Andrea Hoffmann

In vivo RNAi-mediated silencing of TAK1 decreases inflammatory Th1 and Th17 cells through targeting of myeloid cells.

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 system able to target MPS cells and to silence TAK1 impacts on pathogenic T effector cells in autoimmunity.

Effects of Murine and Human Bone Marrow-Derived Mesenchymal Stem Cells on Cuprizone Induced Demyelination

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.

Osseointegration by bone morphogenetic protein-2 and transforming growth factor beta2 coated titanium implants in femora of New Zealand white rabbits

Background: Intramembranous bone formation is essential in uncemented joint replacement to provide a mechanical anchorage of the implant. Since the discovery of bone morphogenic proteins (BMPs) by Urist in 1965, many studies have been conducted to show the influence of growth factors on implant ingrowth. In this study, the influence of bone morphogenetic protein-2 (rhBMP-2) and transforming growth factor β2 (TGF-β2) on implant osseointegration was investigated. Materials and Methods: Thirty-two titanium cylinders were implanted into the femoral condyles of both hind legs of New Zealand White Rabbits. Four experimental groups were investigated: controls without coating, a macromolecular copolymer + covalently bound BMP-2, adsorbed BMP-2, and absorbed BMP-2+TGF-β2. All samples were analyzed by ex vivo high-resolution micro-computed-tomography after 28 days of healing. Bone volume per total volume (BV/TV) was recorded around each implant. Afterward, all samples were biomechanically tested in a pull-out setup. Results: The highest BV/TV ratio was seen in the BMP-2 group, followed by the BMP-2+TGF-β2 group in high-resolution micro-computed-tomography. These groups were significantly different compared to the control group (P < 0.05). Copolymer+BMP-2 showed no significant difference in comparison to controls. In the pull-out setup, all groups showed higher fixation strength compared to the control group; these differences were not significant. Conclusions: No differences between BMP-2 alone and a combination of BMP-2+TGF-β2 could be seen in the present study. However, the results of this study confirm the results of other studies that a coating with growth factors is able to enhance bone implant ingrowth. This may be of importance in defect situations during revision surgery to support the implant ingrowth and implant anchorage.

Molecular and cellular characteristics of human and non-human primate multipotent stromal cells from the amnion and bone marrow during long term culture.

IntroductionMultipotent 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.MethodsMSCs 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.ResultsHuman 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.ConclusionsLittle 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.

Mesenchymal stem cells do not exert direct beneficial effects on CNS remyelination in the absence of the peripheral immune system

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.

Comparison of in vitro‐cultivation of human mesenchymal stroma/stem cells derived from bone marrow and umbilical cord

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

Grid-like surface structures in thermoplastic polyurethane induce anti-inflammatory and anti-fibrotic processes in bone marrow-derived mesenchymal stem cells

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.

Differential magnesium implant corrosion coat formation and contribution to bone bonding

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.

Induction of neuronal-like phenotype in human mesenchymal stem cells by overexpression of Neurogenin1 and treatment with neurotrophins

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.

Attachment of nanoparticulate drug-release systems on poly(ε-caprolactone) nanofibers via a graftpolymer as interlayer

Electrospun poly(ε-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.