Elif Guzel

Three-Dimensional Ingrowth of Bone Cells Within Biodegradable Cryogel Scaffolds in Bioreactors at Different Regimes

Three-dimensional cell ingrowth within biodegradable cryogel scaffolds made of cross-linked 2-hydroxyethyl methacrylate (HEMA)-lactate-dextran with interconnected macropores was studied in bioreactors at different regimes (static, perfusion, and compression-perfusion). An osteoblast-like cell line (MG63) was used in these studies. The samples taken after selected times from the bioreactors were examined by microscopy techniques (light, SEM, TEM, and laser scanning confocal). The cell culture conditions were found to have a significant impact not only on the cell morphology, such as the extent of cell attachment and ingrowth, but also on cellular activities. Dynamic conditions (perfusion and/or compression) greatly improved cell ingrowth and extracellular matrix (ECM) synthesis. Alkaline phosphatase activity results confirmed the positive effect of dynamic conditions on bone cells.

Focused RF hyperthermia using magnetic fluids

Heat therapies such as hyperthermia and thermoablation are very promising approaches in the treatment of cancer. Compared with available hyperthermia modalities, magnetic fluid hyperthermia (MFH) yields better results in uniform heating of the deeply situated tumors. In this approach, fluid consisting of superparamagnetic particles (magnetic fluid) is delivered to the tumor. An alternating (ac) magnetic field is then used to heat the particles and the corresponding tumor, thereby ablating it. However, one of the most serious shortcomings of this technique is the unwanted heating of the healthy tissues. This results from the magnetic fluid diffusion from the tumor to the surrounding tissues or from incorrect localization of the fluids in the target tumor area. In this study, the authors demonstrated that by depositing appropriate static (dc) magnetic field gradients on the alternating (ac) magnetic fields, focused heating of the magnetic particles can be achieved. A focused hyperthermia system was implemented by using two types of coils: dc and ac coils. The ac coil generated the alternating magnetic field responsible for the heating of the magnetic particles; the dc coil was used to superimpose a static magnetic field gradient on the alternating magnetic field. In this way, focused heating of the particles was obtained in the regions where the static field was dominated by the alternating magnetic field. In vitro experiments showed that as the magnitude of the dc solenoid currents was increased from 0 to 1.8 A, the specific absorption rate (SAR) of the superparamagnetic particles 2 cm apart from the ac solenoid center decreased by a factor of 4.5, while the SAR of the particles at the center was unchanged. This demonstrates that the hyperthermia system is capable of precisely focusing the heat at the center. Additionally, with this approach, shifting of the heat focus can be achieved by applying different amounts of currents to individual dc solenoids. In vivo experiments were performed with adult rats, where magnetic fluids were injected percutaneously into the tails (with homogeneous fluid distribution inside the tails). Histological examination showed that, as we increased the dc solenoid current from 0.5 to 1.8 A, the total burned volume decreased from 1.6 to 0.2 cm3 verifying the focusing capability of the system. The authors believe that the studies conducted in this work show that MFH can be a much more effective method with better heat localization and focusing abilities.

Tissue responses to novel tissue engineering biodegradable cryogel scaffolds: An animal model

Biodegradable macroporous cryogels with highly open and interconnected pore structures were produced from dextran modified with oligo L-lactide bearing hydroxyethylmethacrylate (HEMA) end groups in moderately frozen solutions. Tissue responses to these novel scaffolds were evaluated in rats after dorsal subcutaneous implantation, iliac submuscular implantation, auricular implantation, or in calvarial defect model. In no case, either necrosis or foreign body reaction was observed during histological studies. The cryogel scaffolds integrated with the surrounding tissue and the formation of a new tissue were accompanied with significant ingrowth of connective tissue cells and new blood vessels into the cryogel. The tissue responses were significantly lower in auricular and calvarial implantations when compared with the subcutanous and the submuscular implantations. The degradation of the scaffold was slower in bone comparing to soft tissues. The biodegradable cryogels are highly biocompatible and combine extraordinary properties including having soft and elastic nature, open porous structure, and very rapid and controllable swelling. Therefore, the cryogels could be promising candidates for further clinical applications in tissue regeneration.

Bidirectional Interaction Between Unfolded-Protein-Response Key Protein HSPA5 and Estrogen Signaling in Human Endometrium

The human endometrium is a dynamic tissue that undergoes cyclic changes under the influence of steroid hormones as well as numerous local paracrine and autocrine factors. Heat shock 70 kDa protein (HSPA5; also known as GRP78/BiP), a molecular chaperone within the endoplasmic reticulum, plays crucial roles in normal cellular processes as well as in stress conditions, in which it is a central regulator for the unfolded protein response (UPR). We hypothesized that HSPA5 expression level is variable throughout the menstrual cycle in human endometrium and that estrogen signaling cross-talks with UPR signaling by interacting with HSPA5. HSPA5 expression throughout the menstrual cycle was evaluated in vivo in normal human endometrium. Using in vitro techniques, we then assessed the bidirectional regulation of HSPA5 and estrogen signaling in human endometrial glandular (Ishikawa) and stromal cells (ESC). HSPA5 immunoreactivity in endometrial glandular and stromal cells was cycle-dependent, and was significantly higher in phases of the menstrual cycle when estradiol (E2) levels are known to be the lowest compared with the rest of the cycle (P < 0.001). E2 did not affect HSPA5 expression after 8–24 h incubation in Ishikawa cells and ESC in vitro. However, tunicamycin-induced HSPA5 expression was significantly lowered in these cells when pretreated with E2 (P < 0.01 and P < 0.05, respectively). On the other hand, tunicamycin decreased E2 up-regulated alkaline phosphatase activity (P < 0.001). In conclusion, there is cycle-dependent HSPA5 expression with a possible inverse correlation between HSPA5 expression and E2 levels in human endometrium. We suggest that estrogen signaling cross-talks with the UPR cascade by interacting with HSPA5, as supported by our in vitro findings.

