Abstract Mesenchymal stem cells are multipotent cells able to differentiate into different mesenchymal lineages. Studies in the past had suggested that two of these mesenchymal differentiation ...directions, the chondrogenic and the myogenic differentiation, are negatively regulated by the transcription factor NF-κB. Although osteogenic differentiation has been extensively studied, the influence of NF-κB on this differentiation lineage was not subject of detailed analyses in the past. We have analyzed the consequences of TNF-α treatment and genetic manipulation of the NF-κB pathway for osteogenic differentiation of hMSCs. Treatment of hMSCs during differentiation with TNF-α activates NF-κB and this results in enhanced expression of osteogenetic proteins like bone morphogenetic protein2 (BMP-2) and alkaline phosphatase (ALP). In addition, enhanced matrix mineralization was observed. The direct contribution of the NF-κB pathway was confirmed in cells that express a constitutively active version of the NF-κB-inducing kinase IKK2 (CA-IKK2). The IKK2/NF-κB-induced BMP-2 up-regulation results in the enhancement of RUNX2 and Osterix expression, two critical regulators of the osteogenic differentiation program. Interestingly, a genetic block of the NF-κB pathway did not interfere with osteogenic differentiation. We conclude that TNFα mediated NF-κB activation, although not absolutely required for BMP-2 expression and matrix mineralization nevertheless supports osteogenic differentiation and matrix mineralization by increasing BMP-2 expression. Our results therefore suggest that NF-κB activation may function in lineage selection during differentiation of hMSCs by fostering osteogenic differentiation at the expense of other differentiation lineages.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Abstract Hexagonally arranged Gold nanoparticles with controllable diameters and inter-particle distances were deposited on thick SiO2 layers on top of Si wafers and used as masks during subsequent ...reactive ion etching. In this way, arrays of nanopillars are obtained with well-defined diameters (10/30 nm), inter-pillar distances (50–120 nm) and heights (20–35 nm), all on the nanoscale. Such nanotopographies served as substrate for multipotent mesenchymal stromal cells (MSC) and human osteoblasts (OB) allowing to study cellular responses to purely topographically patterned interfaces. Focus was put on adhesion, proliferation and differentiation of the cells. It turned out experimentally that adhesion is comparable for both cell types practically independent of topographical details at the substrate surface. Topography induced proliferation enhancement, however, is again independent of geometrical details in case of MSC, but significantly sensitive to pillar height in case of OB with a clear preference towards short nanopillars (20 nm). A high sensitivity to topographic details is also observed for osteogenic differentiation of MSC, in that case with a preference towards higher nanopillars (50 nm). The present experimental data also allow the important conclusion that cell proliferation and differentiation can be optimized simultaneously by fine-tuning nanoscaled topographical parameters.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Bone has a high capacity for self-renewal and repair. Prolonged local secretion of interleukin 1β (IL-1β), however, is known to be associated with severe bone loss and delayed fracture healing. Since ...induction of bone resorption by IL-1β may not sufficiently explain these pathologic processes, we investigated, in vitro, if and how IL-1β affects migration of multipotent mesenchymal stromal cells (MSC) or osteoblasts. We found that homogenous exposure to IL-1β significantly diminished both nondirectional migration and site-directed migration toward the chemotactic factors platelet-derived growth factor (PDGF)-BB and insulin like growth factor 1 (IGF-1) in osteoblasts. Exposure to a concentration gradient of IL-1β induced an even stronger inhibition of migration and completely abolished the migratory response of osteoblasts toward PDGF-BB, IGF-1, vascular endothelial growth factor A (VEGF-A) and the complement factor C5a. IL-1β induced extracellular signal-regulated kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinases (JNK) activation and inhibition of these signaling pathways suggested an involvement in the IL-1β effects on osteoblast migration. In contrast, basal migration of MSC and their migratory activity toward PDGF-BB was found to be unaffected by IL-1β. These results indicate that the presence of IL-1β leads to impaired recruitment of osteoblasts which might influence early stages of fracture healing and could have pathological relevance for bone remodeling in inflammatory bone disease.
