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A practical way to prepare primer human chondrocyte culture
(Elsevier Science Bv, 2016) İşyar, Mehmet; Yılmaz, İbrahim; Şirin, Duygu Yaşar; Yalçın, Sercan; Güler, Olcay; Mahiroğulları, Mahir
Biological cartilage repair is one of themost important targets for orthopedic surgeons currently. For this purpose, it is mandatory to know how to prepare a chondrocyte culture. In this study, our purpose was to introduce a method enabling orthopedic surgeons to practice their knowledge and skills on molecular experimental setup at cellular level, based on our experiences from previous pilot studies. Thus, we believe it will encourage orthopedic surgeons. (C) 2016 Prof. PK Surendran Memorial Education Foundation. Published by Elsevier, a division of Reed Elsevier India, Pvt. Ltd. All rights reserved.
A Study of the Effects of Metformin, a Biguanide Derivative, on Annulus Fibrosus and Nucleus Pulposus Cells
(Turkish Neurosurgical Soc, 2020) Kaya, Yasin Emre; Karaarslan, Numan; Yılmaz, İbrahim; Şirin, Duygu Yaşar; 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.
A Study on the Effects of Direct Factor Xa Inhibitors and Direct Thrombin Inhibitors on Human Primary Chondrocyte Cultures
(2019) Kaya, Yasin Emre; Akalan, Hande; Yılmaz, İbrahim; Karaarslan, Numan; Şirin, Duygu Yaşar; 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.
Alloreactive memory B cell detection by flow cytometric cross match using polyclonally activated memory B cell culture supernatants
(Elsevier, 2022) Akalan, Hande; Şirin, Duygu Yaşar; Yılmaz, İpek; Ata, Pınar; Kara, Veli Melih; Taşdemir, Nicel; Bilgen, Türker; Titiz, Mesut İzzet
In addition to alloantibodies, alloreactive memory B cell (mBC) evaluation has a potential for immunological risk assessment during transplantation processes. For the alloreactive mBCs evaluation currently, direct Flow Cytometric (FC) analysis using the HLA tetramer staining is an option. Evaluation of alloantibodies produced by the polyclonally stimulated alloreactive mBCs in in vitro culture system seems to be another useful approach, but this needs further downstream applications. In this study, we investigated the usefulness of the Flow Cytometric Cross Match (FCXM-supernatant) in which in vitro polyclonally activated mBCs culture supernatants and potential donor's lymphocytes being used for the mBC detection. FCXM-supernatant assays were performed between culture supernatants of polyclonally activated mBCs obtained from 4 allosensitized multiparous women and 14 renal transplant patients, and their non-alloimmunized spouses' or donors' lymphocytes, and vice versa. HLA typing was performed by SSP method. Anti-HLA antibodies produced by in vitro activated alloreactive mBCs were also evaluated by the Luminex assays. The success of in vitro polyclonal activation of mBCs was evaluated by a total IgG ELISA test and antibody secreting cell analyses by FC. Donor specific alloreactive mBCs were detected by FCXM-supernatant in 45% of the 18 allosensitized cases. Detection rate was 85% (6 out of 7) in the strongly allosensitized cases. No alloreactive mBCs was detected in control cases without allosensitization. FCXMsupernatant negative results of the allosensitized cases were related to low level of allosensitization and insufficient polyclonal stimulation evaluated by total IgG antibody tests of the supernatants. We herein report a practical methodology for alloreactive mBC detection as a donor specific manner using the FCXM-supernatant assay so that this would easily be transformed into a routine test performed in tissue typing laboratories.
Are biological agents toxic to human chondrocytes and osteocytes?
