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Abdominal trauma (AT) is of major global importance, particularly because the civil, terroristic, and military traumatic potential of blast injuries has increased. The consequences of blunt abdominal injuries are highly variable and frequently underestimated or even overlooked. However, the underlying path mechanisms and subsequent innate immune response remain poorly understood. Therefore, we investigated the spatiotemporal local and systemic effects of a standardized blast-induced blunt AT on the intestine and innate immune response. In an established AT model, 66 male C57Bl6 mice were anesthetized and exposed to either a single blast wave centered on the epigastrium or control treatment (sham). At 2, 6, or 24 hours after trauma induction, animals were sacrificed. In 16 of 44 (36%) AT animals, one or more macroscopically visible injuries of the intestine were observed. Epithelial damage was detected by histological analysis of jejunum and ileum tissue samples, quantified by the Chiu score and by increased plasma concentrations of the intestinal fatty acid-binding protein, an enterocyte damage marker. Moreover, in the early posttraumatic period, elevated syndecan-1, claudin-5, and mucin-2 plasma levels also indicated alterations in the gut-blood barrier. Increased levels of pro-inflammatory cytokines such as TNF and macrophage inflammatory protein 2 in tissue homogenates and plasma indicate a systemic immune activation after blunt AT. In conclusion, we detected early morphological intestinal damage associated with high, early detectable intestinal fatty acid-binding protein plasma levels, and a considerable time- and dose-dependent impairment of the gut-blood barrier in a newly established mouse model of blunt AT. It appears to be a sufficient model for further studies of the intestinal immunopathophysiological consequences of AT and the evaluation of novel therapeutic approaches.

IntroductionWe previously showed that attenuated glucocorticoid receptor (GR) function in mice (GRdim/dim) aggravates systemic hypotension and impairs organ function during endotoxic shock. Hemorrhagic shock (HS) causes impaired organ perfusion, which leads to tissue hypoxia and inflammation with risk of organ failure. Lung co-morbidities like chronic obstructive pulmonary disease (COPD) can aggravate tissue hypoxia via alveolar hypoxia. The most common cause for COPD is cigarette smoke (CS) exposure. Therefore, we hypothesized that affecting GR function in mice (GRdim/dim) and pre-traumatic CS exposure would further impair hemodynamic stability and organ function after HS.MethodsAfter 3 weeks of CS exposure, anesthetized and mechanically ventilated GRdim/dim and GR+/+ mice underwent pressure-controlled HS for 1h via blood withdrawal (mean arterial pressure (MAP) 35mmHg), followed by 4h of resuscitation with re-transfusion of shed blood, colloid fluid infusion and, if necessary, continuous intravenous norepinephrine. Acid–base status and organ function were assessed together with metabolic pathways. Blood and organs were collected at the end of the experiment for analysis of cytokines, corticosterone level, and mitochondrial respiratory capacity. Data is presented as median and interquartile range.ResultsNor CS exposure neither attenuated GR function affected survival. Non-CS GRdim/dim mice had a higher need of norepinephrine to keep target hemodynamics compared to GR+/+ mice. In contrast, after CS exposure norepinephrine need did not differ significantly between GRdim/dim and GR+/+ mice. Non-CS GRdim/dim mice presented with a lower pH and increased blood lactate levels compared to GR+/+ mice, but not CS exposed mice. Also, higher plasma concentrations of some pro-inflammatory cytokines were observed in non-CS GRdim/dim compared to GR+/+ mice, but not in the CS group. With regards to metabolic measurements, CS exposure led to an increased lipolysis in GRdim/dim compared to GR+/+ mice, but not in non-CS exposed animals.ConclusionWhether less metabolic acidosis or increased lipolysis is the reason or the consequence for the trend towards lower catecholamine need in CS exposed GRdim/dim mice warrants further investigation.

We previously demonstrated marked lung-protective properties of the H

Abdominal trauma (AT) is of major global importance, particularly with the increased potential for civil, terroristic, and military trauma. The injury pattern and systemic consequences of blunt abdominal injuries are highly variable and frequently underestimated or even missed, and the pathomechanisms remain still poorly understood. Therefore, we investigated the temporal-spatial organ and immune response after a standardized blast-induced blunt AT. Anesthetized mice were exposed to a single blast wave centered on the epigastrium. At 2, 6, or 24 h after trauma, abdominal organ damage was assessed macroscopically, microscopically, and biochemically. A higher degree of trauma severity, determined by a reduction of the distance between the epigastrium and blast inductor, was reflected by a reduced survival rate. The hemodynamic monitoring during the first 120 min after AT revealed a decline in the mean arterial pressure within the first 80 min, whereas the heart rate remained quite stable. AT induced a systemic damage and inflammatory response, evidenced by elevated HMGB-1 and IL-6 plasma levels. The macroscopic injury pattern of the abdominal organs (while complex) was consistent, with the following frequency: liver > pancreas > spleen > left kidney > intestine > right kidney > others > lungs and was reflected by microscopic liver and pancreas damages. Plasma levels of organ dysfunction markers increased during the first 6 h after AT and subsequently declined, indicating an early, temporal impairment of the function on a multi-organ level. The established highly reproducible murine blunt AT, with time- and trauma-severity-dependent organ injury patterns, systemic inflammatory response, and impairment of various organ functions, reflects characteristics of human AT. In the future, this model may help to study the complex immuno-pathophysiological consequences and innovative therapeutic approaches after blunt AT.

