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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.

Mesenchymal stromal cells (MSCs) are a promising cell source for tissue engineering and regenerative medicine. In our lab, we found that MSC preparations from bone marrow of many different donors had a limited capacity of in vitro differentiation into osteogenic and chondrogenic lineages&mdash, a capacity claimed to be inherent to MSCs. The current study was designed to test the hypothesis that the amount of heparin used as anticoagulant during bone marrow harvest had an inhibitory influence on the in vitro differentiation capacity of isolated MSCs. Bone marrow was obtained from the femoral cavity of twelve donors during total hip arthroplasty in the absence or presence of heparin. No coagulation was observed in the absence of heparin. The number of mononuclear cells was independent of heparin addition. Isolated MSCs were characterized by morphology, population doubling times, expression of cell surface antigens and in vitro differentiation. Results of these analyses were independent of the amount of heparin. Transcriptome analyses of cells from three randomly chosen donors and quantitative realtime PCR (qRT-PCR) analysis from cells of all donors demonstrated no clear effect of heparin on the transcriptome of the cells. This excludes heparin as a potential source of disparate results.

Smad8 is a transcriptional regulator that participates in the intracellular signaling pathway of the transforming growth factor-β (TGF-β) family. Full-length Smad8 is an inactive protein in the absence of ligand stimulation. The expression of a truncated version of the protein lacking the MH1 domain (cSmad8) revealed constitutive activity in genetically engineered mesenchymal stem cells and, in combination with BMP-2, exhibited a tendon cell-inducing potential. To further explore function and applicability of Smad8 in regenerative medicine recombinant production is required. Herein, we further engineered cSmad8 to include the transactivation signal (TAT) of the human immunodeficiency virus (HIV) to allow internalization into cells. TAT-hcSmad8 was produced in endotoxin-free ClearColi® BL21 (DE3), refolded from inclusion bodies (IBs) and purified by Heparin chromatography. Analysis of TAT-hcSmad8 by thermal shift assay revealed the formation of a hydrophobic core. The presence of mixed α-helixes and β-sheets, in line with theoretical models, was proven by circular dichroism. TAT-hcSmad8 was successfully internalized by C3H10T1/2 cells, where it was mainly found in the cytoplasm and partially in the nucleus. Finally, it was shown that TAT-hcSmad8 exhibited biological activity in C3H10T1/2 cells after co-stimulation with BMP-2.

Background aims Mesenchymal stroma/stem-like cells (MSCs) are a popular cell source and hold huge therapeutic promise for a broad range of possible clinical applications. However, to harness their full potential, current limitations in harvesting, expansion and characterization have to be overcome. These limitations are related to the heterogeneity of MSCs in general as well as to inconsistent experimental protocols. Here we aim to compare in vitro methods to facilitate comparison of MSCs generated from various tissues. Methods MSCs from 3 different tissues (bone marrow, dental pulp, adipose tissue), exemplified by cells from 3 randomly chosen donors per tissue, were systematically compared with respect to their in vitro properties after propagation in specific in-house standard media, as established in the individual laboratories, or in the same commercially available medium. Results Large differences were documented with respect to the expression of cell surface antigens, population doubling times, basal expression levels of 5 selected genes and osteogenic differentiation. The commercial medium reduced differences in these parameters with respect to individual human donors within tissue and between tissues. The extent, size and tetraspanin composition of extracellular vesicles were also affected. Conclusions The results clearly demonstrate the extreme heterogeneity of MSCs, which confirms the problem of reproducibility of results, even when harmonizing experimental conditions, and questions the significance of common parameters for MSCs from different tissues in vitro.

PURPOSE: There is a plethora of studies on recombinant human bone morphogenetic protein-2 (rhBMP-2) application and delivery systems, but surprisingly few reports address the biophysical properties of the protein which are of crucial importance to develop effective delivery systems or to solve general problems related to rhBMP-2 production, purification, analysis and application. METHODS:The solubility, stability and bioactivity of rhBMP-2 obtained by renaturation of E. coli derived inclusion bodies was assessed at different pH and in different buffer systems using (dynamic) light scattering and thermal shift assays as well as intrinsic fluorescence measurements and luciferase based bioassays. RESULTS: rhBMP-2 is poorly soluble at physiological pH and higher. The presence of divalent anions further decreases the solubility even under acidic conditions. Thermal stability analyses revealed that rhBMP-2 precipitates are more stable compared to the soluble protein. Moreover, correctly folded rhBMP-2 is also bioactive as precipitated protein and precipitates readily dissolve under appropriate buffer conditions. Once properly formed rhBMP-2 also retains biological activity after temporary exposure to high concentrations of chaotropic denaturants. However, care should be taken to discriminate bioactive rhBMP-2 precipitates from misfolded rhBMP-2 aggregates, e.g. resolvability in MES buffer (pH 5) and a discrete peak in thermoshift experiments are mandatory for correctly folded rhBMP-2. CONCLUSIONS: Our analysis revealed that E. coli derived rhBMP-2 precipitates are not only bioactive but are also more stable compared to the soluble dimeric molecules. Knowledge about these unusual properties will be helpful to design improved delivery systems requiring lower amounts of rhBMP-2 in clinical applications.

