Correction: Abughanam, G., et al. Mesenchymal Stem Cells Extract (MSCsE)-Based Therapy Alleviates Xerostomia and Keratoconjunctivitis Sicca in Sjogren's Syndrome-Like Disease. Int. J. Mol. Sci. 2019, 20, 4750.

The authors wish to make the following corrections to this paper [...].

Cancer stem cells enrichment with surface markers CD271 and CD44 in human head and neck squamous cell carcinomas.

Head and neck squamous cell carcinoma (HNSCC) has a poor 5-year survival rate of 50%. One potential reason for treatment failure is the presence of cancer stem cells (CSCs). Several cell markers, particularly CD44, have been used to isolate CSCs. However, isolating a pure population of CSC in HNSCC still remains a challenging task. Recent findings show that normal oral stem cells were isolated using CD271 as a marker. Thus, we investigated the combined use of CD271 and CD44 to isolate an enriched subpopulation of CSCs, followed by their characterization in vitro, in vivo, and in patients' tissue samples. Fluorescent-activated cell sorting was used to isolate CD44+/CD271+ and CD44+/CD271- from two human HNSCC cell lines. Cell growth and self-renewal were measured with MTT and sphere/colony formation assays. Treatment-resistance was tested against chemotherapy (cisplatin and 5-fluorouracil) and ionizing radiation. Self-renewal, resistance, and stemness-related genes expression were measured with qRT-PCR. In vivo tumorigenicity was tested with an orthotopic immunodeficient mouse model of oral cancer. Finally, we examined the co-localization of CD44+/CD271+ in patients' tissue samples. We found that CD271+ cells were a subpopulation of CD44+ cells in human HNSCC cell lines and tissues. CD44+/CD271+ cells exhibited higher cell proliferation, sphere/colony formation, chemo- and radio-resistance, upregulation of CSCs-related genes, and in vivo tumorigenicity when compared to CD44+/CD271- or the parental cell line. These cell markers showed increased expression in patients with the increase of the tumor stage. In conclusion, using both CD44 and CD271 allowed the isolation of CSCs from HNSCC. These enriched CSCs will be more relevant in future treatment and HNSCC progression studies.

Mesenchymal Stem Cells Extract (MSCsE)-Based Therapy Alleviates Xerostomia and Keratoconjunctivitis Sicca in Sjogren's Syndrome-Like Disease.

Sjogren's syndrome (SS) is an autoimmune disease that manifests primarily in salivary and lacrimal glands leading to dry mouth and eyes. Unfortunately, there is no cure for SS due to its complex etiopathogenesis. Mesenchymal stem cells (MSCs) were successfully tested for SS, but some risks and limitations remained for their clinical use. This study combined cell- and biologic-based therapies by utilizing the MSCs extract (MSCsE) to treat SS-like disease in NOD mice. We found that MSCsE and MSCs therapies were successful and comparable in preserving salivary and lacrimal glands function in NOD mice when compared to control group. Cells positive for AQP5, AQP4, α-SMA, CK5, and c-Kit were preserved. Gene expression of AQP5, EGF, FGF2, BMP7, LYZ1 and IL-10 were upregulated, and downregulated for TNF-α, TGF-ß1, MMP2, CASP3, and IL-1ß. The proliferation rate of the glands and serum levels of EGF were also higher. Cornea integrity and epithelial thickness were maintained due to tear flow rate preservation. Peripheral tolerance was re-established, as indicated by lower lymphocytic infiltration and anti-SS-A antibodies, less BAFF secretion, higher serum IL-10 levels and FoxP3+ Treg cells, and selective inhibition of B220+ B cells. These promising results opened new venues for a safer and more convenient combined biologic- and cell-based therapy.

Cross-contamination of the human salivary gland HSG cell line with HeLa cells: A STR analysis study.

