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Avatar Biotechnologies for Oral Health and Healthy Longevity Research Unit, Department of Research Affairs, Faculty of Dentistry, Chulalongkorn University, Bangkok, ThailandInternational Graduate Program in Oral Biology, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand
Avatar Biotechnologies for Oral Health and Healthy Longevity Research Unit, Department of Research Affairs, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand
Avatar Biotechnologies for Oral Health and Healthy Longevity Research Unit, Department of Research Affairs, Faculty of Dentistry, Chulalongkorn University, Bangkok, ThailandDepartment of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Songkhla, Thailand
Avatar Biotechnologies for Oral Health and Healthy Longevity Research Unit, Department of Research Affairs, Faculty of Dentistry, Chulalongkorn University, Bangkok, ThailandDepartment of Clinical Pathology, Faculty of Medicine, Navamindradhiraj University, Bangkok, Thailand
Faculty of Dentistry, National University of Singapore, Singapore, SingaporeCentre for Advanced 2D Materials, National University of Singapore, Singapore, SingaporeDepartment of Materials Science and Engineering, College of Design and Engineering, National University of Singapore, Singapore, SingaporeORCHIDS: Oral Care Health Innovations and Designs Singapore, National University of Singapore, Singapore, Singapore
Avatar Biotechnologies for Oral Health and Healthy Longevity Research Unit, Department of Research Affairs, Faculty of Dentistry, Chulalongkorn University, Bangkok, ThailandDepartment of Research Affairs, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
Corresponding author: Avatar Biotechnologies for Oral Health and Healthy Longevity Research Unit, Department of Research Affairs, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand.
Avatar Biotechnologies for Oral Health and Healthy Longevity Research Unit, Department of Research Affairs, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand
Hyposalivation and severe dry mouth syndrome are the most common complications in patients with head and neck cancer (HNC) after receiving radiation therapy. Conventional treatment for hyposalivation relies on the use of sialogogues such as pilocarpine; however, their efficacy is constrained by the limited number of remnant acinar cells after radiation. After radiotherapy, the salivary gland (SG) secretory parenchyma is largely destroyed, and due to the reduced stem cell niche, this gland has poor regenerative potential. To tackle this, researchers must be able to generate highly complex cellularized 3D constructs for clinical transplantation via technologies, including those that involve bioprinting of cells and biomaterials. A potential stem cell source with promising clinical outcomes to reserve dry mouth is adipose mesenchymal stem cells (AdMSC). MSC-like cells like human dental pulp stem cells (hDPSC) have been tested in novel magnetic bioprinting platforms using nanoparticles that can bind cell membranes by electrostatic interaction, as well as their paracrine signals arising from extracellular vesicles. Both magnetized cells and their secretome cues were found to increase epithelial and neuronal growth of in vitro and ex vivo irradiated SG models. Interestingly, these magnetic bioprinting platforms can be applied as a high-throughput drug screening system due to the consistency in structure and functions of their organoids. Recently, exogenous decellularized porcine ECM was added to this magnetic platform to stimulate an ideal environment for cell tethering, proliferation, and/or differentiation. The combination of these SG tissue biofabrication strategies will promptly allow for in vitro organoid formation and establishment of cellular senescent organoids for aging models, but challenges remain in terms of epithelial polarization and lumen formation for unidirectional fluid flow. Current magnetic bioprinting nanotechnologies can provide promising functional and aging features to in vitro craniofacial exocrine gland organoids, which can be utilized for novel drug discovery and/or clinical transplantation.
