Molecular Mechanisms Involved in Neural Substructure Development during Phosphodiesterase Inhibitor Treatment of Mesenchymal Stem Cells.

Stem cells are highly important in biology due to their unique innate ability to self-renew and differentiate into other specialised cells. In a neurological context, treating major injuries such as traumatic brain injury, spinal cord injury and stroke is a strong basis for research in this area. Mesenchymal stem cells (MSC) are a strong candidate because of their accessibility, compatibility if autologous, high yield and multipotency with a potential to generate neural cells. With the use of small-molecule chemicals, the neural induction of stem cells may occur within minutes or hours. Isobutylmethyl xanthine (IBMX) has been widely used in cocktails to induce neural differentiation. However, the key molecular mechanisms it instigates in the process are largely unknown. In this study we showed that IBMX-treated mesenchymal stem cells induced differentiation within 24 h with the unique expression of several key proteins such as Adapter protein crk, hypoxanthine-guanine phosphoribosyltransferase, DNA topoisomerase 2-beta and Cell division protein kinase 5 (CDK5), vital in linking signalling pathways. Furthermore, the increased expression of basic fibroblast growth factor in treated cells promotes phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase (MAPK) cascades and GTPase-Hras interactions. Bioinformatic and pathway analyses revealed upregulation in expression and an increase in the number of proteins with biological ontologies related to neural development and substructure formation. These findings enhance the understanding of the utility of IBMX in MSC neural differentiation and its involvement in neurite substructure development

Valproic Acid Promotes Early Neural Differentiation in Adult Mesenchymal Stem Cells Through Protein Signalling Pathways.

Regenerative medicine is a rapidly expanding area in research and clinical applications. Therapies involving the use of small molecule chemicals aim to simplify the creation of specific drugs for clinical applications. Adult mesenchymal stem cells have recently shown the capacity to differentiate into several cell types applicable for regenerative medicine (specifically neural cells, using chemicals). Valproic acid was an ideal candidate due to its clinical stability. It has been implicated in the induction of neural differentiation; however, the mechanism and the downstream events were not known. In this study, we showed that using valproic acid on adult mesenchymal stem cells induced neural differentiation within 24 h by upregulating the expression of suppressor of cytokine signaling 5 (SOCS5) and Fibroblast growth factor 21 (FGF21), without increasing the potential death rate of the cells. Through this, the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway is downregulated, and the mitogen-activated protein kinase (MAPK) cascade is activated. The bioinformatics analyses revealed the expression of several neuro-specific proteins as well as a range of functional and structural proteins involved in the formation and development of the neural cells

Fibrinogen adsorption and platelet adhesion to silica surfaces with stochastic nanotopography

In this study, the effect of surface nanoscale roughness on fibrinogen adsorption and platelet adhesion was investigated. Nanorough silica surfaces with a low level of surface roughness (10 nm Rrms) were found to support the same level of fibrinogen adsorption as the planar silica surfaces, while nanorough silica surfaces with higher levels of surface roughness (15 nm Rrms) were found to support significantly less fibrinogen adsorption. All surfaces analyzed were found to support the same level of platelet adhesion; however, platelets were rounded in morphology on the nanorough silica surfaces while platelets were spread with a well-developed actin cytoskeleton on the planar silica. Unique quartz crystal microbalance with dissipation monitoring (QCM-D) responses was observed for the interactions between platelets and each of the surfaces. The QCM-D data indicated that platelets were more weakly attached to the nanorough silica surfaces compared with the planar silica. These data support the role of surface nanotopography in directing platelet-surface interactions even when the adsorbed fibrinogen layer is able to support the same level of platelet adhesion

A Molecular Analysis of Cytokine Content across Extracellular Vesicles, Secretions, and Intracellular Space from Different Site-Specific Adipose-Derived Stem Cells