OBJECTIVES: The human salivary gland (HSG) cell line, labeled as a submandibular ductal cell line, is commonly used as in vitro models to study radiation therapy, Sjögren's syndrome, pleomorphic adenoma, mucocele, epithelial-to-mesenchymal transition, and epigenetics. However, the American Type Culture Collection (ATCC) has recently released a list of cross-contaminated cell lines that included HSG. Despite this notice, some research laboratories still use HSG as a salivary cell model. Therefore, this study examined the authenticity of HSG sampled from three different laboratories. METHODS: DNA was extracted from HSG and additional salivary cell lines (NS-SV-AC, NS-SV-DC, A253, HSY) and submitted for cell line authentication with short tandem repeat (STR) analysis. RESULTS: All HSG samples had STR profiles indicating >80% match with HeLa in both the ATCC and Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) databases. This confirmed that HSG sampled from three different laboratories and HSY shared a common ancestry (host) with HeLa, whereas NS-SV-AC, NS-SV-DC, and A253 had unique STR profiles. CONCLUSION: Short tandem repeat analysis revealed that HSG was contaminated by the HeLa cell line. Furthermore, because genotyping of the original HSG cell line was not performed during its establishment, it will be difficult to authenticate an uncontaminated sample of HSG.

Treatment for salivary gland hypofunction at both initial and advanced stages of Sjögren-like disease: a comparative study of bone marrow therapy versus spleen cell therapy with a 1-year monitoring period.

BACKGROUND AIMS: Non-obese diabetic mice (NOD) exhibit autoimmune Sjögren-like disease (SS-like). We reported previously that a combined-therapy consisting of immuno- and cell-based therapy rescued NOD from SS-like. However, therapies tested to date on NOD mice were aimed at the initial phase of SS-like. It is unknown whether therapies are effective in restoring salivary function when given at an advanced phase of SS-like. METHODS: The efficacy of two therapies (bone marrow versus spleen cells) was compared head-to-head for halting/reversing salivary hypofunction at two critical time points of SS-like (7-week-old NOD with normal saliva output and 20-week-old NOD with minimal saliva). NOD mice were divided into four groups: (i) control, (ii) complete Freund's adjuvant (CFA), (iii) bone marrow transplants with CFA or (iv) spleen cell transplants with CFA. Mice were monitored 8-12 months after therapy. RESULTS: Both cell therapies were effective during the initial phase of SS-like; salivary flow rates were maintained between 80-100% of pre-symptomatic levels. Spleen cell therapy was better than bone marrow when administered in the initial phase of SS-like. When cell therapies were given at an advanced phase of SS-like (20 weeks and older), salivary flow rates improved but were at best 50% of pre-symptomatic levels. Both cell therapies decreased tumor necrosis factor-α, transforming growth factor-ß1 levels and T and B cells while increasing epidermal growth factor and regulatory T cells. Elevated serum epidermal growth factor levels were measured in spleen-treated mice. CONCLUSIONS: A therapeutic effect in advanced phase disease, albeit in mice, holds promise for humans in which Sjögren syndrome is generally not diagnosed until a late stage.

GDFs promote tenogenic characteristics on human periodontal ligament-derived cells in culture at late passages.

Tendon/ligament injures are leading disabilities worldwide. The periodontal ligament (PDL) connects teeth to bone, and is comparable to a tendon/ligament-to-bone insertion. PDL-derived cells (PDLCs) express both osteo/cementogenesis and teno/ligamentogenesis genes. However, an efficient method to induce a tenogenic differentiation of PDLCs has not been thoroughly examined. Therefore, this study tested if growth/differentiation factors (GDFs) enhanced tenogenic characteristics of human PDLCs, as a potential cell source for tendon/ligament engineering. Results demonstrated recombinant GDF-5/GDF-7 inhibited alkaline phosphatase (ALP) activity of PDLCs from passage 3 to 6, while GDF-5 enhanced ALP in dental pulp-derived cells and mesenchymal stem cells. GDF-5 (particularly at 10 ng/ml concentration) induced high expression of both early (scleraxis) and mature (tenomodulin, aggrecan, collagen3) tenogenic genes in P4-6 PDLCs, while inhibiting expression of specific transcription-factors for osteogenic, chondrogenic and adipogenic differentiation. Exogenous GDFs might lead PDLCs being expanded in culture during several passages to highly useful cell source for tendon/ligament engineering.

