Epithelial cell changes in salivary glands

Salivary gland epithelial cells (SGECs) play a critical role in maintaining salivary homeostasis by performing functions, such as Na+ reabsorption, K+ excretion, regulation of saliva volume and osmolality and modulation of water reabsorption or excretion. These functions are mediated by the Ca²⁺-activated sodium-potassium channels. Additionally, SGECs express MHC class II molecules and co-stimulatory molecules such as CD86 and CD80 on their surfaces. These molecules interact with CD28 on T lymphocytes to facilitate their activation (Ref. Reference Kapsogeorgou, Moutsopoulos and Manoussakis23). Aberrant SGECs can actively present self-antigens or indirectly promote self-antigen release through apoptosis. In a study using salivary gland epithelial cells derived from patients with SS, tumour necrosis factor (TNF) was found to induce an increase in apoptosis in these cells. Elevated TNF levels result in impaired muscarinic-like signalling, mislocalisation of aquaporin (AQP), abnormal distribution of salivary mucins (MUC5B and MUC7) outside the basement membrane, expression of autoantigens and simultaneous stimulation of autoantibody production by B lymphocytes (Ref. Reference Kawakami24).

Proper salivary secretion depends on the binding of M-choline neurotransmitters, such as acetylcholine, to muscarinic-like receptors. Activation of the PIP2 (phosphatidylinositol 4,5 diphosphate) pathway by modulating intracellular calcium ion levels triggers IP3R (inositol triphosphate receptor) signalling. However, in patients with SS, elevated levels of TNF inhibit muscarinic-receptor signalling and disrupt this pathway. This disruption impairs the function of IP3R calcium channels in the adenohypophysis (Ref. Reference Burlage25), thereby compromising its normal physiological activity. Moreover, disruption of the IP3R signalling pathway alters intracellular calcium levels, thereby affecting molecular exchange in the aquaporin (AQP) pathway. In particular, only a limited amount of AQP5 on the parietal membrane of acinar cells is activated by calcium ions, resulting in incomplete transport of intracellular water molecules across the parietal membrane. In addition, water molecules may also be incorrectly transported from water channels at different locations within acinar cells (e.g. basement membrane) due to abnormal distribution of AQP5. Thus, abnormal AQP5 expression and distribution in the acini of the salivary glands may lead to insufficient saliva secretion.

Additionally, these cells induce the production of various inflammatory factors, including CXCL8, TNF, IFNα, β, IL-6 and IL-1β, and express and expose self-antigens (e.g. SSA/Ro, M receptors, parotid secretory proteins, salivary protein 1 and carbonic anhydrase 6) (Ref. Reference Verstappen26). SS affects not only the follicular cells of the salivary gland but also compromises the structure and function of ductal cells to some extent. This is evidenced by an increase in apoptosis, particularly through mechanisms involving Fas/FasL and IFN-γ, or poly (I:C) induced apoptosis following loss of nesting (i.e. apoptosis induced by detachment from contact with the extracellular matrix or neighbouring cells) (Ref. Reference Fang27). Furthermore, salivary gland epithelial cells (SGECs) express various Toll-like receptors (TLRs), such as TLR1, 2, 3, 4 and 7, as well as other pattern recognition receptors (PRRs), at the initiation of SS. Among these TLR3, SSA/Ro60, SSA/Ro52 and SSB/La autoantigens induce the expression of type I IFNR (IFN receptors), TNFR(TNF receptors) and other cytokine receptors that transmit inflammatory signals (Ref. Reference Maimets28). These autoantigens also secrete a variety of cytokines such as IL-1, IL-6, IL-7, IL-18, TNF and BAFF, that can directly contribute to glandular damage. Additionally, they secrete chemokines, including CXCL8, CXCL10, CXCL12 and CXCL13, that attract inflammatory cells to the gland and contribute to damage (Ref. Reference Hai29). The resulting inflammatory environment surrounding the glands diminishes the proliferation and differentiation capacity of salivary gland epithelial cells (SGECs) and promotes cellular senescence.

Activation of Innate Immunity

The pathogenesis of pSS is not well understood; however, it is widely recognised as a multi-step disorder triggered by environmental, genetic and inflammatory pathway interactions. The established genetic predisposition to SS lies in a variant of the tumour necrosis factor α inducible protein 3 (TNFAIP3) gene, the deficiency of which can induce an inflammatory response of macrophages to nontuberculous mycobacteria (NTMs), increasing the antigen repertoire of the mycobacterium and making the body more susceptible to autoimmune diseases. Paratuberculosis (MAP), a type of NTM, is also thought to be an environmental trigger for many autoimmune diseases through its cross-reactivity with heat shock protein 65 (hsp65) with host-specific proteins.