The Myc gene has been implicated in the pathogenesis of most types of human cancerous tumors. Myc/Max activates large numbers of pro-tumor genes; however it also induces anti-proliferation genes. When anti-proliferation genes are activated by Myc, cancer cells can only survive if they are downregulated. Hepatocellular carcinoma (HCC) specific intronic long noncoding antisense (lnc-AS) RNA, the EVA1A-AS gene, is located within the second intron (I2) of the EVA1A gene (EVA-1 homolog A) that encodes an anti-proliferation factor. Indeed, EVA1A, but not EVA1A-AS, is expressed in normal liver. Depletion of EVA1A-AS suppressed cell proliferation of HepG2 cells by upregulation of EVA1A. Overexpression of EVA1A caused cell death at the G2/M phase via microtubule catastrophe. Furthermore, suppressed EVA1A expression levels are negatively correlated with differentiation grade in 365 primary HCCs, while EVA1A-AS expression levels are positively correlated with patient survival. Notably, both EVA1A and EVA1A-AS were activated by the Myc/Max complex. Eva1A-AS is transcribed in the opposite direction near the 3′splice site of EVA1A I2. The second intron did not splice out in a U2 dependent manner and EVA1A mRNA is not exported. Thus, the Myc/Max dependent anti-proliferating gene, EVA1A, is controlled by Myc/Max dependent anti-sense noncoding RNA for HCC survival.

Medical treatment of certain diseases and biomedical implants are tending to use delivery systems on the nanoscale basis for biologically active factors including drugs (e. g. antibiotics) or growth factors. Nanoparticles are a useful tool to deliver bioactive substances of different chemical nature directly to the site where it is required in the patient. Here we developed three innovative delivery systems based on different polysaccharides in order to induce a sustained release of TGF-β

The use of neurotrophic factors as therapeutic agents for neurodegenerative diseases is considered as an approach aimed at restoring and maintaining neuronal function in the peripheral and central nervous system. Since the neuroprotective effect is depending on chronic delivery of the neurotrophic factors a sustained application, e.g., via cell‐based delivery is necessary. Human mesenchymal stem cells (hMSCs) were lentivirally modified to overexpress brain‐derived neurotrophic factor (BDNF) and to express fluorescent marker genes for easy visualization. Since genetically modified cells should be site‐specifically retained (e.g., by encapsulation) in the patients to avoid adverse effects the cells were additionally differentiated to chondrocytes to hypothetically improve their vitality and survival in a delivery matrix. Different polycations for lentiviral transduction were investigated for their efficiency. The success of differentiation was determined by analysis of chondrocyte marker genes and the neuroprotective effect of BDNF‐overexpressing cells was exemplarily investigated on neurons of the peripheral auditory system. The genetically modified hMSCs overexpressed BDNF from under 1 to 125 ng ml−1 day−1 depending on the donor and transfection method. Using protamine sulfate the transfection efficacy was superior compared to the use of polybrene. The BDNF secreted by the MSCs was significantly neuroprotective in comparison to the relevant controls even though the produced mean concentrations were lower than the effective concentrations for recombinant industrially produced proteins described in literature. The presented system of BDNF‐overexpressing hMSCs is neuroprotective and is therefore considered as a promising method for sustained delivery of proteins in therapeutically relevant amounts to degenerating neuronal structures.

Musculoskeletal dysfunctions are highly prevalent due to increasing life expectancy. Consequently, novel solutions to optimize treatment of patients are required. The current major research focus is to develop innovative concepts for single tissues. However, interest is also emerging to generate applications for tissue transitions where highly divergent properties need to work together, as in bone-cartilage or bone-tendon transitions. Finding medical solutions for dysfunctions of such tissue transitions presents an added challenge, both in research and in clinics. This review aims to provide an overview of the anatomical structure of healthy adult entheses and their development during embryogenesis. Subsequently, important scientific progress in restoration of damaged entheses is presented. With respect to enthesis dysfunction, the review further focuses on inflammation. Although molecular, cellular and tissue mechanisms during inflammation are well understood, tissue regeneration in context of inflammation still presents an unmet clinical need and goes along with unresolved biological questions. Furthermore, this review gives particular attention to the potential role of a signaling mediator protein, transforming growth factor beta-activated kinase-1 (TAK1), which is at the node of regenerative and inflammatory signaling and is one example for a less regarded aspect and potential important link between tissue regeneration and inflammation.