Calcitonin gene-related peptide (CGRP) and procalcitonin, which are overexpressed in sepsis, exert distinct immunomodulatory effects mediated through the CGRP receptor. The CGRP receptor antagonist olcegepant improves survival in murine sepsis. This study evaluated whether CGRP receptor antagonism is similarly beneficial in a porcine model of polymicrobial sepsis.We conducted a prospective randomised, controlled, investigator-blinded trial in adult pigs of either sex, that were anaesthetised and ventilated before sepsis was induced by polymicrobial (autologous) faecal peritonitis. After the onset of early septic shock (systolic blood pressure90 mm Hg or10% decline from baseline MAP), pigs were resuscitated (i.v. fluid/antibiotics/vasopressors) and randomised to receive either i.v. olcegepant (n=8) or vehicle control (n=8). The primary outcome was time to death, euthanasia required up to 72 h after surgery (according to predefined severe cardiorespiratory failure), or both. Secondary outcomes included haemodynamic changes, and systemic as well as organ inflammation (mRNA expression).Septic shock developed 8.7 h (inter-quartile range, 5.8-11.1 h) after the onset of faecal peritonitis. Olcegepant worsened survival, with 6/8 pigs randomised to the control group surviving 72.0 h (50.9-72.0 h), compared with 3/8 pigs receiving olcegepant surviving 51.3 h (12.5-72.0 h; P=0.01). At 48 h, lower MAP and higher cardiac output occurred in pigs receiving olcegepant. Cardiac, hepatic, and renal injury was not different between pigs randomised to receive olcegepant or vehicle. Olcegepant reduced mRNA expression of several inflammation-related cytokines and CD68CGRP receptor antagonism with olcegepant was not beneficial in this porcine model of polymicrobial sepsis, which closely mimics human sepsis.

As a response to pro-inflammatory signals mesenchymal stem cells (MSCs) secrete agents and factors leading to lymphocyte recruitment, counteracting inflammation, and stimulating immunosuppression. On a molecular level, the signalling mediator TGF-β-activated kinase 1 (TAK1) is activated by many pro-inflammatory signals, plays a critical role in inflammation and regulates innate and adaptive immune responses as well. While the role of TAK1 as a signalling factor promoting inflammation is well documented, we also considered a role for TAK1 in anti-inflammatory actions exerted by activated MSCs. We, therefore, investigated the capacity of lipopolysaccharide (LPS)-treated murine MSCs with lentivirally modulated TAK1 expression levels to recruit lymphocytes. TAK1 downregulated by lentiviral vectors expressing TAK1 shRNA in murine MSCs interfered with the capacity of murine MSCs to chemoattract lymphocytes, indeed. Analysing a pool of 84 secreted factors we found that among 26 secreted cytokines/factors TAK1 regulated expression of one cytokine in LPS-activated murine MSCs in particular: interleukin-6 (IL-6). IL-6 in LPS-treated MSCs was responsible for lymphocyte recruitment as substantiated by neutralizing antibodies. Our studies, therefore, suggest that in LPS-treated murine MSCs the inflammatory signalling mediator TAK1 may exert anti-inflammatory properties via IL-6. Supplementary Information The online version contains supplementary material available at 10.1007/s11010-021-04180-8.

Polycaprolactone (PCL) fiber mats with different surface modifications were functionalized with a chitosan nanogel coating to attach the growth factor human bone morphogenetic protein 2 (BMP-2). Three different hydrophilic surface modifications were compared with regard to the binding and in vitro release of BMP-2. The type of surface modification and the specific surface area derived from the fiber thickness had an important influence on the degree of protein loading. Coating the PCL fibers with polydopamine resulted in the binding of the largest BMP-2 quantity per surface area. However, most of the binding was irreversible over the investigated period of time, causing a low release in vitro. PCL fiber mats with a chitosan-graft-PCL coating and an additional alginate layer, as well as PCL fiber mats with an air plasma surface modification boundless BMP-2, but the immobilized protein could almost completely be released. With polydopamine and plasma modifications as well as with unmodified PCL, high amounts of BMP-2 could also be attached directly to the surface. Integration of BMP-2 into the chitosan nanogel functionalization considerably increased binding on all hydrophilized surfaces and resulted in a sustained release with an initial burst release of BMP-2 without detectable loss of bioactivity in vitro.

Acute exposure to high concentrations of H2S causes severe brain injury and long-term neurological disorders. The mechanisms of H2S-induced neurodegeneration are not known. To better understand the cellular and molecular mechanisms of H2S-induced neurodegeneration we used a broad-spectrum proteomic analysis approach to search for key molecules in H2S-induced neurotoxicity. Mice were subjected to acute whole body exposure of up to750 ppm of H2S. The H2S-treated group showed behavioral motor deficits and developed severe lesions in the inferior colliculus (IC), part of the brainstem. The IC was microdissected for proteomic analysis. Tandem mass tags (TMT) liquid chromatography mass spectrometry (LC-MS/MS)-based quantitative proteomics was applied for protein identification and quantitation. LC-MS/MS was able to identify 598, 562, and 546 altered proteomic changes for day 1 (2 h post H2S exposure), day 2, and day 4 of H2S exposure, respectively. Mass spectrometry data were analyzed by Perseus 1.5.5.3 statistical analysis, and gene ontology heat map clustering. Quantitative real-time PCR was used to confirm some of the H2S-dependent proteomics changes. Taken together, acute exposure to H2S induced behavioral motor deficits along with progressive neurodegeneration including disruption of several biological processes in the IC such as cellular morphology, energy metabolism, and calcium signaling. The obtained broad-spectrum proteomics data may provide important clues to elucidate mechanisms of H2S-induced neurotoxicity.HighlightsMice exposed to H2S recapitulated H2S-induced neurotoxicity manifested in humans.The IC in the mouse brain is the most sensitive to H2S-induced neurodegeneration.Proteomic expressions of key proteins were validated at transcription level.Several biological pathways were dysregulated by H2S exposure.

