Covalent linkage of sulfated hyaluronan to the collagen scaffold Mucograft® enhances scaffold stability and reduces proinflammatory macrophage activation in vivo.

Sulfated glycosaminoglycans (sGAG) show interaction with biological mediator proteins. Although collagen-based biomaterials are widely used in clinics, their combination with high-sulfated hyaluronan (sHA3) is unexplored. This study aims to functionalize a collagen-based scaffold (Mucograft®) with sHA3 via electrostatic (sHA3/PBS) or covalent binding to collagen fibrils (sHA3+EDC/NHS). Crosslinking without sHA3 was used as a control (EDC/NHS Ctrl). The properties of the sHA3-functionalized materials were characterized. In vitro growth factor and cytokine release after culturing with liquid platelet-rich fibrin was performed by means of ELISA. The cellular reaction to the biomaterials was analyzed in a subcutaneous rat model. The study revealed that covalent linking of sHA3 to collagen allowed only a marginal release of sHA3 over 28 days in contrast to electrostatically bound sHA3. sHA3+EDC/NHS scaffolds showed reduced vascular endothelial growth factor (VEGF), transforming growth factor beta 1 (TGF-ß1) and enhanced interleukin-8 (IL-8) and epithelial growth factor (EGF) release in vitro compared to the other scaffolds. Both sHA3/PBS and EDC/NHS Ctrl scaffolds showed a high proinflammatory reaction (M1: CD-68+/CCR7+) and induced multinucleated giant cell (MNGC) formation in vivo. Only sHA3+EDC/NHS scaffolds reduced the proinflammatory macrophage M1 response and did not induce MNGC formation during the 30 days. SHA3+EDC/NHS scaffolds had a stable structure in vivo and showed sufficient integration into the implantation region after 30 days, whereas EDC/NHS Ctrl scaffolds underwent marked disintegration and lost their initial structure. In summary, functionalized collagen (sHA3+EDC/NHS) modulates the inflammatory response and is a promising biomaterial as a stable scaffold for full-thickness skin regeneration in the future.

In Vivo Efficacy of Neutrophil-Mediated Bone Regeneration Using a Rabbit Calvarial Defect Model.

Reconstruction of bone due to surgical removal or disease-related bony defects is a clinical challenge. It is known that the immune system exerts positive immunomodulatory effects on tissue repair and regeneration. In this study, we evaluated the in vivo efficacy of autologous neutrophils on bone regeneration using a rabbit calvarial defect model. Methods: Twelve rabbits, each with two surgically created calvarial bone defects (10 mm diameter), were randomly divided into two groups; (i) single application of neutrophils (SA-NP) vs. SA-NP control, and (ii) repetitive application of neutrophils (RA-NP) vs. RA-NP control. The animals were euthanized at 4 and 8 weeks post-operatively and the treatment outcomes were evaluated by micro-computed tomography, histology, and histomorphometric analyses. Results: The micro-CT analysis showed a significantly higher bone volume fraction (bone volume/total volume) in the neutrophil-treated groups, i.e., median interquartile range (IQR) SA-NP (18) and RA-NP (24), compared with the untreated controls, i.e., SA-NP (7) and RA-NP (14) at 4 weeks (p < 0.05). Similarly, new bone area fraction (bone area/total area) was significantly higher in neutrophil-treated groups at 4 weeks (p < 0.05). Both SA-NP and RA-NP had a considerably higher bone volume and bone area at 8 weeks, although the difference was not statistically significant. In addition, immunohistochemical analysis at 8 weeks revealed a higher expression of osteocalcin in both SA-NP and RA-NP groups. Conclusions: The present study provides first hand evidence that autologous neutrophils may have a positive effect on promoting new bone formation. Future studies should be performed with a larger sample size in non-human primate models. If proven feasible, this new promising strategy could bring clinical benefits for bone defects to the field of oral and maxillofacial surgery.

Multinucleated Giant Cells Induced by a Silk Fibroin Construct Express Proinflammatory Agents: An Immunohistological Study.