Apparent diffusion coefficient ratio between axillary lymph node with primary tumor to detect nodal metastasis in breast cancer patients.

PURPOSE: To evaluate the ratios of apparent diffusion coefficient (ADC) calculated from diffusion weighted imaging (DWI) between axillary lymph nodes with primary breast tumor lesions in the detection of axillary lymph nodes metastasis in breast cancer patients. MATERIALS AND METHODS: A total of 36 patients with breast tumors and their axillary lymph nodes were included in this study for MR image scan. The ADC values were calculated using DW-MR imaging software. The ADC values and the ADC ratios of axillary lymph nodes in patients with primary breast lesion were compared among benign and metastatic axillary lymph nodes. All the diagnosis were confirmed by histopathological examination. RESULTS: The mean ADC value of metastatic lymph nodes was significantly lower than those of benign lymph nodes (0.787 × 10(-3) mm(2)/s ± 0.145 versus 1.043 × 10(-3) mm(2)/s ± 0.257) (P < 0.05). In addition, ADC ratio of metastatic lymph nodes with breast lesion was significantly lower than those of benign lymph nodes with breast tumor lesions (0.986 ± 0.17 versus 1.375 ± 0.417) (P < 0.05). Once the ADC ratio was fixed at 0.889 × 10(-3) mm(2)/s, the sensitivity, specificity, and accuracy were 82.22%, 82.35% and 82.28%, respectively. The cutoff of ADC ratio was at 1.097 × 10(-3) mm(2)/s, the sensitivity, specificity, and accuracy can be up to 84.44%, 88.24%, and 86.08%. CONCLUSION: ADC value and ADC ratio could be used as a reliable parameter to detect the axillary lymph nodes metastasis in breast cancer patients, and ADC ratio has a higher accuracy.

Simulation training for medical emergencies of dental patients: A review of the dental literature.

In recent years, due to the aging of the population, the number of dental patients with comorbidities such as hypertension and diabetes has increased. Although it has been reported that these patients are increasingly developing medical emergencies during their dental treatments, many dental providers still do not possess the skills to manage medical emergencies appropriately. Simulation training is essential to improve this situation however, there is no report describing how to conduct an effective simulation in detail for dental office medical emergencies. The purpose of this review is to provide information on simulations that is effective and practical. The authors will highlight the key characteristics for providing effective simulation trainings, such as the selection of simulators, simulation locations, instructors, debriefings, methods for evaluating educational effectiveness, and the use of telesimulation as a method for simulation training due to the global COVID-19 pandemic. In addition, this review provides recommendations on tailoring an ideal simulation training course for those who wish to create one. The authors hope that this review will promote the spread of effective simulation training and in turn, contribute to improving the medical safety of dental patients.

Unbiased proteomic analysis detects painful systemic inflammatory profile in the serum of nerve-injured mice.

ABSTRACT: Neuropathic pain is a complex, debilitating disease that results from injury to the somatosensory nervous system. The presence of systemic chronic inflammation has been observed in patients with chronic pain but whether it plays a causative role remains unclear. This study aims to determine the perturbation of systemic homeostasis by an injury to peripheral nerve and its involvement in neuropathic pain. We assessed the proteomic profile in the serum of mice at 1 day and 1 month after partial sciatic nerve injury (PSNL) or sham surgery. We also assessed mouse mechanical and cold sensitivity in naïve mice after receiving intravenous administration of serum from PSNL or sham mice. Mass spectrometry-based proteomic analysis revealed that PSNL resulted in a long-lasting alteration of serum proteome, where most of the differentially expressed proteins were in inflammation-related pathways, involving cytokines and chemokines, autoantibodies, and complement factors. Although transferring sham serum to naïve mice did not change their pain sensitivity, PSNL serum significantly lowered mechanical thresholds and induced cold hypersensitivity in naïve mice. With broad anti-inflammatory properties, bone marrow cell extracts not only partially restored serum proteomic homeostasis but also significantly ameliorated PSNL-induced mechanical allodynia, and serum from bone marrow cell extracts-treated PSNL mice no longer induced hypersensitivity in naïve mice. These findings clearly demonstrate that nerve injury has a long-lasting impact on systemic homeostasis, and nerve injury-associated systemic inflammation contributes to the development of neuropathic pain.

