Introduction

Salivary glands (SGs) are indispensable for oral health, facilitating lubrication, enzymatic digestion, and microbial defense through saliva secretion. However, dysfunction in SGs can occur due to aging, polypharmacy, autoimmune conditions, and anti-cancer treatments, significantly impacting speech, digestion, and oral health. For instance, radiation therapy for head and neck squamous cell carcinoma (HNSCC) often damages SGs, leading to hyposalivation and xerostomia (dry mouth). Current treatments offer only temporary relief, presenting a significant challenge for the 1.1 million new global cases of head and neck cancer diagnosed annually.

A promising solution involves biopsying healthy SG tissue before radiation therapy to develop a tissue-engineered substitute for autologous re-implantation, aiming to restore salivary function. However, SGs' complex structure and multiple cell types pose significant challenges for replacement. Many SG engineering approaches involve encapsulating SG-derived epithelial cells in hydrogel matrices, yet spatial control remains limited. Recent advancements in three-dimensional (3D) organ building blocks, such as spheroids or organoids, have been achieved using magnetic bioassembly and levitation of adult stem cells, offering innovative solutions for SG tissue engineering. 

Innovation Paths from 2D to 3D SG Culture

Traditional two-dimensional (2D) cell cultures have been fundamental in cell biology and drug discovery for decades. In this system, cells are grown as a monolayer on glass or plastic dishes, providing an easy approach to studying cell behavior and high-throughput screening. However, 2D models do not accurately recapitulate the natural architecture of tissues or the complex cell-cell, and cell–ECM interactions crucial for cell behaviors such as proliferation, differentiation, and migration. These limitations highlight the need for more physiologically relevant models, leading to the development of 3D culture techniques.

Organoids, categorized as 3D mini-organs, are created from primary cells or stem cells with specific signaling cues. They can be formed using various 3D culture techniques, such as nonadhesive substrates promoting cell aggregation, embedding cells within ECM-like hydrogel matrices, or tagging cells with nanoparticles for magnetic assembly into specific shapes. SG organoids, derived from primary adult SG stem/progenitor cells or pluripotent stem cells, require a 3D matrix to mimic the ECM environment, supporting cell attachment, proliferation, and differentiation.

3D Scaffold Materials for SG Tissue Engineering

The replication of the intricate mechanical, physiochemical, and biological properties of SG tissues necessitates advanced 3D scaffold materials. These materials are broadly classified into naturally-derived scaffolds, synthetic polymers, and hybrid materials.

Naturally-derived Scaffold Biomaterials

Matrigel, a solid hydrogel derived from mouse sarcoma cell cultures, mimics the native ECM, supporting SG branching morphogenesis. Despite its widespread use, Matrigel's animal origin and lot-to-lot variation challenge clinical translation. Laminin, another key ECM component, plays a crucial role in SG development but can lead to adverse effects, making the entire laminin-1 sequence unsuitable for clinical applications. However, certain peptides derived from laminin enhance SG spheroid formation and function. Additionally, dECM from SG can serve as a naturally-derived biomaterial, supporting primary SG cell adhesion and SG-like tissue formation.

Synthetic Polymers Scaffolds

Synthetic polymers like polyethylene glycol (PEG) and poly-lactic-co-glycolic acid (PLGA) offer customized mechanical properties suitable for cell growth and differentiation. These materials provide a xenogenic-free, reproducible environment crucial for large-scale production. However, encapsulating individual cells in hydrogels may not form SG structures, necessitating pre-assembled multicellular spheroids. Synthetic polymers like polyacrylamide and polyvinylidene fluoride (PVDF) enhance branching morphogenesis and promote SG cell behavior.

Hybrid Scaffold Materials

Hybrid scaffolds combine natural and synthetic polymers to enhance cell attachment and differentiation. For example, polymer disks coated with ECM peptides support SG cells' adhesion and growth. Coating synthetic scaffolds with natural components like Matrigel or laminin has shown enhanced SG cell proliferation and adhesion, offering promising solutions despite potential challenges in biocompatibility and mechanical properties.

Magnetic 3D Bioassembly Platforms for SG Organoid Fabrication

The magnetic 3D bioassembly platform is a groundbreaking technology for creating functional SG organoids. This platform utilizes MNPs, which are incorporated into cells through electrostatic interactions. Once magnetized, cells are detached from their surfaces and suspended in culture media. External magnetic fields are then used to manipulate the cells into desired 3D structures.

Fig. 1 SG organoid biofabrication workflow utilizing two different magnetic 3D bioassembly platforms (Klangprapan J.; et al. 2024).

Magnetic 3D Bioassembly (M3DB): In this method, magnetic drives placed under the culture plate induce cell aggregation. This technique allows for precise spatial control over the formation of 3D structures and promotes the synthesis of ECM by the cells themselves.

Magnetic 3D Levitation (M3DL): In this approach, magnetic drives are placed on top of the culture plate, concentrating cells at the air-liquid interface. This technique enhances nutrient diffusion and accelerates cell aggregation, promoting the formation of tight junctions and epithelial spheroids.

Both methods enable the production of dense, spatially organized cellular constructs capable of synthesizing ECM without the need for engineered substrates. The resulting organoids can be analyzed using various assays, including cytotoxicity tests, immunohistochemistry, and western blotting.

Applications in SG Tissue Engineering

Magnetic 3D bioassembly strategies offer several advantages for SG tissue engineering. These techniques provide spatial control over cell placement, allowing for the creation of complex 3D architectures that mimic the native SG structure. The ability to generate matrix-free 3D structures eliminates the limitations associated with external scaffolds, promoting cell-cell interactions and endogenous ECM production. Moreover, the scalability and reproducibility of these approaches make them suitable for high-throughput applications in regenerative medicine.

Recent advancements in magnetic 3D bioassembly have demonstrated the potential for creating functional SG organoids. These mini-organs can be utilized for studying SG development, disease modeling, and drug screening. Additionally, the combination of magnetic 3D bioassembly with other techniques, such as decellularized ECM (dECM) and plant molecular farming cues, has shown promise in enhancing the functionality and regenerative potential of SG tissue constructs.

In conclusion, magnetic 3D bioassembly platforms represent a cutting-edge approach to SG tissue engineering. These strategies offer spatial control, scalability, and reproducibility, enabling the creation of functional 3D tissue constructs that mimic the native SG structure. The integration of magnetic 3D bioassembly with other innovative techniques holds great promise for advancing SG regenerative therapies and addressing the challenges associated with SG dysfunction.

Conclusion

The magnetic 3D bioassembly platform represents a significant advancement in the field of tissue engineering, particularly for complex structures like SG organoids. By enabling precise spatial control and promoting endogenous ECM synthesis, this technology offers new possibilities for regenerative medicine and SG transplantation. While challenges remain, ongoing research and optimization of this platform hold great promise for improving SG function and addressing the limitations of current treatments. As the field continues to evolve, magnetic 3D bioassembly may become a cornerstone of personalized medicine and regenerative therapies.