The use of flaps for management of deep sternal wound complications: A systematic review and meta-analysis.

BACKGROUND: Many options are available for reconstruction after deep sternal wound infections. However, these options have not been critically appraised. The aim of this systematic review and meta-analysis was to assess the existing evidence on sternal rewiring versus flap reconstruction and pectoralis major muscle flaps (PMFs) versus greater omental flaps (GOFs). METHODS: A systematic review and meta-analysis was performed. CENTRAL, MEDLINE and EMBASE were searched. Outcomes of interest included mortality, treatment failure and length of hospital stay (LOS). RESULTS: Fourteen studies were included. Nine studies compared flaps to rewiring, reporting on 618 patients. Patients treated with flaps had significantly lower mortality compared with patient treated with rewiring (Risk ratio [RR] 0.42, 95% confidence interval [CI]: 0.23-0.77, P < 0.01). Flap patients had significantly lower treatment failure compared with those who were treated with rewiring (RR 0.22, 95% CI: 0.14-0.37, P < 0.01). No statistically significant differences were observed in LOS between patients treated with flaps compared those treated with rewiring (standard mean difference -0.84, 95% CI: -1.91 to 0.24, P = 0.13). Five studies compared PMF with GOF, reporting on 599 patients. No statistically significant differences were found in mortality (RR 0.63, 95% CI: 0.24-1.68, P = 0.36), LOS (standard mean difference -14.52, 95% CI: -42.00 to 12.96, P = 0.30) or treatment failure (RR 1.37, 95% CI: 0.31-6.07, P = 0.68) in patients treated with PMF compared with patients treated with GOF. CONCLUSIONS: Flap-based reconstruction demonstrated improved mortality and treatment outcomes compared to sternal rewiring. However, no significant differences were observed in outcomes between the PMF- and GOF-based reconstructions.

Vacuum-assisted closure therapy for the management of deep sternal wound complications: A systematic review and meta-analysis.

BACKGROUND: Vacuum-assisted closure (VAC) therapy has become a popular treatment option for wound healing. The aim of this meta-analysis was to assess the use of VAC therapy as a bridge before the definitive treatment for the management of deep sternal wound complications. METHODS: A systematic literature review and meta-analysis were performed in PubMed and Embase. Outcomes of interest included mortality, treatment failure, length of hospital stay (LOS), length of intensive care unit (ICU) stay and cost of treatment. RESULTS: Twenty-two studies involving 1980 patients were included in the quantitative synthesis of this meta-analysis. Patients treated with VAC had significantly lower overall mortality [1738 patients; Risk ratio [RR] = 0.36 (95% confidence interval [CI]: 0.25, 0.51)], treatment failure [1210 patients; RR = 0.26 (95% CI: 0.19, 0.37)], LOS [498 patients; (standard mean difference = -0.44 (95% CI: -0.81, -0.07)] and ICU stay [309 patients; (standard mean difference = -0.34 (95% CI: -0.67, -0.01)] compared to that of non-VAC patients. VAC therapy was associated with reduced cost of treatment per patient compared with that of non-VAC therapies (reductions of 3600 USD, 6000 USD and 8983 USD in the reported studies). CONCLUSIONS: VAC therapy as an adjunct in the definitive treatment of patients with deep sternal wound complications was associated with lower mortality, treatment failure, LOS, ICU stay and cost of treatment when compared with a non-VAC approach. Randomised controlled trials would be essential to confirm these findings.

The Effects of Chronological Age on the Chondrogenic Potential of Mesenchymal Stromal Cells: A Systematic Review.

Tissue engineering and cell therapy for regenerative medicine have great potential to treat chronic disorders. In musculoskeletal disorders, mesenchymal stromal cells (MSCs) have been identified as a relevant cell type in cell and regenerative strategies due to their multi-lineage potential, although this is likely to be a result of their trophic and immunomodulatory effects on other cells. This PRISMA systematic review aims to assess whether the age of the patient influences the chondrogenic potential of MSCs in regenerative therapy. We identified a total of 3027 studies after performing a search of four databases, including Cochrane, Web of Science, Medline, and PubMed. After applying inclusion and exclusion criteria, a total of 14 papers were identified that were reviewed, assessed, and reported. Cell surface characterization and proliferation, as well as the osteogenic, adipogenic, and chondrogenic differentiation, were investigated as part of the analysis of these studies. Most included studies suggest a clear link between aged donor MSCs and diminished clonogenic and proliferative potential. Our study reveals a heterogeneous and conflicting range of outcomes concerning the chondrogenic, osteogenic, and adipogenic potential of MSCs in relation to age. Further investigations on the in vitro effects of chronological age on the chondrogenic potential of MSCs should follow the outcomes of this systematic review, shedding more light on this complex relationship.