Remyelination is the natural repair mechanism in demyelinating disorders such as multiple sclerosis (MS) and it was proposed that it might protect from axonal loss. For unknown reasons, remyelination is often incomplete or fails in MS lesions and therapeutic treatments to enhance remyelination are not available. Recently, the transplantation of exogenous mesenchymal stem cells (MSC) has emerged as a promising tool to enhance repair processes. This included the animal model experimental autoimmune encephalomyelitis (EAE), a commonly used model for the autoimmune mechanisms of MS. However, in EAE it is not clear if the beneficial effect of MSC derives from a direct influence on brain resident cells or if this is an indirect phenomenon via modulation of the peripheral immune system. The aim of this study was to determine potential regenerative functions of MSC in the toxic cuprizone model of demyelination that allows studying direct effects on de- and remyelination without the influence of the peripheral immune system. MSC from three different species (human, murine, canine) were transplanted either intraventricularly into the cerebrospinal fluid or directly into the lesion of the corpus callosum at two time points: at the onset of oligodendrocyte progenitor cell (OPC) proliferation or the peak of OPC proliferation during cuprizone induced demyelination. Our results show that MSC did not exert any regenerative effects after cuprizone induced demyelination and oligodendrocyte loss. During remyelination, MSC did not influence the dynamics of OPC proliferation and myelin formation. In conclusion, MSC did not exert direct regenerative functions in a mouse model where peripheral immune cells and especially T lymphocytes do not play a role. We thus suggest that the peripheral immune system is required for MSC to exert their effects and this is independent from a direct influence of the central nervous system.

Mesenchymal stem cells (MSCs) are regarded as an immune privileged cell type with numerous regeneration-promoting effects. The in vivo behavior of MSC and underlying mechanisms leading to their regenerative effects are largely unknown. The aims of this study were to comparatively investigate the in vivo behavior of canine (cMSC), human (hMSC), and murine MSC (mMSC) following intra-cerebroventricular transplantation. At 7 days post transplantation (dpt), clusters of cMSC, hMSC, and mMSC were detected within the ventricular system. At 49 dpt, cMSC-transplanted mice showed clusters mostly consisting of extracellular matrix lacking transplanted MSC. Similarly, hMSC-transplanted mice lacked MSC clusters at 49 dpt. Xenogeneic MSC transplantation was associated with a local T lymphocyte-dominated immune reaction at both time points. Interestingly, no associated inflammation was observed following syngeneic mMSC transplantation. In conclusion, transplanted MSC formed intraventricular cell clusters and exhibited a short life span in vivo. Xenogeneically in contrast to syngeneically transplanted MSC triggered a T cell-mediated graft rejection indicating that MSCs are not as immune privileged as previously assumed. However, MSC may mediate their effects by a "hit and run" mechanism and future studies will show whether syngeneically or xenogeneically transplanted MSCs exert better therapeutic effects in animals with CNS disease.

Rotator cuff tear is the most frequent tendon injury in the adult population. Despite current improvements in surgical techniques and the development of grafts, failure rates following tendon reconstruction remain high. New therapies, which aim to restore the topology and functionality of the interface between muscle, tendon and bone, are essentially required. One of the key factors for a successful incorporation of tissue engineered constructs is a rapid ingrowth of cells and tissues, which is dependent on a fast vascularization. The dorsal skinfold chamber model in female BALB/cJZtm mice allows the observation of microhemodynamic parameters in repeated measurements in vivo and therefore the description of the vascularization of different implant materials. In order to promote vascularization of implant material, we compared a porous polymer patch (a commercially available porous polyurethane based scaffold from Biomerix™) with electrospun polycaprolactone (PCL) fiber mats and chitosan-graft-PCL coated electrospun PCL (CS-g-PCL) fiber mats in vivo. Using intravital fluorescence microscopy microcirculatory parameters were analyzed repetitively over 14 days. Vascularization was significantly increased in CS-g-PCL fiber mats at day 14 compared to the porous polymer patch and uncoated PCL fiber mats. Furthermore CS-g-PCL fiber mats showed also a reduced activation of immune cells. Clinically, these are important findings as they indicate that the CS-g-PCL improves the formation of vascularized tissue and the ingrowth of cells into electrospun PCL scaffolds. Especially the combination of enhanced vascularization and the reduction in immune cell activation at the later time points of our study points to an improved clinical outcome after rotator cuff tear repair. © 2020 Gniesmer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Homozygous loss of SMN1 causes spinal muscular atrophy (SMA), the most common and devastating childhood genetic motor-neuron disease. The copy gene SMN2 produces only ∼10% functional SMN protein, insufficient to counteract development of SMA. In contrast, the human genetic modifier plastin 3 (PLS3), an actin-binding and -bundling protein, fully protects against SMA in SMN1 -deleted individuals carrying 3–4 SMN2 copies. Here, we demonstrate that the combinatorial effect of suboptimal SMN antisense oligonucleotide treatment and PLS3 overexpression—a situation resembling the human condition in asymptomatic SMN1 -deleted individuals—rescues survival (from 14 to >250 days) and motoric abilities in a severe SMA mouse model. Because PLS3 knockout in yeast impairs endocytosis, we hypothesized that disturbed endocytosis might be a key cellular mechanism underlying impaired neurotransmission and neuromuscular junction maintenance in SMA. Indeed, SMN deficit dramatically reduced endocytosis, which was restored to normal levels by PLS3 overexpression. Upon low-frequency electro-stimulation, endocytotic FM1-43 (SynaptoGreen) uptake in the presynaptic terminal of neuromuscular junctions was restored to control levels in SMA-PLS3 mice. Moreover, proteomics and biochemical analysis revealed CORO1C, another F-actin binding protein, whose direct binding to PLS3 is dependent on calcium. Similar to PLS3 overexpression, CORO1C overexpression restored fluid-phase endocytosis in SMN-knockdown cells by elevating F-actin amounts and rescued the axonal truncation and branching phenotype in Smn-depleted zebrafish. Our findings emphasize the power of genetic modifiers to unravel the cellular pathomechanisms underlying SMA and the power of combinatorial therapy based on splice correction of SMN2 and endocytosis improvement to efficiently treat SMA.

