Advances in the application of iron oxide nanoparticles (IONs and SPIONs) in three-dimensional cell culture systems.

BACKGROUND: The field of tissue engineering has remarkably progressed through the integration of nanotechnology and the widespread use of magnetic nanoparticles. These nanoparticles have resulted in innovative methods for three-dimensional (3D) cell culture platforms, including the generation of spheroids, organoids, and tissue-mimetic cultures, where they play a pivotal role. Notably, iron oxide nanoparticles and superparamagnetic iron oxide nanoparticles have emerged as indispensable tools for non-contact manipulation of cells within these 3D environments. The variety and modification of the physical and chemical properties of magnetic nanoparticles have profound impacts on cellular mechanisms, metabolic processes, and overall biological function. This review article focuses on the applications of magnetic nanoparticles, elucidating their advantages and potential pitfalls when integrated into 3D cell culture systems. This review aims to shed light on the transformative potential of magnetic nanoparticles in terms of tissue engineering and their capacity to improve the cultivation and manipulation of cells in 3D environments.

Identifying potential immuno-oncology targets in salivary gland mucoepidermoid carcinoma based on inflammatory status and treatment response.

BACKGROUND: Mucoepidermoid carcinoma is a rare salivary gland malignant tumour. This study aimed to investigate inflammatory and immune signatures of mucoepidermoid carcinoma by identifying potential proteo-transcriptomic biomarkers towards the development of precision immuno-oncology treatment strategies. METHODS: A total of 30 biopsies obtained from patients diagnosed with mucoepidermoid carcinoma between 2013 and 2022 were analysed after H&E staining for scoring of histological inflammatory stroma subtypes and inflammatory hotspots with QuPath. Multiplex immunofluorescence staining and NanoString nCounter PanCancer IO 360™ panel were used to assess stroma and tumour inflammation signatures in high grade mucoepidermoid carcinoma cases in the tumour microenvironment via proteomics and transcriptomics, respectively. RESULTS: Inflammatory cells within the histological inflammatory stroma inflammatory (HIS-INF/hot) tumour neighbourhoods were greater compared to the histological inflammatory stroma-immune desert (HIS-ID/cold) (p = 0.001). A similar trend was observed between treatment non-responders and responders in stroma neighbourhoods (p = 0.0625) and in stroma-to-interface inflammatory hotspots (p = 0.0081), indicating an augmented inflammatory response in hot tumours and non-responders. Furthermore, there were striking differences in the expression of pan-immune leukocyte marker CD45 between responders and non responders particularly in the tumour neighbourhoods (p = 0.0341), but such were not robust for PD-1 and macrophage fractions. Additionally, transcriptomic analysis revealed key differences in leukocyte activation profiles between responders and non-responders. CONCLUSION: This preliminary report unveils the importance of assessing immune leukocyte cellular fractions and pathways for future prognostic biomarker discoveries in mucoepidermoid carcinoma as per the involvement of CD45-driven inflammatory and immune mediators in high grade mucoepidermoid carcinoma in non-responders to treatment. These findings will potentially contribute to the development of novel personalised immunotherapies.

Alteration of extracellular matrix proteins in atrophic periodontal ligament of hypofunctional rat molars.

OBJECTIVES: The aim of this study was to investigate the effect of mechanical force on possible dynamic changes of the matrix proteins deposition in the PDL upon in vitro mechanical and in vivo occlusal forces in a rat model with hypofunctional conditions. MATERIALS AND METHODS: Intermittent compressive force (ICF) and shear force (SF) were applied to human periodontal ligament stem cells (PDLSCs). Protein expression of collagen I and POSTN was analyzed by western blot technique. To establish an in vivo model, rat maxillary molars were extracted to facilitate hypofunction of the periodontal ligament (PDL) tissue of the opposing mandibular molar. The mandibles were collected after 4-, 8-, and 12-weeks post-extraction and used for micro-CT and immunohistochemical analysis. RESULTS: ICF and SF increased the synthesis of POSTN by human PDLSCs. Histological changes in the hypofunctional teeth revealed a narrowing of the PDL space, along with a decreased amount of collagen I, POSTN, and laminin in perivascular structures compared to the functional contralateral molars. CONCLUSION: Our results revealed that loss of occlusal force disrupts deposition of some major matrix proteins in the PDL, underscoring the relevance of mechanical forces in maintaining periodontal tissue homeostasis by modulating ECM composition.