Principles of management of hand fractures.

The optimal management of hand fractures requires a multidisciplinary approach. Initial assessment should include a thorough medical history and clinical examination, followed by appropriate radiological imaging. These are crucial in determining the appropriate management. Following joint stabilisation to allow fractures to unite, early mobilisation is needed to maximise the functional restoration of the hand. In this review, the principles of operative and non-operative management of these injuries are discussed.

Distinct spatiotemporal contribution of morphogenetic events and mechanical tissue coupling during Xenopus neural tube closure.

Neural tube closure (NTC) is a fundamental process during vertebrate development and is indispensable for the formation of the central nervous system. Here, using Xenopus laevis embryos, live imaging, single-cell tracking, optogenetics and loss-of-function experiments, we examine the roles of convergent extension and apical constriction, and define the role of the surface ectoderm during NTC. We show that NTC is a two-stage process with distinct spatiotemporal contributions of convergent extension and apical constriction at each stage. Convergent extension takes place during the first stage and is spatially restricted at the posterior tissue, whereas apical constriction occurs during the second stage throughout the neural plate. We also show that the surface ectoderm is mechanically coupled with the neural plate and its movement during NTC is driven by neural plate morphogenesis. Finally, we show that an increase in surface ectoderm resistive forces is detrimental for neural plate morphogenesis.

Somitic mesoderm morphogenesis is necessary for neural tube closure during Xenopus development.

Neural tube closure is a fundamental process during vertebrate embryogenesis, which leads to the formation of the central nervous system. Defective neural tube closure leads to neural tube defects which are some of the most common human birth defects. While the intrinsic morphogenetic events shaping the neuroepithelium have been studied extensively, how tissues mechanically coupled with the neural plate influence neural tube closure remains poorly understood. Here, using Xenopus laevis embryos, live imaging in combination with loss of function experiments and morphometric analysis of fixed samples we explore the reciprocal mechanical communication between the neural plate and the somitic mesoderm and its impact on tissue morphogenesis. We show that although somitic mesoderm convergent extension occurs independently from neural plate morphogenesis neural tube closure depends on somitic mesoderm morphogenesis. Specifically, impaired somitic mesoderm remodelling results in defective apical constriction within the neuroepithelium and failure of neural tube closure. Last, our data reveal that mild abnormalities in somitic mesoderm and neural plate morphogenesis have a synergistic effect during neurulation, leading to severe neural tube closure defects. Overall, our data reveal that defective morphogenesis of tissues mechanically coupled with the neural plate can not only drastically exacerbate mild neural tube defects that may arise from abnormalities within the neural tissue but can also elicit neural tube defects even when the neural plate is itself free of inherent defects.

Chronological Age Affects MSC Senescence In Vitro-A Systematic Review.

The use of mesenchymal stromal cells (MSCs) in regenerative medicine and tissue engineering is well established, given their properties of self-renewal and differentiation. However, several studies have shown that these properties diminish with age, and understanding the pathways involved are important to provide regenerative therapies in an ageing population. In this PRISMA systematic review, we investigated the effects of chronological donor ageing on the senescence of MSCs. We identified 3023 studies after searching four databases including PubMed, Web of Science, Cochrane, and Medline. Nine studies met the inclusion and exclusion criteria and were included in the final analyses. These studies showed an increase in the expression of p21, p53, p16, ROS, and NF-κB with chronological age. This implies an activated DNA damage response (DDR), as well as increased levels of stress and inflammation in the MSCs of older donors. Additionally, highlighting the effects of an activated DDR in cells from older donors, a decrease in the expression of proliferative markers including Ki67, MAPK pathway elements, and Wnt/ß-catenin pathway elements was observed. Furthermore, we found an increase in the levels of SA-ß-galactosidase, a specific marker of cellular senescence. Together, these findings support an association between chronological age and MSC senescence. The precise threshold for chronological age where the reported changes become significant is yet to be defined and should form the basis for further scientific investigations. The outcomes of this review should direct further investigations into reversing the biological effects of chronological age on the MSC senescence phenotype.

Basement membrane remodelling regulates mouse embryogenesis.

