Differentially expressed genes related to lymph node metastasis in advanced laryngeal squamous cell cancers.

Understanding the molecular mechanisms and gene expression in laryngeal squamous cell carcinoma (LSCC) may explain its aggressive biological behavior and regional metastasis pathways. In the present study, patients with locally advanced LSCC tumors were examined for differential gene expression in the normal mucosa (non-tumoral mucosa), tumors and lymph node tissues. The aim was to identify possible predictive genes for lymph node metastasis. A total of 16 patients who had undergone total laryngectomy with neck dissection for advanced LSCC were randomly selected from a hospital database: Eight of the patients had lymph node metastasis (Group 1) and the other eight patients did not have metastasis (Group 2). Overall survival (OS), disease-free survival (DFS) and disease-specific survival (DSS) were analyzed. For each patient, paraffin-embedded tissue samples were collected from non-tumoral mucosa, tumoral lesions and lymph node tissues. RNA was extracted from the tissue samples and used for complementary DNA synthesis, and microarray analysis was subsequently performed on each sample. Gene expression levels were determined in each specimen, and Groups 1 and 2 were compared and statistically analyzed. The microarray results for lymph node metastasis-positive and -negative groups, indicated the differential expression of 312 genes in the lymph nodes, 691 genes in the normal mucosal tissue and 93 genes in the tumor tissue. Transgelin (TAGLN) and cofilin 1 (CFL1) were identified as possible target genes and validated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The RT-qPCR results for TAGLN and CFL1 supported the microarray data. OS, DFS and DSS times were longer in Group 2 than in Group 1 (P=0.002, 0.015 and 0.009, respectively). In addition, TAGLN and CFL1 were associated with DFS and DSS. On the basis of these results, it is suggested that TAGLN and CFL1 expression may play an important role in the pathogenesis of regional metastasis and poor prognosis in advanced LSCC.

Fabrication of a multi-layered decellularized amniotic membranes as tissue engineering constructs.

As a promising approach in tissue engineering, decellularization has become one of the mostly-studied research areas in tissue engineering thanks to its potential to bring about several advantages over synthetic materials since it can provide a 3-dimensional ECM structure with matching biomechanical properties of the target tissue. Amniotic membranes are the tissues that nurture the embryos during labor. Similarly, these materials have also been proposed for tissue regeneration in several applications. The main drawback in using amniotic membranes is the limited thickness of these materials since most tissues require a 3D matrix for an enhance regeneration. In order to prevent this limitation, here we report a facile fabrication methodology for multilayered amniotic membrane-based tissue constructs. The amniotic membranes of Wistar albino rats were first decellularized with the physical and chemical methods and utilized as scaffolds. Secondly, the prepared decellularized membranes were sutured to form a multilayered 3D structure. Within the study, 7 groups including control (PBS), were prepared based on physical and chemical decellularization methods. UV exposure and freezing techniques were used as a physical decellularization methods while hypertonic medium and SDS (sodium dodecyl sulfate) protocols were used as chemical decellularization methods. The combinations of both protocols were also used. In groups, A was the control and group B was applied just UV. In group C was applied UV and freezing. In addition to UV and freezing, in group D was applied hypertonic solution while group E was applied SDS (0.03 %). In group F was applied UV, freezing, hypertonic solution and SDS (0.03 %). In group G was applied UV, hypertonic solution, SDS (0.03 %) and freezing, respectively. Based on the histological and quantitative analyses, F and G groups were found as the most efficient decellularization protocols in rat amniotic membranes. Then, group F and G decellularized amniotic membranes were used to form scaffolds and thus-formed matrices were further characterized in vitro cell culture studies and mechanical tests. Cytotoxicity analyses performed using MTT showed a good cell viability in F and G groups scaffolds. The percentage viability rate was higher in G group (81.3 %) compared to F (75.33 %) and also cell viability in G group was found more meaningful according to p value which was obtained 0.007. Cellular adhesions after in vitro cell culture and morphology of scaffolds were evaluated by scanning electron microscopy (SEM). It was observed that the cells cultivated in equal amounts of tissue scaffolds were higher in the F compared to that observed in group G. The mechanical testing with 40 N force revealed 0.77 mm displacement in group F while it was 0.75 mm in group G. Moreover, according to force-controlled test, 2.9 mm displacement of F group and 1.2 mm displacement of G group was measured. As a result, this study shows that the multilayered decellularized amniotic membrane scaffolds support cell survival and adhesion and can form a flexible biomaterial with desired handling properties.