Clonogenic neural stem cells (NSCs) are self-renewing cells that maintain the capacity to differentiate into brain-specific cell types, and may also replace or repair diseased brain tissue. NSCs can ...be directly isolated from fetal or adult nervous tissue, or derived from embryonic stem cells. Here, we describe the efficient conversion of human adult bone marrow stromal cells (hMSC) into a neural stem cell-like population (hmNSC, for human marrow-derived NSC-like cells). These cells grow in neurosphere-like structures, express high levels of early neuroectodermal markers, such as the proneural genes NeuroD1, Neurog2, MSl1 as well as otx1 and nestin, but lose the characteristics of mesodermal stromal cells. In the presence of selected growth factors, hmNSCs can be differentiated into the three main neural phenotypes: astroglia, oligodendroglia and neurons. Clonal analysis demonstrates that individual hmNSCs are multipotent and retain the capacity to generate both glia and neurons. Our cell culture system provides a powerful tool for investigating the molecular mechanisms of neural differentiation in adult human NSCs. hmNSCs may therefore ultimately help to treat acute and chronic neurodegenerative diseases.
Multipotent mesenchymal stromal cells (MSC) exert immune-modulatory effects and support tissue regeneration in various local trauma models. In case of a polytrauma, high amounts of danger-associated ...molecular patterns are released, leading to a systemic increase of inflammatory mediators. The influence of such a complex inflammatory microenvironment on human MSC is mainly unknown so far. Therefore, we investigated the effects of a defined serum-free polytrauma "cocktail" containing IL beta, IL6, IL8 and the anaphylatoxins C3a and C5a, in concentrations corresponding to those measured in the blood of polytrauma patients, on human MSC in vitro. The polytrauma cocktail induced directed migration of MSC with C3a representing its major soluble chemoattractive agent. Furthermore, the polytrauma cocktail and IL1beta upregulated the expression of MMP1 indicating a potential role of IL1beta to enhance MSC migration in the tissue context. COX2, PTGES and TSG6 were also found to be upregulated upon stimulation with the polytrauma cocktail or IL1beta, but not through other single factors of the polytrauma cocktail in pathophysiologically relevant concentrations. An RNA expression array of 84 inflammation-related genes revealed that both the polytrauma cocktail and IL1beta induced C3, CSF1, TLR3 and various chemokines without major qualitative or quantitative differences. These results indicate that IL1beta is a crucial mediator of the polytrauma cocktail in terms of immune-modulation and MMP1 expression. Thus, upon encountering the primary sterile, inflammatory milieu of a polytrauma, endogenous or systemically transfused MSC might be able to migrate to sites of injury, secrete TSG6 and PGE2 and to influence macrophage biology as observed in local trauma models.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background:
Sport injuries of the knee often lead to posttraumatic arthritis. In addition to direct damage of the cartilage, trauma-associated intra-articular bleeding may cause hemarthrosis. Both ...blood exposure and trauma are known to induce cell death and inflammation and to enhance proteoglycan release in cartilage.
Hypothesis:
Blood exposure increases chondrocyte death as well as inflammatory and degenerative processes in traumatized cartilage.
Study Design:
Controlled laboratory study.
Methods:
Human macroscopically intact osteoarthritic (OA) cartilage explants were impacted by a drop-tower system (0.59 J) and cultivated with or without 10% blood. Interactive effects were studied concerning cell survival, gene expression, and the release of mediators over 24 hours and 96 hours. To evaluate the effects of trauma and hemarthrosis in vivo, a newly established blunt cartilage trauma model in the rabbit was used. Treatment of the knee joints of mature New Zealand White rabbits consisted of the following groups: control (C), arthrotomy (A), arthrotomy with cartilage trauma (AT; 1.0 J), and arthrotomy with cartilage trauma and blood injection (ATH). After 1 and 12 weeks, inflammatory mediators in the synovial fluid and histological changes of the cartilage were determined, and immunohistological staining was performed.