Tissue sculpting during development has been attributed mainly to cellular events through processes such as convergent extension or apical constriction1,2. However, recent work has revealed roles for basement membrane remodelling in global tissue morphogenesis3-5. Upon implantation, the epiblast and extraembryonic ectoderm of the mouse embryo become enveloped by a basement membrane. Signalling between the basement membrane and these tissues is critical for cell polarization and the ensuing morphogenesis6,7. However, the mechanical role of the basement membrane in post-implantation embryogenesis remains unknown. Here we demonstrate the importance of spatiotemporally regulated basement membrane remodelling during early embryonic development. Specifically, we show that Nodal signalling directs the generation and dynamic distribution of perforations in the basement membrane by regulating the expression of matrix metalloproteinases. This basement membrane remodelling facilitates embryo growth before gastrulation. The establishment of the anterior-posterior axis8,9 further regulates basement membrane remodelling by localizing Nodal signalling-and therefore the activity of matrix metalloproteinases and basement membrane perforations-to the posterior side of the embryo. Perforations on the posterior side are essential for primitive-streak extension during gastrulation by rendering the basement membrane of the prospective primitive streak more prone to breaching. Thus spatiotemporally regulated basement membrane remodelling contributes to the coordination of embryo growth, morphogenesis and gastrulation.

Morphogenesis of extra-embryonic tissues directs the remodelling of the mouse embryo at implantation.

Mammalian embryos change shape dramatically upon implantation. The cellular and molecular mechanism underlying this transition are largely unknown. Here, we show that this transition is directed by cross talk between the embryonic epiblast and the first extra-embryonic tissue, the trophectoderm. Specifically, we show via visualisation of a Cdx2-GFP reporter line and pharmacologically mediated loss and gain of function experiments that the epiblast provides FGF signal that results in differential fate acquisition in the multipotent trophectoderm leading to the formation of a tissue boundary within this tissue. The trophectoderm boundary becomes essential for expansion of the tissue into a multi-layered epithelium. Folding of this multi-layered trophectoderm induces spreading of the second extra-embryonic tissue, the primitive endoderm. Together, these events remodel the pre-implantation embryo into its post-implantation cylindrical shape. Our findings uncover how communication between embryonic and extra-embryonic tissues provides positional cues to drive shape changes in mammalian development during implantation.

Determining Temporal and Spatial Expression of Calpains in Amphibians.

Calpains are a family of calcium-dependent intracellular cysteine proteases that regulate important physiological processes by substrate cleavage. Despite the fact that Calpains have been identified in the Xenopus genome, their expression patterns and role have not been characterized. Therefore, herein, we describe two methods to determine temporal and spatial expression of Calpain 2 during Xenopus development, namely, RT-PCR and whole-mount in situ hybridization (WISH). In addition, indirect immunofluorescence (IF) is described to determine translocation to the plasma membrane, which correlates with activity levels of Calpain 2.

Methods of Calpain Inhibition to Determine the Role of Calpains in Embryo Development in Amphibians.

Calpains are a family of calcium-dependent intracellular cysteine proteases that regulate important physiological processes by substrate cleavage. Despite the fact that the role of calpains in cell migration and other processes has been extensively studied in vitro, the same does not apply to cell migration and morphogenetic events during embryogenesis, in vivo. Herein, we describe the use of three different methods to selectively block calpain activity in vivo in order to investigate the impact on Xenopus gastrulation and neurulation, namely, a calpain inhibitor, a dominant negative, and a morpholino antisense oligonucleotide (MO). We also provide methods to determine the effectiveness of the calpain inhibition and effect on cell fate specification and morphogenetic movements, during embryogenesis in vivo.

Author Correction: Sequential formation and resolution of multiple rosettes drive embryo remodelling after implantation.

In the version of this Article originally published, the first name of author Guangdun Peng was spelled incorrectly as Guangdum. This has now been amended in all versions of the Article.

Sequential formation and resolution of multiple rosettes drive embryo remodelling after implantation.

The morphogenetic remodelling of embryo architecture after implantation culminates in pro-amniotic cavity formation. Despite its key importance, how this transformation occurs remains unknown. Here, we apply high-resolution imaging of embryos developing in vivo and in vitro, spatial RNA sequencing and 3D trophoblast stem cell models to determine the sequence and mechanisms of these remodelling events. We show that cavitation of the embryonic tissue is followed by folding of extra-embryonic tissue to mediate the formation of a second extra-embryonic cavity. Concomitantly, at the boundary between embryonic and extra-embryonic tissues, a hybrid 3D rosette forms. Resolution of this rosette enables the embryonic cavity to invade the extra-embryonic tissue. Subsequently, ß1-integrin signalling mediates the formation of multiple extra-embryonic 3D rosettes. Podocalyxin exocytosis leads to their polarized resolution, permitting the extension of embryonic and extra-embryonic cavities and their fusion into a unified pro-amniotic cavity. These morphogenetic transformations of embryogenesis reveal a previously unappreciated mechanism for lumen expansion and fusion.