The innate immune system plays a crucial role in the initiation of the inflammatory process, but the amplification and persistence of the autoimmune process require a constant interaction between the innate immune system and the adaptive immune system. The inflammatory process of pSS involves a variety of immune cells, including dendritic cells (DCs), macrophages, natural killer cells (NKs) and mast cells (MCs). Research has demonstrated that in advanced disease models, salivary glands exhibit infiltration foci containing B-cell germinal centres primarily composed of B cells, T cells, DCs and macrophages. These cells act as APCs, capable of taking up self-antigens released in response to salivary gland cell injury (e.g. viral or bacterial infection, mechanical damage) and presenting them to CD4⁺ T cells via MHC (Ref. Reference Luckashenak30). Furthermore, macrophages in patients with primary SS exhibit high IL-18 expression, and the degree of macrophage infiltration is strongly correlated with salivary gland enlargement (Ref. Reference Zhou and McNamara31). According to another study, peripheral blood NK cell levels were found to decrease in patients with SS; however, NK cell activity, along with the expression of NK cell activity receptor CD2 and natural cytotoxicity receptor (NKP62), increased (Refs. Reference Lin32, Reference Jingjing33). Gene sequence studies have indicated that polymorphisms in the NCR3 promoter are linked to susceptibility to pSS(Primary Sjögren’s syndrome) because NCR3 encodes the activation receptor (NKp30) in NK cells (Ref. Reference Rusakiewicz34). DCs and NK cells interact via NKp30, promoting the secretion of IL-12 and IFN, thereby driving inflammation. Additionally, NK-like T-cell subset has been identified, capable of directly secreting IL-17 and IFN-γ. In patients with SS and salivary glands of NOD mice, an increase in the NK cell number is observed. This increase may be attributed to the migration of NK cells from peripheral blood to salivary gland tissues (Ref. Reference Roescher35). With the development of SS, the number of NK-like T cells returns to normal levels, and their function aligns with that of CD3⁺ T cells and CD56⁺ NK cells.

T-lymphocyte activation

The process of intrinsic immune activation described above indicates that following injury to salivary gland cells, autoantigens are released, captured by APCs and subsequently presented to T cells. In patients with SS, 60%–70% of the lymphocytes infiltrating the exocrine glands are T lymphocytes, with more than 70% of these being CD4⁺ T cells. Studies have demonstrated that CD4⁺ T cells can differentiate into various subpopulations, including helper T (Th) cells (Th1, Th2 and Th17), regulatory T cells (Tregs) and follicular helper T cells (Tfh), each playing a distinct role in immune responses. These T-cell subpopulations secrete characteristic cytokines and produce specific activators that modulate the immune system (Ref. Reference Yura and Hamada21).

The study found that patients with SS exhibited an increase in Th1 cells within their salivary glands, whereas the Th2 cell population remained unchanged, resulting in an elevated Th1/Th2 ratio (Ref. Reference Mitsias36). Conversely, the number of Th1 cells in the peripheral blood of patients with SS exhibited a notable decline compared to that in healthy controls, whereas Th2 levels remained constant, resulting in a decreasing trend in the Th1/Th2 ratio (Ref. Reference Van Woerkom37). This trend may be attributed to the migration of Th1 cells from the peripheral blood to the salivary gland tissue, that has been suggested to reflect a systemic immune response (Ref. Reference An38). Th1 cells play a crucial role in the pathogenesis of SS, primarily accumulating in the early stages of the disease, prior to the formation of germinal centres. In contrast, Th2 cells are more prevalent during the infiltrative stage of SS, which occurs after the development of germinal centres. Th1-type cells in the salivary glands of patients with SS predominantly secrete IFN-γ, TNF-α and other inflammatory factors (Ref. Reference Jiang39). Elevated levels of these factors have been detected using fluorescent probes and immunohistochemistry, indicating a potential association between SS progression and elevated levels of Th1-cell-related inflammatory factors (Refs. Reference Shi40, Reference Singh and Cohen41). The study also identified an association between polymorphisms in STAT4 and IL-12 genes and the pathogenesis of SS through GWAS(Genome-Wide Association Studies) (Ref. Reference Imgenberg-Kreuz42). STAT4 is a transcription factor crucial for Th1 cell differentiation, promoting their development and function. Additionally, IL-12 plays a key role by stimulating Th1 and NK cells to secrete IFN-γ, thereby amplifying the Th1-mediated immune response. Elevated IFN-γ levels enhance antigen presentation by histone S via MHC II molecules, facilitating the generation of autoantibodies and immune complexes and contributing to the autoimmune process (Ref. Reference Hamm‐Alvarez43). Notably, NOD mice deficient in IFN-γ or its receptor do not exhibit SS-like manifestations (Ref. Reference Meng44), whereas mice with elevated IL-12 levels develop lesions resembling those observed in SS (Refs. Reference Cha45, Reference Zou46).