Periostin-integrin interaction regulates force-induced TGF-ß1 and α-SMA expression by hPDLSCs.

OBJECTIVE: Periostin (PN), a major matricellular periodontal ligament (PDL) protein, modulates the remodeling of the PDL and bone, especially under mechanical stress. This study investigated the requirement of PN-integrin signaling in force-induced expression of transforming growth factor-beta 1 (TGF-ß1) and alpha-smooth muscle actin (α-SMA) in human PDL stem cells (hPDLSCs). METHODS: Cells were stimulated with intermittent compressive force (ICF) using computerized controlled apparatus. Cell migration was examined using in vitro scratch assay. The mRNA expression was examined using real-time polymerase chain reaction. The protein expression was determined using immunofluorescent staining and western blot analysis. RESULTS: Stimulation with ICF for 24 h increased the expression of PN, TGF-ß1, and α-SMA, along with increased SMAD2/3 phosphorylation. Knockdown of POSTN (PN gene) decreased the protein levels of TGF-ß1 and pSMAD2/3 upon force stimulation. POSTN knockdown of hPDLSCs resulted in delayed cell migration, as determined by a scratch assay. However, migration improved after seeding these knockdown cells on pre-PN-coated surfaces. Further, the knockdown of αVß5 significantly attenuated the force-induced TGF-ß1 expression. CONCLUSION: Our findings indicate the importance of PN-αVß5 interactions in ICF-induced TGF-ß1 signaling and the expression of α-SMA. Findings support the critical role of PN in maintaining the PDL's tissue integrity and homeostasis.

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).

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.

Orofacial Pain, Musical Performance and Associated Coping Behaviors, Psychological Distress and Disability among Asian Young Adults.

Musicians often report orofacial pain (OFP) and performance-related psychological distress related to occupational neuromuscular overuse, but to date, no study has been performed in Asian musicians to assess these factors. This study evaluated OFP, psychological distress, coping behaviors, and disability among Asian musical performers. A total of 201 participants in Singaporean music ensembles were surveyed from which 159 met the inclusion criteria for vocalists or instrumentalist musicians (mean age 20.26 ± 2.20 years). Self-administered questionnaires assessed musical practices, jaw/neck pre-conditioning exercises, pain-related temporomandibular disorders (TMD), OFP descriptors, pain chronicity and disability, coping behaviors and psychological distress. Univariate and multi-variate analyses were carried out. OFP, while performing, was more than two-fold higher in instrumentalists when compared to vocalists (41.4-48% vs. 17.2%, p = 0.002). A similar trend occurred for OFP that progresses while playing (p = 0.035) and for persistent OFP that reduces playing (p = 0.001). There were no differences in psychological distress, pain coping and disability between groups. Vocalists were found to practice jaw/neck pre-conditioning exercises more frequently (75% vs. 4-12.9% in instrumentalists, p < 0.0001). While performing, Asian vocalists reported less OFP when compared to instrumentalists. Future prospective studies are needed to confirm if pre-conditioning exercises play a protective role against OFP in vocalists.

Biofabrication, biochemical profiling, and in vitro applications of salivary gland decellularized matrices via magnetic bioassembly platforms.