(Bmc, 2015) İşyar, Mehmet; Bilir, Bülent; Yılmaz, İbrahim; Çakmak, Selami; Şirin, Duygu Yaşar; Yıldırım Güzelant, Aliye; Mahiroğulları, Mahir
Purpose: The aim of the present study is to investigate the effects of biological agents (BAs) on human chondrocytes and osteocytes in vitro. Methods: Primary cell cultures obtained from gonarthrosis patients were divided into four groups, two of which were designated as control cultures of chondrocyte and osteocyte, and the other two groups were exposed to BAs administered via the culture medium. Cultured cells were characterized by immunophenotyping. Before and after administration of the agents, the cultures were observed by inverted and environmental scanning electron microscopy (ESEM). The number of live cells and the proliferation rate were monitored by MTT assay. Results: Rituximab and adalimumab were the least toxic agents to chondrocytes, whereas adalimumab and etanercept were to osteocytes. Conclusion: During periods of intense active inflammation, the concentration of the preferred BAs after inhibition of inflammation needs to be emphasized when their effects on cartilage and bone tissue are considered at the cellular level if the clinical practice is to continue.
Are chondrocytes damaged when rheumatologic inflammation is suppressed?
(Taylor & Francis Ltd, 2017) Yıldırım Güzelant, Aliye; İşyar, Mehmet; Yılmaz, İbrahim; Şirin, Duygu Yaşar; Çakmak, Selami; Mahiroğulları, Mahir
Aim: The use of biological agents (BAs) for treating diseases such as rheumatoid arthritis (RA), spondyloarthropathy, and systemic lupus erythematosus to reduce inflammation has been fruitful. Especially as part of the increasing number of studies on the intra-articular application of BAs, the effects of BAs on cartilage have been widely investigated. In the present study, the effects of rituximab, abatacept, and adalimumab, all approved antirheumatic agents, on human primary chondrocytes were investigated comparatively and on the molecular level through viability, proliferation, and toxicity analyses. Materials and methods: Osteochondral tissues from the distal femur and proximal tibia were resected during total knee arthroplasty from patients (n=3) with confirmed gonarthrosis in whom all medical or conservative treatments had failed. Standard human primary chondrocyte cell culturing was carried out. Immunophenotyping was performed on the cells that adhered to the flask, and their chondrotoxicity was observed using a flow cytometry device. Images of the cells showing chondrotoxicity were analyzed using invert and environmental scanning microscopes, and microimages were obtained. The MTT-enzyme linked immunosorbent assay was performed to observe the toxic effects of BAs on the proliferation of chondrocytes at 24 and 48h. The results were analyzed using the number of cells and proliferation; statistical comparisons among the groups were carried out using one-way ANOVA. The alpha significance level was set at <0.01. Results: These pharmaceutical agents were chondrotoxic, especially on viability and proliferation (p=0.0000). Conclusion: BAs are generally used during active inflammation, and following the management of inflammation, their dosage should be determined taking into consideration their cellular-level toxic effects on chondrocytes.
Are radio-contrast agents commonly used in discography toxic to the intact intervertebral disc tissue cells?
(Wiley, 2019) Karaarslan, Numan; Yılmaz, İbrahim; Özbek, Hanefi; Şirin, Duygu Yaşar; Kaplan, Necati; Çalışkan, Tezcan; Ateş, Özkan
In 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.
Are the leading drugs against Staphylococcus aureus reallytoxic to cartilage?
