Duygu Yasar Sirin

Are the leading drugs against Staphylococcus aureus really toxic to cartilage

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 exposed to 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.

Are biological agents toxic to human chondrocytes and osteocytes

PurposeThe aim of the present study is to investigate the effects of biological agents (BAs) on human chondrocytes and osteocytes in vitro.MethodsPrimary 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.ResultsRituximab and adalimumab were the least toxic agents to chondrocytes, whereas adalimumab and etanercept were to osteocytes.ConclusionDuring 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.

A practical way to prepare primer human chondrocyte culture

Biological cartilage repair is one of the most 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.

Are chondrocytes damaged when rheumatologic inflammation is suppressed

Abstract 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 48 h. 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.

In vitro analysis of a novel controlled release system designed for intratympanic administration of N-acetylcysteine: a preliminary report.

The aim of this in-vitro experimental study was to design a novel drug delivery system that may permit controlled release of N-acetylcysteine (NAC) following intratympanic administration. The system was composed of two different solutions that attained a hydrogel form within seconds after getting into contact with each other. The authors performed swelling, pH and temperature tests and analysis of controlled release of NAC from this novel controlled release system. For the structure and porosity analysis of the hydrogel, an environmental scanning electron microscope (SEM) was used. The diameter of designed hydrogel showed an increase when pH was increased. In addition, in comparison to acidic values, the pore diameter of the hydrogel increased significantly especially in physiological level. The increase in the pore diameter was also directly proportional to the increase in temperature. Spectrophotometric analysis showed that the amount of NAC released into the medium was statistically significant (p=0.038, t=-2.18, 95% CI; DF: 27). SEM analysis of the samples revealed a smooth surface topography and numerous porous structures. The authors are of the opinion that the designed hydrogel may be used as an alternative method for intratympanic delivery of NAC for otoprotective purposes. The disadvantages of intratympanic injection of the drug in its liquid form, including leakage through eustachian tube, restraining the patient in an uncomfortable position, necessity for repetitive injections and dose dependent inflammation of the middle ear epithelium, may also be avoided. Further in vivo studies should be conducted to assess its tolerability and effectivity.

The association between different molecular weights of hyaluronic acid and CHAD, HIF-1α, COL2A1 expression in chondrocyte cultures

The aim of the present study was to investigate the effects of three different formulations of hyaluronic acid (HA): Low molecular weight (MW) Sinovial One®, medium MW Viscoplus® and high MW Durolane®, on chondrocyte proliferation and collagen type II (COL2A1), hypoxia-inducible factor 1α (HIF-1α) 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-1α 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-1α 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.

Effect of naproxen on proliferation and differentiation of primary cell cultures isolated from human cartilage tissue

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 µM). Surface morphologies of the cells were compared using inverted light and environmental scanning electron microscopy. Treatment groups were compared by analysis of variance with Tukeys 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.

Are We Economically Efficient Enough to Increase the Potential of in Vitro Proliferation of Osteoblasts by Means of Pharmacochemical Agents

Background: The aim of this study was to test the necessity of using expensive and unaccesible pharmacological-chemical agents in the proliferation of bone tissue cultures and in the induction of mineralized matrix formation to increase the osteogenic effect. Methods: For this purpose, human primary cell cultures were prepared and then divided into two groups. Whereas the cells in group I were fed with an osteoblast stimulator medium containing Dulbecco’s Modified Eagle Medium (DMEM) and β-glycerophosphate, the cells in group II were fed with DMEM containing dexamethasone and 2-phospho-L-ascorbic acid trisodium salt. Both groups were evaluated in terms of viability, toxicity, and proliferation and then compared in terms of cell surface morphology through inverted light and environmental scanning electron microscopy. In addition to immunoflow cytometric analyses, the effects of alkaline phosphatase activities were evaluated using the spectrophotometric method to examine the osteoblastic activities. Costs were calculated in the currency of the European Union (Euros). The Tukey Honestly Significant Difference test was used to reach the statistical evaluation of the data after the analysis of variance. Results: It was reported that the level of the alkaline phosphates was higher in group I compared to group II. It was observed that the surface morphology quality, the number of living cells, and proliferation were higher in group II and that the results were deemed statistically significant. Conclusion: It was found that the 2-phospho-L-ascorbic acid trisodium salt and dexamethasone mixture was as effective as the expensive commercial kits on the osteogenic effect on human primary bone tissue.

Are radio-contrast agents commonly used in discography toxic to the intact intervertebral disc tissue cells?

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.

Evaluation of the effects of pregabalin on chondrocyte proliferation and CHAD, HIF-1α, and COL2A1 gene expression

Introduction The aim of the present study is to investigate the effects of pregabalin (PGB) on chondrocyte proliferation and collagen type II (COL2A1), hypoxia-inducible factor 1-α (HIF-1α), and chondroadherin (CHAD) gene expression in osteoarthritic chondrocytes. Material and methods Standard primary chondrocyte cultures were prepared using osteochondral tissues that were surgically obtained from 6 patients with gonarthrosis. Cell morphology was evaluated using an inverted microscope, and cell death and proliferation were determined through MTT analysis, which was confirmed by AO/PI staining and statistically evaluated. The expression levels of CHAD, COL2A1, and HIF-1α genes were assessed using gene-specific TaqMan Gene Expression Assays. Results MTT analyses showed that PGB administration did not have a negative or toxic effect on cell viability and proliferation in cultured chondrocytes (p < 0.001), but in our morphological evaluation extracellular matrix development was observed to be weaker in cultures treated with PGB. After 24 h of treatment, COL2A1, HIF-1α, and CHAD gene expression decreased in the groups to which PGB was applied compared to gene expression before the experiment (at 0 h); at 48 h, CHAD and HIF-1α expression increased to the same level as the control group, but the expression of COL2A1 continued to decrease. Conclusions Further studies need to be conducted with more participants to prove that there is a negative correlation between extracellular matrix formation and PGB administration. Our preliminary data show that even at low doses and over short-term administration, PGB may affect chondrocyte cells at the gene-expression level.