Fatma Helvacioglu

Synovial cell culture and tissue engineering of a tendon synovial cell biomembrane

Restrictive adhesions are a common complication of tendon injury and repair in the hand, resulting in severe dysfunction. Creating a barrier between the repair sites and surrounding tissue layers may prevent adhesions. We present the first stage in the process of developing a synovial biomembrane for this purpose. Synovial cells harvested from the Achilles tendon sheath and the knee joint of a Wistar albino rat were cultured for 2 weeks in culture medium, and then impregnated into a collagen type 1 matrix for another 2 weeks. Cells originating from both tendon and synovium demonstrated cell growth and layer formation on the surfaces of the matrix 2 weeks after impregnation. Alcian blue staining using Scotts method demonstrated the presence of acidic mucopolysaccharide, indicating hyaluronic acid (HA) production. This provides indirect evidence of functioning synovial cells on the membrane. It is possible to culture synovial cells and engineer a synoviocyte-collagen membrane that synthesizes endogenous HA. Application of this biomembrane to tendon repair sites may help to prevent adhesions after tendon repairs. Evaluation of this method on in vivo models is required.

Bio-engineered synovial membrane to prevent tendon adhesions in rabbit flexor tendon model.

During tendon injuries, the tendon sheath is also damaged. This study aims to test effectiveness of engineered tendon synovial cell biomembrane on prevention of adhesions. Forty New Zealand Rabbits enrolled into four study groups. Engineered synovial sheath was produced by culturing cell suspension on fabricated collagen matrix membrane. Study groups were: tendon repair (group A), tendon repair zone covered with plane matrix (Group B), synovial suspension injection into the zone of repair over matrix (Group C), and biomembrane application (Group D). Biomechanical evaluations of tendon excursion, metacarpophalangeal and proximal interphalangeal joints range of motion, H&E and Alcian Blue with neutral red staining, and adhesion formation graded for histological assessments were studied. Ten non-operated extremities used as control. Tendon excursions and range of motions were significantly higher and close to control group for Group D, p < 0.05. Adhesion formation was not different among Groups C, D, and Control, p > 0.005. Hyaluronic acid synthesis was demonstrated at groups C and D at the zone of injury. Application of synovial cells into the tendon repair zone either by cell suspension or within a biomembrane significantly decreases the adhesion formation. Barrier effect of collagen matrix and restoration of hyaluronic acid synthesis can explain the possible mechanism of action.

Effects of alendronate and pamidronate on apoptosis and cell proliferation in cultured primary human gingival fibroblasts

Data arising from the recent literature directed the researchers to study on the degree and extent of bisphosphonate toxicity on oral mucosa in further detail. The aim of this study is to determine the half maximal inhibitory concentration of pamidronate (PAM) and alendronate (ALN) on human gingival fibroblasts in vitro using 3-[4.5-thiazol-2-yl]-2.5-diphenyltetrazolium bromide (MTT) assay and to evaluate the effects of both agents on the proliferation and apoptotic indices. Cells used in the study were generated from human gingival specimens and divided into alendronate (n = 240), PAM (n = 240), and control groups (n = 60). Based on the MTT assay results, 10−4, 10−5, 10−6, and 10−7 M concentrations of both drugs were administered and the effects were evaluated for 6, 12, 24, 48, or 72 h periods. An indirect immunofluorescence technique was used to evaluate apoptotic (anti-caspase 3) and proliferation (anti-Ki67) indices. Toxicity of both PAM and ALN was found to be the most potent at 10−4–10−5 M range. The apoptotic index of PAM group was found to be significantly higher than ALN group for all concentrations especially at 24 h incubation time (p < 0.05). The decrease in the proliferation index was found similar in first 48 h for both drugs; however, after 72 h of incubation decrease in proliferation index in PAM group was found to be significantly higher (p < 0.05). Micromolar concentrations of not only PAM but also ALN rapidly affect cells generated from human oral gingival tissue by inducing apoptosis together with inhibition of proliferation. Cytotoxic effects of both ALN and PAM on primary human gingival fibroblasts, which cause significant changes in apoptotic and proliferative indices as shown in this in vitro study, suggests that the defective epithelialization of oral mucosa is possibly a major factor on the onset of bisphosphonate-related osteonecrosis of the jaw cases.