Trending three-dimensional tissue engineering platforms developed via biofabrication and bioprinting of exocrine glands are on the rise due to a commitment to organogenesis principles. Nevertheless, a proper extracellular matrix (ECM) microarchitecture to harbor primary cells is yet to be established towards human salivary gland (SG) organogenesis. By using porcine submandibular gland (SMG) biopsies as a proof-of-concept to mimic the human SG, a new decellularized ECM bioassembly platform was developed herein with varying perfusions of sodium dodecyl sulfate (SDS) to limit denaturing events and ensure proper preservation of the native ECM biochemical niche. Porcine SMG biopsies were perfused with 0.01%, 0.1%, and 1% SDS and bio-assembled magnetically in porous polycarbonate track-etched (PCTE) membrane. Double-stranded DNA (dsDNA), cell removal efficiency, and ECM biochemical contents were analyzed. SDS at 0.1% and 1% efficiently removed dsDNA (< 50 ng/mg) and preserved key matrix components (sulfated glycosaminoglycans, collagens, elastin) and the microarchitecture of native SMG ECM. Bio-assembled SMG decellularized ECM (dECM) perfused with 0.1-1% SDS enhanced cell viability, proliferation, expansion confluency rates, and tethering of primary SMG cells during 7 culture days. Perfusion with 1% SDS promoted greater cell proliferation rates while 0.1% SDS supported higher acinar epithelial expression when compared to basement membrane extract and other substrates. Thus, this dECM magnetic bioassembly strategy was effective for decellularization while retaining the original ECM biochemical niche and promoting SMG cell proliferation, expansion, differentiation, and tethering. Altogether, these outcomes pave the way towards the recellularization of this novel SMG dECM in future in vitro and in vivo applications.

Magnetic bioassembly platforms for establishing craniofacial exocrine gland organoids as aging in vitro models.

A multitude of aging-related factors and systemic conditions can cause lacrimal gland (LG) or salivary gland (SG) hypofunction leading to degenerative dry eye disease (DED) or dry mouth syndrome, respectively. Currently, there are no effective regenerative therapies that can fully reverse such gland hypofunction due to the lack of reproducible in vitro aging models or organoids required to develop novel treatments for multi-omic profiling. Previously, our research group successful developed three-dimensional (3D) bioassembly nanotechnologies towards the generation of functional exocrine gland organoids via magnetic 3D bioprinting platforms (M3DB). To meet the needs of our aging Asian societies, a next step was taken to design consistent M3DB protocols to engineer LG and SG organoid models with aging molecular and pathological features. Herein, a feasible step-by-step protocol was provided for producing both LG and SG organoids using M3DB platforms. Such protocol provided reproducible outcomes with final organoid products resembling LG or SG native parenchymal epithelial tissues. Both acinar and ductal epithelial compartments were prominent (21 ± 4.32% versus 42 ± 6.72%, respectively), and could be clearly identified in these organoids. Meanwhile, these can be further developed into aging signature models by inducing cellular senescence via chemical mutagenesis. The generation of senescence-like organoids will be our ultimate milestone aiming towards high throughput applications for drug screening and discovery, and for gene therapy investigations to reverse aging.

Shear Stress Enhances the Paracrine-Mediated Immunoregulatory Function of Human Periodontal Ligament Stem Cells via the ERK Signalling Pathway.

Relevant immunomodulatory effects have been proposed following allogeneic cell-based therapy with human periodontal ligament stem cells (hPDLSCs). This study aimed to examine the influence of shear stress on the immunosuppressive capacity of hPDLSCs. Cells were subjected to shear stress at different magnitudes (0.5, 5 and 10 dyn/cm2). The expression of immunosuppressive markers was evaluated in shear stress-induced hPDLSCs using qRT-PCR, western blot, enzyme activity and enzyme-linked immunosorbent assays. The effects of a shear stress-derived condition medium (SS-CM) on T cell proliferation were examined using a resazurin assay. Treg differentiation was investigated using qRT-PCR and flow cytometry analysis. Our results revealed that shear stress increased mRNA expression of IDO and COX2 but not TGF-ß1 and IFN-γ. IDO activity, kynurenine and active TGF-ß1 increased in SS-CM when compared to the non-shear stress-derived conditioned medium (CTL-CM). The amount of kynurenine in SS-CM was reduced in the presence of cycloheximide and ERK inhibitor. Subsequently, T cell proliferation decreased in SS-CM compared to CTL-CM. Treg differentiation was promoted in SS-CM, indicated by FOXP3, IL-10 expression and CD4+CD25hiCD127lo/- subpopulation. In conclusion, shear stress promotes kynurenine production through ERK signalling in hPDLSC, leading to the inhibition of T cell proliferation and the promotion of Treg cell differentiation.

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.

Development of high-throughput lacrimal gland organoid platforms for drug discovery in dry eye disease.