Saliva plays a crucial role in taste, bolus formation, mastication, and swallowing. Additionally, saliva maintains a biochemical environment that hydrates and protects the oral mucosa from environmental challenges. This secretory fluid has antibacterial, antiviral, and antifungal properties, maintains a stable pH, and prevents dental demineralization [
]. Reduction of salivary flow can cause persistent tooth decay, oral infections, and oral mucosal pain. Salivary gland (SG) hypofunction and dry mouth syndrome (also named xerostomia) are the most long-standing complications of radiation therapy (RT) in head and neck cancer (HNC) patients. Dry mouth symptoms are also associated with several systemic diseases including diabetes mellitus, Sjögren's syndrome, thyroid disease, granulomatous diseases, and other immunological conditions [
]. Treatment of HNC patients requires a multidisciplinary approach, in which RT is considered the primary modality or an adjunct to surgical treatment [
]. Radiation injury is generally classified into acute and late effects. Acute symptoms occur during or within a few weeks after treatment. This damage is predominant in tissues composed of fast-proliferating cells (i.e., oral mucosa). On the contrary, RT late consequential effects arise months or years later and tend to occur in tissues with slow cellular turnover like the SG [
Salivary glands (SG) are one of the most radiosensitive tissues, and its self-regenerative potential is low as they are composed of highly differentiated cells in their acinar and ductal epithelial compartments [
]. A SG is a complex organ consisting of an acinar epithelial compartment surrounded by myoepithelial cells, a ductal network, a neural network (with parasympathetic and sympathetic nerves), and a vascular system [
]. Together, these components work on a concerted manner to maintain the proper function of SG. When SG is in the radiation field, RT injures the acinar cells, blood vessels, and surrounding nerves. Salivary hypofunction is directly proportional to the area exposed to RT and the radiation dose [
In the last decade, new RT modalities such as three-dimensional conformal radiation therapy (3DCRT), intensity-modulated radiation therapy (IMRT), and proton radiation therapy, have been used to mitigate bystander injuries towards normal adjacent tissues (like the SG) [
Intensity-modulated radiotherapy reduces radiation-induced morbidity and improves health-related quality of life: results of a nonrandomized prospective study using a standardized follow-up program.
]. Affected cancer patients display a loss of 80% of total secretory acinar cells and the remaining epithelial cells are prominently ductal, both of which have very slow turnover and regenerative potential [
]. Thus, recent regenerative medicine efforts are aiming to replace and repair the acinar secretory compartment.
In general, three main strategies have been used to repair the injured SG: the transplantation of autologous SG spheres (or salispheres), stem cells and/or their bioactive lysates, and the transfer of bioengineered SG organoids [
]. Recently, many reports using 3D culture systems and bioprinting of SG organoids have been published and such will be reviewed in the next sections. Organoids from bioprinting platforms have exhibited promising outcomes for clinical transplantation, disease modeling, and high-throughput drug screening. This review focuses on the promises and challenges of organoid biofabrication for craniofacial exocrine glands like the SG, particularly those pertaining to magnetic bioprinting platforms.
2. Stem/progenitor cell sources for SG regeneration
2.1 Stem/progenitor cells in SG development
During SG development, stem/progenitor cells express different markers. There are differentiation events from pluripotent stem cells at the embryonic stage to fully differentiated cells in the mature and functional SG organ [
]. During these morphogenetic events, transit-amplifying cells actively participate. For example, pre-invaginating oral epithelium expresses Keratin 5 (K5) and the Transcription Factor SRY-Box 2 (SOX2), which turns into Keratin 14 (K14)+/K5+ co-expressed cells in early stage of SG development [
]. Specifically, when lineage tracing of K14+ cells was performed, it was discovered that during the early stage of SG development (E10.5 - E12.5), K14+ cells are responsible for giving rise to the entire epithelial compartment including acinar, ductal, and myoepithelial cells [
]. Interestingly, Athwal and colleagues found that SOX2+ oral epithelial cells at E9 - E11 contribute to all epithelial cells of SG at E13, including SOX10+ cells [
]. In addition, SOX10+ cells at E13, a transit-amplifying population from SOX2+ oral epithelium, were found to give rise to the entire parenchyma until E16 stage [
]. An increase in the proliferation of cells (Ki67+) was observed in the edge of acini and duct within 3 days after deligation of the main duct of the submandibular gland (SMG) of Wistar rats [
]. In addition, in swine models, proliferated cells positive with CD49f and intracellular laminin were discovered in ductal and periductal areas 10 days after deligation [
]. A 24-month-follow up study in patients treated with radiation therapy (RT) showed that parotid gland (PG) stimulated saliva flow can spontaneously recover up to 35% of pre-RT levels with a radiation dose of 38 Gy [