Isolation, Culture, and Characterization of Primary Salivary Gland Cells.

Primary cells are an essential tool for in vitro studies and are obtained directly from living tissues or organs. They closely mimic the physiological state and maintain in vivo functions for short periods of time under optimal conditions. Isolation and culture of salivary gland (SG) cells are useful to decipher the various mechanisms involved in salivary gland dysfunction. However, unlike some other primary cell cultures, SG cell cultures from patient-derived tissues present several challenges. They are difficult to obtain, culture, expand, and characterize due to their sensitive heterogenous cell population and limited expansion potential. In addition, the majority of saliva-secreting acinar cells fail to maintain a differentiated state ex vivo for long periods, and eventually succumb to an acinar-to-ductal metaplasia, losing their secretory phenotype and functions. Herein, we describe two detailed protocols for primary SG cell isolation, culture, and expansion from human (or mouse) salivary tissues using serum-free culture media. We also describe the growth kinetics of these primary cells along with their immunocytochemical characterization. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of SG single-cell culture from freshly obtained human or mouse SG tissues. Basic Protocol 2: Preparation of SG explant culture from freshly obtained human or mouse SG tissues.

Investigation of Fusobacterium Nucleatum in saliva and colorectal mucosa: a pilot study.

As evidence has been linking the oral bacterium Fusobacterium nucleatum (F. nucleatum) to colorectal tumorigenesis, we aimed to produce preliminary data on the expression of F. nucleatum in both oral and colorectal body sites in cases diagnosed with colorectal neoplasms (CRN) and CRN-free controls. We conducted a pilot hospital-based case-control study among patients who underwent colonoscopy examination. Saliva samples and biopsies from healthy colon mucosa from CRN cases and CRN-free controls, and from tumors in cases, were collected, as well as data on periodontal condition and potential CRN risk factors. A total of 22 CRN cases and 21 CRN-free controls participated in this study, with a total of 135 biospecimens collected and analyzed by qPCR for detection and quantification of F. nucleatum. The detection rate of F. nucleatum was 95% in saliva samples and 18% in colorectal mucosa specimens. The median (95% CI) salivary F. nucleatum level was 0.35 (0.15-0.82) and 0.12 (0.05-0.65) in case and control groups, respectively, with a Spearman correlation of 0.64 (95% CI 0.2-0.94) between F. nucleatum level in saliva and healthy colorectal mucosa in controls. Our study results support the need for and the feasibility of further studies that aim to investigate the association between oral and colorectal levels of F. nucleatum in CRN cases and controls.Clinical Relevance: Considering the current evidence linking F. nucleatum to colorectal carcinogenesis, investigating the role of oral F. nucleatum expression in its colorectal enrichment is crucial for colorectal cancer screening and prevention avenues.

Microchimerism in salivary glands after blood- and marrow-derived stem cell transplantation.

Blood- and marrow-derived stem cells (BMDSCs) provide disease-ameliorating effects for cardiovascular and autoimmune diseases. Microchimerism from donor BMDSCs has been reported in several recipient tissues. We hypothesized that this finding suggests a potential use of BMDSCs in the treatment of salivary dysfunctions. We investigated the presence of Y chromosome-positive cells in salivary gland biopsies of 5 females who had received a marrow or blood stem cell transplant from male donors. One to 16 years after transplantation, all recipients exhibited scattered Y chromosome-positive cells in the acini, ducts, and stroma of their salivary glands (mean of 1.01%). Potentially, these cells can be markers of transplantation tolerance, contribute to neoplastic epithelial tissues, or engraft at sites of injury. In addition, transplantation of BMDSCs could be used for treatment of Sjögren's syndrome and salivary glands damaged by therapeutic irradiation for cancers of the head and neck.

[Relationship between glucose-6-phosphate dehydrogenase gene mutations and neonatal jaundice in Naning, Guangxi].