Microarray analysis of cartilage: comparison between damaged and non-weight-bearing healthy cartilage.

AIM: A limited healing response to focal cartilage lesions is frequently encountered in the clinical cartilage pathology. This study compares the gene expression patterns of damaged and undamaged regions of cartilage obtained from the same patient with focal cartilage lesions. The aim of this study is to provide new genes and proteins, which may be a potential future target of research. METHODS: During the autologous chondrocyte implantation (MACI) surgery, cartilage tissues (healthy non-weight bearing and Damaged-lesion side) were obtained from 10 patients with knee focal cartilage lesions. The degeneration status of the cartilage was characterized according to ICRS criteria. Whole genome microarray gene expression profiling was performed and some of the differentially regulated genes were validated with RT-PCR. RESULTS: Damaged and undamaged non-weight bearing cartilage showed distinct gene expression profiles. Genes involved in cell signaling, matrix degradation, hypoxia, and the inflammatory response showed significant up- or down-regulation. In the focal lesions, expression of genes such as HIF1α, TIMP-2, EID1, EID2, NCOA3, NBR1, SP100, and HSP90AA1 was significantly higher compared to healthy non-weight bearing cartilage from the same joint, whereas TIMP-4 was lower. CONCLUSION: The genes examined in this study differ distinctly between focal cartilage (ICRS 3-4) lesions and undamaged sites of the same joint. We believe that the data set forth in this study may be used for clinical purposes and be a guide in the development of new biological approaches for therapy.

Development of a Sequential Antibiotic Releasing System for Two-Stage Total Joint Replacement Surgery.

In recent years, there has been an increase in nanoparticle research towards creating better release systems that can maintains the effective dosage over desired periods. Conventional nanoparticle systems have not been successful for this goal. Thus, the aim of this study is to evaluate the sequential release profiles of hybrid materials, combining nanoparticles, hydrogels and bone cement, for the treatment of arthroplasty infections. In this study, Vancomycin, which is one of the most used antibiotics in orthopedics, was loaded to alginate-chitosan nanoparticles. These drug-loaded nanoparticles were dispersed in an alginate gel and the gel covered the polymethylmethacrylate bone cement. After the crosslinking of the gel around the bone cement, the sequential release profile was evaluated for 60 days in vitro. The results of the morphological, chemical characterization and encapsulations studies showed that different loadings of drugs resulted in different encapsulation efficiencies. Although the release profile from the nanoparticles was as expected, the sequential release profile of the combined system has a Fickian type release for a longer time period. In conclusion, the results indicate that combining different release systems can alter the release profile of the system.

Autologous stem cell-derived chondrocyte implantation with bio-targeted microspheres for the treatment of osteochondral defects.

BACKGROUND: Chondral injury is a common problem around the world. Currently, there are several treatment strategies for these types of injuries. The possible complications and problems associated with conventional techniques lead us to investigate a minimally invasive and biotechnological alternative treatment. Combining tissue-engineering and microencapsulation technologies provide new direction for the development of biotechnological solutions. The aim of this study is to develop a minimal invasive tissue-engineering approach, using bio-targeted microspheres including autologous cells, for the treatment of the cartilage lesions. METHOD: In this study, a total of 28 sheeps of Akkaraman breed were randomly assigned to one of the following groups: control (group 1), microfracture (group 2), scaffold (group 3), and microsphere (group 4). Microspheres and scaffold group animals underwent adipose tissue collection prior to the treatment surgery. Mesenchymal cells collected from adipose tissue were differentiated into chondrocytes and encapsulated with scaffolds and microspheres. Osteochondral damage was conducted in the right knee joint of the sheep to create an animal model and all animals treated according to study groups. RESULTS: Both macroscopic and radiologic examination showed that groups 3 and 4 have resulted better compared to the control and microfracture groups. Moreover, histologic assessments indicate hyaline-like cartilage formations in groups 3 and 4. CONCLUSION: In conclusion, we believe that the bio-targeted microspheres can be a more effective, easier, and safer approach for cartilage tissue engineering compared to previous alternatives.

