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Öğe Are radio-contrast agents commonly used in discography toxic to the intact intervertebral disc tissue cells?(Wiley, 2019) Karaarslan, Numan; Yılmaz, İbrahim; Özbek, Hanefi; Yaşar Şirin, Duygu; Kaplan, Necati; Çalışkan, Tezcan; Özdemir, Çiğdem; Akyuva, Yener; Ateş, ÖzkanIn the literature, there have been no studies showing clear results on how radio-contrast pharmaceuticals would affect intact disc tissue cells. In this context, it was aimed to evaluate the effects of iopromide and gadoxetic acid, frequently used in the discography, on intact lumbar disc tissue in pharmaco-molecular and histopathological level. Primary cell cultures were prepared from the healthy disc tissue of the patients operated in the neurosurgery clinic. Except for the control group, the cultures were incubated with the indicated radio-contrast agents. Cell viability, toxicity and proliferation indices were tested at specific time intervals. The cell viability was quantitatively analysed. It was also visually rechecked under a fluorescence microscope with acridine orange/propidium iodide staining. Simultaneously, cell surface morphology was analysed with an inverted light microscope, while haematoxylin and eosin (H&E) staining methodology was used in the histopathological evaluations. The obtained data were evaluated statistically. Unlike the literature, iopromide or gadoxetic acid did not have any adverse effects on the cell viability, proliferation and toxicity (P <0.05). Although this study reveals that radio-contrast pharmaceuticals used in the discography, often used in neurosurgical practice, can be safely used, it should be remembered that this study was performed in an in vitro environment.Öğe Are specific gene expressions of extracellular matrix and nucleus pulposus affected by primary cell cultures prepared from intact or degenerative intervertebral disc tissues?(Turkish Neurosurgical Society, 2019) Karaarslan, Numan; Yılmaz, İbrahim; Özbek, Hanefi; Yaşar Şirin, Duygu; Kaplan, Necati; Akyuva, Yener; Gönültaş, Aylin; Ateş, ÖzkanAIM: To determine the gene expression patterns of nucleus pulposus (NP) in cell cultures obtained from degenerated or intact tissues. MATERIAL and METHODS: Whereas 12 of the cases were diagnosed with lumbar disc herniation and had undergone lumbar microdiscectomy, 12 cases had undergone traumatic intervertebral discectomy and corpectomy, along with discectomy after spinal trauma. NP-specific markers and gene expressions of the reagents of the extracellular matrix in the experimental setup were tested at the 0th, 24th, and 48th hours by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Visual evaluations were simultaneously made in all samples using invert and fluorescence microscopy. Vitality and proliferation analyses were evaluated by UV spectrophotometer. As a method of statistical evaluation, Spearman was used for categorical variants, and the Pearson correlation was used for variants with numerical and plain distribution. RESULTS: No association was found either between the tissue type and times (r=0.000; p=1.000) or between the region that the tissue was obtained from and hypoxia transcription factor-1 alpha (HIF-1 alpha) gene expression (r=0.098; p=0.245). There was no correlation between cell proliferation and chondroadherin (CHAD) expression or between type II collagen (COL2A1) and CHAD gene expressions. It was found that CHAD and HIF-1 alpha gene expressions and HIF-1 alpha and COL2A1 gene expressions affected cell proliferation. CONCLUSION: Cell culture setups are of paramount importance because they may influence the pattern of changes in the gene expressions of the cells used in these setups.Öğe Can a biodegradable implanted bilayered drug delivery system loaded with BMP-2/BMP-12 take an effective role in the biological repair process of bone-tendon injuries? A preliminary report(Hindawi Ltd, 2017) Kömür, Baran; Akyuva, Yener; Karaslan, Numan; İşyar, Mehmet; Gümüştaş, Seyit Ali; Yılmaz, İbrahim; Akkaya, Semih; Şirin, Duygu Yaşar; Mutlu, Çağri Ata; Batmaz, Ahmet Güray; Güler, Olcay; Mahiroğulları, MahirBackground. Use of biodegradable and biocompatible materials in the orthopedic surgery is gaining popularity. In this research, the rate of controlled release of a bilayered prototype biomaterial designed to promote osteoblastic and tenoblastic activity was calculated using pharmacochemical methods. Methods. The first part of the design, composed of a sodium tetraborate, polyvinyl alcohol, and starch based hydrogel, was loaded with bone morphogenic protein-2. The second part which was composed of a sodium tetraborate, polyvinyl alcohol, and chitosan based hydrogel was loaded with bone morphogenic protein-12. Osteochondral and tendon tissue specimens were obtained from patients with a diagnosis of gonarthrosis and primary bone cells and tendon cells