]. Hence, this means saliva flow cannot be fully regained after RT. The recovery level of saliva secretion was influenced by the radiation dose, for example the saliva flow rate was 86% of the original rate for 25 Gy, yet it was lower than 31% for greater than 40 Gy radiation dose [
].Through this technique, researchers have identified certain cell populations carrying SG stem/progenitor cell features. Most of the SG stem/progenitor cells locate in the ductal network and give rise to ductal cells under homeostatic conditions (Fig. 1) [
]. For example, lineage tracing of Keratin 14 (K14) or Keratin 5 (K5) expressing cells revealed that these cells give rise to granular and excretory ductal cells during homeostasis [
]. Furthermore, it was observed that cells expressing K5 or (Axis inhibition protein 2) Axin2 in intercalated duct contributed to acinar cells in both ductal ligation and RT mice models [
]. Specifically, the tracing of Mist1+ cells in all major SG of Mist1CreERT2;R26LacZ mice showed that these differentiated acinar cells underwent continued proliferation to replenish acinar cells during both homeostasis and after duct deligation [
]. Noticeably, several recent studies found SOX2+ or SOX9+ cells are in fact SG acinar progenitors that can replace acinar cells during homeostasis and injured conditions [
]. A study in Sox2-deficient mice revealed that the absence of Sox2 in Krt14+ cells during SG development resulted in a notable impairment of acini formation [
]. Furthermore, researchers observed a marked depletion of SOX10+ acinar progenitor cells and AQP5+ pro-acinar cells, which are essential for normal acinar development and function [
]. In addition, genetically removing Sox2 in SOX2+ cells or ablation of SOX2+ cells using diphtheria toxin caused depletion of AQP5+ but not K18+ cells [
]. These results suggest that SOX2+ cells play a critical role in the maintenance of acinar progenitor cell populations and acinar morphogenesis. Lineage tracing of SOX9+ cells in SMG of adult mice demonstrated that SOX9+ cells give rise to all epithelial cell lineages, but mainly acinar cells [
]. Moreover, after 90 days of RT, mice lacking SOX9+ cells exhibited a significant reduction in saliva flow, Ki67+ cells, as well as an increase in cellular apoptosis, in comparison to wild-type mice [
]. Furthermore, May and colleagues revealed that the orchestration of acinar progenitor towards acinar specification and maturation was controlled by interaction between nerve-derived neuregulin (NRG1) and ERBB3 receptor [
]. In addition, these neural-epithelial signals regulated acinar specification via mTOR2 signaling. Table 1 summarizes the most recent putative markers used to recognize stem cells/progenitors in the adult SG [
]. A large number of markers have been selected to identify and isolate stem/progenitors cells, which will be discussed in the next section.
Fig. 1Schematic structure and compartments of the SG unit and proposed locations of SG stem/progenitor cells. Created with Biorender.com. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
2.3 Stem/progenitor cells isolated from salispheres
Coppes and colleagues are the first research group to isolate SG stem cells from an in vitro floating culture system or salisphere arising from the self-aggregation of primary cells from SG tissues [
]. Briefly, SG tissues are mechanically and enzymatically dissociated into cell clumps and salispheres are formed after 3-5 days in a specific medium composed of Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12), epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), N2, insulin, and dexamethasone [
]. Salispheres were found to include cells expressing Stem cells antigen-1 (Sca-1), Tyrosine-protein kinase Kit (c-kit), RNA-binding Protein Musashi Homologue 1 (Musashi-1), which have been identified as stem/progenitor cell markers. According to this later in vivo transplantation study, only 100-300 c-kit+ cells extracted from salispheres are needed to enhance the function of irradiated SG after transplantation [
]. The quantity of c-kit+ cells is, however, very low in human-derived salispheres. For instance, the percentage of c-kit+ cells is around 0.1% in 51-60-year-old patients, and it decreases with time [
]. Due to this limitation, researchers are now identifying other SG stem cell populations. The CD24hi/CD29hi stem cell population had been used with a relative success for the functional rescue of irradiated glands. After transplantation, this population can restore 46% of saliva flow after 120 days [
]. Despite differences in cellular compartments and stem cell marker expression, the restorative capacity of salispheres arising from different SGs is the same [
More recently, a striking study isolated single cell clones expressing Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) and CD90 from human SG. These clonal SG cells showed both mesenchymal and epithelial characteristics [
]. LGR5+/CD90+ cells have been suggested to be multipotent glandular stem cells (GCS) instead of "progenitors" because these cells temporarily exhibited salivary progenitor characteristics throughout their epithelial differentiation steps. Additionally, transplantation of GCS into irradiated mouse SG models showed an improvement in body weight, glandular weight, saliva flow, and the expression of salivary secretory proteins as well [
]. This study suggested that GCS are located in the inter-secretory end-pieces of the human SG and indicated that most of them did not co-express K5 or K14 [
]. However, more genetic lineage tracing studies are necessary to confirm the precise location of GCS and understand their roles in SG damage.