A previous study found increased numbers of Th17 cells in the peripheral blood, salivary glands and spleens of NOD mice, suggesting that Th17 cells play a pro-inflammatory role in SS pathogenesis (Ref. Reference Hwang47). Th17 cells primarily secrete IL-17A, a cytokine that is implicated in various autoimmune diseases. IL-17A contributes to inflammation and tissue damage by promoting the recruitment of other immune cells and stimulating the release of additional pro-inflammatory cytokines and chemokines. This reinforces the chronic inflammatory environment that is characteristic of SS. In the mouse model of SS, a correlation was observed between IL-17 levels and the sex of the mice, with females exhibiting a significantly greater increase compared to that of males (Ref. Reference Voigt48). Furthermore, the elimination of IL-17 resulted in a more pronounced improvement in symptoms in females than in males. Recent research has also identified CD4⁺CD16⁺RORγT⁺ Th17 lymphocytes as linked to the presence of anti-SSA and/or anti-SSB antibodies and hyperglobulinaemia in SS. Conversely, CD4⁺CD16⁺RORγT⁻ Th17 lymphocytes are associated with disease activity, suggesting that different subsets of Th17 cells play distinct roles in disease pathology and progression (Ref. Reference Li49). Data indicate a decrease in Th17 cell counts in the peripheral blood of patients with SS (Ref. Reference Sudzius50), implying the potential redistribution of these cells to tissues. Tregs, characterised by CD25⁺FOXP3⁺ expression and immunomodulatory properties, suppress effector T-cell activation and maintain homeostasis. However, lower levels of Treg cells were observed in tissues during the inactive phase of SS (Ref. Reference An38). Sex differences remarkably affect Treg cell expression. Oestrogen promotes Treg cell immunosuppression, whereas androgens suppress Treg cell activity. In males, SS predominantly manifests as lacrimal gland inflammation, leading to dry eyes. In contrast, females are more likely to experience salivary gland inflammation, resulting in dry mouth (Ref. Reference Lieberman, Kreiger and Koretzky51).

Tfh cells stimulate B-cell proliferation; however, the degree of this stimulation does not correlate with SS activity. The presence of CCR7 low PD-1 high Tfh cells in the peripheral blood of patients with SS indicates a significant increase in Tfh cell numbers, suggesting their potential contribution to glandular inflammation and influence disease activity. Tfh cells comprise various subtypes, including Tfh1-, Tfh2- and Tfh17-like cells. Notably, an increase in Tfh17-like cells is observed in the peripheral blood (Ref. Reference Li52). Furthermore, the quantity of T follicle regulatory cells (Tfr) in the peripheral blood of individuals with SS significantly elevates, and an increase in the Tfr/Tfh ratio is positively associated with the extent of infiltration in the salivary glands, potentially indicating the presence of ectopic lymphoid tissue (Ref. Reference Kasashima53).

Additionally, transforming growth factor β(TGF-β) particularly induces the differentiation of Th9 cells, which in turn secrete IL-9. This transformation occurs in Th2 cells. IL-9 inhibits Tregs and promotes Th17 cell differentiation and plays a role in the development of B-lymphocyte tumorigenesis in advanced models of SS (Ref. Reference Heim54). Th22 cells are capable of secreting IL-22, IL-13 and TNF-α and can differentiate into Th1 and Th2 cells under specific conditions. Although Th22 cells and IL-9 have been less studied in the context of SS, their relevance in other autoimmune diseases, such as systemic lupus erythematosus (SLE), systemic sclerosis and rheumatoid arthritis (RA), suggests their potential importance in the pathogenesis of autoimmune diseases.