Multinucleated giant cells (MNGCs) are frequently observed in the implantation areas of different biomaterials. The main aim of the present study was to analyze the long-term polarization pattern of the pro- and anti-inflammatory phenotypes of macrophages and MNGCs for 180 days to better understand their role in the success or failure of biomaterials. For this purpose, silk fibroin (SF) was implanted in a subcutaneous implantation model of Wistar rats as a model for biomaterial-induced MNGCs. A sham operation was used as a control for physiological wound healing. The expression of different inflammatory markers (proinflammatory M1: CCR-7, iNos; anti-inflammatory M2: CD-206, CD-163) and tartrate-resistant acid phosphatase (TRAP) and CD-68 were identified using immunohistochemical staining. The results showed significantly higher numbers of macrophages and MNGCs within the implantation bed of SF-expressed M1 markers, compared to M2 markers. Interestingly, the expression of proinflammatory markers was sustained over the long observation period of 180 days. By contrast, the control group showed a peak of M1 macrophages only on day 3. Thereafter, the inflammatory pattern shifted to M2 macrophages. No MNGCs were observed in the control group. To the best of our knowledge, this is study is the first to outline the persistence of pro-inflammatory MNGCs within the implantation bed of SF and to describe their long-term kinetics over 180 days. Clinically, these results are highly relevant to understand the role of biomaterial-induced MNGCs in the long term. These findings suggest that tailored physicochemical properties may be a key to avoiding extensive inflammatory reactions and achieving clinical success. Therefore, further research is needed to elucidate the correlation between proinflammatory MNGCs and the physicochemical characteristics of the implanted biomaterial.

Improved detection of sentinel lymph node metastases allows reliable intraoperative identification of patients with extended axillary lymph node involvement in early breast cancer.

BACKGROUND: An improved procedure that allows accurate detection of negative sentinel lymph node (SLN) and of SLN macrometastases during surgery would be highly desirable in order to protect patients from further surgery and to avoid unnecessary costs. We evaluated the accuracy of an intraoperative procedure that combines touch imprint cytology (TIC) and subsequent frozen section (FS) analysis. 2276 SLNs from 1072 patients with clinical node-negative early breast cancer were evaluated during surgery using TIC. Only cytologically-positive SLN were subsequently analysed with a single FS, preserving cytologically-negative SLN for the final postoperative histological diagnosis. Sensitivity, specificity and the accuracy of this approach were analysed by comparing the results from intra- and postoperative SLN and axillary node evaluation. This intraoperative method displayed 100% specificity for SLN metastases and was significantly more sensitive for prognostically relevant macrometastases (85%) than for micrometastases (10%). Sensitivity was highest for patients with two or more positive LNs (96%) than for those with only one (72%). 98% of the patients with final pN2a-pN3a were already identified during surgery. Patients who received primary axillary lymph node dissection had significantly more frequent metastases in further LNs (44.6%). Sensitivity was highest for patients with luminal-B, HER2+ and triple negative breast cancer and for any subtype if Ki-67 > 40%. TIC and subsequent FS of cytologically-positive SLNs is highly reliable for detection of SLN macrometastases, and allows accurate identification of patients with a high risk of extended axillary involvement during surgery, as well as accurate histological diagnosis of negative SLN.

Thermal treatment at 500 °C significantly reduces the reaction to irregular tricalcium phosphate granules as foreign bodies: An in vivo study.

Evaporation of phosphate species during thermal treatment (> 400 °C) of calcium phosphates leads to the formation of an alkaline layer on their surface. The aim of this study was to evaluate the hypothesis that the biological response of thermally treated calcium phosphates is modified by the presence of such an alkaline layer on their surface. For this purpose, 0.125-0.180 mm α- and ß-tricalcium phosphate (TCP) granules were obtained by crushing and size classification, with some being subjected to thermal treatment at 500 °C. The four types of granules (α-TCP, ß-TCP, α-TCP-500 °C, and ß-TCP-500 °C) were implanted subcutaneously and orthotopically in rats. Sham operations served as control. Subcutaneously, α-TCP and ß-TCP induced significantly more multinucleated giant cells (MNGCs) than calcined granules. Most of the induced MNGCs were TRAP-negative, CD-68 positive and cathepsin K-negative, reflecting a typical indication of a reaction with a foreign body. The vessel density was significantly higher in the α-TCP and ß-TCP groups than it was in the α-TCP-500 °C and ß-TCP-500 °C groups. In the femur model, ß-TCP-500 °C induced significantly more new bone formation than that induced by ß-TCP. The granule size was also significantly larger in the ß-TCP-500 °C group, making it more resistant to degradation than ß-TCP. The MNGC density was higher in the α-TCP and ß-TCP groups than in the α-TCP-500 °C and ß-TCP-500 °C groups, including cathepsin-positive, CD-68 positive, TRAP-positive and TRAP-negative MNGCs. In conclusion, this study confirms that the biological response of calcium phosphates was affected by the presence of an alkaline layer on their surface. Thermally-treated α-TCP and ß-TCP granules produced significantly fewer MNGCs and were significantly less degraded than non-thermally-treated α-TCP and ß-TCP granules. Thermally treating α-TCP and ß-TCP granules shifts the reaction from a foreign body reaction towards a physiological reaction by downregulating the number of induced MNGCs and enhancing degradation resistance.