Cells from the mononuclear phagocyte system (MPS) act as systemic and local amplifiers that contribute to the progression of chronic inflammatory disorders. Transforming growth factor-β–activated kinase 1 (TAK1) is a pivotal upstream mitogen-activated protein kinase-kinase-kinase acting as a mediator of cytokine expression. It remains critical to determine in vivo the implication of TAK1 in controlling the innate immune system. Here, we describe a vehicle tailored to selectively deliver siRNAs into MPS cells after intravenous administration, and validate in vivo the potential of the RNAi-mediated TAK1 knock down for immunomodulation. In a mouse model of immune-mediated inflammatory disorder, we show that anti-TAK1 siRNA lipoplexes efficiently alleviate inflammation, severely impair the downstream c-Jun N-terminal kinase and nuclear factor-κB signaling pathways, and decrease the expression of proinflammatory mediators. Importantly, the systemic TAK1 gene silencing decreases the frequency of Th1 and Th17 cells, both mediating autoimmunity in experimental arthritis, demonstrating the immunomodulatory potential of TAK1. Finally, in vitro inhibition of TAK1 in myeloid cells decreases interferon-γ–producing T cells, suggesting that a delivery sys-tem able to target MPS cells and to silence TAK1 impacts on pathogenic T effector cells in autoimmunity.

Mutations in the receptor tyrosine kinase Ror2 account for Brachydactyly type B and Robinow Syndrome. We have identified two novel factors interacting with the Ror2 intracellular domain. TAK1 (TGF-beta activated kinase 1), a MAP3K, interacts with Ror2 and phosphorylates its intracellular carboxyterminal serine/thronine/proline-rich (STP) domain. This TAK1-dependent phosphorylation of Ror2 induces phosphorylation of tyrosine-residues including a MAPK-like TGY-motif. The TAK1-dependent phosphorylation is enhanced by a second cytosolic factor, PRTB, which interacts with Ror2 and with TAK1 as well. The TAK1-dependent Tyr-phosphorylation of Ror2 is not mediated by the Ror2 tyrosine kinase domain and seems predominantly triggered by cytosolic kinases. Wnt-ligand binding differentially controls the Ror2/TAK1 interaction. Wnt1-binding displaces TAK1 from Ror2 while Wnt3a and Wnt5a are unable to do so thus modifying TAK1's capacity to cause phosphorylation of Ror2. Ror2 seems to act as a Wnt co-receptor enhancing Wnt-dependent canonical pathways while Tyr- and Ser/Thr-phosphorylation of Ror2 negatively controls the efficiency of these pathways. We propose that the level of the Wnt-ligand-regulated phosphorylation by cytosolic factors determines whether Ror2 acts as a stimulator or as an inhibitor of canonical Wnt-signalling.

Magnesium alloys are presently under investigation as promising biodegradable implant materials with osteoconductive properties. To study the molecular mechanisms involved, the potential contribution of soluble magnesium corrosion products to the stimulation of osteoblastic cell differentiation was examined. However, no evidence for the stimulation of osteoblast differentiation could be obtained when cultured mesenchymal precursor cells were differentiated in the presence of metallic magnesium or in cell culture medium containing elevated magnesium ion levels. Similarly, in soft tissue no bone induction by metallic magnesium or by the corrosion product magnesium hydroxide could be observed in a mouse model. Motivated by the comparatively rapid accumulation solid corrosion products physicochemical processes were examined as an alternative mechanism to explain the stimulation of bone growth by magnesium-based implants. During exposure to physiological solutions a structured corrosion coat formed on magnesium whereby the elements calcium and phosphate were enriched in the outermost layer which could play a role in the established biocompatible behavior of magnesium implants. When magnesium pins were inserted into avital bones, corrosion lead to increases in the pull out force, suggesting that the expanding corrosion layer was interlocking with the surrounding bone. Since mechanical stress is a well-established inducer of bone growth, volume increases caused by the rapid accumulation of corrosion products and the resulting force development could be a key mechanism and provide an explanation for the observed stimulatory effects of magnesium-based implants in hard tissue. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 697-709, 2017.