cultures were prepared following treatment with collagenase enzyme. Cell samples were collected from the groups by means of an invert light microscope and environmental scanning electron microscope underwent at the 1st and 21st days. Thelevel of osteogenic differentiation was measured by the activity of alkaline phosphatase. For the statistical evaluation of the obtained data, groups were compared with post hoc Tukey test following analysis of variance. Level of significance was accepted to be <0,01. Results. Both osteogenic and tenogenic stimulation were observed in the cultured specimens. In comparison to the control groups, the rate of proliferation of healthy cells was found to be higher in the groups to which the design was added (P < 0.01). Conclusions. Our research is a preliminary report that describes a study conducted in an in vitro experimental setting. We believe that such prototype systems may be pioneers in targeted drug therapies after reconstructional surgeries.Öğe Delivering growth factors through a polymeric scaffold to cell cultures containing both nucleus pulposus and annulus fibrosus(Turkish Neurosurgical Society, 2019) Akyuva, Yener; Kaplan, Necati; Yılmaz, İbrahim; Özbek, Hanefi; Yaşar Şirin, Duygu; Karaaslan, Numan; Güler, Olcay; Ateş, ÖzkanAIM: To design a novel, polyvinyl alcohol (PVA)-based polymeric scaffold that permits the controlled release of insulin-like growth factor 1 (IGF-1) /bone morphogenetic protein (BMP)-2 following intervertebral disc administration. MATERIAL and METHODS: The drug delivery system was composed of two different solutions that formed a scaffold within seconds of coming into contact with each other. Swelling, pH, and temperature tests and analysis of the controlled release of growth factors (GFs) from this system were performed. The release kinetics of the GFs were determined through enzyme-linked immunosorbent assay (ELISA). Cell proliferation and viability were monitored with microscopy and analyzed using an MTT assay and acridine orange/propidium iodide (AO/PI) staining. Chondroadherin (CHAD), hypoxia inducible factor-1 alpha (HIF-1 alpha), and collagen type II (COL2A1) gene expressions were determined with quantitative real-time polymerase chain reaction (qRT-PCR) analysis to show the effects of IGF-1/BMP-2 administration on annulus fibrosus cell (AFC)/nucleus pulposus cell (NPC) cultures. For the statistical evaluation of the obtained data, experimental groups were compared with a post hoc Tukey's test following an analysis of variance. RESULTS: The scaffold allowed for the controlled release of IGF-1 and BMP-2 in different time intervals. It was observed that as the application time increased, the number of cells and the degree of extracellular matrix development increased in AFC/NPC cultures. AO/PI staining and an MTT analysis showed that cells retained their specific morphology and continued to proliferate. It was observed that HIF-1 alpha and CHAD expression increased in a time-dependent manner, and no COL2A1 expression in the AFC/NPC cultures was observed. CONCLUSION: The designed scaffold may be used as an alternative method for intervertebral disc administration of GFs after further in vivo studies. Such prototype scaffolds may be an innovative technology in targeted drug therapies after reconstructive neurosurgical interventions.Öğe Does transcription factor, induced by daptomycin and vancomycin, affect HIF-1?, Chondroadherin, and COL2A1?(Inonu University School of Medicine, 2018) Karaarslan, Numan; Yılmaz, İbrahim; Yaşar Şirin, Duygu; Özbek, Hanefi; Kaya, Yasin Emre; Akyuva, Yener; Kaplan, Necati; Doğan, Mustafa; Gümüştaş, Seyit Ali; Ateş, Özkan; Erdem, İlknurAim: In this study, it was firstly aimed to investigate the effect of Daptomycin (DAP) on the proliferation in Vancomycin (VCM)-administered primary chondrocyte cultures and non-drug-administered primary chondrocyte cultures. Our second objective was to investigate the effects of DAP and VCM on the NP-specific marker protein chondroadherin (CHAD), which is associated with spinal cord and dorsal column growth, on the transcription factor-1 alpha (HIF-1?), which is induced by hypoxia, and on a type II collagen (COL2A1), which is also known to play a significant role in the development of extracellular matrix, at the pharmaco-molecular level. Material and Methods: Standard human primary chondrocyte cultures were established. DAP and VCM were added to the samples. In all groups, molecular analysis was performed at 0th, 24th and 48th hours. In addition, the surface morphology of the cells was evaluated. Results: Changes in cell morphology and cell death in cultures were observed 24 hours after administration of antibiotics to cell cultures. It was observed that drug administration was associated with the cell viability and that cell viability rate for two antibiotics was similar at the 0th and 48th hours. The expression of three genes decreased at the 24th hour in the experimental group where DAP was administered. Conclusion: Thanks to this molecular-based research, it should not be forgotten that DAP and VCM active pharmacological agents, especially used in the treatment of Methicillin-resistant Staphylococcus aureus induced surgical infections, have a negative effect on human chondrocyte and ECM components.