Aims/Purpose: The purpose of this research was to evaluate the decellularization efficiency of various individual protocols and their combinations for bovine cornea intended for ocular surface biomaterial applications. Methods: Two fundamental decellularization protocols, namely sodium chloride (NaCl) and Ammonium & Triton X‐100 were taken as benchmark and their combinations with freeze–thaw and lyophilization, were assessed whether, the decellularization efficiency improved ( n = 3). Overall, protocols that were investigated were as follows: (1) Lyophilization followed by NaCl, (2) Freeze–Thaw followed by NaCl, (3) Lyophilization, Ammonium and Triton X‐100 (1%), (4) Ammonium and Triton X‐100 (5%), (5) Freeze–thaw, Ammonium and Triton X‐100 (1%), (6) Lyophilization, Ammonium and Triton X‐100 (5%), (7) Freeze–thaw and (8) NaCl. These methods were assessed using gel electrophoresis for qualitative DNA analysis and NanoDrop for quantitative DNA content. Efficient decellularization was achieved in the presence of DNA content below 50 ng/mg ECM dry weight and <200 bp DNA fragment length. Results: Gel electrophoresis revealed DNA content over 200 bp in groups 1, 2,7 and 8 while other groups fulfilled the criterion for efficient decellularization. DNA content as measured by NanoDrop indicated efficient decellularization in all groups, except group 7. Conclusions: Our study provides valuable insights into the effectiveness of combined and individual decellularization protocols for bovine cornea. Our findings suggest, that NaCl and its combinations with lyophilization and freeze–thaw yield in ineffective decellularization. On the other hand, Ammonium and Triton X‐100, individually or in combinations, result in favourable decellularization.

Aims/Purpose: In this study we aimed to investigate miRNAs associated with scarless wound healing in limbal‐derived stomal‐mesenchymal stem cells (hLMSC) and orbital adipose tissue‐derived stem cells (hOA‐MSC) treated individually with vitamin C and exosomes obtained after vitamin C treatment. Methods: Isolation and characterization of hLMSCs and hOA‐MSCs were performed. MTT assay was employed to determine the ideal non‐cytotoxic and proliferative concentration of vitamin C (vit C). The selected concentration was applied to the cell culture medium of hLMSCs and hOA‐MSCs (vit C‐treated) followed by procurement of secreted exosomes (vit C‐exo) and their characterization. These exosomes were then used to enrich cell culture media of naïve hLMSCs and hOA‐MSCs. miR29, miR107, miR155, and miR543 miRNAs and Transforming Growth Factor Beta 1 (TGFβ1), TGFβ3 were analysed by Real‐Time PCR in both vit C treated and vit C‐exo‐treated cells. Results: Vit C‐exo and vit C‐treated hLMSCs demonstrated slight reductions in TGFβ1 and TGFβ3 expressions compared to controls. In contrast, a modest increase in TGFβ1 and TGFβ3 levels was noted in hOA‐MSCs after vit‐C and vit C‐exo treatment. After vit C‐exo application, expression of miR155 and miR107 increased in hLMSCs and decreased in hOA‐MSCs. An opposite trend was observed with miR543 expression with an increase in hOA‐MSCs and a decrease in hLMSCs. miR29 associated with fibrosis was dramatically increased in hOA‐MSCs, but not in hLMSCs. Conclusions: In conclusion, we show that miRNA and RNA contents of treated mesenchymal stem cells may vary depending on the origin of tissue. These results suggest that vitamin C could promote a relatively more pronounced anti‐fibrotic character in hOA‐MSCs. The authors acknowledge the support from Eskisehir Osmangazi University, Scientific Research Projects (ESOGU‐BAP Grant ID: TOA‐2022‐2458).

