Biomimetic Extracellular Matrix Nanofibers Electrospun with Calreticulin Promote Synergistic Activity for Tissue Regeneration.

In recognition of the potential of calreticulin (CRT) protein in enhancing the rate and quality of wound healing in excisional animal wound models, this study was to incorporate CRT via polyblend electrospinning into polycaprolactone (PCL)/type 1 collagen (Col1) nanofibers (NFs; 334 ± 75 nm diameter) as biomimetic extracellular matrices to provide a novel mode of delivery and protection of CRT with enhanced synergistic activities for tissue regeneration. Release kinetic studies using fluoresceinated CRT (CRT-FITC) polyblend NFs showed a burst release within 4 h reaching a plateau at 72 h, with further intervals of release upon incubation with fresh phosphate buffered saline for up to 8 weeks. By measuring fluorescence during the first 4 h of release, CRT-FITC-containing NFs were shown to protect CRT from proteolytic digestion (e.g., by subtilisin) compared to CRT-FITC in solution. CRT incorporated into NFs (CRT-NFs) also showed retention of biological activities and potency for stimulating proliferation and migration of human keratinocytes and fibroblasts. Fibroblasts seeded on CRT-NFs, after 2 days, showed increased amounts of fibronectin, TGF-ß1, and integrin ß1 in cell lysates by immunoblotting. Compared to NFs without CRT, CRT-NFs supported cell responses consistent with greater cell polarization and increased laminin-5 deposition of keratinocytes and a more motile phenotype of fibroblasts, as suggested by vinculin-capping F-actin fibers nonuniformly located throughout the cell body and the secretion of phosphorylated focal adhesion kinase-enriched migrasomes. Altogether, CRT electrospun into PCL/Col1 NFs retained its structural integrity and biological functions while having additional benefits of customizable loading, protection of CRT from proteolytic degradation, and sustained release of CRT from NFs, coupled with innate physicochemical cues of biomimetic PCL/Col1 NFs. Such synergistic activities have potential for healing recalcitrant wounds such as diabetic foot ulcers.

Return to Ballet Progression for Dancers After Hip Arthroscopy for Instability or Femoroacetabular Impingement Syndrome.

ABSTRACT: Ballet dancers may be predisposed to hip injuries because of the unique demands placed on the hips during dance training and performance. Hip arthroscopy can be used to address several of these symptomatic disorders, including hip instability and femoroacetabular impingement syndrome (FAIS). After hip arthroscopy, ballet dancers undergo a rehabilitation program to allow for healing, range of motion restoration, and progressive strengthening. Once patients complete the standard postoperative therapy program, a paucity of information is available to guide dancers back to the advanced hip movements involved in ballet. Therefore, the purpose of this clinical commentary is to present a stepwise rehabilitation protocol with return to ballet progression for dancers undergoing hip arthroscopy for instability or FAIS. Particular emphasis is placed on movement-specific exercises for ballet performers, and objective clinical metrics, to guide return to dance progression.

Polyphosphazenes enable durable, hemocompatible, highly efficient antibacterial coatings.

Biocompatible antibacterial coatings are highly desirable to prevent bacterial colonization on a wide range of medical devices from hip implants to skin grafts. Traditional polyelectrolytes are unable to directly form coatings with cationic antibiotics at neutral pH and suffer from high degrees of antibiotic release upon exposure to physiological concentrations of salt. Here, novel inorganic-organic hybrid polymer coatings based on direct layer-by-layer assembly of anionic polyphosphazenes (PPzs) of various degrees of fluorination with cationic antibiotics (polymyxin B, colistin, gentamicin, and neomycin) are reported. The coatings displayed low levels of antibiotic release upon exposure to salt and pH-triggered response of controlled doses of antibiotics. Importantly, coatings remained highly surface active against Escherichia coli and Staphylococcus aureus, even after 30 days of pre-exposure to physiological conditions (bacteria-free) or after repeated bacterial challenge. Moreover, coatings displayed low (<1%) hemolytic activity for both rabbit and porcine blood. Coatings deposited on either hard (Si wafers) or soft (electrospun fiber matrices) materials were non-toxic towards fibroblasts (NIH/3T3) and displayed controllable fibroblast adhesion via PPz fluorination degree. Finally, coatings showed excellent antibacterial activity in ex vivo pig skin studies. Taken together, these results suggest a new avenue to form highly tunable, biocompatible polymer coatings for medical device surfaces.