Öğe Effects of etanercept, a tumor necrosis factor receptor fusion protein, on primary cell cultures prepared from intact human intervertebral disc tissue(Spandidos Publications, 2019) Çalışkan, Tezcan; Şirin, Duygu Yaşar; Karaarslan, Numan; Yılmaz, İbrahim; Özbek, Hanefi; Akyuva, Yener; Kaplan, Necati; Kaya, Yasin Emre; Şimşek, Abdullah Talha; Güzelant, Aliye Yıldırım; Ateş, ÖzkanThe aim of the present study was to investigate the effects of etanercept (ETA), a tumor necrosis factor (TNF) inhibitor, on human cell cultures prepared from intact intervertebral disc tissue. ETA is used as a treatment for cases of rheumatoid arthritis, psoriatic arthritis, axial spondyloarthritis and ankylosing spondylitis accompanied by moderate or severe joint pain. ETA was applied to primary cell cultures [annulus fibrosus and nucleus pulposus (NP) from intact intervertebral disc tissue]. Cell cultures without ETA treatment served as the control group. Morphological and quantitative molecular analyses of the two groups were performed. The number of viable cells and cell proliferation decreased in the ETA-treated cultures as compared with those in the control group. Furthermore, in the treatment group, the chondroadherin gene, an NP-specific marker, was not expressed after 24 h. By contrast, the cartilage oligo matrix protein was expressed 24, 48 and 72 h post-ETA treatment, while its expression was significantly lower than that in the control group. In addition, the expression of interleukin-1 beta, as well as matrix metallopeptidase-7 and -19, was markedly decreased. Overall, the cell proliferation and gene expression in the ETA-treated cells were significantly different from those in the control group (P<0.05). These results suggest that the treatment duration and dosage of TNF inhibitors, which are used to suppress active inflammation, should be considered in the clinical setting. These biological agents may delay the healing of intervertebral disc tissue damage by slowing cell proliferation and altering gene expression via anabolic and catabolic pathways.Öğe Investigation of the effect of dipyrone on cells isolated from intervertebral disc tissue(Spandidos Publications, 2019) Akgün, Feride Sinem; Şirin, Duygu Yaşar; Yılmaz, İbrahim; Karaarslan, Numan; Özbek, Hanefi; Şimşek, Abdullah Talha; Kaya, Yasin Emre; Kaplan, Necati; Akyuva, Yener; Çalışkan, Tezcan; Ateş, ÖzkanThe present study aimed to evaluate the effects of dipyrone, an indispensable analgesic, anti-pyretic and anti-spasmodic used in emergency departments, on nucleus pulposus and annulus fibrosus cells in vitro. After surgical biopsy, primary cell cultures were prepared from intact intervertebral disc tissues. Dipyrone was administered to the cultures in the experimental groups except for the control group. The data obtained were statistically evaluated. The proliferation was identified to be suppressed via MTT analysis. The gene expression profile of the intervertebral disc cells in the dipyrone-treated groups was significantly changed. The expression of chondroadherin, cartilage oligo matrix protein, interleukin-1 beta and metalloproteinase (MMP)-19 genes were decreased, but MMP-13 and MMP-7 genes expressions were increased, as determined via reverse transcription-quantitative PCR. AO/PI staining revealed that no apoptotic or other type of cell death was detectable after administration of dipyrone does not mean that the drug is innocuous. The occurrence of cellular senescence and/or the halt of cell proliferation may also be important mechanisms underlying the adverse inhibitory effects of dipyrone. Therefore, prior to administering dipyrone in clinical practice, all possible adverse effects of this drug should be considered.Öğe Is it possible to treat osteosarcoma using oligonucleotides confined into controlled release drug delivery systems?