Aims/Purpose: Melatonin is a time‐and‐concentration dependent pro and antiinflammatory agent that also regulates cell proliferation, migration, angiogenesis, fibrosis, and extracellular matrix synthesis. This study aims to assess the effect of exosomes obtained from melatonin‐treated human limbal mesenchymal stem cells (hLMSCs) on naive hLMSCs and ascertain their influence on antifibrotic and proregenerative pathways involved in corneal scarring. Methods: hLMSCs were harvested from the eye bank of Eskişehir Osmangazi University Hospital and were subsequently isolated and characterized. We then treated hLMSCs (Mel‐treated hLMSCs) with varying concentrations of melatonin followed by isolation and characterization of the procured exosomes. These exosomes were added to enrich cell culture media of naïve hLMSCs (Mel‐exo‐treated hLMSCs) to examine their antifibrotic and proregenerative effects. The expression of miR155, miR29 Transforming Growth Factor Beta 1 (TGFβ1), TGFβ3, Peroxisome Proliferator‐Activated Receptor Gamma (PPARγ), and Alpha‐Smooth Muscle Actin (α‐SMA) miRNA and genes were compared between Mel‐treated hLMSCs and Mel‐exo‐treated hLMSCs by using Real‐Time PCR. Results: TGFβ3 expression increased dramatically in response to Mel‐exo, while the increase in TGFβ1 expression was only minimal in correlation with levels of miR155 expression. Likewise, PPARγ expression was increased while an inhibition was observed in α‐SMA expression and miR29 expression in Mel‐exo‐treated hLMSCs compared to Mel‐treated hLMSCs. Conclusions: Melatonin treatment of hLMSCs resulted in expression of exosomes that could drive naïve hLMSCs in favour of antifibrotic and proregenerative effects as evidenced by higher TGFβ3 and PPARγ expressions and inhibition of α‐SMA. This approach could prove beneficial in cornea and ocular surface tissue engineering applications. The authors acknowledge the support from Eskisehir Osmangazi University, Scientific Research Projects (Grant ID: TOA‐2022‐2458).

To investigate the antiproliferative properties of exopolysaccharides (EPS) on human colorectal adenocarcinoma cell line (Caco-2) and the regulation of MUC5AC gene expression, the antiproliferative effect of EPS isolated from D36 strain was determined by MTT test and the regulation of MUC5AC gene expression was examined using Real-Time PCR. Enterococcus faecium D36 (E. faecium D36) were characterized by Ribotyping analysis. Some biochemical methodologies were preliminarily used to characterize the probiotic potential of E. faecium D36, including morphological, cultural, and physiological characteristics. EPS isolated from E. faecium D36 strain has an antiproliferative effect on Caco-2 cell line, and mucin gene (MUC5AC) expression levels decreased. These results suggest that EPS isolated from E. faecium D36 strain might be a source for a novel anticancer agent. Based on our results, it is believed that EPS obtained from E. faecium D36 can be used as a protective and therapeutic substance during the early stages of cancer, especially colon cancer. EPS affects colon cancer by reducing the invasion ability of cancer by decreasing MUC5AC expression. These findings are thought to shed light on future in vivo studies.

This study aimed to investigate the differences between the exosomal microRNA-127-5p expression profiles of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) and human synovial fluid-derived mesenchymal stem cells (hSF-MSCs) during chondrogenesis in terms of regenerative treatment of cartilage. Synovial fluid-derived mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, and human fetal chondroblast cells (hfCCs) were directed to chondrogenic differentiation. Alcian Blue and Safranin O stainings were performed to detect chondrogenic differentiation histochemically. Exosomes derived from chondrogenic differentiated cells and their exosomes were isolated and characterized. microRNA-127-5p expressions were measured by Quantitative reverse transcription PCR (qRT-PCR). Significantly higher levels of microRNA-127-5p expression in differentiated hAT-MSCs exosomes, similar to human fetal chondroblast cells, which are the control group in the chondrogenic differentiation process, were observed. hAT-MSCs are better sources of microRNA-127-5p than hSF-MSCs for stimulating chondrogenesis or in the regenerative therapy of cartilage-related pathologies. hAT-MSCs exosomes are rich sources of microRNA-127-5p and can be an essential candidate for cartilage regeneration treatments.

Melatonin has a role in the cell survival signaling pathways as a candidate for secondary stroke prevention. Therefore, in the present study, the coordination of ipsilateral and contralateral hemispheres to evaluate delayed post-acute effect of melatonin was examined on recovery of the cell survival and apoptosis after stroke. Melatonin was administered (4 mg/kg/day) intraperitoneally for 45 days, starting 3 days after 30 min of middle cerebral artery occlusion. The genes and proteins related to the cell survival and apoptosis were investigated by immunofluorescence, western blotting, and RT-PCR techniques after behavioral experiments. Melatonin produced delayed neurological recovery by improving motor coordination on grip strength and rotarod tests. This neurological recovery was also reflected by high level of NeuN positive cells and low level of TUNEL-positive cells suggesting enhanced neuronal survival and reduced apoptosis at the fifty-fifth day of stroke. The increase of NGF, Nrp1, c-jun; activation of AKT; and dephosphorylation of ERK and JNK at the fifty-fifth day showed that cell survival and apoptosis signaling molecules compete to contribute to the remodeling of brain. Furthermore, an increase in the CREB and Atf-1 expressions suggested the melatonin's strong reformative effect on neuronal regeneration. The contralateral hemisphere was more active at the latter stages of the molecular and functional regeneration which provides a further proof of principle about melatonin's action on the promotion of brain plasticity and recovery after stroke.