Co-culture Model for Cutaneous Wound Healing to Assess a Porous Fiber-Based Drug Delivery System.

In vitro tissue-engineered cell culture models are an essential instrument to investigate physiological and pathophysiological wound healing mechanisms and to evaluate new beneficial wound dressing materials and therapeutics to identify possible drug targets and to improve regeneration processes in nonhealing and chronic wounds. In this study, the authors established an in vitro model for cutaneous wound healing, based on primary human dermal microvascular endothelial cells (HDMEC) and primary human dermal fibroblasts (HDF) to study wound healing-associated processes. Co-cultivation of HDMEC and HDF results in the formation of microvessel-like structures in long-term co-cultures. The proposed in vitro co-culture model can be easily modified by adding macrophages to simulate the process of inflammation, thus allowing in vitro investigation of pathophysiological wound healing processes present in nonhealing wounds. Furthermore, the beneficial in vitro wound healing model was used to evaluate a porous fiber-based drug delivery dressing material consisting of melt-spun porous fibers that were filled with a hydrogel carrier (gellan gum) containing vascular endothelial growth factor (VEGF). Angiogenic capability was chosen as functional parameter for improved wound healing, and release of deposited VEGF from the dressing material was evaluated up to 7 days of cultivation. The experiments demonstrated that the porous fiber-based drug delivery dressing material for dermal wound healing with incorporated VEGF strongly enhances the process of angiogenesis in the in vitro co-culture model through a release of VEGF over 7 days of cultivation. In conclusion, tissue-engineered human skin equivalents could contribute significantly to the understanding and improvement of drug releasing dressing materials in the context of treating chronic wounds.

Cholinergic signaling controls immune functions and promotes homeostasis.

Acetylcholine (ACh) was created by nature as one of the first signaling molecules, expressed already in procaryotes. Based on the positively charged nitrogen, ACh could initially mediate signaling in the absence of receptors. When evolution established more and more complex organisms the new emerging organs systems, like the smooth and skeletal muscle systems, energy-generating systems, sexual reproductive system, immune system and the nervous system have further optimized the cholinergic signaling machinery. Thus, it is not surprising that ACh and the cholinergic system are expressed in the vast majority of cells. Consequently, multiple common interfaces exist, for example, between the nervous and the immune system. Research of the last 20 years has unmasked these multiple regulating mechanisms mediated by cholinergic signaling and thus, the biological role of ACh has been revised. The present article summarizes new findings and describes the role of both non-neuronal and neuronal ACh in protecting the organism from external and internal health threats, in providing energy for the whole organism and for the individual cell, controling immune functions to prevent inflammatory dysbalance, and finally, the involvement in critical brain functions, such as learning and memory. All these capacities of ACh enable the organism to attain and maintain homeostasis under changing external conditions. However, the existence of identical interfaces between all these different organ systems complicates the research for new therapeutic interventions, making it essential that every effort should be undertaken to find out more specific targets to modulate cholinergic signaling in different diseases.

Biomimetic strategy towards gelatin coatings on PET. Effect of protocol on coating stability and cell-interactive properties.

