Trehalose versus carboxymethylcellulose oral spray for relieving radiation-induced xerostomia in head and neck cancer patients: a randomized controlled trial.

BACKGROUND: The aim of this study was to investigate the effect of trehalose oral spray to relieve radiation-induced xerostomia on a randomized controlled trial (RCT). METHODS: Prior to RCT, the effect of trehalose (5-20%) on the epithelial growth of fetal mouse salivary gland (SG) explants was evaluated to confirm if 10% trehalose exerted the best epithelial outcomes. Participants who completed radiotherapy for head and neck cancer (HNC) treatment were enrolled in a double-blind RCT, according to inclusion and exclusion criteria as per the CONSORT statement. The experimental group (n = 35) received 10% trehalose spray, while the control group (n = 35) received carboxymethylcellulose (CMC) spray to apply intra-orally 4 times/day for 14 days. Salivary pH and unstimulated salivary flow rate were recorded pre- and post-interventions. The Xerostomia-related Quality of Life scale (XeQoLs) was filled, and scores assessed post-interventions. RESULTS: In the SG explant model, pro-acinar epithelial growth and mitosis was supported by 10% topical trehalose. As for RCT outcomes, salivary pH and unstimulated salivary flow rate were significantly improved after use of 10% trehalose spray when compared to CMC (p < 0.05). Participants reported an improvement of XeQoLs dimension scores after using trehalose or CMC oral sprays in terms of physical, pain/discomfort, and psychological dimensions (p < 0.05), but not social (p > 0.05). When comparing between CMC and trehalose sprays, XeQoLs total scores were not statistically different (p > 0.05). CONCLUSIONS: The 10% trehalose spray improved salivary pH, unstimulated salivary flow rate, and the quality-of-life dimensions linked with physical, pain/discomfort, and psychological signs. The clinical efficacy of 10% trehalose spray was equivalent with CMC-based saliva substitutes for relieving radiation-induced xerostomia; therefore, trehalose may be suggested in alternative to CMC-based oral spray.(Thai Clinical Trials Registry; https://www.thaiclinicaltrials.org/ TCTR20190817004).

Magnetic bioassembly platforms towards the generation of extracellular vesicles from human salivary gland functional organoids for epithelial repair.

Salivary glands (SG) are exocrine organs with secretory units commonly injured by radiotherapy. Bio-engineered organoids and extracellular vesicles (EV) are currently under investigation as potential strategies for SG repair. Herein, three-dimensional (3D) cultures of SG functional organoids (SGo) and human dental pulp stem cells (hDPSC) were generated by magnetic 3D bioassembly (M3DB) platforms. Fibroblast growth factor 10 (FGF10) was used to enrich the SGo in secretory epithelial units. After 11 culture days via M3DB, SGo displayed SG-specific acinar epithelial units with functional properties upon neurostimulation. To consistently develop 3D hDPSC in vitro, 3 culture days were sufficient to maintain hDPSC undifferentiated genotype and phenotype for EV generation. EV isolation was performed via sequential centrifugation of the conditioned media of hDPSC and SGo cultures. EV were characterized by nanoparticle tracking analysis, electron microscopy and immunoblotting. EV were in the exosome range for hDPSC (diameter: 88.03 ± 15.60 nm) and for SGo (123.15 ± 63.06 nm). Upon ex vivo administration, exosomes derived from SGo significantly stimulated epithelial growth (up to 60%), mitosis, epithelial progenitors and neuronal growth in injured SG; however, such biological effects were less distinctive with the ones derived from hDPSC. Next, these exosome biological effects were investigated by proteomic arrays. Mass spectrometry profiling of SGo exosomes predicted that cellular growth, development and signaling was due to known and undocumented molecular targets downstream of FGF10. Semaphorins were identified as one of the novel targets requiring further investigations. Thus, M3DB platforms can generate exosomes with potential to ameliorate SG epithelial damage.

Epigallocatechin-3-Gallate Protects Pro-Acinar Epithelia Against Salivary Gland Radiation Injury.