BACKGROUND:Thiadiazole has attracted a great deal of interest as a versatile heterocycle for the discovery and development of potent anticancer agents. Thiadiazole derivatives exert potent antitumor activity against a variety of human cancer cell lines through various mechanisms.OBJECTIVE:The goal of this work was to design and synthesize thiadiazole-based anticancer agents with anti-angiogenic activity.METHODS:N-aryl-2-[(5-(aryl)amino-1,3,4-thiadiazol-2-yl)thio]acetamides (4a-r) were synthesized via the reaction of 5-(aryl)amino-1,3,4-thiadiazole-2(3H)-thiones with N-(aryl)-2-chloroacetamides in the presence of potassium carbonate. The compounds were investigated for their cytotoxic effects on three cancer (A549, HepG2, SH-SY5Y), two normal (HUVEC and 3T3-L1) cell lines using MTT and WST-1 assays. In order to examine whether the compounds have anti-angiogenic effects or not, HUVECs were cultured on matrigel matrix to create a vascular-like tube formation.RESULTS:Compounds 4d, 4m and 4n were more effective on A549 human lung adenocarcinoma cells than cisplatin. The IC50 values of compounds 4d, 4m and 4n for A549 cell line were found to be 7.82 ± 0.4, 12.5 ± 0.22, 10.1 ± 0.52 μM, respectively when compared with cisplatin (IC50= 20 ± 0.51 μM), whilst their IC50 values for HUVEC cell line were determined as 138.7 ± 0.84, 78 ± 0.44, 177.6 ± 0.2 μM, respectively after 48 h of the treatment. The concentrations (10-20-50 μM) of compounds 4d, 4e, 4l, 4m, 4n, 4q and 4r were found to inhibit vascular like tube formation.CONCLUSION:According to their anticancer and anti-angiogenic effects, compounds 4d, 4m and 4n may be potential anticancer agents for further in vivo studies.

The complex process behind the brain topology, which has been extensively studied for the last ten years, is still unclear. Therefore, neural tissue engineering studies are needed to better understand cortical folds. With the development of 4-dimensional (4D) bioprinters using cell-loaded smart materials, a promising path has been opened in the mimicry of the neural tissue. In our study, we review the usage areas of 4D printers, which have been developing in recent years, in modelling brain tissue. As a result of development of smart materials printed with 3-dimensional (3D) printers caused emerging of 4D printers, rapidly. Smart materials can change their properties based on physical, chemical and biological stimuli, and this change can be a reversible process. Cell-loaded printed smart materials should have little effect on cell viability of both the incoming stimulus and the physical change. It is also important that the material used is non-toxic and the solvent is suitable for cell viability. On the other hand, hydrogels are frequently studied to mimic the complex neural network of neural tissue. Agents that affect the crosslinking or degree of crosslinking of hydrogels can be easily controlled and changed. In addition, studies with neural stem cells have shown that hydrogels have a supportive effect on the proliferation and maturation of neural stem cells. Since the folding time, strength and location of smart materials cannot be known precisely, it can be an advantage of 4D bioprinters as it can be controlled and studied whether the results of the stress on the cells in this region will affect other cells. It is an ideal methodology to study the effect of cortical folding on neural stem cells, especially thanks to the ease of experimental manipulations provided by 4D bioprinters. It is expected that 4D bioprinters will be adopted and rapid developments will occur in the multidisciplinary field of tissue engineering of brain tissue in the near coming years.