Results:
The in vitro studies revealed a significant additional or synergistic effect of blood exposure on trauma-induced chondrocyte death, interleukin (IL)–1β and prostaglandin-E2 (PGE2) release, and matrix metalloproteinase (MMP)/pro-MMP level. Singular arthrotomy in vivo induced a temporary inflammation. Histologically, cartilage trauma caused significant OA changes that were not aggravated by an additional hemarthrosis. Trauma led to a persistent deposition of terminal complement complex (TCC), being enhanced by hemarthrosis. However, trauma-induced formation of osteophytes and arthrotomy-induced elevation of tumor necrosis factor–α release were reduced by hemarthrosis.
Conclusion:
While blood exposure clearly aggravated trauma-induced OA processes in the in vitro model, a singular blood injection revealed heterogeneous effects in vivo, enhancing TCC deposition but reducing trauma-induced osteophyte formation while the histological score of traumatized cartilage was not further impaired.
Clinical Relevance:
The results of this study indicate that a singular, limited bleeding event might not exacerbate early trauma-induced cartilage degeneration in joint injuries. An early removal of intra-articular blood may not prevent the final resulting cartilage damage.
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FSPLJ, NUK, OILJ, SAZU, UKNU, UL, UM, UPUK
Insulin-like growth factors (IGFs) are known to be key regulators of bone growth, remodeling, and repair. Since all these processes depend on the recruitment of cells with the potential to be ...committed to the osteoblastic lineage, we studied possible effects of IGF-I and -II on migration of human mesenchymal progenitor cells (MPC) using a modified Boyden chamber assay. The results were compared to those of primary osteoblasts and in vitro-osteogenic-differentiated MPC. IGF-I and -II stimulated cell migration of all these cell populations in a dose-dependent manner from 1 to 100
ng/mL. The maximal chemotactic index (CI) was 4–5 for MPC and primary osteoblasts and about 3 for in vitro-differentiated MPC. Checkerboard analysis revealed that IGFs stimulated true directed cell migration (chemotaxis) and not simply chemokinesis. Addition of an antibody against the type I IGF receptor (αIR3) completely abolished (MPC) or markedly reduced (primary osteoblasts) the chemotactic effects of each of the IGFs. IGFBP-3 itself had no direct effect, while IGFBP-5 stimulated MPC migration at concentrations of 80 and 160
ng/mL. Parallel application of IGFBP-3 had borderline inhibitory effects while the addition of 40
ng/mL of IGFBP-5 enhanced the chemotactic effect of IGF-I on MPC. In conclusion, our results show that IGF-I and -II are chemotactic factors for MPC and indicate that IGFBP-5 both modulates the IGF-I effect and directly stimulates migration of human mesenchymal progenitor cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Implant surfaces should ideally be designed to promote the attachment of target tissue cells; at the same time, they should prevent bacterial adhesion, achievable through modification strategies ...comprising three lines of defense. As the first criterion, selective adhesion can be realized by means of non-adhesive coatings that can be functionalized with small peptides, thereby supporting osteogenic cell attachment for implants in bone contact but not bacterial adhesion. The second line of defense, defined by bacterial survival, quorum sensing and biofilm formation, can be addressed by various antimicrobial substances that can be leaching or non-leaching. The possibility of a third line of defense, the disruption of an established biofilm, is just emerging. Since microorganisms are quite “ingenious” at finding ways to overcome a certain line of defense, the most promising solution might be a combination of all these antibacterial strategies. Coating systems that allow such different approaches to be combined are scarce. However, ultrathin multifunctional NCO-sP(EO-stat-PO)-based layers may represent a promising platform for such an integrated approach.
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NUK, OILJ, SAZU, UKNU, UL, UM, UPUK
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