Abnormal B-lymphocyte activation

The aberrant activation of B cells in patients with SS is likely driven by the activation of various T-cell subsets, including Th9, Th17 and Tfh cells, either through the release of proinflammatory mediators or direct stimulation. This process is critical in the pathogenesis of SS, resulting in hypergammaglobulinaemia and autoantibody production, including anti-nucleoprotein, anti-SSA/Ro and anti-SSB/La antibodies. Elevated levels of these autoantibodies are linked to more severe complications in SS, with higher antibody titres correlating with increased severity of disease manifestations (Ref. Reference Heim54).

Viral infection is considered to be one of the key initiating factors for SS and is associated with the type I IFN pathway, that is commonly upregulated in patients with SS (Ref. Reference Lucchesi, Pitzalis and Bombardieri55). Innate immune cells release type I interferon, inducing autocrine and paracrine secretion of B lymphocyte stimulating factor (BAFF), leading to the activation of B lymphocytes and the production of autoantibodies. In SS, serum autoantibodies and immune complexes further stimulate IFNI expression, thereby creating a positive feedback loop. This loop amplifies B lymphocyte activation and perpetuates ongoing IFNI production, thereby exacerbating the autoimmune response and contributing to disease progression (Ref. Reference Båve56). BAFF extends the lifespan of circulating B cells by binding to their receptors, inhibiting intracellular apoptotic pathways, thereby promoting cell survival (Ref. Reference Wang57). It has also been shown to potentially elevate transitional B-cell levels and initiate autoimmune reactions (Ref. Reference Thiel58). Activation of B-cell receptor signalling serves as another pathway for promoting B-cell survival, proliferation, functional differentiation and migration, offering insights into the localisation of B-cell signalling in SS. The formation of immune complexes through anti-SSA antibody binding to antigens activates TLR and Fc receptors, leading to the production of IFNI (Ref. Reference Bodakçi59). However, the precise role of these antibodies in the development of SS remains unclear.

In SS, B cells contribute to the pathology by secreting pro-inflammatory cytokines. TLR-activated B cells produce a range of cytokines, including pro-inflammatory mediators such as IL-6 and TNF-α, and anti-inflammatory cytokines such as IL-10 (Ref. Reference Qi60). B cells also exhibit a regulatory function through the production of IL-35, composed of IL-12p35/EBI3 (Ref. Reference Shen61). Studies have indicated that patients with pSS exhibit lower levels of IL-35 produced by B cells, suggesting that SS pathogenesis may involve an imbalance between pro- and anti-inflammatory B cells.

Recent studies have demonstrated that regulatory B cells (Breg) modulate immune responses by releasing TGFβ1 and IL-10 (Ref. Reference Fang27). Bregs influence other immune cells either indirectly or directly through the secretion of various cytokines, ultimately contributing to the suppression of inflammatory reactions. In glandular models of autoimmune diseases, the number and/or function of IL-10-secreting (regulatory) B cells tend to be reduced (Ref. Reference Kroese62). However, recent evidence indicates that IL-10-secreting B cells are not functionally impaired (Ref. Reference Lund and Randall63), and a significant increase in Breg concentration in the peripheral blood of patients with SS suggests that Bregs may be involved in the pathogenesis of dry syndrome. Further research is required to explore the association between SS activity and germinal centre, where activated B lymphocytes undergo processes such as somatic hypermutation, BCR editing, antibody affinity maturation and differentiation into plasma and memory B cells. Patients with SS exhibit elevated levels of Tfh cells and DCs in the salivary glands, which influence germinal centre formation and aid in the differentiation of B cells into memory B cells and plasma cells (Ref. Reference Szabo64). Additionally, approximately 20%–25% of people with SS have ectopic germinal centres in their salivary gland tissues (Ref. Reference Lee65).