In this study, the mouse neuroblastoma cell line Neuro-2a was analyzed for expression of angiotensin II receptors. Reverse-transcriptase polymerase chain reaction (RT-PCR) showed that Neuro-2a cells express mRNA of angiotensin II (AngII) receptor subtypes AT1A, AT1B, and AT2. Analysis of Neuro-2a cells by Western blotting revealed AT1 and AT2 receptor protein expression. The predominant molecular weights were determined to be 50.4 kDa for the AT1 receptor and 62.4 kDa for the AT2 receptor. Observation of AT1 and AT2 receptor localization within Neuro-2a cells using immunocytochemistry showed distribution similar to other G-protein coupled receptors with diffuse distribution in the cytosol, perinuclear enrichment and accumulation of receptors on the outer cellular periphery with extension into the neurites. Furthermore, we observed InsP3 formation following AngII induction that could be abolished in presence of the AT1A receptor antagonist losartan. The results clearly show expression of the AngII receptor types AT1A and AT2 in the Neuro-2a cell line. We conclude that Neuro-2a cells represent an interesting model cell line for study of mechanisms that control the interplay between these receptors.

For the treatment of patients with multiple sclerosis there are no regenerative approaches to enhance remyelination. Mesenchymal stem cells (MSC) have been proposed to exert such regenerative functions. Intravenous administration of human MSC reduced the clinical severity of experimental autoimmune encephalomyelitis (EAE), an animal model mimicking some aspects of multiple sclerosis. However, it is not clear if this effect was achieved by systemic immunomodulation or if there is an active neuroregeneration in the central nervous system (CNS). In order to investigate remyelination and regeneration in the CNS we analysed the effects of intravenously and intranasally applied murine and human bone marrow-derived MSC on cuprizone induced demyelination, a toxic animal model which allows analysis of remyelination without the influence of the peripheral immune system. In contrast to EAE no effects of MSC on de- and remyelination and glial cell reactions were found. In addition, neither murine nor human MSC entered the lesions in the CNS in this toxic model. In conclusion, MSC are not directed into CNS lesions in the cuprizone model where the blood-brain-barrier is intact and thus cannot provide support for regenerative processes.

The high recurrence rate of secondary cataract (SC) is caused by the intrinsic differentiation activity of residual lens epithelial cells after extra-capsular lens removal. The objective of this study was to identify changes in the microRNA (miRNA) expression profile during mouse SC formation and to selectively manipulate miRNA expression for potential therapeutic intervention. To model SC, mouse cataract surgery was performed and temporal changes in the miRNA expression pattern were determined by microarray analysis. To study the potential SC counterregulative effect of miRNAs, a lens capsular bag in vitro model was used. Within the first 3 wks after cataract surgery, microarray analysis demonstrated SC-associated expression pattern changes of 55 miRNAs. Of the identified miRNAs, miR-184 and miR-204 were chosen for further investigations. Manipulation of miRNA expression by the miR-184 inhibitor (anti-miR-184) and the precursor miRNA for miR-204 (pre-miR-204) attenuated SC-associated expansion and migration of lens epithelial cells and signs of epithelial to mesenchymal transition such as α-smooth muscle actin expression. In addition, pre-miR-204 attenuated SC-associated expression of the transcription factor Meis homeobox 2 (MEIS2). Examination of miRNA target binding sites for miR-184 and miR-204 revealed an extensive range of predicted target mRNA sequences that were also a target to a complex network of other SC-associated miRNAs with possible opposing functions. The identification of the SC-specific miRNA expression pattern together with the observed in vitro attenuation of SC by anti-miR-184 and pre-miR-204 suggest that miR-184 and miR-204 play a significant role in the control of SC formation in mice that is most likely regulated by a complex competitive RNA network.

The treatment of choice for profound sensorineural hearing loss (SNHL) is direct electrical stimulation of spiral ganglion cells (SGC) via a cochlear implant (CI). The number and excitability of SGC seem to be critical for the success that can be achieved via CI treatment. However, SNHL is associated with degeneration of SGC. Long-term drug delivery to the inner ear for improving SGC survival may be achieved by functionalisation of CI electrodes with cells providing growth factors. Therefore, the capacity of brain-derived neurotrophic factor (BDNF)-secreting NIH3T3 cells grown on cylindrically shaped silicone elastomers (SE) to exert local and sustained neuroprotective effects was assessed in vitro and in vivo. An in vitro model to investigate adhesion and cell growth of lentivirally modified NIH3T3 cells synthesising BDNF on SE was established. The bioactivity of BDNF was characterised by co-cultivation of SGC with cell-coated SE. In addition, cell-coated SE were implanted into deafened guinea pigs. The recombinant NIH3T3 cells proliferated on silicone surfaces during 14 days of cultivation and expressed significantly increasing BDNF levels. Enhanced survival rates and neurite outgrowth of SGC demonstrated the bioactivity of BDNF in vitro. Implantation of SE with adhering BDNF-secreting NIH3T3 cells into the cochleae of systemically deafened guinea pigs induced a significant increase in SGC survival in comparison to SE without cell coating. Our data demonstrate a novel approach of cell-based long-term drug delivery to support SGC survival in vitro and in vivo. This therapeutic strategy--once transferred to cells suitable for clinical application--may improve CI performance.