OBJECTIVE: To study the correlation between glucose-6-phosphate dehydrogenase (G-6-PD) activities and three common mutations of G-6-PD gene G1388A, G1376T and A95G and investigate the effects of G-6-PD gene mutations on neonatal jaundice in Nanning, Guangxi. METHODS: One hundred and twenty-four neonates from Nanning, Guangxi, with hyperbilirubinemia were enrolled. The ARMS-PCR and PCR/REA methods were used to determine G-6-PD gene mutations. G-6-PD activities were measured using the NBT method. The incidence of acute bilirubin encephalopathy and the peak bilirubin concentration 72 hrs after birth were compared between the neonates with different genotypes and between the G-6-PD mutation and normal groups. The risk of blood serum bilirubin >340 mumol/L was evaluated by logistic regression analysis. RESULTS: Of the 124 cases, gene mutations were found in 37 cases, including G1388A (n=20), G1376T (n=14), A95G (n=4) and G1388A+A95G (n=1). Five cases (25%) showed normal G-6-PD activities in the G1388A gene mutation group and 4 (29%) had normal G-6-PD activities in the G1376T G1388A gene mutation group. All of 4 cases of A95G G1388A gene mutation showed a deficiency of G-6-PD activities. There were no significant differences in the incidence of acute bilirubin encephalopathy and the peak bilirubin concentration 72 hrs after birth between the G1388A and G1376T G1388A gene mutation groups. The incidence of acute bilirubin encephalopathy, the peak bilirubin concentration 72 hrs after birth and the risk of serum bilirubin >340 micromol/L in the G-6-PD mutation group were not different from the normal group. CONCLUSIONS: G1388A, G1376T and A95G are common G-6-PD gene mutations in Nanning, Guangxi. The false negative results may be received when the NBT method is used for diagnosis of G-6-PD deficiency. There are similar effects on the incidence of acute bilirubin encephalopathy and the peak bilirubin concentration 72 hrs after birth between different gene mutation groups. G-6-PD gene mutations alone may not contribute to the development of acute bilirubin encephalopathy and the changes of peak bilirubin concentration 72 hrs after birth and the risk of serum bilirubin >340 micromol/L.

Cell culture of differentiated human salivary epithelial cells in a serum-free and scalable suspension system: The salivary functional units model.

Saliva aids in digestion, lubrication, and protection of the oral cavity against dental caries and oropharyngeal infections. Reduced salivary secretion, below an adequate level to sustain normal oral functions, is unfortunately experienced by head and neck cancer patients treated with radiotherapy and by patients with Sjögren's syndrome. No disease-modifying therapies exist to date to address salivary gland hypofunction (xerostomia, dry mouth) because pharmacotherapies are limited by the need for residual secretory acinar cells, which are lost at the time of diagnosis, whereas novel platforms such as cell therapies are yet immature for clinical applications. Autologous salivary gland primary cells have clinical utility as personalized cell therapies, if they could be cultured to a therapeutically useful mass while maintaining their in vivo phenotype. Here, we devised a serum-free scalable suspension culture system that grows partially digested human salivary tissue filtrates composing of acinar and ductal cells attached to their native extracellular matrix components while retaining their 3D in vivo spatial organization; we have coined these salivary spheroids as salivary functional units (SFU). The proposed SFU culture system was sub-optimal, but we have found that the cells could still survive and grow into larger salivary spheroids through cell proliferation and aggregation for 5 to 10 days within the oxygen diffusion rates in vitro. In summary, by using a less disruptive cell isolation procedure as the starting point for primary cell culture of human salivary epithelial cells, we demonstrated that aggregates of cells remained proliferative and continued to express acinar and ductal cell-specific markers.

Cell extracts from spleen and adipose tissues restore function to irradiation-injured salivary glands.