A 6-month in vivo study of polymer/mesenchymal stem cell constructs for cranial defects

Two biodegradable polymers, poly(L-lactide) and poly(ε-caprolactone) were blended (50/50) and used to produce polymeric scaffolds by the dual porogen approach using a salt leaching technique to create pores within the matrix, while supercritical-CO 2 treatment was used to enhance the interconnectivity and to remove impurities from synthesis steps. The scaffolds were highly porous (porosity >90%) with interconnected pore morphologies. These biodegradable scaffolds were evaluated in Sprague Dawley rats for osteoconductive properties over a 6-month period. Bone specimens were analyzed after 1, 3, and 6 months, for bone healing and tissue response. The cortical bone remodeling by controlled osteoblastic and osteoclastic activities as well as the bone marrow elements recovery were semi-quantitatively examined for each group. Excellent integration and biocompatibility behavior was observed in all groups. No adverse tissue responses were observed.

Endoplasmic Reticulum Stress and Homeostasis in Reproductive Physiology and Pathology

The endoplasmic reticulum (ER), comprises 60% of the total cell membrane and interacts directly or indirectly with several cell organelles i.e., Golgi bodies, mitochondria and proteasomes. The ER is usually associated with large numbers of attached ribosomes. During evolution, ER developed as the specific cellular site of synthesis, folding, modification and trafficking of secretory and cell-surface proteins. The ER is also the major intracellular calcium storage compartment that maintains cellular calcium homeostasis. During the production of functionally effective proteins, several ER-specific molecular steps sense quantity and quality of synthesized proteins as well as proper folding into their native structures. During this process, excess accumulation of unfolded/misfolded proteins in the ER lumen results in ER stress, the homeostatic coping mechanism that activates an ER-specific adaptation program, (the unfolded protein response; UPR) to increase ER-associated degradation of structurally and/or functionally defective proteins, thus sustaining ER homeostasis. Impaired ER homeostasis results in aberrant cellular responses, contributing to the pathogenesis of various diseases. Both female and male reproductive tissues undergo highly dynamic cellular, molecular and genetic changes such as oogenesis and spermatogenesis starting in prenatal life, mainly controlled by sex-steroids but also cytokines and growth factors throughout reproductive life. These reproductive changes require ER to provide extensive protein synthesis, folding, maturation and then their trafficking to appropriate cellular location as well as destroying unfolded/misfolded proteins via activating ER-associated degradation mediated proteasomes. Many studies have now shown roles for ER stress/UPR signaling cascades in the endometrial menstrual cycle, ovarian folliculogenesis and oocyte maturation, spermatogenesis, fertilization, pre-implantation embryo development and pregnancy and parturition. Conversely, the contribution of impaired ER homeostasis by severe/prolong ER stress-mediated UPR signaling pathways to several reproductive tissue pathologies including endometriosis, cancers, recurrent pregnancy loss and pregnancy complications associated with pre-term birth have been reported. This review focuses on ER stress and UPR signaling mechanisms, and their potential roles in female and male reproductive physiopathology involving in menstrual cycle changes, gametogenesis, preimplantation embryo development, implantation and placentation, labor, endometriosis, pregnancy complications and preterm birth as well as reproductive system tumorigenesis.

Stem cell suspension injected HEMA-lactate-dextran cryogels for regeneration of critical sized bone defects

Abstract HEMA-Lactate-Dextran cryogel scaffolds were produced by cryogelation. Mesencyhmal stem cells (MSC) were isolated from rat bone marrow. Critical sized cranial bone defects were created in rat cranium. Stem cells were injected inside the macropores of the cryogel scaffolds prepared from HEMA-Lactate-Dextran possessing the same dimensions with the defect and placed in the cranial bone. The cryogels placed in the defect without stem cells served as control. After selected time intervals the experimental sites were removed from the animals and new bone formation and tissue integration were investigated by histological analysis. The in vivo results exhibited osseous tissue integration within the implant and mineralized functionally stable bone restoration of the cranial defects. Tissue formation started in the macrospores of the scaffold starting from periphery to the center. A significant ingrowth of connective tissue cells and new blood vessels allowed new bone formation. Histological data demonstrated that new bone per total defect area ratio, were not significantly different in “scaffold-stem cells” group compared to that of “scaffold only” group on all time points. However, the blood vessel density was significantly higher in “scaffold-stem cells” group comparing to that of the “scaffold only” group on day 30. “Scaffold-stem cells” given group gave better tissue response score when compared to “scaffold only” group on day 180.