A fast and simple approach for immobilization using copolymers as interlayers is reported. The synthesized copolymers form stable self-assembled layers on implant materials like, e.g., titanium in a simple coating/drying/washing sequence and have functional groups which can bind proteins from an aqueous solution. The copolymer films have been characterized via ellipsometry and contact angle measurements and were tested for biocompatibility. An immunoassay was used to determine the amount of BMP2 and demonstrated an approximately 10-fold increase as compared to previously used self-assembled monolayers. A BMP2-responsive cell line with luciferase detection was used to determine the biological activity of the bound signaling protein.

Ligament-to-bone and tendon-to-bone interfaces (entheses, osteotendinous junctions [OTJs]) serve to dissipate stress between soft tissue and bone. Surgical reconstruction of these interfaces is an issue of considerable importance as they are prone to injury and the integration of bone and tendon/ligament is in general not satisfactory. We report here the stem cell-dependent spontaneous formation of fibrocartilaginous and fibrous entheses in heterotopic locations of the mouse if progenitors possess a tenogenic and osteo-/chondrogenic capacity. This study followed the hypothesis that enhanced Bone Morphogenetic Protein (BMP)-signaling in adult mesenchymal stem cells that are induced for tendon formation may overcome the tendon-inherent interference with bone formation and may thus allow the stem cell-dependent formation of tendon-bone interfaces. The tenogenic and osteo-/chondrogenic competence was mediated by the adeno- and/or lentiviral expression of the biologically active Smad8 signaling mediator (Smad8ca) and of Bone Morphogenetic Protein 2 (BMP2). Modified mesenchymal progenitors were implanted in subcutaneous or intramuscular sites of the mouse. The stem cell-dependent enthesis formation was characterized histologically by immunohistological approaches and by in situ hybridization. Transplantation of modified murine stem cells resulted in the formation of tendinous and osseous structures exhibiting fibrocartilage-type OTJs, while, in contrast, the viral modification of primary human bone marrow-derived mesenchymal stromal/stem cells showed evidence of fibrous tendon-bone interface formation. Moreover, it could be demonstrated that Smad8ca expression alone was sufficient for the formation of tendon/ligament-like structures. These findings may contribute to the establishment of stem cell-dependent regenerative therapies involving tendon/ligaments and to the improvement of the insertion of tendon grafts at bony attachment sites, eventually.

Hypothesis: Recombinantly modified cells deliver neurotrophic factors with the capacity to induce differentiation and the outgrowth of neurites of rat pheochromocytoma cells 12 (PC12) serving as a neuronal model. Background: The benefit of cochlea implant (CI) is depending, among other factors, on the number of surviving spiral ganglion neurons (SGN). Studies have shown that the external application of neurotrophic factors in combination with electrical stimulation increases the survival rate of SGN after ototrauma. Therefore, functionalization of electrodes with recombinantly modified cells providing neurotrophic factors to the SGN for inducing survival mechanisms may be an approach to realize drug delivery to the cochlea. Methods: Murine NIH3T3 cells were recombinantly modified with an infectious lentiviral monocistronic and bicistronic system to synthesize glial cell line-derived neurotrophic factor and the green fluorescent protein. Free glial cell line-derived neurotrophic factor from the supernatant of the modified NIH3T3 cells was added to rat PC12, and the neuronal-like outgrowth was determined for 10 days. Results: A significant neuronal-like outgrowth appeared as early as Day 3 after the application of the supernatant. Conclusion: The results indicate that the established in vitro model represents a powerful basic model for determining signal pathways between neuronal-like processing PC12 cells and cellular drug delivery systems.

The benefit achieved by the use of cochlear implants depends among other factors on the number of surviving spiral ganglion cells (SGCs). Neurotrophic factors, especially brain-derived neurotrophic factor (BDNF), have a protective effect on spiral ganglions. Coating of the cochlear implant electrode with BDNF-producing cells may provide long-term delivery of the factor. Therefore, the hypothesis that BDNF-producing fibroblasts can enhance cell survival of cultured SGCs was tested. Lentiviral infection of fibroblasts resulted in BDNF production. Conditioned medium obtained from infected fibroblasts was used for the cultivation of SGCs. As a result, improved survival and neurite outgrowth was observed on SGCs. Our results demonstrate that lentivirally infected fibroblasts produce BDNF that has neurotrophic effects on spiral ganglions.