Evaluation estrogen, progesteron and androgen receptor expressions in corneal epithelium in keratoconus.

PURPOSE: To evaluate the role of sex steroid hormone receptors in corneal epithelium in etiopathogenesis of keratoconus (KC). METHODS: Thirty patients with KC who were planned for corneal collagen-crosslinking and 20 patients who were planned for excimer laser for refractive errors included in this study. Corneal epitheliums were curated mechanically during surgeries. Right eyes were evaluated immunohistochemically and left eyes were evaluated by quantitative polymerase chain reaction (qPCR) to investigate estrogenα, estrogenß, progesterone and androgen receptors. RESULTS: Immunohistochemically, staining for progesterone and androgen receptors did not significantly differ between KC and control groups (p > 0.05). None of the cases had staining for estrogenα and estrogenß receptors. qPCR showed that mRNA expressions of estrogenα and androgen receptors were significantly higher in the KC group (p < 0.001). CONCLUSION: A significantly higher rate of estrogenα and androgen receptor expressions in corneal epithelium from patients with KC through qPCR supports a possible relation between KC and sex steroid hormones.

A device for the functional improvement of lagophthalmos.

A new device was tested on rabbits for the improvement of lagophthalmos, which causes dryness and irritation of the eye and may cause blindness if untreated. In the presented study, 14 rabbits were injected with local anesthetic to induce temporary facial palsy leading to lagophthalmos on one side. To provide functionality to the upper eyelids, ferromagnetic steel plates were either implanted within the eyelid or taped on the eyelid surface. The device detected blinking in the nonparalytic side and moved the anesthetized paralytic eyelid by pulling the steel plate electromagnetically. The control group (n = 5) did not wear the device, and they could not shut their paralytic eyelids. The treatment group with the external placement of the metal plate (n = 4) and the treatment group with the implant (n = 5) wore the device for artificial blinking. All animals were observed during the experiments, and blinking was recorded on digital video. The data collected from video records were analyzed to test the statistical difference of blinking between control and the treated groups. The results showed that the treatment groups could artificially move their paralytic eyelids. Furthermore, the treatment group with the implant showed a noticeable similarity in eyelid position compared with healthy (nonparalytic) eyes.

Oxidative stress and anti-oxidative defence in patients with age-related macular degeneration.

PURPOSE: To evaluate the oxidative stress status and anti-oxidative defence in patients with age-related macular degeneration (AMD). METHODS: A total of 22 patients diagnosed with AMD and 23 age-matched healthy controls were included in the present study. Serum levels of total oxidant status (TOS), total antioxidant status (TAS), total thiol status (TTS) and paraoxonase 1 (PON1) activity were investigated from samples. RESULTS: Significant increase in TOS levels were observed in sera of AMD patients (25.3 ± 12.8) compared to controls (15.0 ± 4.4). TTS (404.3 ± 55.3) and serum PON1 enzyme activities (163.0 ± 65.5) were significantly lower in AMD patients (594.0 ± 64.2) relative to control groups (252.8 ± 132.7). CONCLUSION: The results of the present study show that there is a significant increase in oxidative stress in AMD patients and significant decrease in antioxidant defence, in the total thiol level and in PON1 activity in AMD patients compared with controls. The increased oxidative stress and decreased antioxidant levels may have a synergistic role in AMD development.