Enoxaparin does not affect network formation of adipose tissue–derived microvascular fragments

Dermal substitutes are frequently used for the initial coverage of extensive skin defects. The seeding of these implants with adipose tissue–derived microvascular fragments (ad‐MVF) has recently been shown to accelerate their vascularization and incorporation. In the present study we analyzed whether these processes are affected by a thromboprophylactic therapy with the low molecular weight heparin (LMWH) enoxaparin (enox). Green fluorescent protein (GFP)⁺ ad‐MVF were isolated from enox‐ (8 mg/kg s.c.) and vehicle‐treated (0.9% NaCl s.c.) (C57BL/6‐Tg(CAG‐EGFP)1Osb/J mice and seeded onto Integra matrices. Subsequently, these were implanted into full‐thickness skin defects within dorsal skinfold chambers of enox‐ and vehicle‐treated C57BL/6 wild‐type mice. Repetitive stereomicroscopy and intravital fluorescence microscopy over 2 weeks as well as histological and immunohistochemical analyses on day 14 revealed that enox does not inhibit the reassembly of ad‐MVF into new microvascular networks. In addition, treatment with the anticoagulative compound did not promote implant‐induced hemorrhage formation. Accordingly, Integra matrices in enox‐ and vehicle‐treated animals exhibited a comparable final microvessel density, fraction of GFP⁺ blood vessels originating from seeded ad‐MVF, collagen fiber content, and epithelialization. These novel findings demonstrate that the seeding of dermal substitutes with ad‐MVF may be applied also during thromboprophylactic therapy without affecting implant vascularization and bleeding risk.     

Corrective osteotomy of distal radius malunions using three-dimensional computer simulation and patient-specific guides to achieve anatomic reduction

Malunion is a common complication of distal radius fractures, especially those treated conservatively. In clinical studies, a significant correlation between anatomic reduction and wrist function has been shown. Corrective osteotomy is the preferred treatment for symptomatic cases, notwithstanding the technical challenges. The use of computer simulation improves pre-operative understanding of the three-dimensional deformity. Patient-specific surgical guides, based on precise pre-operative planning, lead to superior perioperative accuracy and reproducibility. The pre-operative planning and surgical technique of distal radius corrective surgery using three-dimensional computer technology are described in detail. The preliminary results demonstrate the excellent clinical and radiographic outcome of this technique.     

Shaping the diversity of Th2 cell responses in epithelial tissues and its potential for allergy treatment

Th2 cells have evolved to protect from large helminth infections and to exert tissue protective functions in response to nonmicrobial noxious stimuli. The initiation, maintenance, and execution of these functions depend on the integration of diverse polarizing cues by cellular sensors and molecular programs as well as the collaboration with cells that are coopted for signal exchange. The complexity of input signals and cellular collaboration generates tissue specific Th2 cell heterogeneity and specialization. In this review, we aim to discuss the advances and recent breakthroughs in our understanding of Th2 cell responses and highlight developmental and functional differences among T cells within the diversifying field of type 2 immunity. We will focus on factors provided by the tissue microenvironment and highlight factors with potential implications for the pathogenesis of allergic skin and lung diseases. Especially new insights into the role of immunometabolism, the microbiota and ionic signals enhance the complexity of Th2 cell regulation and warrant a critical evaluation. Finally, we will discuss how this ensemble of established knowledge and recent breakthroughs about Th2 immunobiology advance our understanding of the pathogenesis of allergic diseases and how this could be exploited for future immunotherapies.     

Effects of ankle–foot braces on medial gastrocnemius morphometrics and gait in children with cerebral palsy

PurposeIn children with cerebral palsy (CP), braces are used to counteract progressive joint and muscle contracture and improve function. We examined the effects of positional ankle–foot braces on contracture of the medial gastrocnemius (MG) and gait in children with CP while referencing to typically developing children.MethodsSeventeen independently ambulant children with CP and calf muscle contracture (age 10.4 ± 3.0y) and 17 untreated typically developing peers (age 9.5 ± 2.6y) participated. Children with CP were analysed before and 16 ± 4 weeks after ankle–foot bracing. MG muscle belly length and thickness, tendon and fascicle length, as well as their extensibility were captured by 2D ultrasound and 3D motion capturing during passive, manually applied stretches. In addition, 3D gait analysis was conducted.ResultsPrior to bracing, the MG muscle–tendon unit in children with CP was 22 % less extensible. At matched amounts of muscle–tendon unit stretch, the muscle belly and fascicles in CP were 7 % and 14 % shorter while the tendon was 11 % longer. Spastic fascicles displayed 32 % less extensibility than controls. Brace wear increased passive dorsiflexion primarily with the knees flexed. During gait, children walked faster and foot lift in swing improved. MG muscle belly and tendon length showed little change, but fascicles further shortened (−11 %) and muscle thickness (−8 %) decreased.ConclusionsUse of ankle–foot braces improves function but may lead to a loss of sarcomeres in series, which could explain the shortened fascicles. To potentially induce gastrocnemius muscle growth, braces may also need to extend the knee or complementary training may be necessary to offset the immobilizing effects of braces.     