Gelatin-modified poly(ethylene terephthalate) (PET) surfaces have been previously realized via an intermediate dopamine coating procedure that resulted in surfaces with superior haemocompatibility compared to unfunctionalized PET. The present study addresses the biocompatibility assessment of these coated PET surfaces. In this context, the stability of the gelatin coating upon exposure to physiological conditions and its cell-interactive properties were investigated. The proposed gelatin-dopamine-PET surfaces showed an increased protein coating stability up to 24 days and promoted the attachment and spreading of both endothelial cells (ECs) and smooth muscle cells (SMCs). In parallel, physisorbed gelatin coatings exhibited similar cell-interactive properties, albeit temporarily, as the coating delaminated within 1 week after cell seeding. Furthermore, no or only minimal immunogenic or inflammatory responses were observed during in vitro cytotoxicity and endotoxicity assessment for all gelatin-modified PET surfaces evaluated. Overall, the combined enhanced biocompatibility reported herein together with the previously proven haemocompatibility show the potential of the gelatin-dopamine-PET surfaces to function as vascular graft candidates.

In vitro evaluation of a biomaterial-based anticancer drug delivery system as an alternative to conventional post-surgery bone cancer treatment.

Patients diagnosed with osteosarcoma are currently treated with intravenous injections of anticancer agents after tumor resection. However, due to remaining neoplastic cells at the site of tumor removal, cancer recurrence often occurs. Successful bone regeneration combined with the control of residual cancer cells presents a challenge for tissue engineering. Cyclodextrins loaded with chemotherapeutic drugs reversibly release the drugs over time. Hydroxyapatite bone biomaterials coated with doxorubicin-loaded cyclodextrin should release the drug with time after implantation directly at the original tumor site and may be a way to eliminate residual neoplastic cells. In the present study, we have carried out in vitro studies to evaluate such a drug-delivery system and have shown that doxorubicin released from cyclodextrin-coated hydroxyapatite retained biological activity and exhibited longer and higher cytotoxic effects on both cancer (osteosarcoma cells) and healthy cells (primary osteoblasts and endothelial cells) compared to biomaterials without cyclodextrin loaded with doxorubicin. Furthermore, doxorubicin released from biomaterials with cyclodextrin moderately induced the expression of tumor suppressor protein p53 whereas p21 expression was similar to control cells. In addition, hypoxic conditions, which occur after implantation until blood-flow to the area is regenerated, protected endothelial cells and primary osteoblasts from doxorubicin-induced cytotoxicity. This chemo-protective effect was far less prominent for the osteosarcoma cells. These findings indicate that a hydroxyapatite-cyclodextrin-doxorubicin chemotherapeutic strategy may enhance the drug-targeting effect on tumor cells while protecting the more sensitive healthy cells for a period of time after implantation. A successful integration of such a drug delivery system might allow healthy cells to initially survive during the doxorubicin exposure period, while eliminating residual neoplastic cells.

Endothelialization and Anticoagulation Potential of Surface-Modified PET Intended for Vascular Applications.

In vascular tissue engineering, great attention is paid to the immobilization of biomolecules onto synthetic grafts to increase bio- and hemocompatibility-two critical milestones in the field. The surface modification field of poly(ethylene terephthalate) (PET), a well-known vascular-graft material, is matured and oversaturated. Nevertheless, most developed methods are laborious multistep procedures generally accompanied by coating instability or toxicity issues. Herein, a straightforward surface modification procedure is presented engineered to simultaneously promote surface endothelialization and anticoagulation properties via the covalent immobilization of gelatin through a photoactivated azide derivative. A complete physicochemical characterization and biological study including cytotoxicity and endotoxin testing are performed. In addition, biocompatibility toward small (diameter ≤ 6 mm) and/or large caliber (diameter ≥ 6 mm) vessels is assessed by micro- and macrovascular endothelial cell assays. Superior bio- and hemocompatibility properties are seen for the gelatin-covalently modified PET surfaces compared to the conventional surface-modification procedures based on physisorption.

Injectable Bone Substitute Material on the Basis of ß-TCP and Hyaluronan Achieves Complete Bone Regeneration While Undergoing Nearly Complete Degradation.

