Decellularized Ear Tissues as Scaffolds for Stem Cell Differentiation

Permanent sensorineural hearing loss is a major medical problem and is due to the loss of hair cells and subsequently spiral ganglion neurons in the cochlea. Since these cells lack the capacity of renewal in mammals, their regeneration would be an optimal solution to reverse hearing loss. In other tissues, decellularized extracellular matrix (ECM) has been used as a mechanical and biochemical scaffold for the induction of stem and other cells toward a target tissue phenotype. Such induced cells have been used for tissue and organ transplants in preclinical animal and human clinical applications. This paper reports for the first time the decellularization of the cochlea and identification of remaining laminin and collagen type IV as a first step in preparing an ECM scaffold for directing stem cells toward an auditory lineage. Fresh ear tissues were removed from euthanized mice, a rat and a human and processed for decellularization using two different detergent extraction methods. Cochleas were imaged with scanning thin-sheet laser imaging microscopy (sTSLIM) and brightfield microscopy. Detergent treatment of fresh tissue removed all cells as evidenced by lack of H&E and DNA staining of the membranous labyrinth while preserving components of the ECM. The organ of Corti was completely removed, as were spiral ganglion neurons, which appeared as hollow sheaths and tubes of basal lamina (BL) material. Cells of the stria vascularis were removed and its only vestige left was its laterally linking network of capillary BL that appeared to “float” in the endolymphatic space. Laminin and type IV collagen were detected in the ECM after decellularization and were localized in vascular, neural and epithelial BL. Further work is necessary to attempt to seed neural and other stem cells into the decellularized ECM to hopefully induce differentiation and subsequent in vivo engraftment into damaged cochleas.     

Neurological symptoms in essential thrombocythemia: impact of JAK2V617F mutation and response to therapy

Patients with essential thrombocythemia (ET) often suffer from neurological symptoms (NS) not ever resulting from previous thrombotic cerebral events (TCE). We reported NS occurred in 282 patients, in order to identify the factors influencing ET‐related NS in the absence of TCE, and the response to therapy. Overall, 116 of 282 patients (41%) presented NS; 101 of them (87%) reported subjective transient and fluctuating NS, without concurrent TCE, which we defined as ET‐related NS, by frequency: cephalalgia, chronic paresthesias, dizziness or hypotension, visual disturbances, and tinnitus. In univariate analysis, ET‐related NS resulted more frequently in young people (P = 0.017) and in females (P = 0.025). We found a higher prevalence of JAK2V617F mutation in ET‐related NS patients (P = 0.021). In multivariate analysis, gender (P = 0.024) and JAK2V617F mutation (P = 0.041) remained significantly associated with the development of ET‐related NS, with a risk of about four times higher for JAK2V617F‐mutated patients (OR = 3.75). Ninety‐seven of 101 patients with ET‐related NS received an antiplatelet (AP) agent at the time of NS, whereas only selected high‐risk ET‐related NS patients were treated with a cytoreductive drug, according to the published guidelines and similarly to patients without NS. We observed that only 32 of 97 (33%) patients with ET‐related NS achieved a complete response after AP treatment. Among the 65 non‐responder patients, 36 (55.4%) improved NS after the introduction of cytoreductive therapy; therefore, the addition of cytoreductive treatment should be considered in this setting.     

C-reactive protein,platelets, and patent ductus arteriosus

The association between inflammation, platelets, and patent ductus arteriosus (PDA) has not been studied so far. The purpose of this study was to evaluate whether C-reactive protein (CRP) is related to low platelet count and PDA. This was a retrospective study of 88 infants with a birth weight ≤1500 g and a gestational age ≤30 weeks. Platelet count, CRP, and an echocardiogram were assessed in all infants. The subjects were matched by sex, gestational age, and birth weight. Differences were compared using the χ², t-test, or Mann–Whitney U-test, as appropriate. Significant variables were entered into a logistic regression model. The association between CRP and platelets was evaluated by correlation and regression analysis. Platelet count (167 000 vs. 213 000 µl^?1, p = 0.015) was lower and the CRP (0.45 vs. 0.20 mg/dl, p = 0.002) was higher, and the platelet count correlated inversely with CRP (r = ?0.145, p = 0.049) in the infants with vs. without PDA. Only CRP was independently associated with PDA in a logistic regression model (OR 64.1, 95% confidence interval 1.4–2941, p = 0.033).     

Age-related differences in distractor interference on line bisection

Using a bisection paradigm, we investigated age-related differences in susceptibility to distractor interference. Older and younger participants were asked to bisect a horizontal line flanked by a pair of distractors, placed in either left or right hemispace. The results showed that (1) in both groups the distractors interfered with line bisection so that the localization of subjective midpoint was selectively shifted away from their position; (2) the shifting of subjective midpoint was greater in the older than in the younger group when the distractors were placed in the left hemispace. We suggest that the increase of the bisection bias in the older group depends on changes in attentional mechanisms involved in inhibiting irrelevant information.     

Effect of a one‐step self‐etch adhesive on endogenous dentin matrix metalloproteinases

Degradation of the hybrid layer created in dentin by dentin adhesives is caused by enzyme activities present within the dentin matrix that destroy unprotected collagen fibrils. The aim of the present study was to evaluate the effect of a one‐step self‐etch adhesive system on dentinal matrix metalloproteinases 2 and 4 (MMP‐2 and MMP‐9, respectively) using in situ zymography and an enzymatic activity assay. The null hypothesis tested was that there are no differences in the activities of dentinal MMPs before and after treatment with a one‐step adhesive system. The MMP‐2 and MMP‐9 activities in dentin treated with the one‐step adhesive, Adper Easy Bond, were quantified using an enzymatic activity assay system. The MMP activities within the hybrid layer created by the one‐step adhesive tested were also evaluated using in situ zymography. The enzymatic assay revealed an increase in MMP‐2 and MMP‐9 activities after treatment with adhesive. In situ zymography indicated that gelatinolytic activity is present within the hybrid layer created with the one‐step self‐etch adhesive. The host‐derived gelatinases were localized within the hybrid layer and remained active after the bonding procedure. It is concluded that the one‐step self‐etch adhesive investigated activates endogenous MMP‐2 and MMP‐9 with the dentin matrix, which may cause collagen degradation over time.     

Atomic force microscopy for biomechanical and structural analysis of human dermis: A complementary tool for medical diagnosis and therapy monitoring

Skin mechanical properties are usually measured considering the entire skin thickness and very little is known about the mechanical behaviour of individual skin layers. We propose atomic force microscopy (AFM) as a tool to quantify nanoscale changes in the biomechanical properties and ultrastructure of human papillary dermis exposed to different mechanical and physical stimuli. Samples from 3 human skin biopsies were studied: one stretched by obesity, one subjected to a high level of sun exposure and normal skin as control. Slices of the papillary dermis layer were harvested at controlled depths from each skin biopsy and 25 μm² areas of each slice were imaged and D‐periodicity of collagen fibres measured by AFM, together with their stiffness. Standard histological analysis was also carried out to correlate biochemical properties and their distribution with stiffness and topography. We obtained similar stiffness values between the sample affected by obesity and the control sample at any depth level into the dermis, while the sun‐exposed sample presented a significantly lower stiffness. Additionally, all samples presented an increase in the stiffness at higher depths into the papillary dermis layer. Collagen fibres close to the epidermis of sample affected either by obesity and sun exposure—the former even more than the latter—are thicker and present a larger D‐period than those in the control sample. Our results open the possibility to use structural and mechanical analysis based on AFM as a complementary tool for medical diagnosis and therapy monitoring.