Cytokines are multifunctional small proteins that have a vital influence on inflammatory states of tissues and play a role in signalling and cellular control mechanisms. Cytokine expression has primarily been viewed as a form of direct secretion of molecules through an active transportation; however, other forms of active transport such as extracellular vesicles are at play. This is particularly important in stem cells where signalling molecules are key to communication managing the levels of proliferation, migration, and differentiation into mature cells. This study investigated cytokines from intracellular content, direct cellular secretions, and extracellular vesicles from adult adipose-derived stem cells isolated from three distinct anatomical locations: abdomen, thigh, and chin. The cells were cultured investigated using live cell microscopy, cytokine assays, and bioinformatics analysis. The cytokines quantified and examined from each sample type showed a distinct difference between niche areas and sample types. The varying levels of TNF-alpha, IL-6 and IL-8 cytokines were shown to play a crucial role in signalling pathways such as MAPK, ERK1/2 and JAK-STAT in cells. On the other hand, the chemotactic cytokines IL-1rn, Eotaxin, IP-10 and MCP-1 showed the most prominent changes across extracellular vesicles with roles in noncanonical signalling. By examining the local and tangential roles of cytokines in stem cells, their roles in signalling and in regenerative mechanisms may be further understood.</jats:p

Quantitative Proteomic Profiling of Small Molecule Treated Mesenchymal Stem Cells Using Chemical Probes.

The differentiation of human adipose derived stem cells toward a neural phenotype by small molecules has been a vogue topic in the last decade. The characterization of the produced cells has been explored on a broad scale, examining morphological and specific surface protein markers; however, the lack of insight into the expression of functional proteins and their interactive partners is required to further understand the extent of the process. The phenotypic characterization by proteomic profiling allows for a substantial in-depth analysis of the molecular machinery induced and directing the cellular changes through the process. Herein we describe the temporal analysis and quantitative profiling of neural differentiating human adipose-derived stem cells after sub-proteome enrichment using a bisindolylmaleimide chemical probe. The results show that proteins enriched by the Bis-probe were identified reproducibly with 133, 118, 126 and 89 proteins identified at timepoints 0, 1, 6 and 12, respectively. Each temporal timepoint presented several shared and unique proteins relative to neural differentiation and their interactivity. The major protein classes enriched and quantified were enzymes, structural and ribosomal proteins that are integral to differentiation pathways. There were 42 uniquely identified enzymes identified in the cells, many acting as hubs in the networks with several interactions across the network modulating key biological pathways. From the cohort, it was found by gene ontology analysis that 18 enzymes had direct involvement with neurogenic differentiation

Gold nanoparticles improve metabolic profile of mice fed a high-fat diet

© 2018 The Author(s). Background: Obesity is a high risk for multiple metabolic disorders due to excessive influx of energy, glucose and lipid, often from a western based diet. Low-grade inflammation plays a key role in the progression of such metabolic disorders. The anti-inflammatory property of gold compounds has been used in treating rheumatoid arthritis in the clinic. Previously we found that pure gold nanoparticles (AuNPs, 21 nm) also possess anti-inflammatory effects on the retroperitoneal fat tissue following intraperitoneal injection, by downregulating tumor necrosis factor (TNF) α. However, whether such an effect can change the risk of metabolic disorders in the obese has not been well studied. The study employed C57BL/6 mice fed a pellet high fat diet (HFD, 43% as fat) that were treated daily with AuNPs [low (HFD-LAu) or high (HFD-HAu) dose] via intraperitoneal injection for 9 weeks. In the in vitro study, RAW264.7 macrophages and 3T3-L1 adipocytes were cultured with low and high concentrations of AuNPs alone or together. Results: The HFD-fed mice showed a significant increase in fat mass, glucose intolerance, dyslipidemia, and liver steatosis. The HFD-LAu group showed an 8% reduction in body weight, ameliorated hyperlipidemia, and normal glucose tolerance; while the HFD-HAu group had a 5% reduction in body weight with significant improvement in their glucose intolerance and hyperlipidemia. The underlying mechanism may be attributed to a reduction in adipose and hepatic local proinflammatory cytokine production, e.g. TNFα. In vitro studies of co-cultured murine RAW264.7 macrophage and 3T3-L1 adipocytes supported this proposed mechanism. Conclusion: AuNPs demonstrate a promising profile for potential management of obesity related glucose and lipid disorders and are useful as a research tool for the study of biological mechanisms

