Bioprinting salivary gland models and their regenerative applications

Salivary glands (SGs) play a crucial role in the oral cavity, facilitating essential functions such as lubrication, enzymatic digestion, and bacteriostasis through saliva secretion. Dysfunction in the SGs, triggered by factors like aging, polypharmacy side effects, autoimmune conditions, and anti-cancer therapies, can impact speech, digestion, and oral health. For instance, radiation therapy for head and neck squamous cell carcinoma (HNSCC) often damages SG, causing hyposalivation and xerostomia (dry mouth). Current treatments provide only temporary relief, posing a significant challenge for the 1.1 million new head and neck cancer cases (HNSCC included) that are diagnosed annually worldwide [1].

To address this issue, a biopsy of healthy SG tissue before radiation therapy could enable the ex vivo development of a tissue-engineered substitute for autologous re-implantation, aiming to restore salivary function. However, SGs, with their complex structure and multiple cell types, present challenges for replacement [2, 3]. Many existing SG engineering approaches involve encapsulating SG-derived epithelial cells in hydrogel matrices; yet, spatial control is limited, especially when mimicking the branched architecture of native SG [4–7]. Fine epithelial layers of ducts and acini, being exceptionally thin, are challenging to recreate synthetically. In 2018, our research group developed for the first time innervated three-dimensional culture (3D) organ building blocks of the SG, spheroids or organoids, using magnetic bioassembly and levitation of adult stem cells from the dental pulp and SG primary stem/progenitor cells, respectively [8, 9]. Earlier, we have provided the SG research field a very comprehensive review of our magnetic 3D bioassembly (bioprinting and levitation) strategies and a state-of-the-art summary of the current regenerative therapies for the salivary gland up to the year 2022 [10]. However, several reports of novel 3D approaches for regenerative medicine and SG tissue engineering were published since then, hence, this review is pertinent [11–17]. Recently, our group was able to effectively expand SG primary cells in the short-term with high proliferative rate when using plant molecular farming-derived cues in combination with hyaluronic acid/alginate hydrogels [13] or cell sheets on top of decellularized extracellular matrix (dECM) bioassembled in porous polymers [11]. Efforts have been made by our team to modify SG ECM properties and improve the cell-matrix interface, impacting cluster morphology and the distribution of key proteins. Yet, achieving spatial control in 3D organ building blocks remains a hurdle, particularly in replicating the intricate structure of SG. In the evolving field of 3D cell culture and bioprinting, significant progress has been made in the formulation of biomaterials at a mesoscale, typical of cell aggregates or tissues at early development [8, 9, 17, 18]. These mesoscale biomaterials can serve as scaffolds for in vitro production of microtissues, organoids, spheroids or assembloids capable of performing key physiological functions resembling those of native tissues. A “modular” approach to microtissue engineering involves arranging distinct hydrogel compartments, loaded with different types of cells, into a biomimetic scaffold. This strategy offers well-defined initial conditions for tissue growth and maturation, including cell proliferation and differentiation into functional tissue. More importantly, compartmentalization enables not only 3D cell patterning but also biopolymer spatial patterning, allowing for the imposition of varying physicochemical cues, such as local gradients in matrix stiffness or molecular protein content. However, these strategies may not be enough to avoid a central necrotic core in these tissue constructs if matrices or cells are too compact or well-packed to allow for proper nutrient mass transfer.

Since 2018, our research group has been developing magnetic 3D bioassembly platforms to produce micro-modular cellular clusters to synthesize their own ECM (internally) and meet the needs of microtissue engineering [8, 9, 11, 13, 14, 19]. Our group has ongoing efforts to fabricate SG organoids through magnetic 3D bioassembly platforms to create matrix-free, reproducible, scalable and functional 3D mini-organ prototypes for regenerative studies combining cells with dECM and plant molecular farming cues focused on SG transplantation and EV therapies for SG regeneration [11, 13, 20]. This review will go over the relevant benefits and challenges of these new platforms.

Organoids, categorized as three-dimensional (3D) mini-organs, created from primary cells or stem cells in a culture dish in the presence of signaling cues specific to that particular organ. To enhance the physiological microenvironment and enable the 3D arrangement of cells, researchers have developed 3D culture techniques. These techniques can be broadly categorized into three types: (1) those using nonadhesive substrates that promote cell–cell interactions, resulting in the aggregation of cells into spheroids (without external hydrogel support), (2) those embedding cells within an ECM-like hydrogel matrix for providing external support, (3) those that involve tagging cells with nanoparticles to render them magnetic and then assemble such cells into a specific shape/morphology via magnetic fields (dot, disc, or ring-like magnets) to force cell-cell interactions that ultimately lead to endogenous ECM production in culture (hence, no external substrates, hydrogels or ECM are needed).

