Yoon Young Go

Promotion of osteogenic differentiation by amnion/chorion membrane extracts

Background The amniotic membrane is a favorable biomaterial to apply in the field of tissue engineering because of its unique biological properties. Human amniotic membranes consist of 2-layered sheets containing numerous growth factors, cytokines and other bioactive substances. Methods In this study, we explored the potential of amnion membrane extracts (AME) and amnion/chorion membrane extracts (A/CME) to promote osteogenic differentiation of osteoblast-like (MG-63) cells. MG-63 cells were cultured in osteogenic induction medium (OIM) with or without 100 µg/mL of AME or A/CME. To determine the early and late differentiation of osteogenesis, alkaline phosphatase (ALP) activity and calcium deposition were measured at 3, 7, 10 and 24 days. Expression of specific genes associated with osteogenic differentiation, including osteocalcin (OCN), osteopontin (OPN), runt domain-containing transcription factor (Runx2) and osterix (OSX) was also determined. Results In vitro experiments demonstrated that A/CME increased ALP activity, osteogenic gene expression and mineralization under osteogenic-inducing conditions. Notably, we found that A/CME contained growth factors related to osteogenesis, including fibroblast growth factors and transforming growth factors, which potentially promoted osteogenic differentiation of MG-63 cells to a greater extent than AME. Conclusions These results indicate that A/CME is capable of providing growth factors and other substrates for osteogenic differentiation, which significantly increased the efficacy of osteogenesis in MG-63 cells. Taken together, the results of this study suggest that human A/CME is a promising biomaterial with therapeutic potential in bone regeneration applications.

Differential effects of amnion and chorion membrane extracts on osteoblast-like cells due to the different growth factor composition of the extracts

Human amniotic membrane extracts contain numerous growth factors and bioactive substances. However, osteogenic effects of amnion and chorion membrane extracts (AME and CME, respectively) on osteoblasts are unclear. In this study, we explored the ability of AME and CME to promote the osteogenic differentiation of osteoblast-like MG-63 cells. MG-63 cells were cultured in osteogenic induction medium (OIM) with or without exogenous AME and CME. CME enhanced the osteogenic differentiation of MG-63 cells compared with AME, as indicated by increased mineralization; alkaline phosphatase activity; and mRNA expression of osteogenic marker genes encoding integrin-binding sialoprotein (IBSP), RUNX2, OSTERIX, and osteocalcin (OCN). Interestingly, AME and CME contained different combinations of osteogenesis-related growth factors, including basic fibroblast growth factor (bFGF), transforming growth factor beta-1 (TGFβ-1), and epidermal growth factor (EGF), which differentially regulated the osteogenic differentiation of MG-63 cells. bFGF and TGFβ-1 present in CME positively regulated the osteogenic differentiation of MG-63 cells, whereas EGF present in AME negatively regulated the differentiation of MG-63 cells. Moreover, exogenous treatment of EGF antagonized CME-induced mineralization of extracellular matrix on MG-63 cells. We compared the osteogenic efficacy of CME with that of BMP2, bFGF, and TGFβ-1 alone or their combinations. We observed that CME greatly enhanced osteogenesis by providing a conductive environment for the differentiation of MG-63 cells. Together, our results indicated that human AME and CME exerted differential effects on osteogenesis because of the presence of different compositions of growth factors. In addition, our results highlighted a new possible strategy of using CME as a biocompatible therapeutic material for bone regeneration.

In vitro Multi-Species Biofilms of Methicillin-Resistant Staphylococcus aureus and Pseudomonas aeruginosa and Their Host Interaction during In vivo Colonization of an Otitis Media Rat Model

Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) are known to cause biofilm-related infections. MRSA and PA have been frequently isolated from chronically infected wounds, cystic fibrosis, chronic suppurative otitis media (CSOM), and from indwelling medical devices, and these bacteria co-exist; however, their interaction with each-other or with the host is not well known. In this study, we investigated MRSA and PA multi-species biofilm communities in vitro and their interaction with the host during in vivo colonization using an OM rat-model. In-vitro biofilm formation and in-vivo colonization were studied using CV-microtiter plate assay and OM rat-model respectively. The biofilms were viewed under scanning electron microscope and bacteria were enumerated using cfu counts. The differential gene expressions of rat mucosa colonized with single or multi-species of MRSA or PA were studied using RNA-sequencing of total transcriptome. In multi-species in-vitro biofilms PA partially inhibited SA growth. However, no significant inhibition of MRSA was detected during in-vivo colonization of multi-species in rat bullae. A total of 1,797 genes were significantly (p < 0.05) differentially expressed in MRSA or PA or MRSA + PA colonized rat middle ear mucosa with respect to the control. The poly-microbial colonization of MRSA and PA induced the differential expression of a significant number of genes that are involved in immune response, inflammation, signaling, development, and defense; these were not expressed with single species colonization by either MRSA or PA. Genes involved in defense, immune response, inflammatory response, and developmental process were exclusively up-regulated, and genes that are involved in nervous system signaling, development and transmission, regulation of cell growth and development, anatomical and system development, and cell differentiation were down-regulated after multi-species inoculation. These results indicate that poly-microbial colonization induces a host response that is different from that induced by single species infection.