Taken together, many studies have suggested that murine and human SG-specific stem/progenitor cell-based therapy is an effective strategy to partially regenerate the damaged SG. Thus, further studies are necessary to determine whether human SG-specific stem/progenitor cells possess similar or greater regenerative properties in large animal models (porcine) before human clinical trials are initiated. Still, the number of SG stem/progenitor cells may turn out to be insufficient for autologous transplantation in elderly subjects with dry mouth. Hence, non-specific SG stem cell therapies are necessary in aging glands and such are discussed in the next section.
3. Applications of non-specific SG stem cells in SG regeneration
Mesenchymal stem cells (MSC) were initially investigated from bone marrow (BM) by Friedenstein in 1974 [
A CXCL5- and bFGF-dependent effect of PDGF-B-activated fibroblasts in promoting trafficking and differentiation of bone marrow-derived mesenchymal stem cells.
]. Therefore, MSC may be suitable to restore function in SG RT-induced damage.
3.1 Bone marrow-derived mesenchymal stem cells
Bone marrow-derived cells (BMC) containing many different types of non-specific stem/progenitor cells such as BMMSC, hematopoietic stem cells, and endothelial progenitor cells, can potentially regenerate SG function according to recent studies [
]. Therefore, a recent study used more homogenous BMMSC sub-populations to transplant into irradiated SG. Transplantation of BMMSC increased saliva flow, gland weight, and lag time significantly compared to phosphate buffered saline (PBS) injection. Moreover, BMMSC have protective effects against RT-induced damage, and may transdifferentiate into secretory acinar cells [
]. Interestingly, the culture method can affect the success of stem cell therapy. A study compared normoxic (O2: 21%) and hypoxic (O2: 1%) as culture conditions. This study indicated that BMMSC preconditioned with 1% O2 upregulated binding of stromal derived-cell factor 1 and C-X-C chemokine receptor type 4 (SDF1-CXCR4) and B-cell lymphoma 2 (Bcl2), which are essential in cell migration and survival. Moreover, the activity of α-amylase in the hypoxic group increased significantly compared to the normoxic group after four weeks of transplantation [
Another promising type of adult stem cells is adipose-derived MSCs (AdMSC) which can be collected by a relatively simple procedure. Their potential is, interestingly, unaffected by the age of donor. Moreover, adipose tissues comprise a greater density of MSC than BM [
]. A study transplanting human AdMSC by systemic administration indicated that human AdMSC could migrate to injured lesions via circulation and become engrafted into SG tissues. The injection of hAdMSC exhibited improvement in saliva flow rate, and a reduction of damaged tissues at 12 weeks after irradiation [
]. The combination of AdMSC and platelet-rich fibrin extract (PRFe) resulted in effective outcomes for the injured SGs, while AdMSC or PRFe only slightly recovered the tissue structure and function [
Intraglandular transplantation of adipose-derived stem cells combined with platelet-rich fibrin extract for the treatment of irradiation-induced salivary gland damage.