A bioinformatics-based analysis of endochondral bone formation model detected several genes upregulated in this process. Among these genes the dickkopf homolog 3 (Dkk3) was upregulated and further studies showed that its expression affects in vitro and in vivo osteogenesis. This study indicates a possible role of Dkk3 in regulating bone formation. Introduction: Endochondral bone formation is a complex biological process involving numerous chondro- genic, osteogenic, and angiogenic proteins, only some of which have been well studied. Additional key genes may have important roles as well. We hypothesized that to identify key genes and signaling pathways crucial for bone formation, a comprehensive gene discovery strategy should be applied to an established in vivo model of osteogenesis. Materials and Methods: We used in vivo implanted C3H10T1/2 cells that had been genetically engineered to express human bone morphogenetic protein-2 (BMP2) in a tetracycline-regulated system that controls osteo- genic differentiation. Oligonucleotide microarray data from the implants (n 4 repeats) was analyzed using coupled two-way clustering (CTWC) and statistical methods. For studying the effects of dickkopf homolog 3 (Dkk3) in chondrogenesis and osteogenesis, C3H10T1/2 mesenchymal progenitors were used. Results: The CTWC revealed temporal expression of Dkk3 with other chondrogenesis-, osteogenesis-, and Wnt-related genes. Quantitative RT-PCR confirmed the expression of Dkk3 in the implants. C3H10T1/2 cells that expressed Dkk3 in the presence of BMP2 displayed lower levels of alkaline phosphatase and collagen I mRNA expression than control C3H10T1/2 cells that did not express Dkk3. Interestingly, the levels of collagen II mRNA expression, Alcian blue staining, and glucose aminoglycans (GAGs) production were not influenced by Dkk3 expression. In vivo CT and bioluminescence imaging revealed that co-expression of Dkk3 and BMP2 by implanted C3H10T1/2 cells induced the formation of significantly lower quantities of bone than cells expressing only BMP2. Conclusions: A bioinformatics analysis enabled the identification of Dkk3 as a pivotal gene with a novel function in endochondral bone formation. Our results showed that Dkk3 might have inhibitory effects on osteogenesis, but no effect on chondrogenesis, indicating that Dkk3 plays a regulatory role in endochondral bone formation. Further mechanistic studies are required to reveal the mechanism of action of Dkk3 in endochondral bone formation.

1. The differential identification of the angiotensin AT 1 A and AT 1 B receptor subtypes is impaired by the existing > 96% homology of both receptors. In the present study, we characterized two polyclonal rabbit peptide antibodies, namely α-AT 1 A and α-AT 1 B , that recognize the C-terminal region of mouse AT 1 A and AT 1 B receptors, respectively. 2. In immunoblotting, both antibodies detected two major AT 1 receptor-specific bands at sizes of 72.5 and 87.6 kDa in mouse tissues and in Neuro-2a cell lysates. In immunohistochemistry, antibodies demonstrated AT 1 receptor-specific staining in renal proximal and distal tubules, as well as in kidney glomeruli. In addition, both antibodies stained AT 1 receptors in Neuro-2a cells with G-protein receptor typical distribution. Dot-blot and ELISA analysis of the α-AT 1 A antibody showed 2.5- to fourfold higher selectivity for its AT 1 A receptor target peptide (1A-PEP) compared with the non-specific AT 1 B receptor peptide (1B-PEP). In contrast, the α-AT 1 B antibody showed high binding affinity towards its target peptide 1B-PEP, but also demonstrated high cross-reactivity for the non-specific peptide 1A-PEP (1.4- to twofold in ELISA and dot-blot analysis). In contrast with the lack of recognition by the α-AT 1 B antibody, the α-AT 1 A antibody selectively recognized the AT 1 A receptor fused to red fluorescence protein in transiently transfected Chinese hamster ovary cells. 3. In summary, we have generated two new peptide antibodies to the mouse AT 1 A and AT 1 B receptors (α-AT 1 A and α-AT 1 B ), of which the α-AT 1 A antibody has the capability to distinguish AT 1 A receptor types in immunological approaches.

Because of the lack of pharmacological approaches, molecular genetic methods have been required to differentiate between angiotensin type 1(AT 1 ) receptor subtypes AT 1a and AT 1b . RNA interference is a new tool for the study of gene function, producing specific downregulation of protein expression. In this study, we used the small hairpin RNA (shRNA) cassette method to screen target sites for selectively silencing AT 1a or AT 1b receptor subtypes in cultured Neuro-2a cells using real-time RT-PCR. For in vivo functional studies, we used C57BL mice with arterial telemetric probes and computerized licking monitors to test the effect of adenovirus carrying the DNA sequence coding AT 1a shRNA (Ad-AT 1a -shRNA). Ad-AT 1a -shRNA was injected into the lateral ventricle (intracerebroventricular) or the brain stem nucleus tractus solitaries/dorsal vagal nucleus (NTS/DVN) with measurement of water intake, blood pressure (BP), and heart rate (HR) for up to 20 days after injection. Tissue culture studies verified the specificity and the efficiency of the constructs. In animal studies, β-galactosidase staining and Ang receptor binding assays showed expression of shRNA and downregulation of Ang AT 1 receptors in the subfornical organ and NTS/DVN by >70%. Intracerebroventricular injection of Ad-AT 1a -shRNA increased water intake with no effect on BP or HR. In contrast, microinjection of Ad-AT 1a -shRNA into NTS/DVN caused a decrease in BP with no effect on HR or water intake. Results demonstrate the use of the RNA interference method in site-directed silencing of gene expression and provide a method for the in vivo study of Ang AT 1 receptor function.