The Small Molecule DAM Inhibitor, Pyrimidinedione, Disrupts Streptococcus pneumoniae Biofilm Growth In Vitro.

Streptococcus pneumoniae persist in the human nasopharynx within organized biofilms. However, expansion to other tissues may cause severe infections such as pneumonia, otitis media, bacteremia, and meningitis, especially in children and the elderly. Bacteria within biofilms possess increased tolerance to antibiotics and are able to resist host defense systems. Bacteria within biofilms exhibit different physiology, metabolism, and gene expression profiles than planktonic cells. These differences underscore the need to identify alternative therapeutic targets and novel antimicrobial compounds that are effective against pneumococcal biofilms. In bacteria, DNA adenine methyltransferase (Dam) alters pathogenic gene expression and catalyzes the methylation of adenine in the DNA duplex and of macromolecules during the activated methyl cycle (AMC). In pneumococci, AMC is involved in the biosynthesis of quorum sensing molecules that regulate competence and biofilm formation. In this study, we examine the effect of a small molecule Dam inhibitor, pyrimidinedione, on Streptococcus pneumoniae biofilm formation and evaluate the changes in global gene expression within biofilms via microarray analysis. The effects of pyrimidinedione on in vitro biofilms were studied using a static microtiter plate assay, and the architecture of the biofilms was viewed using confocal and scanning electron microscopy. The cytotoxicity of pyrimidinedione was tested on a human middle ear epithelium cell line by CCK-8. In situ oligonucleotide microarray was used to compare the global gene expression of Streptococcus pneumoniae D39 within biofilms grown in the presence and absence of pyrimidinedione. Real-time RT-PCR was used to study gene expression. Pyrimidinedione inhibits pneumococcal biofilm growth in vitro in a concentration-dependent manner, but it does not inhibit planktonic cell growth. Confocal microscopy analysis revealed the absence of organized biofilms, where cell-clumps were scattered and attached to the bottom of the plate when cells were grown in the presence of pyrimidinedione. Scanning electron microscopy analysis demonstrated the absence of an extracellular polysaccharide matrix in pyrimidinedione-grown biofilms compared to control-biofilms. Pyrimidinedione also significantly inhibited MRSA, MSSA, and Staphylococcus epidermidis biofilm growth in vitro. Furthermore, pyrimidinedione does not exhibit eukaryotic cell toxicity. In a microarray analysis, 56 genes were significantly up-regulated and 204 genes were significantly down-regulated. Genes involved in galactose metabolism were exclusively up-regulated in pyrimidinedione-grown biofilms. Genes related to DNA replication, cell division and the cell cycle, pathogenesis, phosphate-specific transport, signal transduction, fatty acid biosynthesis, protein folding, homeostasis, competence, and biofilm formation were down regulated in pyrimidinedione-grown biofilms. This study demonstrated that the small molecule Dam inhibitor, pyrimidinedione, inhibits pneumococcal biofilm growth in vitro at concentrations that do not inhibit planktonic cell growth and down regulates important metabolic-, virulence-, competence-, and biofilm-related genes. The identification of a small molecule (pyrimidinedione) with S. pneumoniae biofilm-inhibiting capabilities has potential for the development of new compounds that prevent biofilm formation.

Antimicrobial and Antibiofilm Effects of Human Amniotic/Chorionic Membrane Extract on Streptococcus pneumoniae.