Differentiation and regeneration in salivary gland tissue engineering

Mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent adult stem cells derived from various tissues. They can be expanded in vitro and have the potential to differentiate into various cell types, including bone, cartilage, bone marrow, skeletal muscle and neuronal cells. MSCs demonstrate remarkable immunomodulatory properties, including the ability to inhibit differentiation and maturation of DCs and impair their antigen presentation capabilities (Ref. Reference Liu83). Moreover, MSCs help reduce inflammation by producing IL-6 and granulocyte-macrophage colony-stimulating factors and attenuate TLR2-mediated signalling in resident macrophages. The potential mechanism of action may involve suppression of Th17 cell differentiation while promoting Treg cell proliferation, downregulating the expression of pro-inflammatory cytokines including IL-17A, IL-17F, IFN-γ, IL-6 and TNF-α (Ref. Reference Ma84), while simultaneously enhancing the secretion of anti-inflammatory factors TGF-β and IL-10, thereby restoring the Th17/Treg cell imbalance (Ref. Reference Choi85). Additionally, MSCs have been shown to suppress the proliferation of resting NK cells stimulated by IL-2, and partially inhibit the proliferation of activated NK cells, both of which are critical for the development of SS (Ref. Reference Chen86). MSCs have been successfully isolated from minor human salivary glands and cultured to form tissue-engineered salivary glands. Upon vivo transplantation, ductal branch-like structures were observed, suggesting a potentially innovative approach for restoring salivary gland function using salivary gland tissue-specific stem cells. Furthermore, transplanted MSCs were found to restore the function of salivary glands that were damaged in mice with SS (Ref. Reference Pringle87). MSCs can be effectively isolated and expanded in vitro from human parotid and submandibular glands, demonstrating their potential for differentiation into adipocytes, osteoblasts and chondrocytes, particularly amylase-expressing cells. However, researchers have observed diminished differentiation capacity of salivary gland MSCs derived from SS patients, suggesting that autologous MSC transplantation may be less efficacious (Ref. Reference Wang, Wang and Hua88). Certain clinical trials have demonstrated that patients with SS can effectively tolerate allogeneic umbilical cord mesenchymal stem cells (UMSCs) (Table 1). These patients experienced symptomatic relief, reduced anti-SSA/SSB antibody levels and minimal adverse side effects (Ref. Reference Waterman68).

Table 1.

Research of bioengineering in pSS

Table 1. Long description

The table consists of three columns: Cells/Material, Results, and References.

* Row 1: Salivary gland stem/progenitor cells. Results state basal cells and I D s within the E D s of salivary glands and c-K I T super plus cells serve as stem/progenitor sources. References 38, 66, 67.

* Row 2: U C M S C s. Results show they alleviate S S symptoms, reduce anti-S S A/S S B antibodies in blood, and have minimal side effects. Reference 68.

* Row 3: h D P S C s. Results describe them as easily isolated, exhibiting self-renewal, immune regulation, and multi-lineage differentiation to alleviate decreased saliva in S S. Reference 69.

* Row 4: E S C s. Results note they are pluripotent, proliferate infinitely, and have genetic stability. References 70 to 72.

* Row 5: i P S C s. Results indicate they inhibit A P C s and T f h cells, reducing pro-inflammatory cytokines like B/plasma cells and T h cells. References 73 to 75.

* Row 6: Matrigel. Results state it supports organoid culture and differentiation into salivary gland organoids, increasing saliva secretion in recipients. References 76, 77.

* Row 7: Gel M A. Results highlight high biocompatibility, degradability, cell adhesion, and superior angiogenesis. Reference 78.

* Row 8: H A. Results show H A hydrogel promotes organization into microspheres with a central lumen similar to large adenoid structures. Reference 79.

* Row 9: Decellularization E C M. Results explain that three-dimensional E C M facilitates S G models and enhances surface interactions for polarized lumens. References 80 to 82.

* Row 10: Growth factors. Results indicate use before transplantation increases K I T super plus cells in vitro. References 66, 99 to 100.

Abbreviations: IDs: Intercalated Duct cells; EDs: Excretory Duct cells; SG: Salivary Gland; UCMSCs: Umbilical cord Mesenchymal Stem Cells; SS: Sjögren’s syndrome; anti-SSA/SSB: anti-Sjögren’s syndrome A/B antibody; hDPSCs: Human dental pulp stem cells; ESCs: Embryonic stem cells; iPSCs: Induced pluripotent stem cells; APCs: Antigen-presenting cells; Tfh: Follicular helper T cells; GelMA: Gelatin Methacryloyl; HA: Hyaluronic acid; ECM: Extracellular matrix.