PURPOSE: In this prospective study, the regenerative potential and pathways of a new injectable bone substitute (IBS) material composed of beta-tricalcium phosphate (ß-TCP) and hyaluronan were investigated for its application in alveolar bone regeneration within extraction sockets. MATERIALS AND METHODS: The bone substitute material was implanted in 44 extraction sockets after removal of teeth not worth preserving in the maxillary and mandibular arches of 21 patients. Four months after augmentation, bone biopsy samples were harvested simultaneously with implant placement for histologic and histomorphometric analysis of tissue reaction and determination of tissue formation (newly formed bone, connective tissue, and remaining IBS) within the augmentation bed. Furthermore, the inserted bone-level implants (C-Tech Esthetic Line) were followed up clinically and radiologically for at least 1 year after prosthetic loading to determine the potential impact of tissue reaction to the IBS on implant stability and performance. RESULTS: The histologic and histomorphometric analyses revealed a gentle tissue reaction with mainly mononuclear and only few multinucleated giant cells within the implantation bed. Histomorphometric analysis revealed mainly newly formed bone tissue (44.92% ± 5.16%) and connective tissue (52.49% ± 6.43%). Only a few remnants of the IBS (2.59% ± 2.05%) could be found. The IBS, with its easy application and fluidity, seemed to be suitable for three-dimensional stable defects such as the intact extraction socket. CONCLUSION: The IBS contributed to an osteoconductive tissue reaction while undergoing a time-controlled degradation. Clinical and radiological follow-up investigation of the implants inserted in the regenerated area revealed that the IBS contributed to a long-term stable implantation bed for dental implants. The appearance of the IBS can be described as a bulk that is formed within the augmentation bed and that promotes new bone formation through an osteoconductive procedure.

Macrophage type modulates osteogenic differentiation of adipose tissue MSCs.

Since the reconstruction of large bone defects remains a challenge, knowledge about the biology of bone healing is desirable to develop novel strategies for improving the treatment of bone defects. In osteoimmunology, macrophages are the central component in the early stage of physiological response after bone injury and bone remodeling in the late stage. During this process, a switch of macrophage phenotype from pro-inflammatory (M1) to anti-inflammatory (M2) is observed. An appealing option for bone regeneration would be to exploit this regulatory role for the benefit of osteogenic differentiation of osteoprogenitor cells (e.g., mesenchymal stem cells; MSCs) and to eventually utilize this knowledge to improve the therapeutic outcome of bone regenerative treatment. In view of this, we focused on the in vitro interaction of different macrophage subtypes with adipose tissue MSCs to monitor the behavior (i.e. proliferation, differentiation and mineralization) of the latter in dedicated co-culture models. Our data show that co-culture of MSCs with M2 macrophages, but not with M1 macrophages or M0 macrophages, results in significantly increased MSC mineralization caused by soluble factors. Specifically, M2 macrophages promoted the proliferation and osteogenic differentiation of MSCs, while M0 and M1 macrophages solely stimulated the osteogenic differentiation of MSCs in the early and middle stages during co-culture. Secretion of the soluble factors oncostatin M (OSM) and bone morphogenetic protein 2 (BMP-2) by macrophages showed correlation with MSC gene expression levels for OSM-receptor and BMP-2, suggesting the involvement of both signaling pathways in the osteogenic differentiation of MSCs.

Multinucleated giant cells in the implant bed of bone substitutes are foreign body giant cells-New insights into the material-mediated healing process.

In addition to macrophages, multinucleated giant cells (MNGCs) are involved in the tissue reaction to a variety of biomaterials. Especially in the case of bone substitute materials it has been assumed that the MNGCs are osteoclasts, based on the chemical and physical similarity of many materials to the calcified matrix and the bony environment in which they are used. However, many studies indicate that these cells belong to the cell line of the foreign body giant cells (FBGCs), which are of "inflammatory origin", although they have been shown to possess both a pro- and also anti-inflammatory phenotype. Moreover, no information is available about their role in the tissue reaction to bone substitute materials. The present study was conducted to analyze the origin of MNGCs in the implant beds of a synthetic and a xenogeneic bone substitute and focused on the application of immunohistochemical methods. Two antibodies against integrin molecules specific for osteoclasts (ß-3 integrin) or FBGCs (ß-2 integrin) were used to distinguish both giant cell types. The results of the present study indicate that the MNGCs induced by both kinds of bone substitutes are FBGCs, as they express only ß-2 integrin in contrast to the osteoclasts outside of the immediate implantation areas, which only demonstrate ß-3 integrin expression. These data give new insight into the tissue reaction to both xenogeneic and synthetic bone substitutes. Based on this new knowledge further research concerning the proteomic profile of the FBGCs especially based on the different physicochemical properties of bone substitutes is necessary. This may show that specific characteristics of bone substitutes may exhibit a substantial influence on the regeneration process via the expression of anti-inflammatory molecules by FBGCs. Based on this information it may be possible to formulate and choose bone substitutes that can guide the process of bone tissue regeneration on the molecular level. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1105-1111, 2017.

