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    A study of the effects of metformin, a biguanide derivative, on annulus fibrosus and nucleus pulposus cells
    (Turkish Neurosurgical Society, 2020) Kaya, Yasin Emre; Karaarslan, Numan; Yılmaz, İbrahim; Yaşar Şirin, Duygu; Akalan, Hande; Özbek, Hanefi
    AIM: To investigate the effects of metformin, a drug used widely for the treatment of type 2 diabetes mellitus, on human primary cell cultures prepared from uninjured segment of disc material intervertebral disk tissues.MATERIAL and METHODS: Primary cell cultures were prepared using the tissues of six patients (three males and three females) who had undergone lumbar microdiscectomy and sequestrectomy. Untreated samples served as the control group, and metformintreated samples served as the experimental group. All the samples were evaluated using an inverted light microscope, acridine orange/propidium iodide staining (AO/PI), and a fluorescence microscope. The cytostatic and cytotoxic effects of metformin, which was administered to the samples using a commercial MTT assay kit, were also evaluated. The data obtained were statistically assessed, and the alpha significance value was accepted as less than 0.05. In addition, for the groups' changes in the expressions of chondroadherin (CHAD), cartilage oligomeric matrix protein (COMP), interleukin-1 beta (IL-1 beta) matrix metalloproteinase 7 (MMP-7), and matrix metalloproteinase 19 (MMP-19), genes related to the extracellular matrix synthesis and degradation were determined using gene-specific TaqMan Gene Expression Assays.RESULTS: The administration of the drug adversely affected nucleus pulposus (NP)/annulus fibrosus (AF) cells and extracellular matrix-like structures. This was statistically significant (p<0.05).CONCLUSION: Clinicians should not disregard the adverse effects of metformin, which is used widely in clinical practice, on the components of intervertebral disk tissues.
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    A study on the effects of direct factor Xa inhibitors and direct thrombin inhibitors on human primary chondrocyte cultures
    (Namık Kemal University, 2019) Kaya, Yasin Emre; Akalan, Hande; Yılmaz, İbrahim; Karaarslan, Numan; Yaşar Şirin, Duygu; Özbek, Hanefi; Ateş, Özkan
    Aim:This study investigates the effects of two direct factor Xa inhibitors, apixaban and rivaroxaban, and a direct thrombin inhibitor, dabigatran, on human primary chondrocytecultures. Materials and Methods:Monolayer cultured chondrocytes were prepared. Cell cultures were treated with dabigatran, apixaban, and rivaroxaban. Cultures without drug treatments served as the control group. Using an inverted light microscope, the cell surface morphology was examined. Cell viability and the toxicity of drugs were evaluated using a commercial assay kit, and the results were confirmed using two nucleic acid binding dyes, acridine orange and propidium iodide. The expressions of cartilage oligomeric protein, matrix metalloproteinase-7, and matrix metalloproteinase-19 were assessed using the real-time polymerase chain reaction analysis. All the analyses were performed within 21 days. The data obtained were statistically evaluated. Results:The administration of the three drugs changed the cell viability, proliferation, and expressions of cartilage oligomeric protein, matrix metalloproteinase-7, and matrix metalloproteinase- 19. The results were statistically significant (P<0.05). Conclusion:Results obtained from in vitro studies may not provide accurate and reliable insight for clinical practices. However, clinicians should know that drugs used for the prevention or treatment of diseases may suppress chondrocyte proliferation and damage the extracellular matrix formation.
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    Does oseltamivir protect human chondrocyte and nucleus pulposus cells from degeneration by inhibiting senescence and proinflammation mediated by the NLRP3 inflammasome and NF-?B?
