An in vitro priming step increases the expression of numerous epidermal growth and migration mediators in a tissue-engineering construct.

An FDA-approved, prototypic, living, bilayered skin construct (BSC) has been used for non-healing wounds. Using this particular construct as proof of principle, we hypothesized that an in vitro 'priming' step may enhance its repertoire of expression of key mediators and genes. The priming step used here was incubation in Dulbecco's modified Eagle's medium (DMEM) for 24 h at 37°C and 5% CO2 , with or without construct meshing. Microarray and ingenuity pathway analysis (IPA) showed that >1000 genes were overexpressed by the priming step, including interleukin 6 (IL-6), which plays important roles in wound healing. Genes highly overexpressed by priming were those involved in epidermal proliferation and migration. Quantitative real-time PCR (qRT-PCR), immunostaining and western blots verified the results. An epiboly assay (epidermal migration over dermis) showed that BSC epiboly was inhibited by IL-6 neutralizing antibody. Back wounds of nude mice were treated with primed or control BSCs for 3 days prior to harvesting; primed BSCs showed a significantly (p = 0.006) greater level of epidermal migration vs unprimed. Our study demonstrates that an in vitro priming step induces wound healing-related genes in the BSC, leading to a construct that could prove more effective in stimulating wound healing. Copyright © 2014 John Wiley & Sons, Ltd.

Increasing maternal body mass index during pregnancy increases neonatal intensive care unit admission in near and full-term infants.

BACKGROUND: Obesity is becoming an increasingly commonplace health problem. Obesity during pregnancy is important because the condition adversely affects not only the mother, but also the developing fetus and the newborn. OBJECTIVE: The primary objective of this study was to evaluate the association between maternal body mass index (mBMI) at the time of delivery and neonatal intensive care unit (NICU) admission of offspring and to analyze the role of possible confounding variables that are often associated with obesity. Comorbidities, such as gestational diabetes mellitus (DM), hypertension (HT) and/or pre-eclampsia (PEC), are more common in more obese mothers, as is a higher association of obesity among non-Caucasian patients. METHODS: Using a retrospective cohort design, 1736 mothers and their singleton live-born at ≥35 weeks' gestation were analyzed for mBMI, maternal conditions of DM, HT and/or PEC, and whether NICU care was required and the reason for NICU admission. RESULTS: NICU admission rate was significantly associated with maternal obesity. In comparing women with mBMI < 30 versus mBMI ≥ 30, OR was 1.39 (p = 0.045); OR increased to 1.76 (p = 0.006) in comparing patients with mBMI ≥ 35. mBMI was significantly associated with an increased rate of maternal DM, HT and PEC (p < 0.05 each); however, NICU admission rate was not correlated with DM, HT or PEC. The relationship between NICU admission and mBMI was significant in Caucasian mothers versus a borderline significance in African-American mothers (p = 0.035 versus p = 0.05). After controlling for neonatal hypoglycemia (NH) as the reason for admission to the NICU, no mBMI-NICU association persisted. The rate of infants with NH increased in higher mBMI groups, independent of maternal DM diagnosis. CONCLUSION: This study demonstrated a significant association between higher mBMI groups and NICU admissions independent of diagnosis of maternal comorbidities. However, accounting for NH eliminating this association suggests a pre-clinical diabetic pathology in obese women that affects newborn outcome. Despite increased percentage of nonwhite mothers in higher mBMI groups, African-American race does not seem to be a significant contributing factor in the increased rate of NICU admission in our population.

Robotic thoracoscopic plication for symptomatic diaphragm paralysis.

Diaphragmatic paralysis is an uncommon condition characterized by significant elevation of a hemidiaphragm, and can cause dyspnea. The goal of diaphragm plication is to improve dyspnea by correcting the dysfunctional movement of a diaphragm during inspiration. Minimally invasive thoracoscopic diaphragm plication has been widely used and has been reported to lead to significant improvements in dyspnea and postoperative pulmonary function. Advantages of thoracoscopic plication compared to open thoracotomy are less postoperative pain and shorter hospitalization, yet technical difficulties due to limited workspace afforded by the ribcage and the elevated hemidiaphragm have been a major drawback in using the thoracoscopic approach for this disorder. We describe our experience with robotic thoracoscopic plication for the treatment of diaphragmatic paralysis. This is, to our knowledge, the first report of this kind.

Evaluation of dermal pericapillary fibrin cuffs in venous ulceration using confocal microscopy.

Dermal pericapillary fibrin is a hallmark of venous disease and is thought to play a pathogenic role in the development of ulceration. However, the actual spatial configuration of pericapillary fibrin is unknown, and it remains unclear whether it truly represents a barrier that can impair physiological exchanges between the blood and dermis. Using confocal microscopy on tissue specimens taken from the edges of venous ulcers in six patients, we report a detailed analysis of dermal pericapillary fibrin deposits. Sections were evaluated with an antibody to human fibrinogen/fibrin and viewed, vertically and horizontally, with confocal microscopy. The distribution of fibrin deposition was highly variable and patchy, with areas of great intensity next to others of marginal intensity. Vertical cut sections showed the highest concentration of fluorescent material next to the lumen of dermal capillaries. Horizontal sections showed that maximal fluorescence was distributed at random. Our findings indicate that fibrin deposits in venous ulcers are patchy and discontinuous around dermal vessels. As such, these deposits are unlikely to act as a true and stable anatomic barrier as originally proposed. However, pericapillary fibrin may still act as a physiological barrier under conditions of poor blood flow where even marginal or patchy fibrin deposition might have a greater effect on the exchange of oxygen and other nutrients between blood and dermis.

Fibroblasts from non-healing human chronic wounds show decreased expression of beta ig-h3, a TGF-beta inducible protein.

BACKGROUND: Increasing evidence shows persistent phenotypic alterations in fibroblasts from non-healing human chronic wounds, which may result in faulty extracellular matrix deposition and keratinocyte migration. We have previously shown that these cells are characterized by morphological changes, low proliferative potential and unresponsiveness to TGF-beta1, and down regulated phosphorylation of Smad 2/3 and p42/44 MAPK from decreased expression of the TGF-beta type II receptor. OBJECTIVE: To identify genes and proteins that may be differentially expressed in chronic wounds and their cultured fibroblasts. METHODS: Differential display analysis with 120 random primer sets was used in fibroblasts from human venous ulcers and acute wounds created on the ipsilateral thighs of the same patients. Positive differential results were confirmed by RT-PCR. Immunohistochemistry of cultured fibroblasts and tissues was used to determine the expression of differentially expressed proteins. RESULTS: A total of 16 differentially expressed genes were identified and cloned. The only candidate gene that was differentially expressed in all patients and confirmed by repeated differential display testing and RT-PCR was beta ig-h3, a TGF-beta-induced gene involved in cell adhesion, migration, and proliferation. Decreased expression of beta ig-h3 in chronic wounds and their fibroblasts was further confirmed by Western blot and immunostaining. CONCLUSION: These findings point to beta ig-h3 as an important gene characterizing the abnormal phenotype of chronic wound fibroblasts. Corrective measures to increase the expression of this protein might have therapeutic potential.