Furthermore, numerous studies have explored the use of various types of stem cells, including bone marrow stromal stem cells (BMSCs) (Table 1), human adipose-derived MSCs, amniotic cells, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and other stem cells, for cell therapy and the restoration of radiation-induced salivary gland damage (Ref. Reference Adine70). These stem cells have demonstrated therapeutic potential in inhibiting apoptosis when administered intravenously. Additionally, they have shown potential in facilitating the recovery of functional salivary gland cells and activating salivary gland stem/progenitor cells by promoting epithelial cell proliferation and differentiation through paracrine effects (Refs. Reference Sumita66, Reference Tran67). The use of BMSCs for salivary gland tissue regeneration is currently being investigated. BMSCs cultured in vitro differentiate into epithelial cells that, when transplanted into atrophied glands, regenerate their glandular function (Ref. Reference Kawakami24). Studies have also demonstrated that human dental pulp stem cells (hDPSCs) (Table 1) can be easily isolated and extracted from dental tissues than from bone marrow tissues. These hDPSCs, typically obtained from discarded third molars following surgery (Ref. Reference Chalisserry69), express mesenchymal lineage markers including CD29, CD90, CD105, CD73 and CD44. Furthermore, these cells demonstrate typical characteristics of mesenchymal stem cells, such as self-renewal, immunoregulatory properties, multilineage differentiation potential and the ability to migrate to injury sites (Ref. Reference Fernandes89). According to a recent study, transplantation of human tooth-exfoliated stem cells (SHED) systematically reduces salivary reduction symptoms caused by autoimmune disorders in SS (Ref. Reference Du73).

ESCs and iPSCs may serve as novel cell sources. Although iPSCs share a similar multidirectional differentiation potential with ESCs, they offer the advantage of being obtained from adult cells in culture, thus eliminating the ethical concerns associated with the use of human embryos. ESCs are totipotent and capable of differentiating into three primary embryonic layers: the ectoderm, mesoderm and endoderm. Their ability to proliferate indefinitely in an undifferentiated state with a normal euploid karyotype makes them a promising cell source for regenerative medicine (Ref. Reference Tanaka and Mishima71). Several clinical trials have utilised human ESCs, including those utilising retinal pigment epithelial cells derived from ESCs, to treat retinal macular degeneration and cardiac progenitor cells derived from ESCs to regenerate the epicardium in areas affected by myocardial infarction (Ref. Reference Ilic and Ogilvie72). However, the role of ESCs in salivary gland repair requires further investigation. Although human ESCs offer advantages such as genetic stability, considerable challenges persist, including clinical complications and transplant rejection, that limit their application in salivary gland regeneration. Therefore, these issues must be addressed to enhance the clinical utility of ESCs in this field. iPSCs, however, exhibit similar proliferative capacity and differentiation potential as ESCs but are derived from a patient’s own somatic cells. Studies of SG tissues have demonstrated that both BM-MSCs and iPSC-derived MSCs inhibit APCs and Tfh cell activation. Given that the pathogenic effects of SS may vary with disease stage and IL-10-producing cell types, both MSC types significantly reduce lymphocyte infiltration in the salivary glands. They also decrease the expression of B/plasma cells, Th cells and marker genes associated with activated APCs. This confirms that iPSC-derived MSCs suppress the expression of pro-inflammatory cytokines crucial for SS progression in LPS-stimulated mouse splenocytes. Recently, researchers have explored the use of iPSC-derived retinal pigment epithelial (RPE) cells for treating advanced age-related macular degeneration (AMD) (Ref. Reference Spurdle, Goldgar and Easton74). However, the high monetary and time costs associated with establishing a patient’s own iPSC, along with the persistent risks of tumorigenesis due to the use of viral vectors and oncogenes in their initial programming, render this procedure unsuitable for everyone; therefore, the medical applications of iPSCs remain limited by these challenges (Ref. Reference González, Boué and Belmonte75).

Salivary Gland Tissue Engineering techniques

The researchers discovered that the proposed technique for the construction of injectable functional salivary gland organoids with regard to salivary gland tissue engineering technology serves two purposes: providing insights into disease pathogenesis and facilitating the testing of novel drugs targeting epithelial function. Several studies have explored salivary gland stem cells (including salivary gland stem/progenitor cells, mesenchymal stem cells, MSC, etc.) and organoids, using orthotopic transplantation to address salivary gland function in pSS. Regarding clinical translation, salivary gland tissue engineering – an integrative approach combining stem cells, nanozymes and bioactive materials – holds significant promise for restoring secretory function through functional organoids. To date, only three registered Phase I/II clinical trials have investigated organoid-based transplantation/cell therapies. A notable example is the successful application of salivary gland organoids in treating radiation-induced xerostomia in patients with head and neck cancer (Ref. Reference Verstegen, Coppes and Beghin90).

