A critical influence of HIF-1 on MMP-9 and Galectin-3 in oral lichen planus.

OBJECTIVE: Oral lichen planus carries a risk for malignancy. The pathogenesis of the disease is mediated by various inflammatory mediators. Several mediators could be responsible for the oncogenic behavior in certain cases. Hypoxia-inducible factor-1a (HIF-1), and its possible correlation to Galactin-3 (Gal-3) and matrix metalloproteinase-9 (MMP-9) over expression represents an important indicator for malignant transformation. The investigation of these factors may present evidence-based information on malignant transformation of the disease. SUBJECTS AND METHODS: The study investigated the expression of HIF-1, Gla-3 and MMP-9 in tissue samples of OLP compared to control subjects of un-inflamed gingival overgrowth. 20 biospecimen were allocated in each group. RESULTS: Immunohistochemical findings of OLP showed immunoreactivity for Galectin 3, HIF1a and MMP-9 by most of the epithelial cells. There was a positive correlation between HIF1α and MMP-9, r = 0.9301 (P-value < 0.00001). A positive correlation was detected between Galectin 3 and MMP-9, r = 0.7292 (P-value = 0.000264) between Galectin 3 and HIF1α, r = 0.5893 (P-value = 0.006252). CONCLUSION: These findings confirm the hypothesis that the adaptive pathways to hypoxia as Gal 3 and MMP-9 expressions and their HIF-1 may play a crucial role in carcinogenesis of OLP.

Photobiostimulation conjugated with stem cells or their secretome for temporomandibular joint arthritis in a rat model.

BACKGROUND: Temporomandibular joint (TMJ) arthritis is a debilitating, challenging condition and different methods have been implicated for its treatment. This study aimed to test the therapeutic potentials of low-level laser therapy (LLLT) associated with adipose derived stem cells (ADSC) or their derived secretome on a murine model induced arthritis. METHODS: Forty eight rats were divided into four groups where group I was the sham control, the rest of animals were subjected to arthritis induction using complete Freund's adjuvant, then divided as follows: group II received phosphate buffered saline (PBS) intraarticular injection and irradiation of 0 j/cm2, group III received ADSCs derived secretome and irradiation of 38 j/cm2, and group IV received ADSCs and irradiation of 38 j/cm2 as well. One and three weeks after treatment, animals were euthanized, and paraffin blocks were processed for histological assessment by hematoxylin and eosin stain with histomorphometrical analysis. Histochemical evaluation of joint proteoglycan content was performed through toluidine blue stain, and immunohistochemical staining by the proinflammatory marker tumor necrosis factor-α (TNF-α) was performed followed by the relevant statistical tests. RESULTS: The arthritis group showed histological signs of joint injury including cartilage atrophy, articular disc fibrosis, irregular osteochondral interface, and condylar bone resorption together with high inflammatory reaction and defective proteoglycan content. In contrast, the treated groups III and IV showed much restoration of the joint structure with normal cartilage and disc thickness. The inflammation process was significantly suppressed especially after three weeks as confirmed by the significant reduction in TNF-α positive immunostaining compared to the arthritic group, and the cartilage proteoglycan content also showed significant increase relative to the arthritic group. However, no significant difference between the results of the two treated groups was detected. CONCLUSION: LLLT conjugated with ADSCs or ADSCs derived secretome can efficiently enhance the healing of arthritic TMJs. Stem cell secretome can be applied as a safe, potent therapy. However, further investigations are required to unravel its mechanism of action and pave its way as a safe, novel, cell free therapy.

Biomimetic Strategy to Enhance Epithelial Cell Viability and Spreading on PEEK Implants.