Assembly of embryonic and extraembryonic stem cells to mimic embryogenesis in vitro.

Mammalian embryogenesis requires intricate interactions between embryonic and extraembryonic tissues to orchestrate and coordinate morphogenesis with changes in developmental potential. Here, we combined mouse embryonic stem cells (ESCs) and extraembryonic trophoblast stem cells (TSCs) in a three-dimensional scaffold to generate structures whose morphogenesis is markedly similar to that of natural embryos. By using genetically modified stem cells and specific inhibitors, we show that embryogenesis of ESC- and TSC-derived embryos-ETS-embryos-depends on cross-talk involving Nodal signaling. When ETS-embryos develop, they spontaneously initiate expression of mesoderm and primordial germ cell markers asymmetrically on the embryonic and extraembryonic border, in response to Wnt and BMP signaling. Our study demonstrates the ability of distinct stem cell types to self-assemble in vitro to generate embryos whose morphogenesis, architecture, and constituent cell types resemble those of natural embryos.

Addressing the Functional Determinants of FAK during Ciliogenesis in Multiciliated Cells.

We previously identified focal adhesion kinase (FAK) as an important regulator of ciliogenesis in multiciliated cells. FAK and other focal adhesion (FA) proteins associate with the basal bodies and their striated rootlets and form complexes named ciliary adhesions (CAs). CAs display similarities with FAs but are established in an integrin independent fashion and are responsible for anchoring basal bodies to the actin cytoskeleton during ciliogenesis as well as in mature multiciliated cells. FAK down-regulation leads to aberrant ciliogenesis due to impaired association between the basal bodies and the actin cytoskeleton, suggesting that FAK is an important regulator of the CA complex. However, the mechanism through which FAK functions in the complex is not clear, and in this study we examined the role of this protein in both ciliogenesis and ciliary function. We show that localization of FAK at CAs depends on interactions taking place at the amino-terminal (FERM) and carboxyl-terminal (FAT) domains and that both domains are required for proper ciliogenesis and ciliary function. Furthermore, we show that an interaction with another CA protein, paxillin, is essential for correct localization of FAK in multiciliated cells. This interaction is indispensable for both ciliogenesis and ciliary function. Finally, we provide evidence that despite the fact that FAK is in the active, open conformation at CAs, its kinase activity is dispensable for ciliogenesis and ciliary function revealing that FAK plays a scaffolding role in multiciliated cells. Overall these data show that the role of FAK at CAs displays similarities but also important differences compared with its role at FAs.

Cell-Autonomous Ca(2+) Flashes Elicit Pulsed Contractions of an Apical Actin Network to Drive Apical Constriction during Neural Tube Closure.

Neurulation is a critical period in all vertebrates and results in the formation of the neural tube, which gives rise to the CNS. Apical constriction is one of the fundamental morphogenetic movements that drives neural tube closure. Using live imaging, we show that apical constriction during the neurulation is a stepwise process driven by cell-autonomous and asynchronous contraction pulses followed by stabilization steps. Our data suggest that contraction events are triggered by cell-autonomous Ca(2+) flashes and are driven by a transient contractile apical pool of actin. In addition, we provide evidence that the cell autonomy and asynchrony of contraction are required for the correct spatial distribution of constriction and, as a result, are critical for tissue morphogenesis. Finally, we identify Calpain2 as a regulator of apical constriction and show that it is required for the stabilization step, but is dispensable during contraction.

Calpain2 protease: A new member of the Wnt/Ca(2+) pathway modulating convergent extension movements in Xenopus.

Calpains are a family of calcium-dependent intracellular cysteine proteases that regulate several physiological processes by limited cleavage of different substrates. The role of Calpain2 in embryogenesis is not clear with conflicting evidence from a number of mouse knockouts. Here we report the temporal and spatial expression of Calpain2 in Xenopus laevis embryos and address its role in Xenopus development. We show that Calpain2 is expressed maternally with elevated expression in neural tissues and that Calpain2 activity is spatially and temporally regulated. Using a Calpain inhibitor, a dominant negative and a morpholino oligonoucleotide we demonstrate that impaired Calpain2 activity results in defective convergent extension both in mesodermal and neural tissues. Specifically, Calpain2 downregulation results in loss of tissue polarity and blockage of mediolateral intercalation in Keller explants without affecting adherens junction turnover. We further show that Calpain2 is activated in response to Wnt5a and that the inhibitory effect of Wnt5a expression on animal cap elongation can be rescued by blocking Calpain2 function. This suggests that Calpain2 activity needs to be tightly regulated during convergent extension. Finally we show that expression of Xdd1 blocks the membrane translocation of Calpain2 suggesting that Calpain2 activation is downstream of Dishevelled. Overall our data show that Calpain2 activation through the Wnt/Ca(2+) pathway and Dishevelled can modulate convergent extension movements.