Distribution of inflammatory cells, adhesion molecules, intermediate filaments, and chemokine receptors in subgroups of nasal polyp patients.

Background The pathogenesis of nasal polyps (NPs) is incompletely understood. The aim of this study was to investigate the distribution of inflammatory cells, adhesion molecules, intermediate filaments, and chemokine receptors in subgroups of NP patients. Methods In total, 35 patients were enrolled (group 1, 10 patients with Samter syndrome; group 2, 10 patients with diffuse polyposis without signs of Samter syndrome; group 3: 5 patients with solitary nasal polyps; group 4, 10 controls). Immunohistochemical staining was performed for CD105, CD106, CD62E, CD4, CD8, CXCR4, CD147, CD90, CD104, BF45, vimentin, pancytokeratin, and muscle-specific actin (MSA) in all patients’ specimens. Results Expression of CD4, CD8, and CD106 were similar between the groups. Number of patients expressing CD4 in groups 1, 2, and 3 were higher than the controls. Number of patients expressing CD8 antigen were significantly higher in all three groups than in the control group. Expression of CD147 in groups 3 and 4 was significantly higher than in groups 1 and 2. CD98 expression was higher in groups 1, 2, and 3 than in group 4. The number of patients expressing vimentin in groups 1, 2, and 3 was significantly higher than in group 4. Immunostaining for pancytokeratin was positive in all patients. Conclusion In conclusion, inflammatory cell, adhesion molecule, intermediate filament, and chemokine receptor profiles in nasal polyps differ among different patient groups and control subjects. Additional specific immunohistochemical studies are necessary for development of more specific immunotherapies.

Genistein inhibits cell proliferation and stimulates apoptosis in human coronary artery endothelial cells.

Background/Aims: Isoflavone genistein is a plant-derived compound structurally similar to estradiol, which behaves weakly estrogenic or anti-estrogenic in a cell- and concentration-dependent manner. Genistein has been hypothesized to have beneficial effects on vascular diseases, although the mechanism has been unclear. Here, we investigated whether genistein may play a role in atherogenesis by regulating human coronary artery endothelial cell (HCAEC) survival. Methods: HCAECs obtained from 48- to 53-year-old women (n = 3) were used and immunocytochemistry, cell proliferation assay and apoptosis assay were carried on HCAECs treated by genistein. Results: Immunocytochemistry confirmed that HCAECs in culture express predominantly ESR2. Cell proliferation assay revealed that following 72 h of genistein treatment, HCAEC proliferation decreased in a concentration-dependent (10-10 to 10-6M) manner compared to control (p < 0.01). The anti-proliferative effect of genistein is inhibited by estradiol. Genistein (10-8M) also induced a time-dependent increase in the number of apoptotic HCAECs after 24-, 48- and 72-hour treatments as detected by TUNEL and morphological analyses. Conclusion: These findings suggest that genistein acts as an anti-proliferative agent on HCAECs. The anti-proliferative and proapoptotic effects of genistein on vascular cells underlie the proposed anti-atherogenic and cardioprotective role of genistein.

The mRNA-binding protein HuR is regulated in the menstrual cycle and repressed in ectopic endometrium.

Cytokines modulate turnover of the endometrium during the menstrual cycle and contribute to the pathogenesis of endometriosis. Gene expression for cytokines is often regulated by proteins that bind to adenosine- and uridine-rich elements (AREs) in their transcripts to stabilize or destabilize bound messenger RNAs (mRNAs). HuR/ELAVL1 is an RNA-binding protein that stabilizes ARE-containing mRNAs. We hypothesized that HuR might play a role in regulating cytokine expression during the menstrual cycle and in endometriosis and characterized the expression and regulation of HuR in eutopic and ectopic human endometrium. Tissue sections obtained from normal (n = 23) and ectopic (n = 16) endometrium were immunostained for HuR, and staining intensity was evaluated by HSCORE. Cultured stromal cells isolated from normal endometrium were treated with vehicle, estradiol (E2), progesterone (P), E2 + P, tumor necrosis factor-α (TNF-α), and interleukin 1β (IL-1β) for 24 hours, and HuR expression was determined by Western blot. HuR immunoreactivity was significantly lower in the early proliferative and late secretory phases (157.5 ± 11.08 and 190.0 ± 15.2, respectively), compared to the mid-late proliferative (270.0 ± 8.0) and early-mid secretory phases (256.6 ± 20.2; P < .01, analysis of variance [ANOVA]). Furthermore, HuR expression was significantly lower in ectopic endometrial cells compared to normal endometrium in mid-late proliferative and early-mid-secretory phases (P < .01). Estrogen, P, or cytokines did not alter HuR expression in cultured endometrial stromal cells. Increased HuR levels in the mid-menstrual phases are likely to contribute to reduced mid-cycle cytokine expression and enhanced cellular survival in eutopic endometrium. In ectopic endometrium, elevated cytokine levels associated with endometriosis likely reduce HuR expression.