Polyetheretherketone (PEEK) is a biocompatible material widely used in spinal and craniofacial implants, with potential use in percutaneous implants. However, its inertness prevents it from forming a tight seal with the surrounding soft tissue, which can lead to infections and implant failure. Conversely, the surface chemistry of percutaneous organs (i.e., teeth) helps establish a strong interaction with the epithelial cells of the contacting soft tissues, and hence a tight seal, preventing infection. The seal is created by adsorption of basement membrane (BM) proteins, secreted by epithelial cells, onto the percutaneous organ surfaces. Here, we aim to create a tight seal between PEEK and epithelial tissues by mimicking the surface chemistry of teeth. Our hypothesis is that collagen I, the most abundant tooth protein, enables integration between the epithelial tissue and teeth by promoting adsorption of BM proteins. To test this, we immobilized collagen I via EDC/NHS coupling on a carboxylated PEEK surface modified using diazonium chemistry. We used titanium alloy (Ti-6Al-4V) for comparison, as titanium is the most widely used percutaneous biomaterial. Both collagen-modified PEEK and titanium showed a larger adsorption of key BM proteins (laminin, nidogen, and fibronectin) compared to controls. Keratinocyte epithelial cell viability on collagen-modified PEEK was twice that of control PEEK and ∼1.5 times that of control titanium after 3 days of cell seeding. Both keratinocytes and fibroblasts spread more on collagen-modified PEEK and titanium compared to controls. This work introduces a versatile and biomimetic surface modification technique that may enhance PEEK-epithelial tissue sealing with the potential of extending PEEK applications to percutaneous implants, making it competitive with titanium.

Genetic Interpretation of the Impacts of Honokiol and EGCG on Apoptotic and Self-Renewal Pathways in HEp-2 Human Laryngeal CD44high Cancer Stem Cells.

Most current larynx cancer therapies are generally aimed at the global mass of tumor, targeting the non-tumorigenic cells, and unfortunately sparing the tumorigenic cancer stem cells (CSCs) that are responsible for sustained growth, metastasis, and chemo- and radioresistance. Phytochemicals and herbs have recently been introduced as therapeutic sources for eliminating CSCs. Therefore, we assessed the anti-tumor effects of two herbal ingredients, the green tea extract "Epigallocatechin-3-gallate (EGCG)" and Honokiol (HNK), on parental cells or CD44high CSCs of the human laryngeal squamous cell carcinoma cell line HEp-2. Results revealed that EGCG had a preeminent apoptotic potential on HEp-2 laryngeal CSCs. HNK conferred higher cytotoxic impacts on parental cells mostly by necrosis induction, especially with higher doses, but apoptosis induction with lower doses was also observed. The Notch signaling pathway genes were more potently suppressed by EGCG than HNK. However, HNK surpassed EGCG in downregulating the ß-catenin and the Sonic Hedgehog signaling pathways genes. On a genetic basis, both agents engaged the BCL-2 family-regulated and caspase-dependent intrinsic apoptotic pathway, but EGCG and HNK triggered apoptosis via p53-independent and p53-dependent pathways, respectively. Taken together, EGCG and HNK eradicated HEp-2 human larynx cancer cells through targeting multiple self-renewal pathways and activating diverse cell death modalities.

Decellularized Extracellular Matrix Composite Hydrogel Bioinks for the Development of 3D Bioprinted Head and Neck in Vitro Tumor Models.

Reinforced extracellular matrix (ECM)-based hydrogels recapitulate several mechanical and biochemical features found in the tumor microenvironment (TME) in vivo. While these gels retain several critical structural and bioactive molecules that promote cell-matrix interactivity, their mechanical properties tend toward the viscous regime limiting their ability to retain ordered structural characteristics when considered as architectured scaffolds. To overcome this limitation characteristic of pure ECM hydrogels, we present a composite material containing alginate, a seaweed-derived polysaccharide, and gelatin, denatured collagen, as rheological modifiers which impart mechanical integrity to the biologically active decellularized ECM (dECM). After an optimization process, the reinforced gel proposed is mechanically stable and bioprintable and has a stiffness within the expected physiological values. Our hydrogel's elastic modulus has no significant difference when compared to tumors induced in preclinical xenograft head and neck squamous cell carcinoma (HNSCC) mouse models. The bioprinted cell-laden model is highly reproducible and allows proliferation and reorganization of HNSCC cells while maintaining cell viability above 90% for periods of nearly 3 weeks. Cells encapsulated in our bioink produce spheroids of at least 3000 µm2 of cross-sectional area by day 15 of culture and are positive for cytokeratin in immunofluorescence quantification, a common marker of HNSCC model validation in 2D and 3D models. We use this in vitro model system to evaluate the standard-of-care small molecule therapeutics used to treat HNSCC clinically and report a 4-fold increase in the IC50 of cisplatin and an 80-fold increase for 5-fluorouracil compared to monolayer cultures. Our work suggests that fabricating in vitro models using reinforced dECM provides a physiologically relevant system to evaluate malignant neoplastic phenomena in vitro due to the physical and biological features replicated from the source tissue microenvironment.