(Elsevier Science London, 2016) Doğan, Mustafa; İşyar, Mehmet; Yılmaz, İbrahim; Bilir, Bülent; Şirin, Duygu Yaşar; Çakmak, Selami; Mahiroğulları, Mahir
Many studies have shown that the toxic effects of local antibiotics on bone and cartilage limit orthopedic surgeons. In this study, we evaluated three antibacterial agents used locally to treat highly mortal and morbid diseases in the field of orthopedics, such as septic arthritis. Are vancomycin, teicoplanin, and linezolid, which are archenemies of Staphylococcus aureus, really toxic to chondrocytes? The purpose of the study was to investigate the effects of antibiotics, which are used against S. aureus, on human chondrocytes in vitro. Primary cell cultures obtained from gonarthrosis patients were divided into two main groups. One of these groups was designated as the control chondrocyte culture. The other group was divided into three subgroups, and each group was exposedto vancomycin, teicoplanin, or linezolid. Cell culture samples were characterized by immunophenotyping following incubation with the three different antibiotics. Before and after the agents were administered, the cultures were subjected to inverted and environmental scanning electron microscopy. The number of live cells and the proliferation rate were monitored with the MTT-assay. We found that vancomycin, teicoplanin, and linezolid do not have chondrotoxic effects. Vancomycin, teicoplanin, and linezolid had no chondrotoxic activity during in vitro culture, which supports the argument that these agents can safely be used in orthopedic surgery, especially against methicillin-resistant S. aureus agents. (C) 2015 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Limited. All rights reserved.
Assessing the negative impact of phenyl alkanoic acid derivative, a frequently prescribed drug for the suppression of pain and inflammation, on the differentiation and proliferation of chondrocytes
(Bmc, 2016) Gümüştaş, Seyit Ali; Yılmaz, İbrahim; İşyar, Mehmet; Şirin, Duygu Yaşar; Batmaz, Ahmet Güray; Uğraş, Ali Akın; Mahiroğulları, Mahir; Çiftçi, Zafer
Background: Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently prescribed to relieve pain and inflammation. These NSAIDs have also analgesic effects and can be administered via oral, injectable, and topical routes. During inflammation, a number of synovial mediators and cytokines are released which decrease the pH level of the synovial fluid. Administration of acidic NSAIDs further decreases the pH levels and hence contributes to the destruction of the cartilage. To our knowledge, no cellular-based study regarding the chondrotoxicity of phenyl alkanoic acid derivatives on NSAIDs was conducted before. Thus, the aim of this pioneering study was to examine the effect of frequently prescribed NSAIDs, a phenyl alkanoic acid derivative, flurbiprofen, on the proliferation and differentiation of human primer chondrocyte cultures in vitro. Methods: Primer chondrocyte cultures were prepared from osteochondral tissue obtained during surgery for gonarthrosis. Samples not exposed to the pharmacological agent were used as the control group. The samples were treated with 1, 10, 100, 250, 500, or 1000 mu M of the agent for 24, 48, and 72 h. The cell viability, toxicity, and proliferation were assessed with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) analysis and prechondrocytic precursor stage-specific embryonic antigen-1 (SSEA-1) expression using a commercial ELISA kit spectrophotometrically. The surface morphology of the samples in each group was compared using an inverted light microscope and an environmental scanning electron microscope (ESEM). An analysis of variance was used to compare between-group differences. Tukey's honest significant difference (HSD) method (95 % confidence interval) was used to evaluate the differences and significance in averages. The alpha significance value was considered < 0.01. Results: Statistically significant cytotoxicity was observed in the treatment groups. NSAID had a significant negative effect on the proliferation and differentiation of chondrocytes as compared to the control group (p < 0.01). Conclusion: Before administering phenyl alkanoic acid derivatives in the clinical setting, their role in suppressing the proliferation and differentiation of chondrocytes should be taken into account. Thus, caution should be given when prescribing these drugs.