Acute and chronic rotator cuff tears remain challenging for therapy. A wide range of therapeutic approaches were developed but re-tears and postoperative complications occur regularly. Especially in elderly people, the natural regeneration processes are decelerated, and graft materials are often necessary to stabilize the tendon-to-bone attachment and to improve the healing process. We here investigated in a small animal model a newly developed electrospun polycaprolactone fiber implant coated with a chitosan-polycaprolactone graft copolymer and compared these implants biomechanically and histologically with either a commercially available porous polyurethane implant (Biomerix 3D Scaffold) or suture-fixed tendons. Fifty-one rats were divided into three groups of 17 animals each. In the first surgery, the left infraspinatus tendons of all rats were detached, and the animals recovered for 4 weeks. In the second surgery, the tendons were fixed with suture material only (suture-fixed group; n = 17), whereas in the two experimental groups, the tendons were fixed with suture material and the polyurethane implant (Biomerix scaffold group; n = 17) or the modified electrospun polycaprolactone fiber implant (CS-g-PCL scaffold group; n=17), respectively. The unaffected right infraspinatus tendons were used as native controls. After a recovery of 8 weeks, all animals were clinically inconspicuous. In 12 animals of each group, repaired entheses were biomechanically tested for force at failure, stiffness, and modulus of elasticity, and in five animals, repaired entheses were analyzed histologically. Biomechanically, all parameters did not differ statistically significant between both implant groups, and the entheses failed typically at the surgical site. However, with respect to the force at failure, the median values of the two implant groups were smaller than the median value of the suture-fixed group. Histologically, the modified polycaprolactone fiber implant showed no acute inflammation processes, a good infiltration with cells, ingrowth of blood vessels and tendinous tissue, and a normal fibrous ensheathment. Further improvement of the implant material could be achieved by additional implementation of drug delivery systems. Therewith, the used CS-g-PCL fiber mat is a promising basic material to reach the goal of a clinically usable graft for rotator cuff tear repair.

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.

This review describes the use of picosecond lasers for the treatment of tattoos, pigmented lesions, scars, and their use in rejuvenation. These devices have delivered enhanced efficacy for the treatment of tattoos and pigmented lesions when compared to the older 40-50 nanosecond devices. The fractional delivery with the picosecond devices have opened up a new method of rejuvenation for photodamaged skin and the treatment of scars. The delivery of these high-energy short pulses have created zones of injury in the skin referred to as areas of laser-induced optical breakdown. These areas of damage appear to produce cytokines and chemokines which result in epidermal and dermal repair and remodeling. The dual use of these devices with the flat and the fractional optics have made these devices useful in many ways that have been unanticipated.

Electrospun poly(e-caprolactone) (PCL) fiber mats are modified using a chitosan grafted with PCL (CS-g-PCL), to improve the biological performance and to enable further modifications. The graft copolymer is immobilized by the crystallization of the PCL grafts on the PCL fiber surface as binding mechanism. In this way, the surface of the fibers is covered with chitosan bearing cationic amino groups, which allow adsorption of oppositely charged nanoparticulate drug-delivery systems. The modification of the fiber mats and the attachment of the drug delivery systems are easy and scalable dip processes. The process is also versatile; it is possible to attach different polymeric and inorganic nanoparticulate drug-release systems of cationic or anionic nature. The modifications are verified using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). As proof of principle, the release of ciprofloxacin from silica nanoparticles attached to the modified fiber mats is shown; however, the method is also suited for other biologically active substances including growth factors. The initial cellular attachment and proliferation as well as vitality of the cells is improved by the modification with CS-g-PCL and is further influenced by the type of the drug delivery system attached. Hence, this method can be used to transfer PCL fiber mats into bioactive implants for in-situ tissue engineering applications.

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.

Aim of the study The induced expression of the transcription factors neurogenin1 (Neurog1) or neuronal differentiation 1 (NeuroD1) has previously been shown to initiate neuronal differentiation in embryonic stem cells (ESC). Human bone marrow-derived mesenchymal stem cells (hBMSCs) are ethically non-controversial stem cells. However, they are not pluripotent. In cochlear implantation, regeneration or replacement of lost spiral ganglion neurons may be a measure for the improvement of implant function. Thus, the aim of the study was to investigate whether the expression of Neurog1 or NeuroD1 is sufficient for induction of neuronal differentiation in hBMSCs. Materials and methods Human BMSCs were transduced with lentivirus expressing NeuroD1 or Neuorg1. Transduced cells were then treated with small molecules that enhanced neuronal differentiation. Markers of neuronal differentiation were evaluated. Results Using quantitative reverse transcription PCR, the up-regulation of transcription factors expressed by developing primary auditory neurons, such as BRN3a (POU4F1) and GATA3 , was quantified after induction of Neurog-1 expression. In addition, the expression of the receptor NTRK2 was induced by treatment with its specific ligand BDNF. The induction of expression of the vesicular glutamate transporter 1 was identified on gene and protein level. NeuroD1 seemed not sufficient to induce and maintain neuronal differentiation. Conclusions Induction of neuronal differentiation by overexpression of Neurog1 initiated important steps for the development of glutamatergic neurons such as the spiral ganglion neurons. However, it seems not sufficient to maintain the glutamatergic spiral ganglion neuron-like phenotype.