A cell extract from whole bone marrow (BM), which we named "BM Soup," has the property to restore saliva secretion to irradiation (IR)-injured salivary glands (SGs). However, BM cell harvesting remains an invasive procedure for the donor. The main objective of this study was to test the therapeutic effect of "Cell Soups" obtained from alternate tissues, such as adipose-derived stromal cells (ADSCs) and spleen cells to repair SGs. BM Soup, Spleen Soup, ADSC Soup, or saline (vehicle control) was injected intravenously into mice with IR-injured SGs (13Gy). Results demonstrated that all three cell soups restored 65-70% of saliva secretion, protected acinar cells, blood vessels, and parasympathetic nerves, and increased cell proliferation. Although protein array assays identified more angiogenesis-related growth factors in ADSC Soup, the length of its therapeutic efficiency on saliva flow was less than that of the BM Soup and Spleen Soup. Another objective of this study was to compare "Fresh" versus "Cryopreserved (-80 °C)" BM Soup. It was found that the therapeutic effect of 12-month "Cryopreserved BM Soup" was comparable to that of "Fresh BM Soup" on the functional restoration of IR-injured SGs. In conclusion, both Spleen Soup and ADSC Soup can be used to mitigate IR-damaged SGs.

Diagnostic value of diffusion-weighted magnetic resonance imaging for local and skull base recurrence of nasopharyngeal carcinoma after radiotherapy.

Tumor recurrence is a major cause of nasopharyngeal carcinoma (NPC) treatment failure. Diffusion-weighted imaging (DWI) is used for a variety of cancers, but few data are available for NPC.The aim of the study was to investigate the DWI features of recurrent NPC after radiotherapy and apparent diffusion coefficient (ADC) thresholds for the diagnosis of recurrent NPC.This was a retrospective study of 160 patients with NPC treated by radiotherapy at the Cancer Hospital affiliated to Guangxi Medical University from May 2012 to March 2015. The patients were divided into the local recurrence (n = 39), fibrosis (n = 51), clivus recurrence (n = 22), and clivus nonrecurrence (n = 48) groups. The patients underwent magnetic resonance imaging (MRI), enhanced MRI, and DWI. Receiver operating characteristics curves were used to determine sensitivity, specificity, and negative predictive values.ADC values were significantly different between the recurrence and fibrosis groups (P < .0001). Using ADC threshold values of 0.887 × 10 mm/s for local recurrence, the area under the curve (AUC) of DWI was 0.967 (87.2% sensitivity and 94.1% specificity), compared with 0.732 for routine MRI (71.8% sensitivity and 74.5% specificity) (P < .001). Using ADC threshold values of 1.018 × 10 mm/s for the diagnosis of clivus recurrent NPC, the AUC of DWI was 0.984 (95.5% sensitivity and 91.7% specificity) compared with 0.558 for routine MRI (63.6% sensitivity and 47.9% specificity) (P < .001).DWI has a higher diagnostic value for recurrent NPC than MRI. DWI can increase the diagnosis sensitivity and specificity of locally recurrent NPC.

Optimal timing and frequency of bone marrow soup therapy for functional restoration of salivary glands injured by single-dose or fractionated irradiation.

Injections of bone marrow (BM) cell extract, known as 'BM soup', were previously reported to mitigate ionizing radiation (IR) injury to salivary glands (SGs). However, the optimal starting time and frequency to maintain BM soup therapeutic efficacy remains unknown. This study tested the optimal starting time and frequency of BM soup injections in mice radiated with either a single dose or a fractionated dose. First, BM soup treatment was started at 1, 3 or 7 weeks post-IR; positive (non-IR) and negative (IR) control mice received injections of saline (vehicle control). Second, BM soup-treated mice received injections at different frequencies (1, 2, 3 and 5 weekly injections). Third, a 'fractionated-dose radiation' model to injure mouse SGs was developed (5 Gy × 5 days) and compared with the single high dose radiation model. All mice (n = 65) were followed for 16 weeks post-IR. The results showed that starting injections of BM soup between 1 and 3 weeks mitigated the effect of IR-induced injury to SGs and improved the restoration of salivary function. Although the therapeutic effect of BM soup lessens after 8 weeks, it can be sustained by increasing the frequency of weekly injections. Moreover, both single-dose and fractionated-dose radiation models are efficient and comparable in inducing SG injury and BM soup treatments are effective in restoring salivary function in both radiation models. In conclusion, starting injections of BM soup within 3 weeks post-radiation, with 5 weekly injections, maintains 90-100% of saliva flow in radiated mice.