Tensile Properties of the Murine Ventral Vertical Midline Incision

In clinical surgery, the vertical midline abdominal incision is popular but associated with healing failures. A murine model of the ventral vertical midline incision was developed in order to study the healing of this incision type.The strength of the wild type murine ventral abdominal wall in the midline was contained within the dermis; the linea alba made a negligible contribution. Unwounded abdominal wall had a downward trend (nonsignificant) in maximal tension between 12 and 29 weeks of age. The incision attained 50% of its final strength by postoperative day 40. The maximal tension of the ventral vertical midline incision was nearly that of unwounded abdominal wall by postwounding day 60; there was no difference in unwounded vs. wounded maximal tension at postwounding day 120.After 120 days of healing, the ventral vertical midline incision in the wild type mouse was not significantly different from age-matched nonwounded controls. About half of the final incisional strength was attained after 6 weeks of healing. The significance of this work was to establish the kinetics of wild type incisional healing in a model for which numerous genotypes and genetic tools would be available for subsequent study

Xenografts for tendon and ligament repair

Collagenous materials, usually of bovine or equine origin, have been popular starting points for the development of xenograft prostheses for tendon and ligament repair. Xenografts are highly attractive as they carry small risk of infectious disease, do not compromise the patient's remaining tissues and may have the 'correct' structure as the component being replaced. Animal studies, on dog, rabbit and chicken, have shown tremendous potential for this use of xenograft material as a tendon replacement. Why, therefore, have xenografts been almost universally a total failure in clinical application? The reasons would appear to be two-fold: the animal models have not been appropriate to the intended clinical use and the cross-linking of xenograft materials has not been optimized. Our work on xenograft, heterograft and autograft tissues indicates that both aspects deserve more attention. Quantitative histology indicates that the extent and type of response to xenograft materials differs widely with degree of cross-linking (glutaraldehyde). Attention must also be given to the join of the graft to the host. For both tendon and ligament the join is a site of particular fragility. Even with adequate strength in the mid-substance, tendon and ligament grafts can, and do, fail at the join. We have investigated a variety of mechanisms for joining tendon to tendon and ligaments to bone. The failures of these methods present some insight into the biology of the repair process involved and into how failure may be avoided in future. Biomaterials (1994) 15, (10) 745-752. © 1994

Application of biomechanics to tissue engineering: A personal view

Cellular biomechanics is an area of study that is receiving more attention as time progresses. The response of cells to their mechanical environment, including biomechanical stimuli, has far-reaching ramifications for the area of tissue engineering, especially for tissues designed to withstand mechanical loading (e.g. bone, cartilage, tendons and ligaments, and arteries). The effects of mechanical stimuli on cells are only recently being examined, and the potential role of mechanical stimuli in tissue engineering is still one that is largely ignored in the design of tissue engineering scaffolds. The relationship of mechanical properties of scaffolds or of mechanical stimuli to cell behavior is complex, but vital to the development of the field. Also, understanding the complex interplay of form and environment on cells involves an increase in our knowledge of how cells react to their total environment including mechanical stimuli and material properties. In order to improve tissue engineering outcomes, a nexus must be developed between the mechanical, biochemical, and biological studies of cellular behavior, in the context of extremely complex systems. © 2008 World Scientific Publishing Company

Optical endpoint sensing in an automatic whole blood clotting timer

Most clotting time estimations are performed manually, although attempts have been made previously to automate them. The two major methods for automatically detecting the formation of the gel-like clot are mechanical (viscometric) and optical. The latter is superior in terms of accuracy of timing and freedom from artefacts but can only be performed on blood plasma. This paper describes a device which combines centrifuging to remove red cells and optical sensing of clot formation into a single operation, therepy giving activated clotting times on a par with those obtained mechanically from whole blood. The system offers the advantage over mechanical sensing that no nondisposable parts come in contact with the blood thereby eliminating e major source of timing errors. The timer works with any liquid coagulation activator, and will also time plasma clotting. The two-chambered design of the cuvette allows the activator to be kept separate from the blood until rotor startup The start of centifugal action mixes the blood and activator and starts the time. Timing is stopped auto matically when the rate of increase of optical density in the plasma, owing to fibrin formation, reaches a predetermined fevel. © 1984 IFMBE