Antioxidant agents are promising pharmaceuticals to prevent salivary gland (SG) epithelial injury from radiotherapy and their associated irreversible dry mouth symptoms. Epigallocatechin-3-gallate (EGCG) is a well-known antioxidant that can exert growth or inhibitory biological effects in normal or pathological tissues leading to disease prevention. The effects of EGCG in the various SG epithelial compartments are poorly understood during homeostasis and upon radiation (IR) injury. This study aims to: (1) determine whether EGCG can support epithelial proliferation during homeostasis; and (2) investigate what epithelial cells are protected by EGCG from IR injury. Ex vivo mouse SG were treated with EGCG from 7.5-30 µg/mL for up to 72 h. Next, SG epithelial branching morphogenesis was evaluated by bright-field microscopy, immunofluorescence, and gene expression arrays. To establish IR injury models, linear accelerator (LINAC) technologies were utilized, and radiation doses optimized. EGCG epithelial effects in these injury models were assessed using light, confocal and electron microscopy, the Griess assay, immunohistochemistry, and gene arrays. SG pretreated with EGCG 7.5 µg/mL promoted epithelial proliferation and the development of pro-acinar buds and ducts in regular homeostasis. Furthermore, EGCG increased the populations of epithelial progenitors in buds and ducts and pro-acinar cells, most probably due to its observed antioxidant activity after IR injury, which prevented epithelial apoptosis. Future studies will assess the potential for nanocarriers to increase the oral bioavailability of EGCG.

Bioprinting Strategies for Secretory Epithelial Organoids.

Novel three-dimensional (3D) biofabrication platforms can allow magnetic 3D bioprinting (M3DB) by using magnetic nanoparticles to tag cells and then spatially arrange them in 3D around magnet dots. Here, we report an M3DB methodology to generate salivary gland-like epithelial organoids from stem cells. These organoids possess a neuronal network that responds to saliva neurostimulants.

Strategies for Developing Functional Secretory Epithelia from Porcine Salivary Gland Explant Outgrowth Culture Models.

Research efforts have been made to develop human salivary gland (SG) secretory epithelia for transplantation in patients with SG hypofunction and dry mouth (xerostomia). However, the limited availability of human biopsies hinders the generation of sufficient cell numbers for epithelia formation and regeneration. Porcine SG have several similarities to their human counterparts, hence could replace human cells in SG modelling studies in vitro. Our study aims to establish porcine SG explant outgrowth models to generate functional secretory epithelia for regeneration purposes to rescue hyposalivation. Cells were isolated and expanded from porcine submandibular and parotid gland explants. Flow cytometry, immunocytochemistry, and gene arrays were performed to assess proliferation, standard mesenchymal stem cell, and putative SG epithelial stem/progenitor cell markers. Epithelial differentiation was induced and different SG-specific markers investigated. Functional assays upon neurostimulation determined α-amylase activity, trans-epithelial electrical resistance, and calcium influx. Primary cells exhibited SG epithelial progenitors and proliferation markers. After differentiation, SG markers were abundantly expressed resembling epithelial lineages (E-cadherin, Krt5, Krt14), and myoepithelial (α-smooth muscle actin) and neuronal (ß3-tubulin, Chrm3) compartments. Differentiated cells from submandibular gland explant models displayed significantly greater proliferation, number of epithelial progenitors, amylase activity, and epithelial barrier function when compared to parotid gland models. Intracellular calcium was mobilized upon cholinergic and adrenergic neurostimulation. In summary, this study highlights new strategies to develop secretory epithelia from porcine SG explants, suitable for future proof-of-concept SG regeneration studies, as well as for testing novel muscarinic agonists and other biomolecules for dry mouth.

Unveiling Stem Cell Heterogeneity Toward the Development of Salivary Gland Regenerative Strategies.

Epithelial damage in the salivary gland (SG) resulting in irreversible dry mouth can be commonly induced by gamma radiation therapy. This radiation depletes the SG stem/progenitor cell niche slowing healing and natural gland regeneration. Biologists have been focused in understanding the development and differentiation of epithelial stem and progenitor cell niches during SG organogenesis. These organogenesis studies gave insights into novel cell-based therapies to recreate the three-dimensional (3D) salivary gland (SG) organ, recapitulate the SG native physiology, and restore saliva secretion. Such therapeutical strategies apply techniques that assemble, in a 3D organotypic culture, progenitor and stem cell lines to develop SG organ-like organoids or mini-transplants. Future studies will employ a combination of organoids, decellularized matrices, and smart biomaterials to create viable and functional SG transplants to repair the site of SG injury and reestablish saliva production.