Assessment of the Impact of Curcumin on Cell Cultures Derived from the Primary Intervertebral Disc Tissue in Humans
(Maltepe Üniversitesi, 2023) Albayrak, Mehmet; Yılmaz, İbrahim; Yüzbaşı, Muharrem Furkan; Akalan, Hande; Şirin, Duygu Yaşar; Karaarslan, Numan; Özbek, Hanefi
Aim: Degenerative disc disease in the lumbar spine is widely observed. Degenerative disc diseases are among the causes of low back pain in older age. Modern drug discovery studies have aimed to identify potential molecules that target multiple pathways with a safer profile against degeneration. This study aimed to evaluate the effects of curcumin, a natural phenolic compound, on primary cell cultures prepared using intervertebral disc (IVD) tissues resected during the surgeries of patients with lumbar disc herniation. Materials and Methods: Primary cell cultures were prepared using human IVD tissues of eight patients. Untreated groups served as the control and curcumin-treated groups as the study sample. In-vitro cytotoxicity analyses were performed in all groups. Acridine orange (AO)/propidium iodide (PI) and Janus Green B staining were performed to evaluate cell surface morphologies. One-way analysis of variance and Tukey HSD, a multiple comparison test, were used to assess the obtained data. Results: Proliferation slightly increased as of 24 h in the curcumin-treated samples, but decreased in the 48 and 72 hour curcumin-treated samples compared to the control samples. The obtained results were statistically significant (p
Biyopolimerler ile uygulanan lökositten fakir veya lökositten zengin trombositten zengin plazmanın kondrosit proliferasyonunda konvansiyonel trombositten zengin plazma uygulamalarına üstünlüğü var mıdır?
(2017) Şirin, Duygu Yaşar; Yılmaz, İbrahim; İşyar, Mehmet; Öznam, Kadir; Mahiroğulları, Mahir
Amaç: Bu çalışmada trombositten zengin plazma (TZP) içeriğindeki lökosit konsantrasyonunun ve TZP'nin ilaç taşıyıcı sistem kullanılarak uygulanmasının in vitro koşullarda kondrosit proliferasyonu üzerine olası etkileri araştırıldı.Hastalar ve yöntemler: Medikal veya konservatif tedaviye yanıt vermemiş ve total diz artroplastisi geçirmiş dokuz erkek hastanın (ort. yaş 65 yıl; dağılım 49-81 yıl) kanı iki formülasyon hazırlamak üzere kullanıldı: Düşük konsantrasyon lökositli TZP (2000-4000 lökosit/µL) saf TZP (S-TZP) olarak, yüksek konsantrasyon lökositli TZP (9000-11000 lökosit/µL) ise lökositten zengin TZP (L-TZP) olarak belirlendi. Numuneler kontrol grubu (grup 1), direkt S-TZP uygulanan kondrosit kültürleri (grup 2), direkt L-TZP uygulanan kondrosit kültürleri (grup 3), S-TZP uygulanan hidrojel ile kültürlenen kondrositler (grup 4) ve L-TZP uygulanan hidrojel ile kültürlenen kondrositler (grup 5) olmak üzere beş gruba ayrıldı. Tüm gruplarda hücre morfolojisi, canlılık ve proliferasyon bir prekondrosit belirteci olan evreye özgü embriyonik antijen 1'in (SSEA-1) ifadesi ile karşılaştırıldı.Bulgular: Maksimum hücre proliferasyonu ve SSEA-1 ifadesi grup 4'te gerçekleşti; SSEA-1 ifadesi ve hücre proliferasyonu arasında istatistiksel olarak anlamlı ilişki vardı.Sonuç: Çalışmamız TZP'nin lökosit konsantrasyonunun ve hidrojel gibi taşıyıcı sistemlerin önemini ve taşıyıcı sistem ile uygulanan L-TZP'nin kıkırdak hasarının tedavisinde TZP'nin konvansiyonel uygulamalarından daha etkin olduğunu gösterdi
Can transcription factors in the intervertebral disc of lopinavir/ritonavir prevent degeneration in the nucleus pulposus by mediating the regulation of inflammation through signaling pathways?
(Verduci Publisher, 2022) Yılmaz, I.; Akalan, Hande; Karaarslan, N.; Şirin, Duygu Yaşar; Kaplan, N.; Doğan, Mustafa; Özbek, H.