Compact Bone-Derived Multipotent Mesenchymal Stromal Cells (MSCs) for the Treatment of Sjogren's-like Disease in NOD Mice.

Compact bone (cortical or dense bone) is among the organs that contain multipotent mesenchymal stromal cells (MSCs). Unlike bone marrow plugs where MSCs were initially isolated, compact bone has minimal (amount of) hematopoietic cells and thus facilitates the MSCs isolation process. In vitro, MSCs from compact bone show multipotency and differentiation into mesenchymal tissues such as bone, adipose, and cartilage, under certain conditions. MSCs therapy has been promising in preclinical and clinical studies against autoimmune diseases. Not only can MSCs replace the lost tissue through their regenerative properties, but they can also control the autoimmune attacks by immunoregulatory cytokines. This protocol describes the use of compact bone-derived MSCs to preserve salivary function (saliva flow/output) in the NOD (nonobese diabetic) mouse model affected with Sjogren's-like disease.

The role of human fibronectin- or placenta basement membrane extract-based gels in favouring the formation of polarized salivary acinar-like structures.

Head and neck cancer patients treated with radiotherapy commonly experience hyposalivation and oral/tooth infections, leading to a reduced quality of life. Clinical management is currently unsatisfactory for dry mouth. Thus, there is a need for growing salivary fluid-secreting (acinar) cells for these patients. However, functionally-grown salivary acinar cells are cultured in Matrigel, a product that cannot be used clinically, owing to its source from a mouse sarcoma. Therefore, finding a gel suitable for clinical use and possessing properties similar to that of Matrigel would allow biopsied salivary cells to be expanded in vitro and transplanted into the mouths of xerostomic patients. This study tested gels made with human placenta basement membrane extract (BME) or fibronectin for the growth and differentiation of human salivary biopsies into acinar cells. We report here that, following expansion of primary human salivary gland epithelial cells (huSGs) in serum-free medium, using these gels (made from human proteins) allowed morphological and functional differentiation of salivary ductal cells into acinar-like cells. These (human) gels gave comparable results to Matrigel, such as differentiation into polarized acinar 3D units or monolayers with tight junction proteins (claudin-1, -2, -3) and exhibiting adequate transepithelial electrical resistance, acinar proteins (AQP5, α-amylase, mucin-1, NKCC1) and acinar adhesion-related cell markers (CD44, CD166). Ultrastructural, mRNA and protein analyses confirmed the formation of differentiated acinar polarized cells. The mitotic activity was highest with human placenta BME gel. This human culture model provided a reproducible approach to studying human salivary cell expansion and differentiation for tissue engineering. Copyright © 2016 John Wiley & Sons, Ltd.

A Simplified and Systematic Method to Isolate, Culture, and Characterize Multiple Types of Human Dental Stem Cells from a Single Tooth.

This chapter describes a simplified method that allows the systematic isolation of multiple types of dental stem cells such as dental pulp stem cells (DPSC), periodontal ligament stem cells (PDLSC), and stem cells of the apical papilla (SCAP) from a single tooth. Of specific interest is the modified laboratory approach to harvest/retrieve the dental pulp tissue by minimizing trauma to DPSC by continuous irrigation, reduction of frictional heat from the bur rotation, and reduction of the bur contact time with the dentin. Also, the use of a chisel and a mallet will maximize the number of live DPSC for culture. Steps demonstrating the potential for multiple cell differentiation lineages of each type of dental stem cell into either osteocytes, adipocytes, or chondrocytes are described. Flow cytometry, with a detailed strategy for cell gating and analysis, is described to verify characteristic markers of human mesenchymal multipotent stromal cells (MSC) from DPSC, PDLSC, or SCAP for subsequent experiments in cell therapy and in tissue engineering. Overall, this method can be adapted to any laboratory with a general setup for cell culture experiments.