A magnetic three-dimensional levitated primary cell culture system for the development of secretory salivary gland-like organoids.

Salivary gland (SG) hypofunction and oral dryness can be induced by radiotherapy for head and neck cancers or autoimmune disorders. These are common clinical conditions that involve loss of saliva-secreting epithelial cells. Several oral complications arise with SG hypofunction that interfere with routine daily activities such as chewing, swallowing, and speaking. Hence, there is a need for replacing these saliva-secreting cells. Recently, researchers have proposed to repair SG hypofunction via various cell-based approaches in three-dimensional (3D) scaffold-based systems. However, majority of the scaffolds used cannot be translated clinically due to the presence of non-human-based substrates. Herein, saliva-secreting organoids/mini-glands were developed using a new scaffold/substrate-free culture system named magnetic 3D levitation (M3DL), which assembles and levitates magnetized primary SG-derived cells (SGDCs), allowing them to produce their own extracellular matrices. Primary SGDCs were assembled in M3DL to generate SG-like organoids in well-established SG epithelial differentiation conditions for 7 days. After such culture time, these organoids consistently presented uniform spheres with greater cell viability and pro-mitotic cells, when compared with conventional salisphere cultures. Additionally, organoids formed by M3DL expressed SG-specific markers from different cellular compartments: acinar epithelial including adherens junctions (NKCC1, cholinergic muscarinic receptor type 3, E-cadherin, and EpCAM); ductal epithelial and myoepithelial (cytokeratin 14 and α-smooth muscle actin); and neuronal (ß3-tubulin and vesicular acetylcholine transferase). Lastly, intracellular calcium and α-amylase activity assays showed functional organoids with SG-specific secretory activity upon cholinergic stimulation. Thus, the functional organoid produced herein indicate that this M3DL system can be a promising tool to generate SG-like mini-glands for SG secretory repair.

Three-Dimensional Bioprinting Nanotechnologies towards Clinical Application of Stem Cells and Their Secretome in Salivary Gland Regeneration.

Salivary gland (SG) functional damage and severe dry mouth (or xerostomia) are commonly observed in a wide range of medical conditions from autoimmune to metabolic disorders as well as after radiotherapy to treat specific head and neck cancers. No effective therapy has been developed to completely restore the SG functional damage on the long-term and reverse the poor quality of life of xerostomia patients. Cell- and secretome-based strategies are currently being tested in vitro and in vivo for the repair and/or regeneration of the damaged SG using (1) epithelial SG stem/progenitor cells from salispheres or explant cultures as well as (2) nonepithelial stem cell types and/or their bioactive secretome. These strategies will be the focus of our review. Herein, innovative 3D bioprinting nanotechnologies for the generation of organotypic cultures and SG organoids/mini-glands will also be discussed. These bioprinting technologies will allow researchers to analyze the secretome components and extracellular matrix production, as well as their biofunctional effects in 3D mini-glands ex vivo. Improving our understanding of the SG secretome is critical to develop effective secretome-based therapies towards the regeneration and/or repair of all SG compartments for proper restoration of saliva secretion and flow into the oral cavity.

Canine hereditary ataxia.

The hereditary ataxias are a group of neurodegenerative diseases that cause a progressive (or episodic) cerebellar ataxia. A large number of different disorders have been described in different breeds of purebred dog, and in some instances, more than one disorder occurs in a single breed, creating a confusing clinical picture. The mutations associated with these disorders are being described at a rapid rate, potentially changing our ability to prevent, diagnose, and treat affected dogs. A breed-related neurodegenerative process should be suspected in any pure bred dog with slowly progressive, symmetric signs of ataxia.

Canine paroxysmal movement disorders.

Paroxysmal dyskinesias are episodic movement disorders characterized by muscle hypertonicity that can produce involuntary movements. Signs emanate from the central nervous system; consciousness is not impaired, ictal electroencephalography is normal, and there are no autonomic signs, distinguishing them from seizure disorders. In humans they are classified into 3 groups, each responding to different therapies. A mutation in the gene for brevican (BCAN) has been identified as the cause of Episodic Falling in Cavalier King Charles spaniels. Further elucidation of the genetic causes will enhance our ability to identify and treat these canine diseases.