]. MSC have properties that prevent allogeneic rejection, such as hypo-immunogenicity, regulation of immune cell activity, and formation of a suppressive microenvironment [
]. Until this date, only one phase 1/2 clinical trial using hAdMSC has been reported in SG hypofunction after radiation therapy. This clinical trial showed that the saliva flow rate increased by 33% after 1 month and 50% after 4 months of hAdMSC intra-glandular injection [
]. The long-term follow-up data is essential to make more definitive conclusions about these promising effects of hAdMSC through time. Despite, this trial proved that intra-glandular injection of hAdMSC is a safe approach and more clinical trials should follow.
3.3 Dental pulp stem cells
The development of the SG requires interacting signals from the epithelium and neural crest-derived mesenchyme. Notably, dental pulp stem cells (DPSC) are lineage of neural crest cells [
]. A recent study showed that human SG (HSG) cell line co-culturing with DPSC in Matrigel formed more mature, larger, and higher number of acinar structures than HSG alone. These findings indicated that DPSC stimulated and increased differentiation of HSG into mature SG tissue. Moreover, DPSC have been considered as a supportive mesenchyme for SG regeneration and tissue engineering [
]. A later study used DPSC populations to differentiate into dental pulp endothelial cells (DPEC) which were then injected into irradiated mouse SG models. The purpose of DPEC transplantation was to regenerate the vascular network in the damaged SG. In fact, mouse SG injected with DPEC partially recovered saliva flow rates (62%) when comparing to non-irradiated control glands; while saliva flow rate of PBS-injected glands was 40% of non-irradiated glands [
The mechanisms underlying MSC therapies in SG hypofunction are still not clearly understood. Initially, it was proposed that tissue and vascular regeneration were occurring after stem cell injections [
]. When investigated thoroughly, however, the low rate of stem cell integration or the small number of newly formed parenchyma and vessels could not entirely explain the observed functional improvement [
]. To date, most of the studies have supported the concept that MSC drive immune modulation and induce regenerative activities via paracrine factors (Fig. 2) [
] were reported to have a protective effect or could partially restore the salivary flow in irradiated SG. The benefits of these paracrine effects have prompted scientists to study the effects of bioactive factors released by adipose-derived and bone marrow-derived MSC. Indeed, a Tran et al. study indicated that BM cell extracts possess the same regenerative effect as BM cells [
]. Further, this research group demonstrated that the mononuclear cell extract (a fraction of BM cells) exhibited the best therapeutic effect compared to the other two fractions composed of granulocytes and red blood cells [
Fig. 2Paracrine effects of MSC. MSCs secrete soluble factors which stimulate angiogenesis (IGF1, HGF, VEGF), endogenous stem cells proliferation and differentiation (FGF7, EGF). Moreover, these paracrine factors inhibit cell apoptosis via Bcl2 and Akt and ameliorate fibrosis by MMP2 and MMP9. Created with Biorender.com. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
In summary, MSC-based therapy may be one very promising option for the treatment of SG hypofunction and dry mouth. Moreover, the application of bioactive factors from the stem cell-derived secretome might be a potential avenue that requires further investigations.
4. Development of SG organoids from 3D culture systems
Up to this date, the majority of regenerative strategies have focused on specific stem cell populations [
]. Yet, this is changing with organoid culture systems from patient-derived primary cells. Sato and colleagues first developed organoid technology for intestine after finding LGR5+ cells as gut stem cells. By using culture media containing laminin-rich Matrigel supplemented with R-spondin, EGF, and Noggin, they could develop intestinal organoids from LGR5+ cells [
] were developed. An organoid must exhibit several characteristics of the native organ including multiple differentiated cell types, organ-specific functions, and similar cellular spatial organization. Due to this native organ resemblance, organoids may have multiple applications for in vitro disease modeling, genetic editing and repair, drug discovery, and tissue regeneration [
Transplantation of a bioengineered mouse SG organ produced from embryonic epithelium and mesenchyme was able to fully restore salivary function in SG deficient mice [
]. This SG organ survived with a complete integration with the native parotid gland duct. Interestingly, the regeneration of the autonomic neuronal networks, such as parasympathetic and sympathetic ones, was also observed [
]. Although the outcome of this study is promising, it is not feasible to be translated to clinical grounds due to ethical concerns raised by the utilization of fetal cells and tissues. In addition, this study was performed with mouse fetal glandular tissues which are readily available, but certain ethical issues prevent the use of human fetal glands for regeneration purposes. However, further studies can target the combination of 3D extracellular matrices (ECM) with primary cells with high turnover properties to produce organoids that develop into a mature gland in the donor microenvironment (Fig. 3). Organoids can be fabricated on 3D matrices from primary adult SG stem/progenitor cells, immortalized SG cells, or even pluripotent stem cells but not all have the same structure and functional characteristics. Table 2 displays a list of cell types that have been used to generate SG organoids.