(Bentham Science Publishers Ltd, 2017) Topuk, Savaş; Akyuva, Yener; Karaaslan, Numan; Mutlu, Çağri Ata; Yılmaz, İbrahim; İşyar, Mehmet; Şirin, Duygu Yaşar; Akkaya, Semih; Özbek, Hanefi; Mahiroğulları, MahirPurpose: The present study aimed to analyze the researches that are at the experimental phase concerning osteosarcoma treatment. The researches included drug delivery systems which allow controlled release and imbue small interfering-/micro-ribonucleic acid. Methods: Without any language preference, we searched US National Library of Medicine National Institutes of Health, Embase, OVID, Cochrane Library database of clinical trials from 1843 to May 25, 2016 and traced all the references of incorporated documents. The data were evaluated using descriptive statistics and the results are shown as frequency (%). Results: We haven't encountered any drug delivery system in which Small interfering ribonucleic acid/ micro ribonucleic acid oligonucleotides were embedded successfully against osteosarcoma. There has been only one research in which hairpin-ribonucleic acid was embedded. Conclusion: It was considered that drug delivery system enabling controlled oligonucleotide release in the treatment period of osteosarcoma was not projected for the clinical use. However, it cannot be neglected that the mentioned experimental studies with regard to osteosarcoma treatment establish the basis of target therapies. The method in question looks promising regarding effective treatment of osteosarcoma in the future.Öğe Pregabalin treatment for neuropathic pain may damage intervertebral disc tissue(Spandidos Publications Ltd, 2018) Karaarslan, Numan; Yılmaz, İbrahim; Yaşar Şirin, Duygu; Özbek, Hanefi; Kaplan, Necati; Kaya, Yasin Emre; Akyuva, Yener; Gürbüz, Mehmet Sabri; Öznam, Kadir; Ateş, ÖzkanThe aim of the present study was to determine whether pharmaceutical preparations with pregabalin (PGB) as an active ingredient, which are widely prescribed by clinicians, exert toxic effects on human primary nucleus pulposus (NP) and annulus fibrosis (AF). Primary human cell cultures were obtained from intact (n=6) and degenerated (n=6) tissues resected from the two groups of patients. Different doses of PGB were applied to these cultures and cells were subjected to molecular analyses at 0, 24 and 48 h. Cell vitality, toxicity and proliferation were assessed using a spectrophotometer. The expression of chondroadherin (CHAD), a (member of the NP-specific protein family), hypoxia-inducible factor-1 alpha (HIF-1 alpha) and type II collagen (COL2A1) was measured using reverse transcription-quantitative polymerase chain reaction. The results revealed that cell intensity increased in a time-dependent manner and cell vitality continued in the cultures without pharmaceuticals. Cell proliferation was suppressed in the PGB-treated cultures independent from the dose and duration of application. PGB was demonstrated to suppress the expression of CHAD and HIF-1 alpha. In contrast, COL2A1 gene expression was not revealed in any experimental group. The present study utilized an in vitro model and the PGB active ingredient used herein may not be representative of clinical applications; however, the results demonstrated that PGB has a toxic effect on NP/AF cell cultures containing primary human intervertebral disc tissue. In summary, the use of pharmacological agents containing PGB may suppress the proliferation and differentiation of NP/AF cells and/or tissues, which should be considered when deciding on an appropriate treatment regime.Öğe The association between different molecular weights of hyaluronic acid and CHAD, HIF-1 alpha, COL2A1 expression in chondrocyte cultures(Spandidos Publications Ltd, 2018) Yaşar Şirin, Duygu; Kaplan, Necati; Yılmaz, İbrahim; Karaarslan, Numan; Özbek, Hanefi; Akyuva, Yener; Kaya, Yasin Emre; Öznam, Kadir; Akkaya, Nuray; Güler, Olcay; Akkaya, Semih; Mahiroğulları, MahirThe aim of the present study was to investigate the effects of three different formulations of hyaluronic acid (HA): Low molecular weight (MW) Sinovial One((R)), medium MW Viscoplus((R)) and high MW Durolane((R)), on chondrocyte proliferation and collagen type II (COL2A1), hypoxia-inducible factor 1 alpha (HIF-1 alpha) and chondroadherin (CHAD) expression in primary chondrocyte cultures. Standard primary chondrocyte cultures were established from osteochondral tissues surgically obtained from 6 patients with gonarthrosis. Cell morphology was evaluated using an inverted light microscope; cell proliferation was determined with a MTT assay and confirmed with acridine orange/propidium iodide staining. Levels of CHAD, COL2A1 and HIF-1 alpha expression were assessed using specific TaqMan gene expression assays. The results demonstrated the positive effect of HA treatment on cell proliferation, which was independent from the MW. COL2A1 expression increased in the medium and high MW HA treated groups. It was observed that HIF-1 alpha expression increased in the high MW treated group alone. CHAD expression increased only in the medium MW HA treated group. Evaluation of gene expression revealed that levels of expression increased as the duration of HA application increased, in the medium and high MW HA treated groups. In terms of increased viability and proliferation, a longer duration of HA application was more effective. Taken together, it may be concluded that the administration of medium and high MW HA may be a successful way of treating diseases affecting chondrocytes in a clinical setting.