Due to its ability to induce de novo bone formation the differentiation factor bone morphogenetic protein 2 (BMP2) is often used to enhance the integration of bone implants. With the aim of reducing possible high dose side-effects and to lower the costs, in order to produce affordable implants, we developed a simple and fast method for the immobilization of BMP2 on silica-based surfaces using silane linkers which carry amino or epoxy functions. We put an especial emphasis on the influence of the nanoscale surface topography of the silica layer. Therefore, we chose glass (for control experiments) and Bioverit® II (as a typical implant base material) as support materials and coated these substrates with unstructured or nanoporous amorphous silica layers for comparison. Immobilized BMP2 was quantified by two different methods: by ELISA and by a cell-based assay for active BMP2. These tests probe for immunologically and biologically active BMP2, respectively. The results show that the amino functionalization is better suited for immobilizing the protein. Strikingly, a considerably higher amount of BMP2 could be immobilized on coated Bioverit® II surfaces compared with coated glass substrates, which was presumably due to the macroscopic roughness of the Bioverit® II substrates. In addition, it was found that the nanoporous silica coatings on Bioverit® II substrates were able to bind more BMP2 than the unstructured ones.

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.

Surfaces of biomaterials often do not have the ideal properties for direct application in vivo. Although titanium and its alloys show a good biocompatibility, in some applications there is still need to improve the osteoblast adhesion to titanium implants. A polymeric surface coating is an ideal solution because the polymer can be adjusted to the needs of the application and can be bound to the surface by the photochemical grafting method. Therefore, 22 different polymers were tested for their compatibility using a murine mesenchymal progenitor cell line and three polymers were identified for which more elaborate investigations are reasonable. It was investigated whether or not the results of the cell culture test can be correlated with, e.g., the wetting properties. Indeed it was found that a contact angle above approx. 45 degrees was necessary for good cell adhesion and proliferation. However, otherwise no clear correlation between the contact angle hysteresis or the functionalities of the polymers and the cell growth was observed.

Cell-mediated therapy is currently considered as a novel approach for many human diseases. Potential uses range from topic applications with the regeneration of confined tissue areas to systemic applications. Stem cells including mesenchymal stroma/stem cells (MSCs) represent a highly attractive option. Their potential to cure or alleviate human diseases is investigated in a number of clinical trials. A wide variety of methods has been established in the past years for isolation, cultivation and characterization of human MSCs as expansion is presently deemed a prerequisite for clinical application with high numbers of cells carrying reproducible properties. MSCs have been retrieved from various tissues and used in a multitude of settings whereby numerous experimental protocols are available for expansion of MSCs in vitro. Accordingly, different isolation, culture and upscaling techniques contribute to the heterogeneity of MSC characteristics and the, sometimes, controversial results. Therefore, this review discusses and summarizes certain experimental conditions for MSC in vitro culture focusing on adult bone marrow-derived and neonatal umbilical cord-derived MSCs in order to enhance our understanding for MSC tissue sources and to stratify different procedures. Copyright © 2016 John WileySons, Ltd.

Hepatocellular carcinoma (HCC) is a frequent form of cancer with a poor prognosis and with limited possibilities of medical intervention. It has been shown that over 100 putative driver genes are associated with multiple recurrently altered pathways in HCC, suggesting that multiple pathways will need to be inhibited for any therapeutic method. mRNA processing is regulated by a complex RNA-protein network that is essential for the maintenance of homeostasis. THOC5, a member of mRNA export complex, has a role in less than 1% of mRNA processing, and is required for cell growth and differentiation, but not for cell survival in normal fibroblasts, hepatocytes and macrophages. In this report, we show that 50% depletion of THOC5 in human HCC cell lines Huh7 and HepG2 induced apoptosis. Transcriptome analysis using THOC5-depleted cells revealed that 396 genes, such as transmembrane BAX inhibitor motif containing 4 (TMBIM4), transmembrane emp24-like trafficking protein 10 (Tmed10) and D-tyrosyl-tRNA deacylase 2 (Dtd2) genes were downregulated in both cell lines. The depletion of one of these THOC5 target genes in Huh7 or HepG2 did not significantly induce cell death, suggesting that these may be fine tuners for HCC cell survival. However, the depletion of a combination of these genes synergistically increased the number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive HCC. It must be noted that the depletion of these genes did not induce cell death in the hepatocyte cell line, THLE-2 cells. THOC5 expression was enhanced in 78% of cytological differentiation grading G2 and G3 tumor in primary HCC. Furthermore, the expression of a putative glycoprotein, Tmed10, is correlated to THOC5 expression level in primary HCCs, suggesting that this protein may be a novel biomarker for HCC. These data imply that the suppression of the multiple THOC5 target genes may represent a novel strategy for HCC therapy.