Fig. 3Clinical and in vitro applications of exocrine gland organoids. Organoids are formed by embedding stem/progenitor cells and specific growth factors in 3D extracellular matrix. The organoids can be ultimately utilized for high-throughput drug testing, disease modeling, and tissue regeneration. Created with Biorender.com. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
A 3D matrix plays a crucial role in organoid development because it will mimic an ECM where cells can attach, proliferate, and differentiate. In addition, ECM-like biomaterials must be biocompatible and biodegradable for transplantation into recipient tissues. Extracellular proteins and growth factors may be added to 3D culture systems to stimulate the growth and maturity of cells. Matrigel is a basement membrane protein mixture extracted from mouse sarcoma, and it is rich in ECM peptides such as laminin, collagen IV, heparan sulfate, and growth factors [
]. However, Matrigel has relevant limitations such as lot-to-lot variations which impairs reproducibility and lack of clinical translation due to its animal origin (Engelbreth-Holm-Swarm mouse sarcoma). Additionally, Matrigel is not manufactured to meet Good Manufacturing Practice (GMP) standards, which are required for clinical translation [
]. Recently, a whole-decellularized SG from rats was investigated as a bio-scaffold and such included its native structures and ECM proteins. The decellularized ECM could support the adhesion of primary SG cells and form SG-like tissues [
]. Other biocompatible and biodegradable materials are listed on Table 2. PEG hydrogel and HA-based hydrogel have been suggested as potential biomaterials that resemble ECM for generating SG organoids. Farach-Carson's research group has modified HA-based hydrogels by incorporating them with basement membrane-derived peptides, including bioactive domains from laminin and perlecan [
]. These modified hydrogels enhanced the proliferation of SG primary progenitor-like cells, and acinar-like cells were ultimately formed upon treatment with β-adrenergic and cholinergic agonists [
]. In addition, the same research group used an immunosuppressed mini-swine model to test the feasibility of transplanting HA-based hydrogel constructs encapsulating human SG primary cells into irradiated SG [
]. Hydrogel-encapsulated human SG cells were able to survive and secrete human α-amylase into the oral cavity.
Growth factors play an important role in proliferation, differentiation, migration, motility, and adhesion of primary cells. Growth factors are significant to successful organ formation and regeneration; therefore, they act as a central element in tissue engineering. Growth factors can be mixed into matrices in the fabrication process of them to reconstruct microenvironment of in vivo tissues, which support organoid formation [
]. During SG development, mesenchyme-derived growth factors such as FGFs, EGFs, and IGFs promote cell proliferation, branching morphogenesis, and differentiation [
]. Hence, these growth factors can be effective in generating salivary organoids. Indeed, FGF10 have been used in the differentiation of 3D SG cultures, since they are able to promote epithelial morphogenesis and organoid function [
]. Moreover, stimulation of SG-like organoids with FGF10 ranging from 40 to 400 ng/ml significantly increased amylase activity of these organoids compared to without FGF10 supplement. More recently, a report found that neuregulin-1 (NRG1), a member of EGF family, can replace EGF in the culture media of SG organoids [
Mimicking the epithelial apicobasal cellular polarity and lumenization of the in vivo SG organ continues to be one of the major challenges in SG organoid biofabrication. The SG secretory parenchyma is formed by polarized acinar and ductal cells, which produce and transport saliva into the lumen in a specific directional manner [
]. Hence, recent studies have incorporated such molecules with synthetic ECM to obtain an epithelial polarity in SG organoids. Perlecan domain IV has been shown to drive the polarity of secretory acinar cells by expressing ZO-1, a tight junction protein [
]. Moreover, Nam and colleagues developed a modified fibrin hydrogel with YIGSR and A99 peptides, which are sequences from the Laminin-1 protein responsible for cell adhesion and migration [
]. This combination promoted lumen formation of in vitro SG spheroids and increased the expression of ZO-1 and E-cadherin in epithelial acinar cells at surgical wound site 8 days after hydrogel transplantation [
]. Recently, a novel approach encapsulated clusters of acinar and intercalated ductal cells within matrix metalloproteinase-degradable hydrogels to develop SG organoids [
Encapsulation of primary salivary gland acinar cell clusters and intercalated ducts (AIDUCs) within Matrix Metalloproteinase (MMP)-degradable hydrogels to maintain tissue structure and function.