Dysfunction and damage of the lacrimal gland (LG) results in ocular discomfort and dry eye disease (DED). Current therapies for DED do not fully replenish the necessary lubrication to rescue optimal vision. New drug discovery for DED has been limited perhaps because in vitro models cannot mimic the biology of the native LG. The existing platforms for LG organoid culture are scarce and still not ready for consistency and scale up production towards drug screening. The magnetic three-dimensional (3D) bioprinting (M3DB) is a novel system for 3D in vitro biofabrication of cellularized tissues using magnetic nanoparticles to bring cells together. M3DB provides a scalable platform for consistent handling of spheroid-like cell cultures facilitating consistent biofabrication of organoids. Previously, we successfully generated innervated secretory epithelial organoids from human dental pulp stem cells with M3DB and found that this platform is feasible for epithelial organoid bioprinting. Research targeting LG organogenesis, drug discovery for DED has extensively used mouse models. However, certain inter-species differences between mouse and human must be considered. Porcine LG appear to have more similarities to human LG than the mouse counterparts. We have conducted preliminary studies with the M3DB for fabricating LG organoids from primary cells isolated from murine and porcine LG, and found that this platform provides robust LG organoids for future potential high-throughput analysis and drug discovery. The LG organoid holds promise to be a functional model of tearing, a platform for drug screening, and may offer clinical applications for DED.

Frequency of Four-dimensional Oral Health Problems across Dental Fields - a Comparative Survey of Slovenian and International Dentists.

OBJECTIVES: To compare the frequency of patients' oral health problems and prevention needs among Slovenian and international dentists with the aim to validate the four oral health-related quality of life (OHRQoL) dimensions across six clinical dental fields in all World Health Organization (WHO) regions. METHODS: An anonymous electronic survey in the English language was designed using Qualtrics software. A probability sampling for Slovenia and a convenience sampling strategy for dentist recruitment was applied for 31 countries. Dentists engaged in six dental fields were asked to categorize their patients' oral health problems and prevention needs into the four OHRQoL dimensions (Oral Function, Orofacial Pain, Orofacial Appearance, and Psychosocial Impact). Proportions of patients' problems and prevention needs were calculated together with the significance of Slovenian and international dentists' differences based on dental fields and WHO regions. RESULTS: Dentists (n=1,580) from 32 countries completed the survey. There were 223 Slovenian dentists (females: 68%) with a mean age (SD) of 41 (10.6) years and 1,358 international dentists (females: 51%) with a mean age (SD) of 38 (10.4). Pain-related problems and prevention needs were the most prevalent among all six dental fields reported by dentists; Slovenian (37%) and 31 countries (45%). According to Cohen, differences between Slovenia, the broader European Region, and 31 countries were considered non-significant (<0.1). CONCLUSION: According to the dentists' responses, the frequency of patients' oral health problems and prevention needs are proportionate between Slovenia and 31 countries, regionally and globally. The four OHRQoL dimensions can be considered universal across all dental fields.

Exogenous LIN28 Is Required for the Maintenance of Self-Renewal and Pluripotency in Presumptive Porcine-Induced Pluripotent Stem Cells.

Porcine species have been used in preclinical transplantation models for assessing the efficiency and safety of transplants before their application in human trials. Porcine-induced pluripotent stem cells (piPSCs) are traditionally established using four transcription factors (4TF): OCT4, SOX2, KLF4, and C-MYC. However, the inefficiencies in the reprogramming of piPSCs and the maintenance of their self-renewal and pluripotency remain challenges to be resolved. LIN28 was demonstrated to play a vital role in the induction of pluripotency in humans. To investigate whether this factor is similarly required by piPSCs, the effects of adding LIN28 to the 4TF induction method (5F approach) on the efficiency of piPSC reprogramming and maintenance of self-renewal and pluripotency were examined. Using a retroviral vector, porcine fetal fibroblasts were transfected with human OCT4, SOX2, KLF4, and C-MYC with or without LIN28. The colony morphology and chromosomal stability of these piPSC lines were examined and their pluripotency properties were characterized by investigating both their expression of pluripotency-associated genes and proteins and in vitro and in vivo differentiation capabilities. Alkaline phosphatase assay revealed the reprogramming efficiencies to be 0.33 and 0.17% for the 4TF and 5TF approaches, respectively, but the maintenance of self-renewal and pluripotency until passage 40 was 6.67 and 100%, respectively. Most of the 4TF-piPSC colonies were flat in shape, showed weak positivity for alkaline phosphatase, and expressed a significantly high level of SSEA-4 protein, except for one cell line (VSMUi001-A) whose properties were similar to those of the 5TF-piPSCs; that is, tightly packed and dome-like in shape, markedly positive for alkaline phosphatase, and expressing endogenous pluripotency genes (pOCT4, pSOX2, pNANOG, and pLIN28), significantly high levels of pluripotent proteins (OCT4, SOX2, NANOG, LIN28, and SSEA-1), and a significantly low level of SSEA-4 protein. VSMUi001-A and all 5F-piPSC lines formed embryoid bodies, underwent spontaneous cardiogenic differentiation with cardiac beating, expressed cardiomyocyte markers, and developed teratomas. In conclusion, in addition to the 4TF, LIN28 is required for the effective induction of piPSCs and the maintenance of their long-term self-renewal and pluripotency toward the development of all germ layers. These piPSCs have the potential applicability for veterinary science.