Differential sensing for the regio- and stereoselective identification and quantitation of glycerides

Glycerides are of interest to the areas of food science and medicine because they are the main component of fat. From a chemical sensing perspective, glycerides are challenging analytes because they are structurally similar to one another and lack diversity in terms of functional groups. Furthermore, because animal and plant fat consists of a number of stereo- and regioisomeric acylglycerols, their components remain challenging analytes for chromatographic and mass spectrometric determination, particularly the quantitation of species in mixtures. In this study, we demonstrated the use of an array of cross-reactive serum albumins and fluorescent indicators with chemometric analysis to differentiate a panel of mono-, di-, and triglycerides. Due to the difficulties in identifying the regio- and stereochemistry of the unsaturated glycerides, a sample pretreatment consisting of olefin cross-metathesis with an allyl fluorescein species was used before array analysis. Using this simple assay, we successfully discriminated 20 glycerides via principal component analysis and linear discriminant analysis (PCA and LDA, respectively), including stereo- and regioisomeric pairs. The resulting chemometric patterns were used as a training space for which the structural characteristics of unknown glycerides were identified. In addition, by using our array to perform a standard addition analysis on a mixture of triglycerides and using a method introduced herein, we demonstrated the ability to quantitate glyceride components in a mixture.Glycerides are the primary component of animal fats and vegetable oils (1). They consist of one, two, or three fatty acids esterified on glycerol, and hence are referred to as mono-, di-, and triglycerides, respectively. The structural diversity of glycerides derives in part from their fatty acid alkyl groups, which can differ in carbon number (i.e., chain length), the degree of unsaturation, the position of olefins, and the configuration of the olefins (i.e., cis/trans). Furthermore, these fatty acid alkyl groups can be connected to the sn-1, -2, or -3 carbons on glycerol. Hence, a variety of regio- and stereoisomers can exist for glycerides, posing a challenge for mass spectrometry (2). Further, because the differences in chain length primarily result from the presence of greater or fewer methylene groups, NMR spectroscopy can be ambiguous (3).The analysis of glycerides is primarily important to the food and nutrition industries for tasks such as authenticating edible oils (4), designing foods with certain physical properties (5), and studying how fats are digested and absorbed (6). In particular, classifying all of the various kinds of regio- and stereoisomers of glycerides is biologically important because lipases, enzymes that catalyze the hydrolysis of glycerides into fatty acids and glycerol, exhibit selectivity based on these features of the glyceride substrates. As examples, the position and configuration of olefins, the identity of fatty acid alkyl groups, as well as their position on glycerol (i.e., sn-1,3 versus sn-2), all contribute to differing biological activity (7, 8). Studying the selectivity of these lipases has applications in understanding diseases, including fat malabsorption disorders, hypercholesterolemia, atherosclerosis, and diabetes (9, 10). Research on metabolic disorders has shown that fatty acid accumulation can exert a toxic or a protective effect on a tissue, depending on the specific tissue type (e.g., liver, cardiac, or skeletal muscle) (11, 12) and health state (e.g., diabetic) (13, 14) as well as on the fatty acids (e.g., saturated or unsaturated) (15). Sequestration of fatty acids by esterification to glycerides is one pathway by which these effects are regulated (16). Thus, a deeper understanding of the distinct roles of the cellular storage of structurally different glycerides in normal and disease states is a desirable avenue of research (17). However, currently only limited information is available about the composition of glycerides in adipose and nonadipose tissue.The most common method of glyceride identification is mass spectrometry (MS) (2, 18). However, as alluded to above, this approach has drawbacks. Because glycerides are neutral molecules, they must be ionized to be analyzed by MS. Saponification can be used to obtain the fatty acids, which are both volatile and charged, thereby facilitating MS analysis, but information about the glyceride structure is lost in this process (18). Electrospray ionization and atmospheric pressure chemical ionization are used to ionize glycerides directly; however, the ion yields are low compared with preionized lipids (19, 20). Furthermore, the ability of a glyceride to be ionized using these methods often varies. For example, ion abundance generally increases with increasing number of double bonds in the fatty acid alkyl chain and can also depend on fatty acid alkyl chain length (21). These significant variations in ion abundance mean that ionization methodologies must be developed and tailored to a specific application to satisfactorily detect each glyceride of interest (19). Finally, these variations render the quantification of glycerides, particularly in a complex mixture, quite challenging when using MS (22).Regio- and stereoisomers further confound the discrimination of glycerides by MS, because isomers share the same mass. Other techniques such as chemical derivitization of the glycerides, ion fragmentation, and specialized HPLC must be coupled with MS to effect differentiation of isomeric species. For example, ozonolysis has been used to cleave the double bonds in unsaturated glycerides before ionization to deduce the positions of double bonds (23). Nonaqueous reverse-phase (NARP)-HPLC can resolve cis/trans isomers of triacylglycerols and double-bond positional isomers after treatment of the olefins with bromine (24). Silver ion chromatography has been used to separate triacylglycerol positional isomers under specifically developed solvent and column temperature conditions (25). Silver cationization as a postcolumn treatment in conjunction with NARP-HPLC and ion fragmentation has also been used for triglyceride positional isomer determination (26, 27) Thus, although these current approaches to glyceride isomer analysis have been successful, they are complicated, labor intensive, time-consuming, and at times inconsistent in their results (26).Because glycerides are structurally very similar to one another, we believed that a differential sensing array-based approach would be most suitable for their classification. Our hypothesis was that if a cross-reactive array could be created that was responsive to the subtle structural differences inherent in glycerides, it could be used to pattern individual glycerides, identify structural features of unknown glycerides, and potentially quantitate glycerides in a mixture. Cross-reactive arrays have been successfully used in a number of sensing applications (28–33). Differential sensing mimics the mammalian senses of olfaction and gustation by detecting the pattern of response of an analyte to a collection of semiselective receptors (34, 35). In mammals, the characteristic pattern for a scent or taste is interpreted and stored by the brain (36). In the laboratory, chemometric routines such as principal component analysis (PCA) and linear discriminant analysis (LDA) are used to extract the relevant information from the array. Both PCA and LDA are multivariate methods that reduce the dimensionality of a data set. PCA does so by finding unbiased orthogonal axes that describe decreasing extents of variance in the data derived from different samples (classes) and repetitions of the samples (37). Any grouping of like samples represents intrinsic similarities between the sample datasets whereas separate classification represents differences in that variable space. LDA classifies samples by calculating discriminant functions that maximize the separation between predetermined classes and minimizes the separation within these classes (38, 39). Thus, LDA is a supervised method, meaning that the classes are provided as inputs into the algorithm. For this reason, a validation method called a leave-one-out cross-validation is used to test the predictive value of the model. Further, LDA can be used to predict the identity of unknowns by identifying which classes in the training set the unknowns most resemble.Therefore, the goal of this project was to develop an array of cross-reactive receptors that could discriminate glycerides. The glycerides selected are shown in Fig. 1. The panel includes commercially available mono-, di-, and triacylglycerols with fatty acid alkyl groups that are relevant to mammalian biology (40). Moreover, the panel consists of examples of each of the following stereo- and regioisomers: (i) cis/trans olefins (D1 and D2; T2 and T3), (ii) differing position of the olefin (T3 and T4), and (iii) differing position of the fatty acid alkyl groups on the glycerol (D5 and D6). Clearly, it would be extremely challenging to create highly selective receptors for each individual glyceride, and thus a differential sensing method seems the only reasonable approach to creating an optical sensing routine to identify and classify these structures.Open in a separate windowFig. 1.Glyceride panel with structures and names.Because glycerides are extremely hydrophobic analytes, we postulated that serum albumins (SAs) would be suitable cross-reactive receptors with which to test our hypothesis. SA is a common plasma protein that binds hydrophobic molecules to transport them through the hydrophilic environment of blood plasma (41). The protein binds a number of endogenous compounds: long-chain fatty acids (K_a = 10⁶–10⁷ M⁻¹) (42), bile acids (K_a = 10³–10⁵ M⁻¹) (43), and steroids (K_a = 10³–10⁵ M⁻¹) (44–46), as well as many drugs, toxins, and fluorophores (41). Despite being composed of fatty acid alkyl groups, glycerides bind less tightly to SAs and in a different location than their fatty acid counterparts (47). The primary sequence of SAs differs between species, which thus exhibit differences in ligand binding (41). Previously, we have used arrays of SAs for the differentiation of other hydrophobic analytes including fatty acids (48), terpenes (49), and plasticizers (50). However, none of these previous studies involved differences between the analyte structures as subtle as glycerides do, nor had we challenged our methods to identify structural aspects of an unknown. Furthermore, we had never implemented a quantitation assay in a complex mixture. Because the binding of ligands to SAs is known to depend on subtle differences in their structure (41), we anticipated that success could be achieved but would be highly dependent upon the signaling modality and potentially analyte prederivitization.Thus, herein we describe a method using SAs to fingerprint glycerides that classifies them as mono-, di-, or triglycerides. The glycerides were further classified based on fatty acid chain length, ester positions on glycerol, and olefin regio- and stereochemistry. For the unsaturated glycerides in the panel, differentiation based on olefin position and stereochemistry was achieved by the use of a pretreatment olefin metathesis. Using the protocols described herein, structural features of unknown glycerides could be identified. Furthermore, the quantitation of trilinolein in a mixture of triglycerides was achieved by application of the standard addition method using a net analyte signal technique (SANAS) presented herein.