Dose‐Dependent Immunohistochemical and Ultrastructural Changes After Oral Methylphenidate Administration in Rat Heart Tissue

Methylphenidate, more commonly known as Ritalin, is a piperidine derivative and is the drug most often used to treat attention deficit/hyperactivity disorder, one of the most common behavioural disorders of children and young adults. Our aims were to investigate dose‐dependent immunohistochemical D2 expression and ultrastructural changes of the rat heart tissue, and to demonstrate possible toxicity of the long‐term and high dose use of the methylphenidate. In this study, 27 female pre‐pubertal Wistar albino rats, divided into three different dose groups (5, 10 and 20 mg/kg) and their control groups, were used. They were treated orally with methylphenidate dissolved in saline solution for 5 days/week during 3 months. At the end of the third month, after perfusion fixation, left ventricle of cardiac tissue was removed. Paraffin, semi‐thin and thin sections were collected and immunohistochemical, terminal deoxynucleotidyl transferase‐mediated Dig‐dUTP nick end labelling assay and ultrastructural studies were performed. In conclusion, we believe that Ritalin is dose‐related affecting dopaminergic system to increase heart rhythm and contraction. Thus, this drug may cause degenerative ultrastructural changes in mitochondrial path.

Prostaglandin F receptor expression in intrauterine tissues of pregnant rats

In this investigation, we studied the expression and localization of rat prostaglandin F (FP) receptor in uterine tissues of rats on gestational Days 10, 15, 18, 20, 21, 21.5 and postpartal Days 1 and 3 using Western blotting analysis, real-time PCR, and immunohistochemistry. A high level of immunoreactivity was observed on gestational Days 20, 21, and 21.5 with the most significant signals found on Day 20. FP receptor protein was expressed starting on gestational Day 15, and a fluctuating unsteady increase was observed until delivery. Uterine FP receptor mRNA levels were low between Days 10 and 18 of gestation (p < 0.05). The transcript level increased significantly on Day 20 and peaked on Day 21.5 just before labor (p < 0.05). There was a positive correlation between FP receptor mRNA expression and serum estradiol levels (rs = 0.78; p < 0.01) along with serum estradiol/progesterone ratios (rs = 0.79; p < 0.01). In summary, we observed an increase FP receptor expression in rat uterus with advancing gestation, a marked elevation of expression at term, and a concominant decrease during the postpartum period. These findings indicate a role for uterine FP receptors in the mediation of uterine contractility at term.

Tobacco-Induced Neuronal Degeneration via Cotinine in Rats Subjected to Experimental Spinal Cord Injury

OBJECTIVES Cigarette smoke contains over 4000 chemicals including well-characterized toxicants and carcinogens, among which is cotinine. Cotinine is the principal metabolite of nicotine that has adverse affects on the microcirculation via vasoconstriction, hypoxia and the wound-healing cascade. Its impact on spinal cord injury (SCI) has not been investigated yet. The aim of the present study is to investigate the cotinine effect on SCI. METHODS 48 male Wistar rats were divided into six groups as follows: sham-control, sham-trauma, vehicle-control, vehicle-trauma, cotinine-control, and cotinine-trauma. Initially, a defined concentration of cotinine blood level was maintained by daily intraperitoneal injection of cotinine for 14 days in the cotinine groups. The concentration was similar to the cotinine dose in the blood level of heavy smokers. Only ethyl alcohol was injected in the vehicle groups during the same period. Then, SCI was performed by a Tator clip. The cotinine groups were compared with rats subjected to vehicle and sham groups by immunohistochemical biomarkers such as glial fibrillary acidic protein (GFAP) and 2,3-cyclic nucleotide 3-phosphodiesterase (CNP) expressions. Electron microscopic examination was also performed. RESULTS GFAP-positive cells were noted to be localized around degenerated astrocytes. Marked vacuolization with perivascular and perineural edema was seen in the cotinin consumption groups. These findings showed the inhibition of regeneration after SCI. Similarly, vacuolization within myelin layers was noted in the cotinine groups, which was detected through reduced CNP expression. CONCLUSION Cotinine, a main metabolite of nicotine, has harmful effects on SCI via GFAP and CNP expression. The findings of the present study support the hypothesis that tobacco causes neuronal degeneration via cotinine.

Antiproliferative and anti-apoptotic effect of astaxanthin in an oxygen-induced retinopathy mouse model

OBJECTIVE To evaluate the impact of intravitreal (IV) and intraperitoneal (IP) astaxanthin (AST) injections on neovascular development (ND), retinal morphology, and apoptotic activity in a C57BL/6J mouse model with hyperoxia-induced retinopathy (HIR). DESIGN C57BL/6J mouse model. METHODS Two negative control groups (n = 6 each; one of which received IV sterile dimethyl sulfoxide [DMSO]) of C57BL/6J-type mice were exposed to room air. The HIR groups included 36 C57BL/6J-type mice exposed to 75% ± 2% oxygen from postnatal day (PD) 7 to PD 12. On PD 12, these mice were randomized into 6 groups (n = 6 each): 2 HIR control groups (one of which received IV-DMSO), 2 IV-AST groups (10 and 100 µg/mL), and 2 IP-AST groups (0.5 and 5 mg/kg). We measured ND by counting neovascular tufts in cross sections and examined histological, ultrastructural changes via light and electron microscopy. Apoptosis was detected using terminal deoxynucleotidyl transferase-mediated nick end-labeling. RESULTS No ND was detected in the negative control groups. ND levels were not significantly different between high- and low-dose AST for either means of administration. However, ND levels were significantly lower in the AST groups, regardless of delivery, compared to the control groups. The means of delivery (IP versus IV) also yielded significant differences in ND. The incidence of mitochondrial dysmorphology and apoptosis were lower in groups receiving AST. CONCLUSIONS AST seems to suppress ND and has anti-apoptotic activity in the HIR mouse model.