Non-neuronal acetylcholine involved in reproduction in mammals and honeybees.

Bacteria and archaea synthesize acetylcholine (ACh). Thus, it can be postulated that ACh was created by nature roughly three billion years ago. Therefore, the wide expression of ACh in nature (i.e., in bacteria, archaea, unicellular organisms, plants, fungi, non-vertebrates and vertebrates and in the abundance of non-neuronal cells of mammals) is not surprising. The term non-neuronal ACh and non-neuronal cholinergic system have been introduced to describe the auto- and paracrine, that is, local regulatory actions of ACh in cells not innervated by neuronal cholinergic fibers and to communicate among themselves. In this way non-neuronal ACh binds to the nicotinic or muscarinic receptors expressed on these local and migrating cells and modulates basic cells functions such as proliferation, differentiation, migration and the transport of ions and water. The present article is focused to the effects of non-neuronal ACh linked to reproduction; data on the expression and function of the non-neuronal cholinergic system in the following topics are summarized: (i) Sperm, granulosa cells, oocytes; (ii) Auxiliary systems (ovary, oviduct, placenta); (iii) Embryonic stem cells as first step for reproduction of a new individual after fertilization; (iv) Larval food as an example of reproduction in insects (honeybees) and adverse effects of the neonicotinoids, a class of world-wide applied insecticides. The review article will show that non-neuronal ACh is substantially involved in the regulation of reproduction in mammals and also non-mammals like insects (honeybees). There is a need to learn more about this biological role of ACh. In particular, we have to consider that insecticides like the neonicotinoids, but also carbamates and organophosphorus pesticides, interfere with the non-neuronal cholinergic system thus compromising for example the breeding of honeybees. But it is possible that other species may also be adversely affected as well, a mechanism which may contribute to the observed decline in biodiversity. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.

Analysis of the in vitro degradation and the in vivo tissue response to bi-layered 3D-printed scaffolds combining PLA and biphasic PLA/bioglass components - Guidance of the inflammatory response as basis for osteochondral regeneration.

The aim of the present study was the in vitro and in vivo analysis of a bi-layered 3D-printed scaffold combining a PLA layer and a biphasic PLA/bioglass G5 layer for regeneration of osteochondral defects in vivo Focus of the in vitro analysis was on the (molecular) weight loss and the morphological and mechanical variations after immersion in SBF. The in vivo study focused on analysis of the tissue reactions and differences in the implant bed vascularization using an established subcutaneous implantation model in CD-1 mice and established histological and histomorphometrical methods. Both scaffold parts kept their structural integrity, while changes in morphology were observed, especially for the PLA/G5 scaffold. Mechanical properties decreased with progressive degradation, while the PLA/G5 scaffolds presented higher compressive modulus than PLA scaffolds. The tissue reaction to PLA included low numbers of BMGCs and minimal vascularization of its implant beds, while the addition of G5 lead to higher numbers of BMGCs and a higher implant bed vascularization. Analysis revealed that the use of a bi-layered scaffold shows the ability to observe distinct in vivo response despite the physical proximity of PLA and PLA/G5 layers. Altogether, the results showed that the addition of G5 enables to reduce scaffold weight loss and to increase mechanical strength. Furthermore, the addition of G5 lead to a higher vascularization of the implant bed required as basis for bone tissue regeneration mediated by higher numbers of BMGCs, while within the PLA parts a significantly lower vascularization was found optimally for chondral regeneration. Thus, this data show that the analyzed bi-layered scaffold may serve as an ideal basis for the regeneration of osteochondral tissue defects. Additionally, the results show that it might be able to reduce the number of experimental animals required as it may be possible to analyze the tissue response to more than one implant in one experimental animal.