As a novel approach for organ transplantation, functional organoids constructed through bioengineering have the potential to replace damaged or aged organs. These proposed organoids, derived from stem cells or organ progenitor cells, are capable of self-assembling into organ-like structures with physiological, functional and embryonic-specific properties. During the development of individual organs, these organoids become functionally and structurally congruent with their target organs. Organoid formation is driven by self-assembly, a process by which a disordered system transforms into an organised structure through the interaction of individual components (Ref. Reference Jeong91). The construction of salivary gland-like organs using tissue engineering techniques requires three key elements: cell-to-cell contact, interaction between cells and extracellular matrix (ECM) components and active 3D scaffolds that facilitate better functionality (Figure 2) (Ref. Reference Hajiabbas92). Studies have shown that salivary gland epithelial stem cells cultured within an extracellular matrix (ECM) can self-organise into tube-like structures. For organoid culture, laminin-rich matrix gels such as Matrigel (Table 1) are widely employed due to their versatility in supporting the growth of nearly all organoid types (Ref. Reference Li93). However, natural Matrigel has inherent limitations: its complex and undefined composition may contain bioactive factors that inadvertently interfere with cell behavior. These drawbacks restrict its utility in mechanistic studies of cell culture, regenerative medicine and drug screening (Ref. Reference Aisenbrey76). To address these challenges, gelatin has emerged as a more standardised, controllable and reproducible alternative, offering well-documented biodegradability and biocompatibility (Ref. Reference S g, MMX, Tan and Kurisawa77). Notably, methacrylated gelatin (GelMA) enables photoinitiated free-radical polymerisation, combining the intrinsic benefits of gelatin – such as high biocompatibility, tunable degradability, cell-adhesive properties and adjustable mechanical strength – with enhanced angiogenic capabilities. As a result, GelMA has attracted growing research interest and found increasingly diverse biomedical applications in recent years (Ref. Reference Huang78). Further in vivo studies have demonstrated that these salivary gland organoid xenografts can survive for extended periods, retain the unique secretory functions of salivary glands, increase salivary secretion and promote functional restoration of salivary glands in recipient animals (Ref. Reference Pringle87).

Figure 2.

Application prospect of subsalivary tissue engineering technology in SS| SG: salivary gland; SS: Sjögren syndrome; Th1、2、17: helper T cell 1、2、17; Treg: regulatory T cells; DCs: dendritic cells; NK: natural killer cells; ECM: extracellular matrix; MSCs: mesenchymal stem cell; ESCs: embryonic stem cell; iPSCs: induced Pluripotent stem cells. Created with www.figdraw.com.

Figure 2. Long description

The flowchart is divided into two horizontal panels.

Top panel: On the left, a pink oval contains S G stem/progenitor cells, M S C s, E S C s, and i P S C s. An arrow points to a silhouette of a human torso labeled S S, highlighting the salivary gland area. Two arrows lead from the silhouette to the right. The top arrow points to a pink oval listing immune effects: Reduce B-cell activation, Inhibition of C D 4 plus T cell proliferation and differentiation, T h 1, T h 2, T h 17 down-arrow, T reg up-arrow, Inhibit D C s presents antigens, and Inhibit N K cells proliferation. The bottom arrow points to a row of cells labeled differentiation into S G E C s.

Bottom panel: On the left, a large blue circle is divided into three sections. The top section contains endothelial cells, acinar cells, nerve cells, vessel cells, and stem cells. The bottom-left section contains active 3 D scaffolds, mixed materials, and decellularized E C M. The bottom-right section contains soluble E C M proteins and growth factors. An arrow points from this circle to a petri dish containing salivary gland organoids with secretory function. A final arrow points to a silhouette of a human torso labeled S S.

