammation of the human salivary glands, while proving the inhibitory effect of tumor necrosis factor-a (TNF- a) on the inflammation of the salivary glands.

The co-culture system allows us to strengthen the study of microbial infections: Pinnock et al. [71] found that compared with traditional culture the mouse oral epithelium organoid model of infection is more capable of displaying the invasion of Porphyromonas gingivalis to host cells. Third, for hereditary diseases, previous studies often cultured the whole organs in vitro to study oral-related hereditary diseases, whereas the recently mature gene-editing technology CRISPR-Cas9 has been successfully applied to organoids derived from patients with cystic fibrosis, successfully correcting CFTR gene and performance functions [72]. This successful example may usher in the use of OM organs in treatment of hereditary dentin disorder, cleft lip, and cleft palate.

Developmental and regenerative medicine

The existing knowledge about OM development is mostly derived from animal models, which will involve inter-species differences and ethical obstacles; however, the faithful reflection of organoids on body tissues provides an opportunity for in-depth understanding of oral developmental processes. For example, in a comprehensive transcriptome analysis of the organoid derived from dental pulp cells, Rosowski et al. [73] found that the activation of Notch and TGF-β pathways during tooth development may be associated with the reshape of the cytoskeleton because this organoid simulates the process of embryonic mesenchymal condensation during early development of human teeth. However, this knowledge will eventually be attributed to the application of regenerative medicine due to the omnipresent scarcity of donated organs. When transplanted into the gland-defective mice body, the salivary gland organoids derived from multipotent stem cells were successfully connected to the recipient’s parotid ducts, and showed a mature salivary gland phenotype and performed a secretory function [29], indicating that the feasibility of transplanting salivary glands organoids that can continuously expand and differentiate into the body to repair and regenerate target organs. In addition, some dental organs will also contribute to the transformation of dental treatment from materials to regeneration. It is worth noting that the restoration and regeneration of dental tissue is not strict with regard to xenogeneic rejection, but requires hardness and wear-resistance of the materials.

Drug screening, personalized treatment, and toxicology

When it comes to traditional drug testing tools, the options are primary/cell line two-dimensional cultures or animal models, the former of which, however, may only represent a certain cell type and cannot simulate the in vivo system, while the latter requires takes account inter-species differences, a substantial commitment of time, high cost, and difficulty in reshaping. Recently, research has confirmed that organoids are viable in terms of drug screening and personalized treatment because of their ability to simulate diseases in the body. Driehuis’s team [44] has established a living biobank of human oral mucosal cancer organoids and they used these organoids to test a group of drugs, finding that the sensitivity is related to tumor-derived mutations, and the organoid response to radiotherapy is associated with the clinical response of the patient. However, since they can assess the sensitivity of drugs, it can also exhibit toxic effects. For instance, at present, nano-resin based on glycidyl methacrylate monomer is the mainstream material for repairing tooth defects, which once entering the oral cavity, will inevitably and continuously release monomers like a sustained-release agent. In view of this, some research teams [74] have used organoids to evaluate and found that triethylene glycol dimethacrylate could reduce the restoration and regeneration of dental pulps and gingival tissues. In addition, radiotherapy, often used after oral cancer surgery, etc., is usually accompanied by the involvement of surrounding normal tissues and side effects, the most common one of which is xerostomia with damaged salivary glands. However, organoids derived from stem cells of glandular tissues demonstrate the super sensitivity of stem cells in tissues to low-dose radiation, indicating the great potential of organoids in evaluating the side effects of radiation.

Discussion

After more than a decade of vigorous development, many excellent organoid systems in terms of OM regions have been established and proved their great potential in basic medicine research and clinical medicine. Except for the cell sources, most of the OMOs are shown to be similar in construction, generally including ECM scaffolds, growth factors, hormones, recombinant proteins, and small molecule inhibitors/agonists. The construction strategy of Driehuis et al. almost comprised these elements, in which they successfully established organoids from tumors in the floor of the mouth, tongue, gingiva, alveolar process, pharynx, larynx, salivary glands, nasal cavity, and neck, and corresponding normal tissues, while succeeding in summarizing disease in terms of genetics, histology, and functions, with the success rate reaching 60%, which indicates that this strategy may be a good general model for OMOs. Despite the excellent results achieved in the development of OMOs, there is still a lot of room for improvement. For example: (1) Organoids are not mature enough, which may be attributed to lack of the innervation of blood vessels, nerves, and immune systems, a problem that may be improved by co-culture systems of endothelium/immune cells or the introduction of neurotransmitters; (2) High production costs and undefined ingredients, such as expensive recombinant proteins, will not be conducive to expanding production and popularization. Besides, some matrix ingredients are often undefined and potentially tumorigenic. Therefore, only by developing more simplified, lower cost, and clearer ingredients can organoid models be better applied to clinical practice; (3) While oral diseases are systemic, existing oral organoid models, on the other hand, generally only reflect the characteristics of the disease in the oral cavity and cannot interact with other organs. However, combining organoids with “organs on a chip” may well realize these linkages. 4. Traditional detection methods seem to be unable to keep up with the pace of development of organoids, such as that seen in activity detection. However, the introduction of single-cell sequencing and gene editing technology into OM organs will help to optimize the simulation of normal and pathological conditions in the body by oral organoids.

Author contribution

MO performed the majority of the writing; QL and XL performed data acquisition; JY performed the Data interpretation and Article design; XM performed the manuscript modification and verification. JY and XM contributed equally to this work and should be considered as co-corresponding authors. All authors read and approved the final manuscript.

Declarations

Conflict of interest

There is no conflict of interest associated with any of the senior author or other coauthors contributing their efforts in this manuscript.

Ethical statement

Ethical approval and consent to participate is not applicable to this article as no data were generated or analyzed during the current study.