    (Verduci Editore s.r.l, 2022) Yılmaz, İbrahim; Akalan, Hande; Öznam, Kadir; Karaarslan, Numan; Yaşar Şirin, Duygu; Özbek, Hanefi
    OBJECTIVE: Recent drug design studies suggest that inflammation is among the most important factors in the development of both intervertebral disc (IVD) degeneration (IVDD) and osteoarthritis (OA) due to cartilage damage. This study aimed to investigate whether the anti-inflammatory drug oseltamivir has a toxic effect on IVD and cartilage tissue cells. It assessed what effect oseltamivir has on hypoxia-inducible factor (HIF)-1 alpha (HIF1?), which plays an important role in anabolic pathways in IVD and cartilage tissue. In addition, the study analyzed whether oseltamivir could inhibit the release of inflammatory interleukin-1 beta (IL-1?) via the nuclear factor kappa-B (NF-?B) signaling pathway by activating the nucleotide-binding oligomerization domain and leucine-rich repeat protein-3 (NLRP3) inflammasome. MATERIALS AND METHODS: Human lumbar IVD (n = 8) tissues were isolated for annulus fibrosus (AF) and nucleus pulposus (NP) primary cell cultures, and human tibial and femoral cartilage tissues (n = 8) were isolated for primary chondrocyte cultures. Untreated groups served as the control and oseltamivir-treated groups as the study sample. Cell viability and cytotoxicity were evaluated at 0, 24, 48, and 72 h in all groups for changes in HIF-1?, IL-1?, NF-?B, and the NLRP3-inflammasome protein expressions using Western blotting. The ? significance value was < 0.05. RESULTS: In the oseltamivir-treated groups, cell proliferation decreased in both AF/NP cell and chondrocyte cultures obtained from IVD cartilage tissues. After Western blotting analysis, changes were observed in the protein expressions of HIF-1?, IL-1?, NF-?B, and the NLRP3 inflammasome in both AF/NP cells and chondrocytes. The results were statistically significant (p < 0.05). CONCLUSIONS: Oseltamivir treatment may be a promising regenerative strategy to manage IVDD and osteoarthritic cartilage tissues.
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    Evaluation of the expression and proliferation of degenerative markers in primary cell cultures obtained from human intervertebral disc tissue
    (Annals of Medical Research Publishing Inc, 2020) Kaya, Yasin Emre; Akalan, Hande; Yılmaz, İbrahim; Karaarslan, Numan; Yaşar Şirin, Duygu; Özbek, Hanefi
    Aim: A major cause of low back pain is disc degeneration. Nevertheless, no specific and reliable markers of the degeneration of the nucleus pulposus (NP) are available. This presented study aimed to examine changes in the expressions of genes in primary cell cultures isolated from intact and degenerated tissues to give insights into the biopathogenesis of intervertebral disc (IVD) tissue. Material and Methods: Tissues of eight patients (n = 8; average age: 41.74 ± 9.86 years) were resected through microdiscectomy, and primary cell cultures were prepared using degenerated disc tissue. The cultured degenerated tissues served as the study group. The samples in the control group comprised the intact tissues of patients (n = 8; average age: 38.68 ± 7.91 years) resected following a trauma. Morphology of the cell surface were evaluated using an inverted light/fluorescent microscopy at 0 and 24 h on days 10 and 21. The expressions of the chondroadherin (CHAD), cartilage oligomeric matrix protein (COMP), interleukin-1 beta (IL-1 beta), and matrix metalloproteinase (MMP)-7 and MMP-19 genes were evaluated using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The data obtained were statistically analyzed. Results: The four genes investigated, except COMP (P > 0.05), changed significantly in primary cell cultures isolated from degenerative IVD tissues. This result was statistically significant (P < 0.05). The gene expressions in the samples derived from intact IVD tissues changed markedly and these changes were associated with proliferation (P < 0.05). Conclusion: Analyzing the changes in gene expression levels associated with IVD should contribute to future studies on the prevention and treatment of such pathologies. The data obtained from the present study will shed light on cellular-based personal targeted therapies through which genetic information can be transmitted to cells.
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    The effects of rivaroxaban, an oral anticoagulant, on human IVD primary cultures
    (Termedia Publishing House Ltd, 2022) Çalışkan, Tezcan; Akalan, Hande; Yılmaz, İbrahim; Karaarslan, Numan; Yaşar Şirin, Duygu; Özbek, Hanefi
    Introduction: The present study aimed to investigate the potential effects of rivaroxaban, an oral anticoagulant that inhibits the effects of factor Xa, on intact intervertebral disc tissue cells and the extracellular matrix (ECM). Material and methods: Rivaroxaban was applied to primary human cell cultures prepared from tissues of the intervertebral disc. Comparative molecular analyses were performed on non-drug-treated control group samples. Descriptive statistics were presented as the mean +/- standard deviation. An analysis of variance test was performed to determine whether there were significant differences in the mean across the groups. When differences across groups were observed, Tukey's honestly significant difference post-hoc test was used for multiple pairwise comparisons. The significance of the obtained data was determined statistically. The alpha significance value was < 0.05. Results: The cells in the control group and in the rivaroxaban-treated group were viable, healthy, and proliferated (p < 0.05). However, the expression levels of the chondroadherin gene (CHAD), cartilage oligo matrix protein (COMP), matrix metalloproteinase (MMP)-13, and MMP-19 genes were changed (p < 0.05). Conclusions: Although rivaroxaban does not suppress cell proliferation due to morphological, biological, and biochemical changes in the intervertebral disc tissue, it may change the expression of genes that are related to ECM maintenance.