TAK1 (transforming growth factor-beta-activated kinase-1), a MAP3K with considerable sequence similarity to Raf-1 and MEKK-1, has been identified as a transforming growth factor-beta/bone morphogenetic protein (BMP)-activated cytosolic component of the MAPK pathways. In this investigation, the molecular interactions between TAK1 and Smad proteins were characterized as well as their influence on BMP-mediated mesenchymal cell differentiation along the osteogenic/chondrogenic pathway. In co-immunoprecipitations we found an interaction of TAK1 with all Smads tested, R-Smads Smads1-5, the co-Smad Smad4, and the inhibitory Smads (I-Smad6 and I-Smad7). Smad interaction with TAK1 takes place through their MH2 domain. This interaction is dependent on the presence of an active kinase domain in TAK1. TAK1 dramatically interferes with R-Smad transactivation in reporter assays and affects subcellular distribution of Smad proteins. Activated TAK1 also interferes with BMP-dependent osteogenic development in murine mesenchymal progenitor cells (C3H10T 1/2). A potential TAK1-mediated apoptosis process could be excluded for these cells. Both synergistic and interfering influences of TAK1 on BMP-mediated Smad-signaling have been reported previously. We suggest that TAK1 is a factor that is involved in the fine-tuning of BMP effects during osteogenic development.

The functional roles of BMP type IA and IB receptors mediating differentiation into the osteogenic and chondrogenic lineage were investigated in the mesenchymal progenitor line C3H10T1/2 in vitro. The capacity of type IA and IB BMP receptors was assessed by the forced expression of the wild-type (wtBMPR-IA or IB) and of the kinase-deficient, dominant-negative form (dnBMPR-IA or -IB) in parental C3H10T1/2 progenitors as well as in C3H10T1/2 progenitors which recombinantly express BMP2 (C3H10T1/2-BMP2) or GDF5 (C3H10T1/2-GDF5). Consistent with the higher endogenous expression rate of BMPR-IA in comparison with BMPR-IB, BMPR-IA plays the dominant role in BMP2-mediated osteo-/chondrogenic development. BMPR-IB moderately influences osteogenic and hardly chondrogenic development. BMPR-IB seems to be unable to efficiently activate downstream signaling pathways upon forced expression. However, a mutation conferring constitutive activity to the BMPR-IB receptor indicates that this receptor possesses the capacity to activate downstream signaling cascades. These results suggest that in mesenchymal progenitors C3H10T1/2 BMPR-IA is responsible for the initiation of the osteogenic as well as chondrogenic development and that BMPR-IA and -IB receptor pathways are well separated in this mesenchymal progenitor line and may not substitute each other. In addition this indicates that type IB and IA BMP receptors may transmit different signals during the specification and differentiation of mesenchymal lineages.

Tissue regeneration requires the recruitment of adult stem cells and their differentiation into mature committed cells. In this study we describe what we believe to be a novel approach for tendon regeneration based on a specific signalling molecule, Smad8, which mediates the differentiation of mesenchymal stem cells (MSCs) into tendon-like cells. A biologically active Smad8 variant was transfected into an MSC line that coexpressed the osteogenic gene bone morphogenetic protein 2 (BMP2). The engineered cells demonstrated the morphological characteristics and gene expression profile of tendon cells both in vitro and in vivo. In addition, following implantation in an Achilles tendon partial defect, the engineered cells were capable of inducing tendon regeneration demonstrated by double quantum filtered MRI. The results indicate what we believe to be a novel mechanism in which Smad8 inhibits the osteogenic pathway in MSCs known to be induced by BMP2 while promoting tendon differentiation. These findings may have considerable importance for the therapeutic replacement of tendons or ligaments and for engineering other tissues in which BMP plays a pivotal developmental role.

The BMP2-dependent onset of osteo/chondrogenic differentiation in the acknowledged pluripotent murine mesenchymal stem cell line (C3H10T1/2) is accompanied by the immediate upregulation of Fibroblast Growth Factor Receptor 3 (FGFR3) and a delayed response by FGFR2. Forced expression of FGFR3 in C3H10T1/2 is sufficient for chondrogenic differentiation, indicating an important role for FGF-signaling during the manifestation of the chondrogenic lineage in this cell line. Screening for transcription factors exhibiting a chondrogenic capacity in C3H10T1/2 indentified that the T-box containing transcription factor Brachyury is upregulated by FGFR3-mediated signaling. Forced expression of Brachyury in C3H10T1/2 was sufficient for differentiation into the chondrogenic lineage in vitro and in vivo after transplantation into muscle. A dominant-negative variant of Brachyury, consisting of its DNA-binding domain (T-box), interferes with BMP2-mediated cartilage formation. These studies indicate that BMP-initiated FGF-signaling induces a novel type of transcription factor for the onset of chondrogenesis in a mesenchymal stem cell line. A potential role for this T-box factor in skeletogenesis is further delineated from its expression profile in various skeletal elements such as intervertebral disks and the limb bud at late stages (18.5 d.p.c.) of murine embryonic development.

This review focuses on the molecular aspects of osteogenesis and chondrogenesis as they relate to cellular and developmental levels. We discuss BMP (bone morphogenetic protein)-mediated signaling pathways for the patterning of skeletal elements and mechanisms for the induction of cartilage and bone formation. We emphasize BMP-mediated initiation of chondrogenesis and osteogenesis and consider BMP-dependent signaling cascades in mesenchymal progenitor cells during the development of the axial and appendicular skeleton and during growth plate and joint formation. In particular, we discuss BMP-signaling mediators of the Smad and TAK family and the modulation of these signals by cross-talk and integration with other signaling pathways.