Background: Streptococcus pneumoniae colonize the human nasopharynx in the form of biofilms. The biofilms act as bacterial reservoirs and planktonic bacteria from these biofilms can migrate to other sterile anatomical sites to cause pneumonia, otitis media (OM), bacteremia and meningitis. Human amniotic membrane contains numerous growth factors and antimicrobial activity; however, these have not been studied in detail. In this study, we prepared amniotic membrane extract and chorionic membrane extract (AME/CME) and evaluated their antibacterial and antibiofilm activities against S. pneumoniae using an in vitro biofilm model and in vivo OM rat model. Materials and Methods: The AME/CME were prepared and protein was quantified using DCTM (detergent compatible) method. The minimum inhibitory concentrations were determined using broth dilution method, and the synergistic effect of AME/CME with Penicillin-streptomycin was detected checkerboard. The in vitro biofilm and in vivo colonization of S. pneumoniae were studied using microtiter plate assay and OM rat model, respectively. The AME/CME-treated biofilms were examined using scanning electron microscope and confocal microscopy. To examine the constituents of AME/CME, we determined the proteins and peptides of AME/CME using tandem mass tag-based quantitative mass spectrometry. Results: AME/CME treatment significantly (p < 0.05) inhibited S. pneumoniae growth in planktonic form and in biofilms. Combined application of AME/CME and Penicillin-streptomycin solution had a synergistic effect against S. pneumoniae. Biofilms grown with AME/CME were thin, scattered, and unorganized. AME/CME effectively eradicated pre-established pneumococci biofilms and has a bactericidal effect. AME treatment significantly (p < 0.05) reduced bacterial colonization in the rat middle ear. The proteomics analysis revealed that the AME/CME contains hydrolase, ribonuclease, protease, and other antimicrobial proteins and peptides. Conclusion: AME/CME inhibits S. pneumoniae growth in the planktonic and biofilm states via its antimicrobial proteins and peptides. AME/CME are non-cytotoxic, natural human product; therefore, they may be used alone or with antibiotics to treat S. pneumoniae infections.

Anti-apoptotic effect of dexamethasone in an ototoxicity model

BackgroundDexamethasone (DEX) is used for the treatment of various inner ear diseases. However, the molecular mechanism of DEX on gentamicin induced hair cell damage is not known. Therefore, this study investigated the protective effect of DEX on gentamicin (GM)-induced ototoxicity and the effect of GM on the expression of apoptosis related genes.MethodsThe protective effects of DEX were measured by phalloidin staining of explant cultures of organ of Corti from postnatal day 2–3 mice with GM-induced hair cell loss. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining was used to detect apoptosis and immunofluorescence was done to analyze the effect of DEX on the expression of apoptosis related genes.ResultsCochlear explant cultures of postnatal day-4-old mice were exposed to 0, 1, 5, 10, 30, 50, and 100 μg/ml DEX and GM during culture. DEX protected from GM-induced hair cell loss in the inner ear of postnatal day 4 mice. To understand the molecular mechanisms by which DEX pre-treatment decreased hair cell loss, the testes of cochlear explant cultures of postnatal day 4 mice were examined for changes in expression of cochlear apoptosis mediators. The pro-apoptotic protein Bax was significantly down-regulated and numbers of apoptotic hair cells were decreased.ConclusionsDEX has a protective effect on GM-induced hair cell loss in neonatal cochlea cultures and the protective mechanism may involve inhibition of the mitochondrial apoptosis pathway. The combination with scaffold technique can improve delivery of DEX into the inner ear to protect GM-induced ototoxicity.

Effect of Lead on Human Middle Ear Epithelial Cells

Lead is a ubiquitous metal in the environment, but no studies have examined lead toxicity on the middle ear. Here, we investigated lead toxicity and its mechanism in human middle ear epithelial cells (HMEECs). Moreover, we investigated the protective effects of amniotic membrane extract (AME) and chorionic membrane extract (CME) against lead toxicity in HMEECs. Cell viability was analyzed using the cell counting kit, and reactive oxygen species (ROS) activity was measured using a cellular ROS detection kit. After lead(II) acetate trihydrate treatment, mRNA levels of various genes were assessed by semiquantitative real-time polymerase chain reaction. Following treatment with AME or CME after lead exposure, the changes in cell viability, ROS activity, and gene expression were analyzed. Exposure to >100 μg/mL of lead(II) acetate trihydrate caused a significant decrease in cell viability and increased ROS production in HMEECs. Lead exposure significantly increased the mRNA expression of genes encoding inflammatory cytokines and mucins. Administration of AME or CME restored cell viability, reduced ROS activity, and ameliorated mRNA levels. Our findings suggest that environmental lead exposure is related to the development of otitis media, and AME and CME may have antioxidative and anti-inflammatory effects against lead toxicity.