Biological scaffolds encompass both natural and synthetic materials. Natural options comprise ECM components such as collagen and fibronectin and substances such as chitosan, alginate and hyaluronic acid (HA). Synthetic materials include poly (ethylene glycol), poly (lactic acid), poly (lactic-co-glycolic acid) (PLGA) and poly (ethylene glycol) copolymers. The composition and structure of scaffolds notably influence cell differentiation and tissue-specific structural formation. Hydrogels can be biomodified to rapidly control the protein gradient (Ref. Reference Fordham79). Human salivary gland cells are encapsulated within an HA hydrogel (Table 1) incorporating a tandem proteoglycan structural domain IV peptide (PlnDIV), facilitating their organisation into microspheres resembling large adenoidal-like structures with a central lumen (Ref. Reference Qi94). However, a static protein gradient exists on the gel surface and remains confined within the 2D structure. By employing gel microfluidics, a more advanced method for the dynamic delivery of soluble signals, microfluidic systems enable precise manipulation of 3D cellular and microenvironments (Ref. Reference Campisi95). Simultaneously, these systems allow for observation and monitoring of how cells and assembled microsphere structures respond to diverse stimuli, thereby facilitating the management of protein gradients on a 3D scale (Table 1) (Ref. Reference Zheng96).

Creating bioscaffold materials from mammalian ECM involves decellularising the source tissue. Previous research indicates that preserving the complex composition and 3D ultrastructure of the ECM is advantageous for constructing 3D models of SG. However, nearly all physical and biochemical decellularisation methods inevitably lead to structural disruptions and alterations in the surface environment and composition. Therefore, developing effective and optimised decellularisation methods is crucial for maintaining both the integrity and biological functionality of ECMs (Ref. Reference Crapo, Gilbert and Badylak80). Biological scaffold materials derived from ECMs often involve tissue or organ decellularisation processes. Whole-organ decellularisation plays a crucial role in supporting the generation of organs in vitro by removing cells that can trigger immune rejection, while preserving the original tissue structure and composition. This method also retains ECM components within the scaffolds, allowing them to provide physicochemical signals that closely mimic physiological conditions. Researchers have recently cultured rat primary SG cells on scaffolds derived from whole-organ decellularised SGs. Scanning electron microscopy observations have shown that these decellularised SG scaffolds support SG cell adhesion and the formation of polarised lumens. Immunofluorescence staining further indicated the expression of various differentiation markers, suggesting that whole-organ decellularised ECM may serve as an effective scaffolding material for the tissue-engineered salivary gland regeneration (Ref. Reference Hussein81). Moreover, the intricate microstructure mimics the physical characteristics of the basement membrane, enhancing surface interactions and promoting the formation of well-defined polarised lumens and highly differentiated salivary epithelial cells (Ref. Reference Soscia82). The biodegradability, porosity, stiffness and strength of the scaffold can influence its effects on cell adhesion, migration and differentiation (Ref. Reference Peters97).

The objectives of tissue-engineered scaffolds are to establish and maintain differentiated cellular phenotypes, ensure formation of functional units and promote branching (Ref. Reference Ozdemir98). These functions depend on the incorporation of specific bioactive factors into the medium or scaffold. Supplements in the cell culture medium include fetal bovine serum, glutamine and antibiotics. Furthermore, the addition of bioactive molecules to basal medium can enhance the prolonged expansion of salivary stem cells in vitro. For instance, laminin-111 promotes the formation of adenoid-like structures and stimulates the expression of AQP5 and K14 (Ref. Reference Maruyama99). Soluble growth factors (Table 1) are crucial for SG development, branching morphogenesis, endbud formation, cell proliferation, differentiation, innervation initiation and angiogenesis. Additionally, a ROCK inhibitor enhances cell growth, survival, proliferation and expression of α-amylase and the Met proto-oncogene (c-Met) in SG cultures (Ref. Reference Tao100).

The major characteristics of human salivary gland tissues include cellular heterogeneity, structural diversity and glandular secretory functions. These aspects pose challenges for the research and development of human salivary gland tissue-engineered organoids. Therefore, optimising the long-term maintenance of stem/progenitor cells and fostering their growth and development in 3D culture is crucial. This approach leverages the cellular, molecular and functional attributes of native tissues (Ref. Reference Fujii101). Continual exploration of biomaterials that can effectively mimic the ECM is imperative to maintain its structure and integrity during the construction of tissue-engineering models (Ref. Reference Tran102). Salivary gland tissue engineering – integrating stem cells, nanozymes and bioactive materials – holds promise for restoring secretory function via functional organoids. To date, only three registered Phase I/II clinical trials have explored organoid-based transplantation/cell therapy. A notable example is the successful use of salivary gland organoids to treat radiation-induced xerostomia in head and neck cancer patients. Currently, researchers are exploring the application of sophisticated biomaterials to address the challenges arising from the absence of biomedical signals, with the aim to enhance the precise release of biomedical stimulators in both temporal and spatial dimensions.