TLR4 stimulation by LPS enhances angiogenesis in a co-culture system consisting of primary human osteoblasts and outgrowth endothelial cells.

The development of new approaches leading to fast and successful vascularization of tissue-engineered constructs is one of the most intensively studied subjects in tissue engineering and regenerative medicine. Recently, TLR4 activation and LPS stimulation of endothelial cells have been reported to promote angiogenesis in a variety of settings. In this study, we demonstrate that TLR4 activation by Ultrapure LPS Escherichia coli 0111:B4 (LPS-EB) significantly enhances microvessel formation in a co-culture system consisting of outgrowth endothelial cells (OECs) and primary human osteoblasts (pOBs). The precise modes of TLR4 action on the process of angiogenesis have also been investigated in this study. Using quantitative fluorescence microscopy in monocultures of OECs and pOBs, it was found that TLR4 activation through LPS-EB upregulates the expression level of TLR4/MYD88 and enhances both angiogenesis and osteogenesis. Furthermore, ELISA and qRT-PCR have shown that the level of two adhesion molecules (ICAM-1 and E-selectin), two cytokines (IL-6 and IL-8) and two growth factors (VEGF and PDGF-BB) related to angiogenesis increase significantly after LPS-EB treatment. This increased understanding of the role of TLR4 in angiogenesis could be of value in various settings related to tissue repair and tissue engineering. Moreover, since LPS and TLR4 agonists improve angiogenesis and osteogenesis, TLR4 agonists (endogenous or synthetic) could be used for angiogenesis intervention in vivo and therefore could be tested for their potential clinical applications in promoting angiogenesis in bone tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.

The role of the intestinal microvasculature in inflammatory bowel disease: studies with a modified Caco-2 model including endothelial cells resembling the intestinal barrier in vitro.

The microvascular endothelium of the gut barrier plays a crucial role during inflammation in inflammatory bowel disease. We have modified a commonly used intestinal cell model based on the Caco-2 cells by adding microvascular endothelial cells (ISO-HAS-1). Transwell filters were used with intestinal barrier-forming Caco-2 cells on top and the ISO-HAS-1 on the bottom of the filter. The goal was to determine whether this coculture mimics the in vivo situation more closely, and whether the model is suitable to evaluate interactions of, for example, prospective nanosized drug vehicles or contrast agents with this coculture in a physiological and inflamed state as it would occur in inflammatory bowel disease. We monitored the inflammatory responsiveness of the cells (release of IL-8, soluble intercellular adhesion molecule 1, and soluble E-selectin) after exposure to inflammatory stimuli (lipopolysaccharide, TNF-α, INF-γ, IL1-ß) and a nanoparticle (Ba/Gd: coprecipitated BaSO4 and Gd(OH)3), generally used as contrast agents. The barrier integrity of the coculture was evaluated via the determination of transepithelial electrical resistance and the apparent permeability coefficient (Papp) of NaFITC. The behavior of the coculture Caco-1/ISO-HAS-1 was compared to the respective monocultures Caco-2 and ISO-HAS-1. Based on transepithelial electrical resistance, the epithelial barrier integrity of the coculture remained stable during incubation with all stimuli, whereas the Papp decreased after exposure to the cytokine mixture (TNF-α, INF-γ, IL1-ß, and Ba/Gd). Both the endothelial and epithelial monocultures showed a high inflammatory response in both the upper and lower transwell-compartments. However, in the coculture, inflammatory mediators were only detected on the epithelial side and not on the endothelial side. Thus in the coculture, based on the Papp, the epithelial barrier appears to prevent a potential inflammatory overreaction in the underlying endothelial cells. In summary, this coculture model exhibits in vivo-like features, which cannot be observed in conventional monocultures, making the former more suitable to study interactions with external stimuli.

Detection of cellular senescence within human invasive breast carcinomas distinguishes different breast tumor subtypes.