Activation of endogenous FAK via expression of its amino terminal domain in Xenopus embryos.

BACKGROUND: The Focal Adhesion Kinase is a well studied tyrosine kinase involved in a wide number of cellular processes including cell adhesion and migration. It has also been shown to play important roles during embryonic development and targeted disruption of the FAK gene in mice results in embryonic lethality by day 8.5. PRINCIPAL FINDINGS: Here we examined the pattern of phosphorylation of FAK during Xenopus development and found that FAK is phosphorylated on all major tyrosine residues examined from early blastula stages well before any morphogenetic movements take place. We go on to show that FRNK fails to act as a dominant negative in the context of the early embryo and that the FERM domain has a major role in determining FAK's localization at the plasma membrane. Finally, we show that autonomous expression of the FERM domain leads to the activation of endogenous FAK in a tyrosine 397 dependent fashion. CONCLUSIONS: Overall, our data suggest an important role for the FERM domain in the activation of FAK and indicate that integrin signalling plays a limited role in the in vivo activation of FAK at least during the early stages of development.

In vivo, site-specific, covalent conjugation of quantum dots to proteins via split-intein splicing.

The ability to target proteins with nanostructures and/or nanodevices in vivo is important for understanding and controlling their biological function. Quantum dots (QDs) serve as an ideal model nanostructure due to their superior optical properties that permit visual confirmation of in vivo targeting and localization and due to their potential as a bio-imaging tool. Here, we describe the site-specific covalent conjugation of quantum dots to target proteins in vivo using an intein-based method. Experimental procedure includes the following steps: (1) fusion of Pleckstrin-homology (PH) domains with the N-terminus half of a split intein (I(N)); (2) conjugation of the C-terminal (I(C)) intein-derived peptide to streptavidin-coated QDs in vitro; and (3) in vivo expression of PH-I(N) following microinjection of PH-I(N) RNA and I(C)-QDs into Xenopus embryos. Intein-splicing results in covalent conjugation of QDs with the C-terminus of the PH domain without interfering with protein localization or function. Such produced QD-PH conjugates could be monitored in real time within live embryos.The use of near infrared-emitting QDs allows monitoring of QD conjugates within the embryo at depths where EGFP is undetectable demonstrating the advantages of QDs for this type of experiment. The reported approach therefore allows the covalent conjugation of QDs or other similar nanostructures to proteins in vivo and the targeting of such nanomaterial to any intracellular compartment or signaling -complex within the cells of the developing embryo.

Split-inteins for simultaneous, site-specific conjugation of quantum dots to multiple protein targets in vivo.

BACKGROUND: Proteins labelled with Quantum Dots (QDs) can be imaged over long periods of time with ultrahigh spatial and temporal resolution, yielding important information on the spatiotemporal dynamics of proteins within live cells or in vivo. However one of the major problems regarding the use of QDs for biological imaging is the difficulty of targeting QDs onto proteins. We have recently developed a DnaE split intein-based method to conjugate Quantum Dots (QDs) to the C-terminus of target proteins in vivo. In this study, we expand this approach to achieve site-specific conjugation of QDs to two or more proteins simultaneously with spectrally distinguishable QDs for multiparameter imaging of cellular functions. RESULTS: Using the DnaE split intein we target QDs to the C-terminus of paxillin and show that paxillin-QD conjugates become localized at focal adhesions allowing imaging of the formation and dissolution of these complexes. We go on to utilize a different split intein, namely Ssp DnaB mini-intein, to demonstrate N-terminal protein tagging with QDs. Combination of these two intein systems allowed us to simultaneously target two distinct proteins with spectrally distinguishable QDs, in vivo, without any cross talk between the two intein systems. CONCLUSIONS: Multiple target labeling is a unique feature of the intein based methodology which sets it apart from existing tagging methodologies in that, given the large number of characterized split inteins, the number of individual targets that can be simultaneously tagged is only limited by the number of QDs that can be spectrally distinguished within the cell. Therefore, the intein-mediated approach for simultaneous, in vivo, site-specific (N- and C-terminus) conjugation of Quantum Dots to multiple protein targets opens up new possibilities for bioimaging applications and offers an effective system to target QDs and other nanostructures to intracellular compartments as well as specific molecular complexes.