Micelle-Coated, Hierarchically Structured Nanofibers with Dual-Release Capability for Accelerated Wound Healing and Infection Control.

Tailoring nanofibrous matrices-a material with much promise for wound healing applications-to simultaneously mitigate bacterial colonization and stimulate wound closure of infected wounds is highly desirable. To that end, a dual-releasing, multiscale system of biodegradable electrospun nanofibers coated with biocompatible micellar nanocarriers is reported. For wound healing, transforming growth factor-ß1 is incorporated into polycaprolactone/collagen (PCL/Coll) nanofibers via electrospinning and the myofibroblastic differentiation of human dermal fibroblasts is locally stimulated. To prevent infection, biocompatible nanocarriers of polypeptide-based block copolymer micelles are deposited onto the surfaces of PCL/Coll nanofibers using tannic acid as a binding partner. Micelle-modified fibrous scaffolds are favorable for wound healing, not only supporting the attachment and spreading of fibroblasts comparable to those on noncoated nanofibers, but also significantly enhancing fibroblast migration. Micellar coatings can be loaded with gentamicin or clindamycin and exhibit antibacterial activity as measured by Petrifilm and zone of inhibition assays as well as time-dependent reduction of cellular counts of Staphylococcus aureus cultures. Moreover, delivery time of antibiotic dosage is tunable through the application of a novel modular approach. Altogether, this system holds great promise as an infection-mitigating, cell-stimulating, biodegradable skin graft for wound management and tissue engineering.

A critical evaluation of liquid chromatography with hybrid linear ion trap-Orbitrap mass spectrometry for the determination of acidic contaminants in wastewater effluents.

Acidic pesticide and pharmaceutical contaminants were pre-concentrated and extracted from wastewater samples (500 mL) using solid-phase extraction. Analyte recoveries were 79-96%, with % RSD values in the range, 1.7-7.4%. Analyte identification and quantification were carried out using liquid chromatography-mass spectrometry (LC-MS) with hybrid linear ion trap (LIT) Orbitrap instrumentation. Using a resolution setting of 30,000 FWHM, full-scan MS analysis was performed using heated electrospray ionization (HESI) in negative mode. The high mass resolution capabilities of the Orbitrap MS were exploited for the determination of trace contaminants allowing facile discrimination between analytes and matrix. The dependant scan functions of the Orbitrap MS using higher collisional dissociation (HCD) and LIT MS were evaluated for the confirmation of analytes at trace concentration levels. Mass accuracy for target contaminants using this method was less than 2 ppm. The limits of quantitation (LOQs) were in the range, 2.1-27 ng/L. The inter-day accuracy and precision were measured over a five-day period at two concentrations. The % relative errors were in the range, 0.30-7.7%, and the % RSD values were in the range, 1.5-5.5%. Using this method, 2,4-D, mecoprop, ibuprofen, naproxene and gemfibrozil were identified in several wastewater treatment plants in Ireland.

Semi-automated liquid chromatography-mass spectrometry (LC-MS/MS) method for basic pesticides in wastewater effluents.

Effluent from wastewater treatment plants have been identified as an important source of micro-organic contaminants in the environment. An online high-performance liquid chromatography-heated electrospray ionization tandem mass spectrometric method was developed and validated for the determination of basic pesticides in effluent wastewaters. Most available methods for pesticide analysis of wastewater samples are time-consuming, require complex clean-up steps and are difficult to automate. The method developed used a simple solid-phase extraction clean-up for salt and lipid reduction. On-line sample pre-concentration was performed using a reversed phase (C(18)) column, and analytes were separated by back-flushing onto an analytical column (C(8)) with detection using QqQ MS. An option to increase MS resolution was exploited to minimize interference from endogenous compounds in the matrix. A better than unit mass resolution was used (Q1 full width half maximum (FWHM) = 0.2 Da and Q3 FWHM = 0.7 Da), which was as rugged as a unit resolution method, and improved signal/noise and better detection limits were achieved for the targeted basic pesticides. This method was applied to the determination of 11 pesticides, including methoxytriazine, chlorotriazines, chloroacetanilides, phenylurea and carbamate pesticides. The percentage recovery values for these pesticides using the online trapping column were within the range, 73-95%, with relative standard deviation (RSD) values <8.9%. The highest concentrations of these pesticides in wastewater effluents in County Cork, Ireland, were simazine (0.51 µg/L), prometon (0.14 µg/L), diuron (0.21 µg/L) and atrazine (0.19 µg/L).