SG organoids are often derived from primary adult SG stem/progenitor cells or pluripotent stem cells (PSCs) using 3D matrix [28, 29]. A 3D matrix is crucial for organoid development, mimicking ECM for cell attachment, proliferation, and differentiation. Cell spheroids can be formed using various 3D culture techniques including rotating culture vessels, hanging drop, spontaneous cell aggregation and magnetic 3D bioassembly [30, 31]. SG primary cells can form salivary functional spheroids in serum-free culture, while preserving native ECM and expressing acinar cells, epithelial polarization, and tight junction proteins. Additionally, salivary spheroids cultured in ECM-derived 3D matrices can maintaining the structural integrity for 10 days or more [32]. However, after 10 days of spheroid culture, cellular apoptosis can occur due to uncontrollable increase in size, lower oxygen diffusion rate and nutrient limitations. Hence, controlling the size of spheroids is challenging, especially when high cell density is needed, often resulting in cell death in the central core [33].

Our research team has successfully produced organoids resembling secretory exocrine organs using both M3DB and M3DL [8, 9, 20, 72]. These bioassembly systems can incorporate various human SG cell types, including epithelial, myoepithelial, neuronal and endothelial cells, into organotypic systems [9]. Our study demonstrated this platform for creating bio-functional SG organoids from human dental pulp stem cells (hDPSC). These cells undergo expansion and specialized differentiation into secretory and innervated SG-like epithelial organoids through cultivation with epithelial and neurogenic differentiated media [8]. Also, hDPSC organoids express specific epithelial markers and display SG secretory functions under muscarinic and adrenergic stimulations. Fibroblast growth factor 10 (FGF10), a crucial signaling cue for SG morphogenesis [73], was supplemented in media to promote hDPSC differentiation towards epithelial and neuronal tissue biofabrication [8]. SG organoids via M3DB showed SG-like epithelia with neuro-functional properties, secreting α-amylase and intracellular calcium mobilization. A recent study utilized porcine primary SG and lacrimal grand (LG) cells on the M3DB platform to create functional craniofacial exocrine gland organoids for in vitro aging models. These organoids expressed functional acinar and ductal units with epithelial progenitors, and successfully induced cellular senescence to mimic the native SG and LG hypofunction representing dry eyes and dry mouth [74]. Notably, the transplantation of organoids rescued both the epithelial bud and neuronal components of ex vivo irradiated SG. Curiously, there was a integration between the neuronal networks of the ex vivo irradiated SG and SG-like organoids [8]. In addition, our group has developed magnetically assembled dECM constructs from porcine SG primary cells. These dECM constructs support primary SG cell proliferation, tethering, and differentiation compared to conventional culture plastic dishes and surfaces coated with basement membrane extract (also called BME) [11].

Later on, the M3DL platform was developed to overcome technical shortcomings such as poor light penetration and nutrient diffusion in the center of organoids [68]. The study used porcine SG primary cells to create in vitro SG-like organoids using M3DL. This organoid had positive SG markers from various cellular SG compartments, including adherens junctions (EpCAM, E-cadherin), ductal epithelial and myoepithelial (Cytokeratin 14 and α-smooth muscle actin), and neuronal (β3-tubulin and vesicular acetylcholine transferase). They also exhibited intracellular calcium activity and α-amylase secretion in response to cholinergic stimulation [10]. In the process of SG organoid development, establishing apicobasal polarity in epithelial cells and forming branched lumenized ducts are crucial for directing salivary flow and facilitating saliva production. Achieving these epithelial polarity properties in SG organoids or mini-glands has proven challenging [46]. Nonetheless, the application of these bioassembly strategies has demonstrated promise in in vivo rodent models through the use of magnets [69]. In this specific in vivo study, the magnetized stem cells were biocompatible and effectively targeted.

Extracellular vesicles (EV) are emerging as nano-based therapeutic approaches in regenerative medicine due to their ability to target specific targets, such as cell proliferation, immunomodulation, and angiogenesis [75–77]. The use of EV combined with biomaterials and bioengineering techniques can enhance tissue repair and facilitate wound healing. Notably, EV lack immune rejection and cytotoxicity, are promptly preserved, and can maintain their bioactive properties for long-term storage towards future SG tissue engineering applications [12, 76]. Furthermore, EV can be accurately quantified and produced in large quantities during cell line in vitro expansion without invasive extraction procedures [78]. This not only leads to cost reduction but also results in shorter therapy durations. The presence of EV in body fluids such as blood, breast milk, and saliva are noteworthy [79–81]. These transport a cargo of proteins, RNA, DNA and lipids, facilitating their exchange or transport between cells [82]. In addition, EV have proteins that shed from the cell membrane as well as intracellular proteins. These proteins play a regulatory role in cell-to-ECM interactions and cell-to-cell communications [83]. Remarkably, EV exert control over cellular processes and can traverse tissue barriers [84]. This characteristic makes them a highly sought-after targeted structure in adult stem cells for the discovery of novel biomolecules, particularly in endothelial and neuronal networks, thereby advancing tissue regeneration.