]. This approach showed that SG organoids expressed ZO-1 and basement membrane proteins that are important for apicobasal polarity such as laminin-1 and collagen IV. Interestingly, lumenized structure can also be observed in these SG organoids but no neuronal network can be found [
Encapsulation of primary salivary gland acinar cell clusters and intercalated ducts (AIDUCs) within Matrix Metalloproteinase (MMP)-degradable hydrogels to maintain tissue structure and function.
In brief, recent tissue engineering research studies have provided many promising methodologies to generate SG organoids from stem/progenitor cells and ECM. However, only a limited number of studies comprehensively evaluated the potential regeneration capacity of SG organoids upon transplantation into irradiated SG.
5. Bioprinting and magnetic bioassembly nanotechnologies
In 2018, our research group has developed a novel magnetic-based 3D culture platform in which cells are tagged with magnetic nanoparticles to organize them into spheroids and mature organoids. This magnetic nanoparticle solution is composed of gold, iron oxide, and poly-L-lysine, which can simply magnetize different cell types via electrostatic interactions at the cell membrane level [
]. In general, after magnetized cells are seeded in the culture media, magnets are placed on top of the plate (levitation platform) or under the plate (bioprinting platform) to accelerate cell aggregation and tight-junction formation and produce epithelial spheroids (Fig. 4). The subsequent 3D cellularized constructs are dense, spatially organized, and can synthesize ECM [
]. Earlier 3D cell culture systems using Matrigel or hydrogels were costly because they involve a long biofabrication process and a time-consuming analysis. For example, these 3D cell culture protocols with Matrigel/hydrogels take 9 – 12 days for the spheroid formation to be completed [
]. This 3D culture system has been utilized in the biofabrication of organized 3D exocrine gland tissue constructs including the mammary gland, pancreas, lacrimal gland (LG), and salivary gland (Table 3). Organoid biofabrication studies using magnetic nanoparticles in mammary gland and pancreas have applied this nanotechnology to produce of tumor organoids, in which multiple cell types were co-cultured to create heterotypic tumor organoids [
Fig. 4Magnetic-based 3D culture platforms. Monolayer cells are incubated overnight before trypsinization for bioprinting purposes. Spheroids can be fabricated by both bioprinting and levitation techniques using a magnetic dot placed at bottom or on top of culture plate, respectively. In addition, magnetized cells can be organized into ductal or vascular constructs using a magnetic ring. Created with Biorender.com. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Noticeably, our research group has successfully fabricated many secretory exocrine-like organoids by both bioprinting and levitation using magnetic bioassembly platforms [
]. Firstly, epithelial organoids were created from magnetized hDPSC, which were expanded and differentiated into secretory and innervated SG-like epithelial organoids by culturing with epithelial differentiation media and neurogenic differentiated media, respectively [
]. These SG-like organoids expressed SG specific epithelial markers such as Aquaporin 5 (AQP5), K5, and K14; and performed SG secretory functions upon cholinergic and adrenergic stimulation. In addition, the epithelial bud and neuronal compartment of the ex vivo irradiated SG were rescued after organoid transplantation. Remarkably, there was a biological integration between the neuronal network of ex vivo irradiated SG and SG-like organoids [
]. Secondly, using porcine SG primary cells as a proof of concept, the magnetic levitation platform was utilized to fabricate SG organoids composed of diverse cell types including epithelial acinar and ductal, myoepithelial, and neuronal cells (Fig. 5) [
]. Next, the secretome of SG-like bioprinted organoids (fabricated from the magnetic bioprinting of hDPSC) was investigated in ex vivo irradiated SG fetal models [
]. In these ex vivo models, SG organoid-derived exosomes could rescue epithelial bud and neuronal growth of irradiated fetal SG. The administration of exosomes was able to promote epithelial bud growth by a 2.4-fold when compared to transplantation of SG organoids [
]. However, our group has not yet addressed the SG biofabrication challenges related to the lack of both large lumens and a robust apicobasal epithelial polarization. Both are relevant to ascertain a unidirectional salivary flow process, and we believe these can be spatially developed and tuned with customized magnetic fields. A neuronal network can be easily formed in these bio-printed SG organoids, but vascularization was not investigated neither actively induced, and this might have an impact on their long-term viability.