The use of autologous cells for the coating of implant surfaces presents a promising tool to attenuate foreign body reaction and inflammation. However, insertion forces that occur especially during implantation of electrodes into the narrow cochlea may strip off cells from the surface. Thus, implant surfaces should be ideally structured in a way that protects the cell coating from mechanical removal during implantation. The structuring of implant surfaces may also direct cells towards desired functions to further enhance their performance and clinical suitability. In this study, grid-like square cavities were generated on thermoplastic polyurethane (TPU) surfaces using a combination of femtosecond laser ablation and replication methods. Afterwards, they were tested as potential scaffolds for human bone marrow-derived mesenchymal stem cells (MSCs) in order to use it on neural prostheses. Structured and non-structured TPU allowed proper adhesion and survival of MSCs. Surface structuring resulted in regulation of over 500 genes. Many of the upregulated genes are known to be involved in anti-inflammatory, anti-fibrotic and wound healing processes whereas genes relevant for mesenchymal differentiation programs were downregulated. The enhanced secretion of two representative factors (prostaglandin E2 and interleukin-1 receptor antagonist, respectively) was confirmed by ELISA and the downregulation of other genes involved in adipogenic and osteogenic differentiation were confirmed by gene expression analysis for a cultivation period of up to 21 days. In addition, mRNA of the surface antigens CD24 and ENDOGLIN (CD105) as representative factors for stemness did not show notable variation between cultivation on structured versus non-structured TPU or between 7 versus 21days of cultivation. Thus, surface topography of TPU seems to be a powerful tool to protect cells from mechanical forces during insertion and to influence cell behaviour.

Introduction Multipotent stromal cells (MSCs) are among the key candidates in regenerative medicine. However variety of MSC sources and general heterogeneity lead to controversial data in functional characterization. Furthermore, despite intensive usage as preclinical animal model, little is known about MSCs of the common marmoset monkey. Methods MSCs derived from placental amnion and bone marrow samples from human and common marmoset were characterized in parallel over 12 passages to monitor similarities and significant differences (p ≤ 0.05, Student’s t-test) in MSC markers and major histocompatibility complex (MHC) class I expression by immunohistochemistry, flow cytometry, real-time PCR, metabolic activity test, with special focus on pluripotency associated genes. Results Human and non-human primate MSCs were characterized for expression of MSC markers and capability of differentiation into mesenchymal lineages. MSCs could be cultured more than 100 days (26 passages), but metabolic activity was significantly enhanced in amnion vs. bone marrow MSCs. Interestingly, MHC class I expression is significantly reduced in amnion MSCs until passage 6 in human and marmoset, but not in bone marrow cells. For MSC markers, CD73 and CD105 levels remain unchanged in amnion MSCs and slightly decline in bone marrow at late passages; CD166 is significantly higher expressed in human MSCs, CD106 significantly lower vs. marmoset. All cultured MSCs showed pluripotency marker expression like Oct-4A at passage 3 significantly decreasing over time (passages 6–12) while Nanog expression was highest in human bone marrow MSCs. Furthermore, human MSCs demonstrated the highest Sox2 levels vs. marmoset, whereas the marmoset exhibited significantly higher Lin28A values. Bisulfite sequencing of the Oct-4 promoter region displayed fewer methylations of CpG islands in the marmoset vs. human. Conclusions Little is known about MSC characteristics from the preclinical animal model common marmoset vs. human during long term culture. Studied human and common marmoset samples share many similar features such as most MSC markers and reduced MHC class I expression in amnion cells vs. bone marrow. Furthermore, pluripotency markers indicate in both species a subpopulation of MSCs with true ‘stemness’, which could explain their high proliferation capacity, though possessing differences between human and marmoset in Lin28A and Sox2 expression.

Tendons and ligaments are related connective tissues that join muscle to bone and bone to bone, respectively. Tendon and ligament injuries are widely distributed clinical problems in society and while healing of such disorders can occur, the original biological properties of the tissue do not return to normal. In this review, recent work on tendon and ligament development and the use of growth factors for successful cellular therapy of tendon and ligament disorders are discussed. In addition, anti-inflammatory concepts for the treatment of tendon and ligament injuries and recent developments in stem cell engineering for tendon and ligament tissues are examined. Lastly, gene transfer strategies for therapeutic applications to heal tendon and ligament disorders are reviewed.