OBJECTIVE: This study was con-ducted to examine whether lopinavir/ritonavir (Lop/r), an HIV protease inhibitor, can improve disc physiology and slow down intervertebral disc (IVD) degeneration through in vitro experimental methods, as well as whether it can suppress inflammation with interleukin-1 beta (IL-113) and sex-determining region Y (SRY) protein-related high-mobility group box genes-9 (SOX9) through hypoxia-inducible factor 1-alpha (HIF-1 alpha) and the nuclear factor kappa B (NF-KB) signaling pathway. The aim was to investigate whether Lop/r application is toxic to IVD cells and the microenvironment simultaneously. PATIENTS AND METHODS: Human primary cell cultures were prepared using herniated IVD tissues obtained from patients with lumbar disc hernia who were unresponsive to conservative and medical treatment, and thereby, were operated on. The untreated culture samples served as control group, and the samples treated with Lop/r served as study group. Microscopic evaluations were performed simultaneously using fluorescent and supravital dyes in all groups. In addition to cell viability, toxicity, and proliferation analysis through a commercial kit, IL-113, SOX9, HIF-1 alpha, and NF-KB protein expressions were evaluated using Western blotting. In the statistical comparison of the obtained data, an alpha value less than 0.05 was considered significant. RESULTS: Cell proliferation decreased in the Lop/r group, but no cell death was observed (p < 0.05). Moreover, at the end of 72 hours after Lop/r application, IL-113 and NF-kB protein expressions decreased by 40% and 52%, respectively, while HIF-1 alpha and SOX9 protein expressions increased by 4% and 59%, respectively (p < 0.05). CONCLUSIONS: Although these data were obtained from an in vitro experimental study, it is believed that these findings could make significant contributions to the pharmaco-regenerative treatment modalities of IVD degeneration. Lop/r suppresses the IL-113 and NF-KB and induces SOX9 and HIF-1 alpha, since these signaling path-ways may be related to human IVD degeneration.
Chondrocyte proliferation, viability and differentiation is declined following administration of methylphenidate utilized for the treatment of attention-deficit/hyperactivity disorder
(Sage Publications Ltd, 2017) Gümüştaş, F.; Yılmaz, I.; Şirin, Duygu Yaşar; Gümüştaş, Seyit Ali; Batmaz, Ahmet Güray; İşyar, Mehmet; Mahiroğulları, Mahir
Purpose: Methylphenidate (MPH) derivative drugs are used because of psychostimulants effects on attention-deficit hyperactivity disorder in children and adults. As far as we know, toxic or anti-proliferative effects of MPH against cartilage tissue were not studied in the literature. The present study was carried out to investigate the possible effects of MPH on the proliferation, viability and differentiation of primary human chondrocytes, in vitro. Methods: Monolayer primary chondrocyte cultures were prepared using osteochondral tissue obtained from patients who underwent a total knee prosthesis operation. Stock solution of MPH was prepared and aliquots having 1-1000 mu M concentrations of the drug was composed. These solutions were applied to the wells containing cultured chondrocyte samples within the well plates. Control groups were composed of pure chondrocyte culture and no solution was added into them. All groups were evaluated at 24, 48 and 72 h in order to determine the possible negative effects of the drug on the chondrocytes. The data were evaluated by Tukey's honestly significantly different test following analysis of variance. Results: In the group where MPH was applied, it was found that viability, proliferation and stage-specific embryonic antigen-1 protein expression were decreased in comparison to the control group. Conclusions: It was emphasized that clinicians should not disregard the fact that this drug might suppress chondrocyte cell proliferation and chondrogenic differentiation.
Delivering Growth Factors through a Polymeric Scaffold to Cell Cultures Containing both Nucleus Pulposus and Annulus Fibrosus
(Turkish Neurosurgical Soc, 2019) Akyuva, Yener; Kaplan, Necati; Yılmaz, İbrahim; Özbek, Hanefi; Şirin, Duygu Yaşar; Karaarslan, Numan; Ateş, Özkan
AIM: 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.
Do we damage nucleus pulposus tissue while treating cerebrovascular ischemic neurological deficits with nimodipine?