Mesenchymal stem cell (MSC) transplantation is a viable model for tissue regeneration; however, its application in clinical settings is intricate and time-consuming. This process involves extended culture times and relies on individual availability. Notably, MSC can release EV, including exosomes and macrovesicles, which act as paracrine cues mediating communication between MSC and target cells. This unique capability potentially mirrors the biological activity of MSC and offers an alternative to whole cell therapy [85, 86] In a recent study, EV secreted from SG-derived MSC could repair the damaged tissue and restore SG hypofunction in obstructive sialadenitis [12]. These EV exhibit anti-inflammatory effects in lipopolysaccharide-induced organoids modelling inflammation and in macrophage polarization by reducing acinar-to-ductal metaplasia. More interestingly, miRNAs from EV have been found to target and regulate certain inflammation-related pathways [12]. Furthermore, EV can be extracted from hDPSC after 24–72 h of culture in serum-free media [87, 88], promoting acinar epithelial repair in a mice model, suggesting efficacy in SG regeneration strategies [16, 84]. Dental literature has reported the functional significance of hDPSC-derived EV, indicating their ability to activate dental pulp tissue regeneration, induce stem cell differentiation [89], and promote angiogenesis [77].

However, the role of exosomes in in vivo SG regenerative models post-radiotherapy remains untested due to their complex biological cues. The International Society for Extracellular Vesicles or ISEV conducted a global survey for Minimal Information for Studies of Extracellular Vesicles (MISEV2023) guidelines [93], in collaboration with our research group and others. This survey was to provide an updated overview of the advantages and limitations of producing, separating, and characterizing EV from various sources, including body fluids, and solid tissues especially cell culture. Cell culture conditions significantly affect the yield, composition, and function of EV [94, 95] Cell cultures may also contain xenogeneic contaminants such as exogenous bovine RNA, EVs and proteins complexes. This contamination limits the clinical applications of EVs, as it can affect their cargo composition [96, 97] Hence, the EV purification method to obtain a high yield and purity of EV needs to be carefully considered.

In the present, we are investigating magnetic 3D bioassembly approaches in SG cancer organoids as a potential avenue. Remarkably, our SG cancer organoids, cultured for two days, exhibit significant expression of SG ductal epithelial markers (Cytokeratin 14), a proliferation marker (Ki67), and cell adherens junction proteins, demonstrating consistent size at 300 µm diameter and reproducibility. With this robust model in place, the next research steps involve advancing towards the testing of radiotherapy platforms, leveraging the potential of the magnetic 3D bioassembly platform to enhance our understanding and therapeutic interventions in SG cancer research. Furthermore, this 3D platform has previously demonstrated its capability to recapitulate the native extracellular matrix in various tissues for modular organ prototyping, including the lung [102], aortic valve [103], adipose tissue [66] and cancer organoids such as breast [104] pancreatic [101] and also glioblastoma [68] (Fig. 5). Organoids, mimicking adult organs in both architecture and function, emerge as invaluable tools for the comprehensive study of tissue repair mechanisms and the intricacies of human diseases.

Magnetic 3D bioassembly nanotechnology plays crucial rules in the SG regeneration field, creating scaled-up, xeno-free, and biocompatible tissue compartments for cell growth, differentiation, and biointegration. These strategies enable co-culture methods for generating SG matrices, cell-derived secretome, and microtissue compartments, and integrating the complexity of human SG components within a 3D architectural framework. In addition, they provide a platform to explore novel surgical techniques using magnetic fields, facilitating the in vivo implantation and stabilization of magnetized SG organoids or mini-glands in sites of injury [69].

Magnetic 3D bioassembly platforms, gold-based MNPs modified with poly-L-lysine and iron oxide, have the potential to bioassemble SG organoids for regenerative applications via organoid transplantation or EV therapies from organoid-derived secretome. In our ongoing studies, SG cancer organoids can be generated via M3DB, and in the future, we expect to use these for high throughput in vitro screening applications with RT fractionated dose regimens. Our research team is currently developing nanoparticles that can serve as potential radiosensitizers to stop the progression of SG cancers.