BACKGROUND/AIMS:Trypan blue is routinely used in cell culture experiments to distinguish between dead cells, which are labelled by trypan blue, and viable cells, which are apparently free of any staining. The assumption that trypan blue labelling is restricted to dead cells derives from the observation that rupture of the plasma membrane correlates with intense trypan blue staining. However, decades ago, trypan blue has been used to trace fluid uptake by viable macrophage-like cells in animals. These studies contributed to the concept of the reticuloendothelial system in vertebrates. Trypan blue itself does not show a fluorescence signal, but trypan blue-labelled proteins do. Therefore, intracellular localization of trypan blue-labelled proteins could give a clue to the entrance pathway of the dye in viable cells.METHODS:We used fluorescence microscopy to visualize trypan blue positive structures and to evaluate whether the bactericide, silver, enhances cellular trypan blue uptake in the brain macrophage-like cell line, BV-2. The pattern of chromatin condensation, visualized by DAPI staining, was used to identify the cell death pathway.RESULTS:We observed that silver nitrate at elevated concentrations (≥ 10 µM) induced in most cells a necrotic cell death pathway. Necrotic cells, identified by pycnotic nuclei, showed an intense and homogenous trypan blue staining. Apoptotic cells, characterized by crescent-like nuclear chromatin condensations, were not labelled by trypan blue. At lower silver nitrate concentrations, most cells were viable, but they showed trypan blue labelling. Viable cells showed a cell-type specific distribution of heterochromatin and revealed a perinuclear accumulation of bright trypan blue-labelled vesicles and, occasionally, a faint homogenous trypan blue labelling of the cytoplasm and nucleus. Amiloride, which prevents macropinocytosis by blocking the Na+ / H+ exchange, suppressed perinuclear accumulation of dye-labelled vesicles. Swelling of cells in a hypotonic solution induced an intense intracellular accumulation of trypan blue. Cells exposed to a hypotonic solution in the presence of 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), which blocks volume-regulated ion channels, prevented labelling of the cytoplasm and nucleus but did not affect labelling of perinuclear vesicles.CONCLUSION:In viable cells trypan blue-labelled vesicles indicate trypan blue uptake by macropinocytosis and trypan blue-labelled cytosol could indicate a further entry pathway for the dye, like activated volume-regulated channels. Accordingly, fluorescence microscopic analysis of trypan blue-labelled cells allows not only a discrimination between necrotic and apoptotic cell death pathway but also a discrimination between the mode of trypan blue uptake in viable cells - via pinocytosis or via activated volume-regulated ion channels - in the same preparation at the single cell level.

Objectives: Post-ischemic inflammation leads to apoptosis as an indirect cause of functional disabilities after the stroke. Melatonin maybe a good candidate for the stroke recovery because of its anti-inflammatory effects. Therefore, we investigated the effect of melatonin on inflammation in the functional recovery of brain by evaluating ipsilesional and contralesional alterations. Materials and Methods: Melatonin (4 mg/kg/day) was intraperitoneally administered into the mice from the 3rd to the 55th day of the post-ischemia after 30 min of middle cerebral artery occlusion. Results: Melatonin produced a functional recovery by reducing the emigration of the circulatory leukocytes and the local microglial activation within the ischemic brain. Overall, the expression of the inflammation-related genes reduced upon melatonin treatment in the ischemic hemisphere. On the other hand, the expression level of the inflammatory cytokine genes raised in the contralateral hemisphere at the 55th day of the post-ischemia. Furthermore, melatonin triggers an increase in the iNOS expression and a decrease in the nNOS expression in the ipsilateral hemisphere at the earlier times in the post-ischemic recovery. At the 55th day of the post-ischemic recovery, melatonin administration enhanced the eNOS and nNOS protein expressions. Conclusions: The present molecular, biological, and histological data have revealed broad anti-inflammatory effects of melatonin in both hemispheres with distinct temporal and spatial patterns at different phases of post-stroke recovery. These outcomes also established that melatonin act recruitment of contralesional rather than of ipsilesional.

Abstract Successful functional recovery from stroke is crucial in the rehabilitation of patients and mainly depends on several ischemia-induced processes, including cell survival and apoptosis. Melatonin is an interesting candidate for secondary stroke prevention due to its effect on signaling pathways. Therefore, we investigated the coordination of ipsilateral and contralateral hemispheres to evaluate delayed post-acute effect of melatonin on recovery of the cell survival and apoptosis after stroke. Melatonin was administered (4 mg/kg/day) intraperitoneally for 45 days, starting 3-day after 30-min of middle cerebral artery occlusion. The genes and proteins related to the cell survival and apoptosis were investigated by immunofluorescence, western blotting and RT-PCR techniques after behavioral experiments. Melatonin produced delayed neurological recovery by improving motor coordination, which was also reflected by enhanced neuronal survival and reduced apoptosis in the post-acute phase of stroke in both contralateral and ipsilateral hemispheres. The increase of NGF, Nrp1, c-jun; activation of AKT; and dephosphorylation of ERK and JNK at the 55th day showed that cell survival and apoptosis signaling molecules compete to contribute to the remodeling of brain. Furthermore, an increase in the CREB and Atf-1 expressions suggested the melatonin's strong reformative effect on neuronal regeneration. The contralateral hemisphere was more active at the latter stages of the molecular and functional regeneration which provides a further proof of principle about melatonin's action on the promotion of brain plasticity and recovery after stroke.