(2018) Karaarslan, Numan; Yılmaz, İbrahim; Şirin, Duygu Yaşar; Baykız, Derya; Demirkiran, Aykut; Ateş, Özkan
Aim: Nimodipine is used to prevent cerebrovascular-originated ischemic neurological deficits, yet its effects on nucleus pulposus (NP) cells or annulus fibrosus (AF) cells weren’t studied. This study aimed to examine nimodipine’s effects on vitality and proliferation of chondroadherin (CHAD), type II collagen (COL2A1), and hypoxia-inducible factor 1 alpha (HIF 1?) gene expression in human primary NP/AF cells.Material and Methods: NP/AF cell cultures obtained from 6 patients who underwent microdiscectomy were treated with 100 µMolar nimodipine and analyzed at 0, 24, and 48 h. Data were evaluated using one-way ANOVA and post-hoc Tukey HSD with 95% confidence interval.Results: We observed suppressed cell proliferation and increased necrosis in nimodipine-treated NP/AF cell cultures, especially degenerated tissue. COL2A1 gene expression wasn’t detected in any experimental groups. CHAD and HIF 1? expression had timedependent decreases in control. CHAD and HIF 1? expression were found to decrease at 24h, but increased at 48h in degenerated tissue. In nimodipine-applied intact tissues, CHAD expression was stable at 24h but 1.62 times higher than control at 48h. HIF 1? levels were lower than control.Conclusion: In nimodipine-treated degenerated AF/NP cultures, CHAD and HIF 1? expressions had time-dependent decreases. However, after complete RT-PCR data evaluation, no correlation between nimodipine application and gene expression occurred.
Does leukocyte-poor or leukocyte-rich platelet-rich plasma applied with biopolymers have superiority to conventional platelet-rich plasma applications on chondrocyte proliferation?
(Turkish Joint Diseases Foundation, 2017) Şirin, Duygu Yaşar; Yılmaz, İbrahim; İşyar, Mehmet; Öznam, Kadir; Mahiroğulları, Mahir
Objectives: This study aims to investigate the possible effects of leukocyte concentration in the content of platelet-rich plasma (PRP) and the administration of PRP using a drug delivery system on chondrocyte proliferation in vitro conditions. Patients and methods: Blood from nine male patients (mean age 65 years; range 49 to 81 years) with advanced stage osteoarthritis who had not responded to medical or conservative treatments and underwent total knee arthroplasty was used to prepare two formulations: PRP with low concentration leukocytes (2000-4000 leukocytes/mu L) was designated as pure PRP (P-PRP), whereas PRP with high concentration leukocytes (9000-11000 leukocytes/mu L) as leukocyte-rich PRP (L-PRP). Samples were divided into five groups as control group (group 1), chondrocyte cultures with P-PRP applied directly (group 2), chondrocyte cultures with L-PRP applied directly (group 3), chondrocytes co-cultured with P-PRP applied hydrogel (group 4), and chondrocytes co-cultured with L-PRP applied hydrogel (group 5). In all groups; cell morphology, viability and proliferation were compared with the expression of stage-specific embryonic antigen-1 (SSEA-1), a precondrocyte marker. Results: Maximum cell proliferation and SSEA-1 expression occurred in group 4, with a statistically significant correlation between SSEA-1 expression and cell proliferation. Conclusion: Our study showed the importance of leukocyte concentration of PRP and efficiency of delivery systems such as hydrogel and that L-PRP administered with a delivery system is more efficient than conventional applications of PRP in the treatment of cartilage damage.
Does Nimodipine, a Selective Calcium Channel Blocker, Impair Chondrocyte Proliferation or Damage Extracellular Matrix Structures?