Human bone morphogenetic protein-2 is a representative of the transforming growth factor-β (TGF-β) superfamily of cytokines. It was produced in high-cell-density cultivations of recombinant Escherichia coli leading to the formation of inclusion bodies with aggregated inactive protein so that the protein had to be solubilized and renatured. Thus, the biological activity of the recombinant protein had to be determined. To avoid time-consuming cell-based assays or radioactive labelling of proteins enzyme-linked immunoreceptor assays were developed. They were based on the specific interaction between the biologically active protein and its receptors, of which the extracellular ligand binding domains were tagged with the Fc part of human IgG and expressed in insect cells. The amount of bound ligand, corresponding to the biologically active recombinant protein, was determined via enzyme-labelled antibodies. Application to various batches of protein showed that not only the amount of active protein could be quantified but also the quality of the protein preparations could be evaluated in significantly shorter analysis times than with conventional cell-based assays.

True tendon regeneration in human patients remains a vision of musculoskeletal therapies. In comparison to other mesenchymal lineages the biology of tenogenic differentiation is barely understood. Specifically, easy and efficient protocols are lacking that might enable tendon cell and tissue differentiation based on adult (stem) cell sources. In the murine mesenchymal progenitor cell line C3H10T½, overexpression of the growth factor bone morphogenetic protein 2 (BMP2) and a constitutively active transcription factor, Smad8 L+MH2, mediates tendon cell differentiation in vitro and the formation of tendon-like tissue in vivo. We hypothesized that during this differentiation secreted factors involved in extracellular matrix formation exert a major impact on tendon development. Gene expression analyses revealed four genes encoding secreted factors that are notably upregulated: periostin, C-type lectin domain family 3 (member b), RNase A4, and follistatin-like 1. These factors have not previously been implicated in tendon biology. Among these, periostin showed a specific expression in tenocytes of adult mouse Achilles tendon and in chondrocytes within the nonmineralized fibrocartilage zone of the enthesis with the calcaneus. Overexpression of periostin alone or in combination with constitutively active BMP receptor type in human mesenchymal stem cells and subsequent implantation into ectopic sites in mice demonstrated a reproducible moderate tenogenic capacity that has not been described before. Therefore, periostin may belong to the factors contributing to the development of tenogenic tissue.

P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid rafts to gain its full functionality.

We have isolated beta-trace protein from cerebrospinal fluid, serum, plasma, and urine samples of normal volunteers and sera and hemofiltrate of patients with chronic renal failure. Blood-derived and urinary beta-trace have significantly higher molecular weights than their cerebrospinal fluid counterpart, the amino acid sequences being identical. Oligosaccharide structural analysis revealed these molecular weight differences to be due to different N-glycosylation. beta-Trace from hemofiltrate and urine has larger sugar chains and concurrently significantly higher sialylation than cerebrospinal fluid-beta-trace which bears truncated "brain-type" oligosaccharide chains (published previously). beta-Trace concentrations were about 40 ng/ml for normal sera and plasma. 2000-6000 ng/ml were measured in sera of dialysis patients whereas in normal human cerebrospinal fluid, beta-trace concentration was about 8000 ng/ml. A reduced amount of 900 ng/ml was found in a single case of hydrocephalus cerebri. The sialylated glycoforms of beta-trace detected in the blood are presumably derived from resorbed cerebrospinal fluid protein whereas beta-TP-molecules bearing asialo-oligosaccharides are absent due to their hepatic clearance. The residual, sialylated beta-TP-species are probably eliminated from the blood via the kidney. This physiological clearance mechanism for the sialylated glycoforms is disturbed in hemodialysis patients resulting in about 100-fold elevated serum concentrations. These results let us suggest beta-trace may become a useful novel diagnostic protein in renal diseases.