Gestational Trophoblastic Neoplasia (GTN) is a term used for a group of malignant gynecological tumors including choriocarcinoma. Low-risk neoplasias can be cured using single agents Methotrexate (MTX) and actinomycin-D(ACD), but in certain cases, decreased responsiveness and serious side effects occur. Therefore, researchers have been attempting to find new treatment modalities. One of the most popular way for increasing cancer patient survival rates is supporting treatment with adjuvant molecules or chemosensitizers. For this purpose, we investigated epigallocatechin-3-gallate (EGCG), a green tea cathecin, and Erlotinib, an EGFR tyrosine kinase inhibitor, as single agents and combined with MTX or ACD. In accordance with this, JAR (human placenta choriocarcinoma) cell line was used as an in vitro model and MTT, LDH, caspase-3 activation, RT-PCR, and Western Blot analyses were performed to investigate the effects of the test materials. Our studies demonstrate that combination of Erlotinib and EGCG with MTX and ACD decreases JAR cell proliferation and metastatic HER2 protein synthesis and increases caspase-3 activation compared to ACD orMTX alone. In addition, significant increase was observed in the apoptotic Bax gene, but no notable protein synthesis occurred in the Western Blot analysis, which suggests that combination of Erlotinib and EGCG with classical chemotherapeutics ACD or MTX may lead the JAR cells to apoptosis, but not by a mitochondrial pathway. All the results indicate that the synergetic effect of Erlotinib and EGCG with classical chemotherapeutics may help to increase patient survival rates of choriocarcinoma, but the detailed mechanism needs further investigation.

We present a case report of intramuscular autotransplantation of the parathyroid cell suspension acquired after total parathyroidectomy. A 15-yr-old female patient who had been undergoing hemodialysis due to chronic renal failure for eight yr was diagnosed with secondary hyperthyroidism and subsequently underwent total parathyroidectomy. The parathyroid cells were acquired from the resected tissues, processed through isolation and cultivation phases, and counted using a cell counter. A total of two million cells were injected into the left deltoid muscle using a 22-gauge needle. After surgery, five and 10 million cells were injected in the fifth and 12 week, respectively. The desired serum levels of parathyroid hormones and calcium were not achieved after the first two transplantations. In addition, there was no regression in the patient's symptoms. However, at four wk after the third transplantation, serum parathyroid hormone level did not decrease to <3 pg/mL, the patient was asymptomatic, and the oral treatment was stopped. Our findings indicate that this new technique is applicable because it is minimally invasive, and it can be easily repeated.

OBJECTIVES:To develop a new parathyroid allotransplant method for the treatment of permanent hypoparathyroidism. MATERIALS AND METHODS:Parathyroid cells 50 × 10(6) derived from a parathyroid hyperplasia patient were transferred to a 61-year-old patient who had thyroidectomy 17 years earlier, allowing to papillary thyroid cancer; he was admitted to our outpatient clinic with symptomatic chronic hypocalcemia. Cell isolation, cryopreservation, and culturing were conducted according to a new protocol. RESULTS:During a follow-up of 5 months, the patient had no complications that could indicate rejection, and clinical symptoms completely resolved without requiring any drug supplementation. CONCLUSIONS:Here, we report a new method, enabling fast and cost-effective parathyroid allotransplant with maintained tissue viability sufficient to treat persistent hypocalcemia.

Objectives: Parathyroid allotransplant is a valuable alternative in treating permanent hypoparathyroidism. However, it is a difficult process that requires several trained staff and advanced laboratory equipment, which makes the costs high. Here, we identify a new parathyroid allotransplant technique.Materials and Methods: After obtaining informed consent from patients, parathyroid cell suspensions obtained from 4 donors who had undergone a subtotal parathyroidectomy owing to chronic renal failure were transplanted in 10 patients with permanent hypoparathyroidism after short-term cell cultivation. Prednisolone were used as immunosuppressant for the first 10 days and discontinued thereafter.Results: Allograft function was observed in 7 patients (70%) at a mean follow-up of 12 months. Daily oral calcium and vitamin D supplementations discontinued totally in 7 patients. No major or minor complication was observed.Conclusions: Our technique is simple, fast, and has a low cost, with a 70% success rate at a mean follow-up of 12 months. It requires few staff, minimal equipment, and short-term immunosuppressant use for maintenance. The technical developments of parathyroid allotransplant, as mentioned in this study, may be important in treating permanent hypoparathyroidism.