Canine hereditary ataxia in old english sheepdogs and gordon setters is associated with a defect in the autophagy gene encoding RAB24.

Old English Sheepdogs and Gordon Setters suffer from a juvenile onset, autosomal recessive form of canine hereditary ataxia primarily affecting the Purkinje neuron of the cerebellar cortex. The clinical and histological characteristics are analogous to hereditary ataxias in humans. Linkage and genome-wide association studies on a cohort of related Old English Sheepdogs identified a region on CFA4 strongly associated with the disease phenotype. Targeted sequence capture and next generation sequencing of the region identified an A to C single nucleotide polymorphism (SNP) located at position 113 in exon 1 of an autophagy gene, RAB24, that segregated with the phenotype. Genotyping of six additional breeds of dogs affected with hereditary ataxia identified the same polymorphism in affected Gordon Setters that segregated perfectly with phenotype. The other breeds tested did not have the polymorphism. Genome-wide SNP genotyping of Gordon Setters identified a 1.9 MB region with an identical haplotype to affected Old English Sheepdogs. Histopathology, immunohistochemistry and ultrastructural evaluation of the brains of affected dogs from both breeds identified dramatic Purkinje neuron loss with axonal spheroids, accumulation of autophagosomes, ubiquitin positive inclusions and a diffuse increase in cytoplasmic neuronal ubiquitin staining. These findings recapitulate the changes reported in mice with induced neuron-specific autophagy defects. Taken together, our results suggest that a defect in RAB24, a gene associated with autophagy, is highly associated with and may contribute to canine hereditary ataxia in Old English Sheepdogs and Gordon Setters. This finding suggests that detailed investigation of autophagy pathways should be undertaken in human hereditary ataxia.

Titanium-enriched hydroxyapatite-gelatin scaffolds with osteogenically differentiated progenitor cell aggregates for calvaria bone regeneration.

Adequate bony support is the key to re-establish both function and esthetics in the craniofacial region. Autologous bone grafting has been the gold standard for regeneration of problematic large bone defects. However, poor graft availability and donor-site complications have led to alternative bone tissue-engineering approaches combining osteoinductive biomaterials and three-dimensional cell aggregates in scaffolds or constructs. The goal of the present study was to generate novel cell aggregate-loaded macroporous scaffolds combining the osteoinductive properties of titanium dioxide (TiO2) with hydroxyapatite-gelatin nanocomposites (HAP-GEL) for regeneration of craniofacial defects. Here we investigated the in vivo applicability of macroporous (TiO2)-enriched HAP-GEL scaffolds with undifferentiated and osteogenically differentiated multipotent adult progenitor cell (MAPC and OD-MAPC, respectively) aggregates for calvaria bone regeneration. The silane-coated HAP-GEL with and without TiO2 additives were polymerized and molded to produce macroporous scaffolds. Aggregates of the rat MAPC were precultured, loaded into each scaffold, and implanted to rat calvaria critical-size defects to study bone regeneration. Bone autografts were used as positive controls and a poly(lactic-co-glycolic acid) (PLGA) scaffold for comparison purposes. Preimplanted scaffolds and calvaria bone from pig were tested for ultimate compressive strength with an Instron 4411(®) and for porosity with microcomputerized tomography (µCT). Osteointegration and newly formed bone (NFB) were assessed by µCT and nondecalcified histology, and quantified by calcium fluorescence labeling. Results showed that the macroporous TiO2-HAP-GEL scaffold had a comparable strength relative to the natural calvaria bone (13.8±4.5 MPa and 24.5±8.3 MPa, respectively). Porosity was 1.52±0.8 mm and 0.64±0.4 mm for TiO2-HAP-GEL and calvaria bone, respectively. At 8 and 12 weeks postimplantation into rat calvaria defects, greater osteointegration and NFB were significantly present in the TiO2-enriched HAP-GEL constructs with OD-MAPCs, compared to the undifferentiated MAPC-loaded constructs, cell-free HAP-GEL with and without titanium, and PLGA scaffolds. The tissue-engineered TiO2-enriched HAP-GEL constructs with OD-MAPC aggregates present a potential useful therapeutic approach for calvaria bone regeneration.