Internalizing problems are associated with oral health-related quality of life in early childhood: Outcomes from an Asian multi-ethnic prospective birth cohort.

Oral health status ideally warrants for a holistic biopsychosocial approach to health and wellness. Little is known about the impact of behavioral problems on oral health-related quality of life (OHRQoL) in children due to the paucity of studies in early childhood, particularly in Asian multi-ethnic populations. This study evaluated the relationship between early child's socioemotional factors and OHRQoL, as well as its association with orofacial pain (OFP) and early childhood caries (ECC) in the Asian GUSTO birth cohort. Mother-child dyads were postnatally assessed at 3 time points. The Child Behavior Checklist (CBCL) was used to assess the child's socioemotional and behavioral problems at age 4-4.5 years together with other validated questionnaires to evaluate maternal anxiety and depression. ECC detection was performed at age 5, and OHRQoL (primary) and OFP (secondary) outcomes were assessed at age 6 from a total of 555 mother-child dyads. After a univariate regression analysis was performed to identify potential predictors and confounders, a multivariate regression model was run with predisposing factors (CBCL internalization and externalization problems, OFP, ECC) and adjusted for confounders (maternal psychosocial states, maternal education) to determine associations with OHRQoL. Results showed an association between CBCL internalization scores and poorer OHRQoL (RR = 1.03, p = 0.033, 95% CI 1.01 to 1.05), although the limited risk ratio may not have a practical applicability in psychosocially healthy children, alike the majority of those evaluated in this cohort. The average OHRQoL overall score among children with OFP was 2.39 times more than those without OFP (OR = 2.39, p < 0.001, 95% CI 2.00 to 2.86). Thus, in early childhood, OFP, and to lesser extent internalizing behaviors, may negatively impact OHRQoL. This study therefore highlights the complex relationship between OHRQoL and its predisposing socioemotional and somatic pain factors, and demands further investigations in clinically relevant populations.

Pediatric patients' reasons for visiting dentists in all WHO regions.

BACKGROUND: Oral Function, Orofacial Pain, Orofacial Appearance, and Psychosocial Impact are the four oral health-related quality of life (OHRQoL) dimensions (4D) or areas in which oral disorders impact pediatric patients. Using their dentists' assessment, the study aimed to evaluate whether pediatric dental patients' oral health concerns fit into the 4D of the Oral Health-Related Quality of Life (OHRQoL) construct. METHODS: Dentists who treat children from 32 countries and all WHO regions were selected from a web-based survey of 1580 international dentists. Dentists were asked if their pediatric patients with current or future oral health concerns fit into the 4D of the Oral Health-Related Quality of Life (OHRQoL) construct. Proportions of all pediatric patients' oral health problems and prevention needs were computed. FINDINGS: Data from 101 dentists treating children only and 523 dentists treating children and adults were included. For 90% of pediatric patients, their current oral health problems fit well in the four OHRQoL dimensions. For 91% of oral health problems they intended to prevent in the future were related to these dimensions as well. Both numbers increased to at least 96% when experts analyzed dentists´ explanations of why some oral health problems would not fit these four categories. CONCLUSIONS: The study revealed the four fundamental components of dental patients, i.e., the four OHRQoL dimensions (Oral Function, Orofacial Pain, Orofacial Appearance, and Psychosocial Impact) are also applicable for pediatric patients, regardless of whether they have current or future oral health concerns, and should be considered when measuring OHRQoL in the pediatric dental patient population.