Effect of creatine on rat sciatic nerve injury: a comparative ultrastructural study

AIM Creatine is an endogenous molecule synthesized in the liver, kidney and pancreas from glycine and arginine and is important for mitochondrial metabolism. It is widely used as a supplement for improving muscle mass and function for many years. As it is expected to prevent apoptosis and diminish oxidative stress, it is also studied in a number of neurodegenerative diseases for its beneficial effect in recent years. We studied the effect of creatine on the peripheral nerve injury in an experimental rat crush injury model to obtain ultrastructural evidence. MATERIAL AND METHODS Animals were randomly divided into 3 groups having 5 animals in each group. Group 1 was the control group, Group 2 the trauma group and Group 3 the trauma+creatine group. The first group served as sham control. In group 2 and group 3, sciatic nerves of the rats received crush injury using aneurysm clips. In group 3, daily 2 g/kg creatine monohydrate was administered via gavage after the trauma. Nerve samples were obtained at the 28th day after trauma for light and electron microscopic evaluation. RESULTS Our comparative analysis results suggest a possible positive effect of creatine supplement on peripheral nerve regeneration as statistical analysis revealed significant differences between group 2 and group 3. Though our finding does not represent a miracle of regenerative support, beneficial usage of creatine is documented in the present study. CONCLUSION Creatine supplement helps to diminish the harmful effects of peripheral nerve crush injury which is also supported by electron microscopy findings.

Histomorphometric and Ultrastructural Evaluation of Long-Term Alpha Lipoic Acid and Vitamin B12 Use After Experimental Sciatic Nerve Injury in Rats.

AIM To analyze the therapeutic effects of long-term alpha lipoic acid (A-LA) and vitamin B12 use via histomorphometric methods and electron microscopy in the transected sciatic nerves of rats. MATERIAL AND METHODS Forty rats were randomized into five groups (n=8/group). In group I, 1 cm segment of sciatic nerve was resected without any other intervention. In group II (sham), following right sciatic nerve transection, primary epineurial anastomosis was performed by placing the edges of the nerve end-to-end. In group III (saline), after right sciatic nerve transection, the ends of the nerves were brought together and closed after application of intraperitoneal physiologic saline. In group IV, 2 mg/kg of alpha lipoic acid and in group V, 2 mg/kg of vitamin B12 was administered intraperitoneally before surgical intervention. RESULTS Histomorphometric and electron microscopic analyses revealed that vitamin B12 did not prevent structural changes, abnormal myelination and g-ratio deviations regarding the functional aspects of the sciatic nerve. Alpha lipoic acid was more effective in restructuring the histomorphometric and structural aspects of the nerve with more myelinated fibers with optimal values (0.55-0.68) than vitamin B12 groups, in which the number of myelinated nerve fibers significantly decreased at optimal intervals (0.55-0.68). CONCLUSION A-LA administration following peripheral nerve transection injury is more effective in promoting nerve healing regarding the structural aspects of the sciatic nerve compared to vitamin B12 and also myelination of nerve fibers by increasing g-values.

Ultrastructural Investigation of Parietal Cells in Fasting-After Fasting and the Effects of Histamin and Gastrin

Objective: The aim of this study is to determine the ultrastructural effect of fasting and applications of histamine and gastrin after fasting in gastric parietal cell. Methods: In this study, nine groups were composed of Swiss albino male rats. Group 1: Control, group 2: fasting for 12 hours, group 3: 12 hours fasting +1hour histamine, group 4: 12 hours fasting + 3min. gastrin, group 5: 12 hours fasting + 3min. DMSO (solvent of gastrin), group 6: 12 hours fasting + 7min. gastrin, group 7: 12 hours fasting + 7min. DMSO, group 8: 12 hours fasting + 15min. gastrin, group 9: 12 hours fasting + 15min. DMSO. At the end of the study time, the tissue samples were passed through routine electron microscopic preparation methods and tissues were embedded in araldite CY212 kit. Thin sections were evaluated on Carl Zeiss EM 900 electron microscope. Results: It was determined in the executed research, by using the electron microscope, that tubulovesicular structures were increased in the fasting group independently of the control group, vacuolar structures were also observed in patches and histamine application did not reveal any difference except for mitochondrial differences in small amounts. However, it was determined that gastrin application increased the cellular degeneration parallel to increasing duration. This transformation was especially observed in nuclear structures, mitochondria and intracytoplasmic canaliculi in the cells.