Microarray Analysis of Gene Expression Alteration in Human Middle Ear Epithelial Cells Induced by Asian Sand Dust

Objectives The primary aim of this study is to evaluate the gene expression profile of Asian sand dust (ASD)-treated human middle ear epithelial cell (HMEEC) using microarray analysis. Methods The HMEEC was treated with ASD (400 µg/mL) and total RNA was extracted for microarray analysis. Molecular pathways among differentially expressed genes were further analyzed. For selected genes, the changes in gene expression were confirmed by real-time polymerase chain reaction. Results A total of 1,274 genes were differentially expressed by ASD. Among them, 1,138 genes were 2 folds up-regulated, whereas 136 genes were 2 folds down-regulated. Up-regulated genes were mainly involved in cellular processes, including apoptosis, cell differentiation, and cell proliferation. Down-regulated genes affected cellular processes, including apoptosis, cell cycle, cell differentiation, and cell proliferation. The 10 genes including ADM, CCL5, EDN1, EGR1, FOS, GHRL, JUN, SOCS3, TNF, and TNFSF10 were identified as main modulators in up-regulated genes. A total of 11 genes including CSF3, DKK1, FOSL1, FST, TERT, MMP13, PTHLH, SPRY2, TGFBR2, THBS1, and TIMP1 acted as main components of pathway associated with 2-fold down regulated genes. Conclusion We identified the differentially expressed genes in ASD-treated HMEEC. Our work indicates that air pollutant like ASD, may play an important role in the pathogenesis of otitis media.

Engineering functional BMP-2 expressing teratoma-derived fibroblasts for enhancing osteogenesis

Bone morphogenetic protein 2 (BMP-2) is considered an effective growth factor for bone formation, and is used for making osteo-inductive scaffolds, but the related clinical investigations have shown low success rates. In this study, we genetically manipulated teratoma-derived fibroblast (TDF) cells by simultaneous introduction of BMP-2 and herpes simplex virus-thymidine kinase (HSV-tk) encoding genes. Self-production of BMP-2 in TDF cells strongly enhanced the alkaline phosphatase (ALP) activity, calcium content, and elevated the mRNA expression of osteogenic marker genes during in vitro osteogenesis. The bone formation volume was also remarkably enhanced in calvarial and femoral critical-size defect models. Ganciclovir (GCV) treatment induced apoptosis in TDF cells co-expressing HSV-tk and BMP-2, implying that HSV-tk suicide gene can modulate the side-effects of stem cell therapy, e.g., development of uncontrollable teratoma and tumor formation. Altogether, our findings revealed a safe and highly efficient technique with potential therapeutic applications for bone regeneration.

Anti-cancer effects of disulfiram in head and neck squamous cell carcinoma via autophagic cell death

Background Disulfiram (DSF), which is used to treat alcohol dependence, has been reported to have anti-cancer effects in various malignant tumors. In this study, we investigated the anti-cancer effects and mechanism of DSF in HNSCC. Methods Head and neck squamous carcinoma cell lines (FaDu and Hep2) were used to analyze the anti-cancer effects of DSF. The anti-cancer effects of DSF were confirmed in vivo using a xenograft tumor model. Results The anti-cancer effects of DSF in HNSCC were found to be copper (Cu) dependent. Specifically, DSF/Cu markedly inhibited HNSCC at a concentration of 1 μM. After DSF/Cu administration, production of reactive oxygen species (ROS) was remarkable starting at 0.5 μM, suggesting that the inhibitory effects of DSF/Cu on HNSCC are mediated through the formation of ROS. The levels of phospho-JNK, phospho-cJun and phospho-p38 were increased after DSF/Cu treatment while levels of phospho-Akt were decreased. These results suggested that the inhibitory effects of DSF/Cu on HNSCC cells involve ROS formation and down-regulation of Akt-signaling. Through these molecular mechanisms, DSF ultimately induce the inhibitory effects on HNSCC cell lines mainly through autophagic cell death, not apoptotic cell death. Lastly, we investigated the clinical relevance of DSF/Cu using a HNSCC xenograft animal model, which showed that tumor growth was remarkably decreased by DSF (50 mg/kg injection). Conclusion In treating patients with HNSCC, DSF may contribute to improved HNSCC patient’s survival. The characteristic anti-cancer effects of DSF on HNSCC may suggest new therapeutic potential for this medication in HNSCC patients.