Salivary gland regeneration: from salivary gland stem cells to three-dimensional bioprinting.

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.

Plant molecular farming-derived epidermal growth factor revolutionizes hydrogels for improving glandular epithelial organoid biofabrication.

Epidermal growth factor (EGF) is a known signaling cue essential towards the development and organoid biofabrication particularly for exocrine glands. This study developed an in vitro EGF delivery platform with Nicotiana benthamiana plant-produced EGF (P-EGF) encapsulated on hyaluronic acid/alginate (HA/Alg) hydrogel to improve the effectiveness of glandular organoid biofabrication in short-term culture systems. Primary submandibular gland epithelial cells were treated with 5 - 20 ng/mL of P-EGF and commercially available bacteria-derived EGF (B-EGF). Cell proliferation and metabolic activity were measured by MTT and luciferase-based ATP assays. P-EGF and B-EGF 5 - 20 ng/mL promoted glandular epithelial cell proliferation during 6 culture days on a comparable fashion. Organoid forming efficiency and cellular viability, ATP-dependent activity and expansion were evaluated using two EGF delivery systems, HA/Alg-based encapsulation and media supplementation. Phosphate buffered saline (PBS) was used as a control vehicle. Epithelial organoids fabricated from PBS-, B-EGF-, and P-EGF-encapsulated hydrogels were characterized genotypically, phenotypically and by functional assays. P-EGF-encapsulated hydrogel enhanced organoid formation efficiency and cellular viability and metabolism relative to P-EGF supplementation. At culture day 3, epithelial organoids developed from P-EGF-encapsulated HA/Alg platform contained functional cell clusters expressing specific glandular epithelial markers such as exocrine pro-acinar (AQP5, NKCC1, CHRM1, CHRM3, Mist1), ductal (K18, Krt19), and myoepithelial (α-SMA, Acta2), and possessed a high mitotic activity (38-62% Ki67 cells) with a large epithelial progenitor population (∼70% K14 cells). The P-EGF encapsulation strikingly upregulated the expression of pro-acinar AQP5 cells through culture time when compared to others (B-EGF, PBS). Thus, the utilization of Nicotiana benthamiana in molecular farming can produce EGF biologicals amenable to encapsulation in HA/Alg-based in vitro platforms, which can effectively and promptly induce the biofabrication of exocrine gland organoids.