Fig. 5Biofabrication process of craniofacial exocrine organoids by magnetic 3D nanotechnology platforms and induction of aging. Created with Biorender.com. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
]. As a reminder, porcine SMG can be used as a proof-of-concept SG model since it shares similar anatomical structures and biological functions with its human counterpart, which is a limitation of rodent models [
]. The magnetically assembled dECM constructs created an ideal environment to support primary SG cell tethering, proliferation, and differentiation compared to conventional polystyrene and basement membrane extract (BME) coated surfaces [
]. Ongoing studies aim to develop a 3D culture platform that combines dECM and magnetic bioprinting technology towards SG cancer organoid biofabrication.
Taken together, these magnetic 3D bioprinting and bioassembly nanotechnology platforms or techniques have important advantages in SG tissue engineering due to the following properties:
(1)
these platforms use a scaffold-free concept in which cells generate their own ECM to support cell growth, differentiation, and biointegration;
(2)
bioassembly of heterotypic 3D cultures combining different types of cells, mimicking the complexity of the native tissues;
(3)
this magnetic nanotechnology can quickly induce cellular assembly into spheroids, which reduces cost and time for culturing and maintaining spheroids;
(4)
the bio-printed SG organoids are reproducible and consistent in size and structure, which is suitable for in vitro drug screening and clinical applications;
(5)
magnetic fields can provide a specific spatial organization of magnetized cells to fabricate complex cellularized constructs with a diversity of shapes.
6. Future directions
In the last two decades, significant advances in the SG regenerative medicine field have been made; however, an effective treatment for dry mouth has not been achieved due to relevant drawbacks. The in vivo applicability of the stem cell-derived secretome and their respective cargo from extracellular vesicles will continue to be a research trend based on the previous promising outcomes in in vitro, ex vivo and in vivo models [
]. Therefore, the combination of dECM and magnetic bioassembly platform should be further studied in the future. However, while the potential benefits of using bio-printed organoids for SG regeneration are exciting, there are still technical challenges to overcome in our bio-printed products, including the development of reproducible and scalable epithelial tissue constructs possessing a large acinar secretory compartment with epithelial polarity and lumenized structures. Such secretory compartment should also be innervated and vascularized for long-term organoid viability and function. Thus, a critical and balanced perspective is required to access the potential of regenerative medicine approaches for treating dry mouth, and alternative approaches such as pharmaceuticals or other regenerative therapies should be considered in parallel.
Declaration of Competing Interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The bioprinting-related research studies are supported by: the National Research Council of Thailand (grant number NRCT5-RSA63001-12) to J.N.F.; the Faculty Research Grant (grant number DRF66033) from Chulalongkorn University Faculty of Dentistry to J.N.F.. T.V.P. was supported by a postgraduate scholarship from Graduate Scholarship Programme for ASEAN or Non-ASEAN Countries (2020 – 2022). This research is supported by the 90th Anniversary of Chulalongkorn University Scholarship under the Ratchadaphiseksomphot Endowment Fund (grant number GCUGR1125651004M). Avatar Biotechnologies for Oral Health and Healthy Longevity Research Unit is funded by the Ratchadaphiseksomphot Endowment Fund, Chulalongkorn University.
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