Oncogene-induced senescence is thought to act as a barrier to tumorigenesis by arresting cells at risk of malignant transformation. Nevertheless, numerous findings suggest that senescent cells may conversely promote tumor progression through the development of the senescence-associated secretome they produce. It is likely that the composition and the physiological consequences mediated by the senescence secretome are dependent on the oncogenes that trigger the senescence program. Breast cancer represents a heterogenous disease that can be divided into breast cancer subtypes due to different subsets of genetic and epigenetic abnormalities. As tumor initiation and progression of these breast cancer subtypes is triggered by diverse oncogenic stimuli, differences in the senescence secretomes within breast tumors might be responsible for tumor initiation, progression, metastasis and therapeutic response. Many studies have addressed the role of senescence as a barrier to tumor progression using murine xenograft models. However, few investigations have been performed to elucidate the degree to which senescent tumor cells are present within untreated human tumors, and if present, whether these senescent tumor cells may play a role in disease progression. In the present study we analysed the appearance of senescent cells within invasive breast cancers. Detection of cellular senescence by the use of SAß-galactosidase (SAß-gal) staining within invasive breast carcinoms from 129 untreated patients revealed differences in the amount of SAß-gal+ tumor cells between breast cancer subtypes. The highest percentages of SAß-gal+ tumor cells were found in HER2-positive and luminal A breast carcinomas whereas triple negative tumors showed either little or no positivity.

Honeybees Produce Millimolar Concentrations of Non-Neuronal Acetylcholine for Breeding: Possible Adverse Effects of Neonicotinoids.

The worldwide use of neonicotinoid pesticides has caused concern on account of their involvement in the decline of bee populations, which are key pollinators in most ecosystems. Here we describe a role of non-neuronal acetylcholine (ACh) for breeding of Apis mellifera carnica and a so far unknown effect of neonicotinoids on non-target insects. Royal jelly or larval food are produced by the hypopharyngeal gland of nursing bees and contain unusually high ACh concentrations (4-8 mM). ACh is extremely well conserved in royal jelly or brood food because of the acidic pH of 4.0. This condition protects ACh from degradation thus ensuring delivery of intact ACh to larvae. Raising the pH to ≥5.5 and applying cholinesterase reduced the content of ACh substantially (by 75-90%) in larval food. When this manipulated brood was tested in artificial larval breeding experiments, the survival rate was higher with food supplemented by 100% with ACh (6 mM) than with food not supplemented with ACh. ACh release from the hypopharyngeal gland and its content in brood food declined by 80%, when honeybee colonies were exposed for 4 weeks to high concentrations of the neonicotinoids clothianidin (100 parts per billion [ppb]) or thiacloprid (8,800 ppb). Under these conditions the secretory cells of the gland were markedly damaged and brood development was severely compromised. Even field-relevant low concentrations of thiacloprid (200 ppb) or clothianidin (1 and 10 ppb) reduced ACh level in the brood food and showed initial adverse effects on brood development. Our findings indicate a hitherto unknown target of neonicotinoids to induce adverse effects on non-neuronal ACh which should be considered when re-assessing the environmental risks of these compounds. To our knowledge this is a new biological mechanism, and we suggest that, in addition to their well documented neurotoxic effects, neonicotinoids may contribute to honeybee colony losses consecutive to a reduction of the ACh content in the brood food.

Influenza virus damages the alveolar barrier by disrupting epithelial cell tight junctions.

A major cause of respiratory failure during influenza A virus (IAV) infection is damage to the epithelial-endothelial barrier of the pulmonary alveolus. Damage to this barrier results in flooding of the alveolar lumen with proteinaceous oedema fluid, erythrocytes and inflammatory cells. To date, the exact roles of pulmonary epithelial and endothelial cells in this process remain unclear.Here, we used an in vitro co-culture model to understand how IAV damages the pulmonary epithelial-endothelial barrier. Human epithelial cells were seeded on the upper half of a transwell membrane while human endothelial cells were seeded on the lower half. These cells were then grown in co-culture and IAV was added to the upper chamber.We showed that the addition of IAV (H1N1 and H5N1 subtypes) resulted in significant barrier damage. Interestingly, we found that, while endothelial cells mounted a pro-inflammatory/pro-coagulant response to a viral infection in the adjacent epithelial cells, damage to the alveolar epithelial-endothelial barrier occurred independently of endothelial cells. Rather, barrier damage was associated with disruption of tight junctions amongst epithelial cells, and specifically with loss of tight junction protein claudin-4.Taken together, these data suggest that maintaining epithelial cell integrity is key in reducing pulmonary oedema during IAV infection.