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.

Generation of Porcine Induced Neural Stem Cells Using the Sendai Virus.

The reprogramming of cells into induced neural stem cells (iNSCs), which are faster and safer to generate than induced pluripotent stem cells, holds tremendous promise for fundamental and frontier research, as well as personalized cell-based therapies for neurological diseases. However, reprogramming cells with viral vectors increases the risk of tumor development due to vector and transgene integration in the host cell genome. To circumvent this issue, the Sendai virus (SeV) provides an alternative integration-free reprogramming method that removes the danger of genetic alterations and enhances the prospects of iNSCs from bench to bedside. Since pigs are among the most successful large animal models in biomedical research, porcine iNSCs (piNSCs) may serve as a disease model for both veterinary and human medicine. Here, we report the successful generation of piNSC lines from pig fibroblasts by employing the SeV. These piNSCs can be expanded for up to 40 passages in a monolayer culture and produce neurospheres in a suspension culture. These piNSCs express high levels of NSC markers (PAX6, SOX2, NESTIN, and VIMENTIN) and proliferation markers (KI67) using quantitative immunostaining and western blot analysis. Furthermore, piNSCs are multipotent, as they are capable of producing neurons and glia, as demonstrated by their expressions of TUJ1, MAP2, TH, MBP, and GFAP proteins. During the reprogramming of piNSCs with the SeV, no induced pluripotent stem cells developed, and the established piNSCs did not express OCT4, NANOG, and SSEA1. Hence, the use of the SeV can reprogram porcine somatic cells without first going through an intermediate pluripotent state. Our research produced piNSCs using SeV methods in novel, easily accessible large animal cell culture models for evaluating the efficacy of iNSC-based clinical translation in human medicine. Additionally, our piNSCs are potentially applicable in disease modeling in pigs and regenerative therapies in veterinary medicine.

Trends in Salivary Gland Tissue Engineering: From Stem Cells to Secretome and Organoid Bioprinting.

Xerostomia or dry mouth are commonly diagnosed in head and neck cancer patients due to salivary gland (SG) epithelial injury after radiotherapy. Regenerative medicine has fetched the opportunity to replace or regenerate the SG epithelia and restore its secretory function. Early adult stem cell transplantation strategies in rodents have recently shown to improve clinical outcomes in radiotherapy-induced xerostomia in Phase 1/2 human trials. Mesenchymal stem cells from adipose tissue are the most promising, although the ones from the labial mucosa, bone marrow, or dental pulp have an attractive therapeutic value after successful findings in ex vivo and in vivo mouse models of SG injury. Emerging approaches using cell-free therapy with cell "extracts", "soups" or secretome components also exhibit favorable outcomes in the same rodent models. When compared to cell-based approaches, extracellular vesicles (EV) from the secretome (i.e., exosomes) can be easily extracted, quantified, and are more stable for long-term storage and use in SG tissue engineering. Additive manufacturing and three-dimensional bioprinting or bioassembly have an important role on generating spheroids or organoids for cell transplantation to ameliorate SG injury. Moreover, organoids can secrete EV, which may have a therapeutic potential worth to explore in future studies. In this review, we will describe the technological advancements and challenges of these different cell-based and cell-free strategies in SG tissue engineering and regeneration. Impact statement Salivary gland (SG)-like innervated epithelial organoids and the secretome produced from stem cells may constitute feasible therapeutic alternatives to regenerate the SG due to their user-friendly, short-lived, consistent, and scalable additive manufacturing processes. Bioprinting such human SG organoids toward in vitro drug discovery may further reduce the incorporation of animal-derived components to the tissue constructs and minimize the use of animal experimentation in SG regeneration. Despite such advancements, transplantation with human adipose-derived mesenchymal stem cells is the only tissue engineering strategy that has reached Phase 1/2 clinical trials and shown to enlarge the serous SG epithelium and improve salivary flow.