Differences in platelet-rich plasma composition influence bone healing.

AIM: Platelet-rich plasma (PRP) is an autologous blood-derived material that has been used to enhance bone regeneration. Clinical studies, however, reported inconsistent outcomes. This study aimed to assess the effect of changes in leucocyte and PRP (L-PRP) composition on bone defect healing. MATERIALS AND METHODS: L-PRPs were prepared using different centrifugation methods and their regenerative potential was assessed in an in-vivo rat model. Bilateral critical-size tibial bone defects were created and filled with single-spin L-PRP, double-spin L-PRP, or filtered L-PRP. Empty defects and defects treated with collagen scaffolds served as controls. Rats were euthanized after 2 weeks, and their tibias were collected and analysed using micro-CT and histology. RESULTS: Double-spin L-PRP contained higher concentrations of platelets than single-spin L-PRP and filtered L-PRP. Filtration of single-spin L-PRP resulted in lower concentrations of minerals and metabolites. In vivo, double-spin L-PRP improved bone healing by significantly reducing the size of bone defects (1.08 ± 0.2 mm3 ) compared to single-spin L-PRP (1.42 ± 0.27 mm3 ) or filtered L-PRP (1.38 ± 0.28 mm3 ). There were fewer mast cells, lymphocytes, and macrophages in defects treated with double-spin L-PRP than in those treated with single-spin or filtered L-PRP. CONCLUSION: The preparation method of L-PRP affects their composition and potential to regenerate bone.

Sulforaphane as a Promising Natural Molecule for Cancer Prevention and Treatment.

Tumorigenicity-inhibiting compounds have been identified in our daily diet. For example, isothiocyanates (ITCs) found in cruciferous vegetables were reported to have potent cancer-prevention activities. The best characterized ITC is sulforaphane (SF). SF can simultaneously modulate multiple cellular targets involved in carcinogenesis, including (1) modulating carcinogen-metabolizing enzymes and blocking the action of mutagens; (2) inhibition of cell proliferation and induction of apoptosis; and (3) inhibition of neo-angiogenesis and metastasis. SF targets cancer stem cells through modulation of nuclear factor kappa B (NF-κB), Sonic hedgehog (SHH), epithelial-mesenchymal transition, and Wnt/ß-catenin pathways. Conventional chemotherapy/SF combination was tested in several studies and resulted in favorable outcomes. With its favorable toxicological profile, SF is a promising agent in cancer prevention and/or therapy. In this article, we discuss the human metabolism of SF and its effects on cancer prevention, treatment, and targeting cancer stem cells, as well as providing a brief review of recent human clinical trials on SF.

Broccoli extract increases drug-mediated cytotoxicity towards cancer stem cells of head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinomas (HNSCC) are malignant neoplasms with poor prognosis. Treatment-resistant cancer stem cell (CSC) is one reason for treatment failure. Considerable attention has been focused on sulforaphane (SF), a phytochemical from broccoli possessing anticancer properties. We investigated whether SF could enhance the chemotherapeutic effects of cisplatin (CIS) and 5-fluorouracil (5-FU) against HNSCC-CSCs, and its mechanisms of action. METHODS: CD44+/CD271+ FACS-isolated CSCs from SCC12 and SCC38 human cell lines were treated with SF alone or combined with CIS or 5-FU. Cell viability, colony- and sphere-forming ability, apoptosis, CSC-related gene and protein expression and in vivo tumour growth were assessed. Safety of SF was tested on non-cancerous stem cells and in vivo. RESULTS: SF reduced HNSCC-CSC viability in a time- and dose-dependent manner. Combining SF increased the cytotoxicity of CIS twofold and 5-FU tenfold, with no effects on non-cancerous stem cell viability and functions. SF-combined treatments inhibited CSC colony and sphere formation, and tumour progression in vivo. Potential mechanisms of action included the stimulation of caspase-dependent apoptotic pathway, inhibition of SHH pathway and decreased expression of SOX2 and OCT4. CONCLUSIONS: Combining SF allowed lower doses of CIS or 5-FU while enhancing these drug cytotoxicities against HNSCC-CSCs, with minimal effects on healthy cells.