(Bentham Science Publ Ltd, 2019) Kaplan, Necati; Yılmaz, İbrahim; Karaarslan, Numan; Kaya, Yasin Emre; Şirin, Duygu Yaşar; Özbek, Hanefi
Background: The study aimed to investigate the effects of the active ingredient, nimodipine, on chondrocyte proliferation and extracellular matrix (ECM) structures in cartilage tissue cells. Methods: Chondrocyte cultures were prepared from tissues resected via surgical operations. Nimodipine was then applied to these cultures and molecular analysis was performed. The data obtained were statistically calculated. Results: Both, the results of the (3-(4,5 dimethylthiazol2-yl)-2,5-diphenyltetrazolium (MTT) assay and the fluorescence microscope analysis [a membrane permeability test carried out with acridine orange/propidium iodide staining (AO/PI)] confirmed that the active ingredient, nimodipine, negatively affects the cell cultures. Conclusion: Nimodipine was reported to suppress cellular proliferation; chondroadherin (CHAD) and hypoxia-inducible factor-1 alpha (HIF-1 alpha) expression thus decreased by 2.4 and 1.7 times, respectively, at 24 hrs when compared to the control group (p < 0.05). Furthermore, type II collagen (COL2A1) expression was not detected (p < 0.05). The risk that a drug prescribed by a clinician in an innocuous manner to treat a patient by relieving the symptoms of a disease may affect the proliferation, differentiation, and viability of other cells and/or tissues at the molecular level, beyond its known side effects or adverse events, should not be forgotten.
Does oseltamivir protect human chondrocyte and nucleus pulposus cells from degeneration by inhibiting senescence and proinflammation mediated by the NLRP3 inflammasome and NF-kappa B?
(Verduci Publisher, 2022) Yılmaz, İbrahim; Akalan, Hande; Öznam, K.; Karaarslan, Numan; Şirin, Duygu Yaşar; 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 alpha), 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 beta) via the nuclear factor kappa-B (NF-kappa 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 alpha, IL-18, NF-kappa B. and the NLRP3-inflammasome protein expressions using Western blotting. The a 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 alpha, IL-1 beta, NF-kappa 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.
Does transcription factor, induced by daptomycin and vancomycin, affect HIF-1?, Chondroadherin, and COL2A1?
(2018) Karaarslan, Numan; Yılmaz, İbrahim; Şirin, Duygu Yaşar; Özbek, Hanefi; Kaya, Yasin Emre; Akyuva, Yener; Doğan, Mustafa; Erdem, İlknur
Aim: 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.
Effect of naproxen on proliferation and differentiation of primary cell cultures isolated from human cartilage tissue
(Spandidos Publ Ltd, 2018) Karaarslan, Numan; Batmaz, Ahmet Güray; Yılmaz, İbrahim; Özbek, Hanefi; Çalışkan, Tezcan; Şirin, Duygu Yaşar; Ateş, Özkan
Non-steroidal anti-inflammatory drugs (NSAIDs) that are applied through oral, injectable or topical routes have been widely used in painful and inflammatory musculoskeletal diseases. The current study aimed to determine whether naproxen, an aryl acetic acid derivative with analgesic and anti-inflammatory effects, has a toxic effect on human chondrocytes. Samples containing monolayer primary chondrocyte cultures were prepared following resection from osteochondral tissues obtained from patients with gonarthrosis. Cell viability, toxicity and proliferation and levels of stage-specific embryonic antigen-1, a precursor to human prechondrocytes, were evaluated spectrophotometrically. The results from the untreated control group were compared with those of the study groups, where naproxen was administered in varying doses (1-1,000 mu M). Surface morphologies of the cells were compared using inverted light and environmental scanning electron microscopy. Treatment groups were compared by analysis of variance with Tukey's honest difference post hoc test. P<0.01 was considered to indicate a statistically significant difference. The research revealed significant changes to proliferation and differentiation of chondrocytes in all treatment groups (P<0.01). Naproxen was demonstrated to suppress chondrocyte proliferation and differentiation, which may be an important factor to consider when prescribing this medication to patients.

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