Human beta-trace protein is a major intrathecally synthesized polypeptide constituent of human cerebrospinal fluid. We have previously shown that this protein is almost quantitatively modified with biantennary complex-type N-linked oligosaccharides which show "brain-type" glycosylation characteristics (Hoffmann,A. et al., J. Neurochem., 63, pp. 2185-2191, 1994). In the present study human beta-trace protein from the cerebrospinal fluid (CSF) of patients with carbohydrate-deficient glycoprotein syndrome (CDGS) due to phosphomannomutase (PMM) deficiency and N-acetyl-glucosaminyltransferase II (GlcNAc-T II) deficiency as well as from control individuals was studied by Western blot analysis. The protein from pooled CSFs was purified by immunoaffinity chromatography. The protein from the five patients with CDGS PMM deficiency showed three protein bands upon SDS-PAGE analysis corresponding to the di-, mono-, and unglycosylated polypeptide forms. Carbohydrate structural analysis of the enzymatically liberated N-glycans was performed applying mapping by HPAEC-PAD, methylation analysis as well as MALD/TOF-MS. Essentially identical oligosaccharide structures were detected in beta-TP from type I patients and control adult pooled CSF. The beta-trace protein from two patients with GlcNAc-T II deficiency showed a single di-N-glycosylated protein band with a significantly lower molecular weight than the di-glycosylated polypeptide from control patients and the beta-trace protein from pooled adult CSF. Beta-TP from GlcNAc-T II deficiency patients shared only three oligosaccharides out of the 13 observed in beta-TP from controls or patients with PMM deficiency. The major oligosaccharide structures of the glycoprotein from patients with GlcNAc-T II deficiency were found to be monoantennary asialo- or monosialylated lactosamine-type chains with proximal fucose and bisecting GlcNAc.

The most widely used technique for isolation of human bone marrow stromal cells (hBMSCs) from bone marrow includes density gradient centrifugation, recovery of the mononuclear cell population, and subsequent isolation of hBMSCs by virtue of their plastic adherence. During subsequent in vitro cultivation, they may lose their original characteristics since in vitro the stem cell niche cannot yet be properly mimicked. To further characterize these culture-induced changes in regard to mRNA and extra- and intracellular protein expression, as well as potential differences between hBMSCs from different donors, we investigated a panel of CD antigens for their presence on in vitro cultured hBMSCs. Interestingly, after culture-induced downregulation of their extracellular expression, both CD146 and CD271 persist intracellularly, which hints at the possibility that culture-induced changes may be reversed by appropriate stimuli. Further, CD34-a protein whose expression on hBMSCs is still controversial-is expressed at the intracellular level in hBMSCs of all donors independently of passage number. CD34 mRNA levels are significantly higher in female than in male donors. In summary, we further elucidate phenotypical changes induced by in vitro culture of hBMSCs, highlight interindividual differences in the phenotype of these cells and for the first time show the intracellular expression of CD34.

For in vitro differentiation of bone marrow-derived mesenchymal stem cells/mesenchymal stromal cells into osteoblasts by 2-dimensional cell culture a variety of protocols have been used and evaluated in the past. Especially the external phosphate source used to induce mineralization varies considerably both in respect to chemical composition and concentration. In light of the recent findings that inorganic phosphate directs gene expression of genes crucial for bone development, the need for a standardized phosphate source in in vitro differentiation becomes apparent. We show that chemical composition (inorganic versus organic phosphate origin) and concentration of phosphate supplementation exert a severe impact on the results of gene expression for the genes commonly used as markers for osteoblast formation as well as on the composition of the mineral formed. Specifically, the intensity of gene expression does not necessarily correlate with a high quality mineralized matrix. Our study demonstrates advantages of using inorganic phosphate instead of beta-glycerophosphate and propose colorimetric quantification methods for calcium and phosphate ions as cost-and time-effective alternatives to X-ray diffraction and Fourier-transform infrared spectroscopy for determination of the calcium phosphate ratio and concentration of mineral matrix formed under in vitro-conditions. We critically discuss the different assays used to assess in vitro bone formation in respect to specificity and provide a detailed in vitro protocol that could help to avoid contradictory results due to variances in experimental design. DFG/SFB/599 DFG/Ho2058/4-1 Studienstiftung des Deutschen Volkes

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 repair of tendon injuries still presents a major clinical challenge to orthopedic medicine. Tendons, like some other tissues, are poorly vascularized and heal slowly. In addition, healing often leads to the formation of fibrous tissue and scar tissue which lack flexibility and biomechanical properties. So the treatment of tendon injuries is challenging. We give an overview of the structure and composition of tendons, pathological states of tendon and natural healing, as well as therapeutic options. We focus in particular on biomaterials that have been specifically developed or suggested for the successful repair of tendon injuries. In addition, we also review factor- and cell-dependent strategies to heal tendon and ligament disorders. Although brief, we hope that this review will be helpful, particularly for those readers who are new to the field of tendon tissue engineering.

Comparative studies of lens and retina regeneration have been conducted within a wide variety of animals over the last 100 years. Although amphibians, fish, birds and mammals have all been noted to possess lens- or retina-regenerative properties at specific developmental stages, lens or retina regeneration in adult animals is limited to lower vertebrates. The present review covers the newest perspectives on lens and retina regeneration from these different model organisms with a focus on future trends in regeneration research.

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.