Insight into Salivary Gland Aquaporins.

The main role of salivary glands (SG) is the production and secretion of saliva, in which aquaporins (AQPs) play a key role by ensuring water flow. The AQPs are transmembrane channel proteins permeable to water to allow water transport across cell membranes according to osmotic gradient. This review gives an insight into SG AQPs. Indeed, it gives a summary of the expression and localization of AQPs in adult human, rat and mouse SG, as well as of their physiological role in SG function. Furthermore, the review provides a comprehensive view of the involvement of AQPs in pathological conditions affecting SG, including Sjögren's syndrome, diabetes, agedness, head and neck cancer radiotherapy and SG cancer. These conditions are characterized by salivary hypofunction resulting in xerostomia. A specific focus is given on current and future therapeutic strategies aiming at AQPs to treat xerostomia. A deeper understanding of the AQPs involvement in molecular mechanisms of saliva secretion and diseases offered new avenues for therapeutic approaches, including drugs, gene therapy and tissue engineering. As such, AQP5 represents a potential therapeutic target in different strategies for the treatment of xerostomia.

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.

Head and neck cancer management and cancer stem cells implication.

Head and neck squamous cell carcinomas (HNSCCs) arise in the mucosal linings of the upper aerodigestive tract and are heterogeneous in nature. Risk factors for HNSCCs are smoking, excessive alcohol consumption, and the human papilloma virus. Conventional treatments are surgery, radiotherapy, chemotherapy, or a combined modality; however, no international standard mode of therapy exists. In contrast to the conventional model of clonal evolution in tumor development, there is a newly proposed theory based on the activity of cancer stem cells (CSCs) as the model for carcinogenesis. This "CSC hypothesis" may explain the high mortality rate, low response to treatments, and tendency to develop multiple tumors for HNSCC patients. We review current knowledge on HNSCC etiology and treatment, with a focus on CSCs, including their origins, identifications, and effects on therapeutic options.

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.

Broccoli extract improves chemotherapeutic drug efficacy against head-neck squamous cell carcinomas.

The efficacy of cisplatin (CIS) and 5-fluorouracil (5-FU) against squamous cell carcinomas of the head and neck (SCCHN) remains restricted due to their severe toxic side effects on non-cancer (normal) tissues. Recently, the broccoli extract sulforaphane (SF) was successfully tested as a combination therapy to target cancer cells. However, the effect of lower doses of CIS or 5-FU combined with SF on SCCHN remained unknown. This study tested the chemotherapeutic efficacies of SF combined with much lower doses of CIS or 5-FU against SCCHN cells aiming to reduce cytotoxicity to normal cells. Titrations of SF standalone or in combination with CIS and 5-FU were tested on SCCHN human cell lines (SCC12 and SCC38) and non-cancerous human cells (fibroblasts, gingival, and salivary cells). Concentrations of SF tested were comparable to those found in the plasma following ingestion of fresh broccoli sprouts. The treatment effects on cell viability, proliferation, DNA damage, apoptosis, and gene expression were measured. SF reduced SCCHN cell viability in a time- and dose-dependent manner. SF-combined treatment increased the cytotoxic activity of CIS by twofolds and of 5-FU by tenfolds against SCCHN, with no effect on non-cancerous cells. SF-combined treatment inhibited SCCHN cell clonogenicity and post-treatment DNA repair. SF increased SCCHN apoptosis and this mechanism was due to a down-regulation of BCL2 and up-regulation of BAX, leading to an up-regulation of Caspase3. In conclusion, combining SF with low doses of CIS or 5-FU increased cytotoxicity against SCCHN cells, while having minimal effects on normal cells.

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.