The Thrsp null mouse (Thrsp(tm1cnm)) and diet-induced obesity.

We created a Thrsp (Spot 14 or S14) null mouse (Thrsp(tm1cnm)) to study the role of Thrsp in de novo lipid synthesis. The Thrsp null mouse exhibits marked deficiencies in de novo lipogenesis in the lactating mammary gland. We now report the Thrsp gene deletion affects body weight and glucose tolerance associated with increased insulin sensitivity. By post-natal day 150 the rate of first generation C57BL/6J backcross Thrsp null mouse weight gain slowed compared to wild type animals. This was due to changes in body fat mass. We studied mice backcrossed for 5 and 11 generations. The weight difference between the null and wild type adult mice diminished with progressive backcross generations. In conclusion the Thrsp gene is involved in the regulation of diet-induced obesity and deletion of Thrsp leads to an improvement in age associated glucose tolerance.

Thyroid hormone regulates oligodendrocyte accumulation in developing rat brain white matter tracts.

Thyroid hormone (TH) is necessary for normal axonal myelination. Myelin basic protein (MBP) is a structural protein essential for myelin function. In this study, we demonstrate that perinatal hypothyroidism regulates MBP mRNA levels via indirect mechanisms. We observed decreased MBP mRNA accumulation in the hypothyroid rat brain at postnatal (PN) d 10 and 50. Acute TH replacement did not rescue hypothyroid MBP mRNA levels at PN5, 10, or 50. TH is necessary for normal intrahemispheric commissure development including the anterior commissure (AC) and the corpus callosum (CC). We determined that perinatal hypothyroidism decreases AC area and cellularity in the developing rat brain by PN10 and 50. In the developing CC, hypothyroidism initially increases area and cellularity by PN5, but then ultimately decreases area and cellularity by PN50. MBP-expressing oligodendrocytes are a recognized target of TH and are responsible for myelination within intrahemispheric commissures. We found that hypothyroidism reduces the number of mature oligodendrocytes within both the AC and CC. This reduction is noted at PN5, 10, and 50 in the AC and by PN10 and 50 in the CC. Together, these data suggest that TH regulates MBP mRNA levels through indirect mechanisms. These data demonstrate the complex mechanisms whereby TH regulates myelination in the developing brain.

Determination of petroleum contamination in shellfish using solid phase micro-extraction with gas chromatography-mass spectrometry.

Headspace solid phase micro-extraction (HS-SPME) has been applied as a sampling technique for the determination of petroleum contamination in shellfish using capillary gas chromatography-mass spectrometry (GC-MS). A poly(dimethylsiloxane) fused silica fibre (100 microm thickness) was found to be satisfactory for the extraction of a range of aliphatic hydrocarbons (HCs) from homogenised shellfish tissues. The SPME conditions, including temperature, salt content, extraction time and desorption temperature, were optimised for a range of aliphatic HCs (C9-C20). A methyl silicone column GC (12 m x 0.20 mm, 0.33 microm layer thickness) was used with a temperature programme from 40 to 260 degrees C and the HCs were determined within a mass range of m/ z=50-550 in electron impact mode. Calibration range was from 10 to 5000 ng/g with linear correlation coefficients ( r(2)) of 0.982 for nonane to 0.997 for octadecane. Detection limits for aliphatic HCs, spiked into shellfish (mussel) tissues, varied from 3.6 ng/g (tetradecane) to 51 ng/g (eicosane) and relative standard deviation (% RSD) values ranged from 1.4% (hexadecane) to 24.3%(eicosane).