Osseointegration of long-term implants is still a problem in orthopaedic surgery. In recent years, several techniques to modify the implant surface to increase bone formation around implants have been described by many authors. Most endoprostheses used in orthopaedic surgery are manufactured from titanium. To understand the process of osseointegration, one has to take into account, that the adhesion of plasma proteins on the surface of titanium implants plays an essential role in the process of implant integration. In the last years, several modifications of implant surfaces (structure, chemistry, surface charge, wettability) have been investigated to improve osseointegration of titanium implants. Furthermore, several cytokines and growth factors have also been suggested to stimulate an implant ingrowth. In this respect, functionalized titanium implant surfaces with bone morphogenetic proteins-2 (BMP-2) as one particular member of the superfamily of transforming growth factor beta (TGF-beta), have proven a potential to stimulate bone formation around implants in different species. In this review the authors provide an overview of surface coatings with BMP-2 and their use in laboratory and experimental settings.

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.

Our aim is to review the recent progress in the management of musculoskeletal disorders. We will cover novel therapeutic approaches based on growth factors, gene therapy and cells, including stem cells, which may be combined with each other as appropriate. We focus mainly on the treatment of soft tissue injuries - muscle, cartilage, and tendon/ligament for both human and animal athletes. The need for innovative strategies results from the fact that despite all efforts, the current strategies for cartilage and tendon/ligament still result in the formation of functionally and biomechanically inferior tissues after injury (a phenomenon called 'repair' as opposed to proper 'regeneration'), whereas the outcome for muscle is more favorable. Innovative approaches are urgently needed not only to enhance the outcome of conservative or surgical procedures but also to speed up the healing process from the very long disabling periods, which is of special relevance for athletes.

Tendons and ligaments are elastic collagenous tissues with similar composition and hierarchical structure, contributing to motion. Their strength is related to the number and size of the collagen fibrils. Collagen fibrils increase in size during development and in response to increased physical demands or training. Tendon disorders are commonly seen in clinical practice and give rise to significant morbidity. Treatment is difficult and patients often suffer from the symptoms for quite a long time. Despite remodelling, the biochemical and mechanical properties of healed tendon tissue never match those of intact tendon. The prerequisite for focussed treatment strategies in the future will be an improved understanding of the molecular events both in the embryo and contributing to regeneration in the adult organism. Novel approaches include the local delivery of growth factors, stem- and tendon-cell-derived therapy, the application of mechanical load and gene-therapeutic approaches based on vehicles encoding selected factors, or combinations of these. Important factors are proteins of the extracellular matrix like the metalloproteinases, growth factors like the bone morphogenetic proteins but also intracellular signalling mediator proteins, such as the Smads and transcription factors from the helix–loop–helix and other families. In this review, we focus specifically on such molecular approaches based on mesenchymal stem cells.

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.

Two different phosphonic acid monolayer films for immobilization of bioactive molecules like the protein BMP-2 on titanium surfaces have been prepared. Monolayers of (11-hydroxyundecyl)phosphonic acid and (12-carboxydodecyl)phosphonic acid molecules were produced by a simple dipping process (the T-BAG method). The terminal functional groups on these monolayers were activated (carbonyl diimidazole for hydroxyl groups and N-hydroxysuccinimide for carboxyl groups) to bind amine containing molecules. The reactivity of the surfaces was investigated using trifluoroethylamine hydrochloride and BMP-2. Each step of the surface modification procedure was characterized by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectroscopy (ToF-SIMS).

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.

Background: Cardiac allograft vascular disease is (CAVD) the most important cause of death following heart transplantation (HTX). Although in the past, researchers focused predominantly on mechanisms of endothelial injury, the possible role of recipient-related and genetically determined factors has not been studied in detail. Methods Stimulated by recent observations in native coronary artery disease, we analyzed the potential impact of angiostensin-converting enzyme (ACE) polymorphism (insertion/deletion [I/D], intron 16) on development and progression of CAVD. We characterized genotype in 146 patients 1 to 12 years after HTX (121 men; mean age, 46.2 ± 11.3 years; observation period, 6.1 ± 3.8 years) and correlated genotype to the onset and progression of CAVD, defined as luminal obstruction > 50%. Results We found allelic frequencies to be 28.8% ( n = 42) for ACE-DD, 49.3% ( n = 72) for ACE-DI, and 21.9% ( n = 32) for ACE-II. Differences in actuarial freedom from vasculopathy were significant 6 years after transplantation, with 84.6% for ACE-II compared with 54.4% for ACE-DD. We observed intermediate results for ACE-DI genotype (77.3%, p = 0.015). Conclusions In this large cohort study, we demonstrated a close relationship between the recipient-related ACE-D genotype and development of advanced CAVD. These observations suggest that gene–environment interactions might be clinically important in coronary vasculopathy after HTX.