Jette Lovmand

The use of combinatorial topographical libraries for the screening of enhanced osteogenic expression and mineralization

Nano- and microstructured surfaces are known to impact on the binding and differentiation of cells, but the detailed basic understanding of the underlying regulatory mechanisms is still scarce, which impedes the rational design of smart biomaterials. Towards a comprehensive analysis of the interplay between topographical parameters such as feature design and lateral and vertical dimensions we here report on a combinatorial screening approach, BioSurface Structure Array (BSSA) of test squares each with a distinct topography. Using such BSSA libraries of 504 topographically distinct surface structures, we have identified combinations of size, gap and height of structures which enhance mineralization as well as the expression of osteogenic markers of a preosteoblastic murine cell line. This generic BSSA screening platform is a versatile technology for the systematic identification of surfaces with specific biological properties, and it may for example be useful for optimizing the design of biomaterials for regulating cellular behaviour.

Large area protein patterning reveals nanoscale control of focal adhesion development.

Focal adhesion development in cells adherent to surface bound fibronectin presented as 200, 500, or 1000 nm diameter circular patches or as homogeneous controls is studied by fluorescence and scanning electron microscopy. Fundamental cellular processes such as adhesion, spreading, focal adhesion and stress fiber formation are shown to be dependent on the spatial distribution of ligands at this scale. Large area samples enable the study of whole cell populations and opens for new potential applications.

Polydopamine/Liposome Coatings and Their Interaction with Myoblast Cells

Surface-mediated drug delivery is a recent concept, where active surface coatings are employed to deliver therapeutic cargo to cells. Herein, we explore the potential of liposomes embedded in polydopamine (PDA) coatings to serve as drug deposits stored on planar substrates. We quantify the PDA growth rate on glass by XPS and show that PDA coatings support myoblast adherence and proliferation. Further, PDA capping layers were deposited on glass substrates precoated with poly(L-lysine) and zwitterionic liposomes. Already thin PDA capping layers render liposome coated surfaces cell adhesive. We experimentally show for the first time, the internalization of a model hydrophobic cargo, that is, fluorescent lipids embedded within the lipid bilayer of liposomes by the cells from the surface. This is evident from the fluorescence exhibited by the cells grown on PDA coatings containing fluorescently labeled liposomes, with the highest fluorescent intensity found in the close proximity of the cell nuclei. The cargo uptake efficiency depends on the thickness of the PDA capping layer and the cell residence time on the coated substrates. Taken together, we demonstrate the first step toward the establishment of a versatile approach using liposomal drug deposits in polymer thin films for surface-mediated drug delivery.

Identification of Distinct Topographical Surface Microstructures Favoring Either Undifferentiated Expansion or Differentiation of Murine Embryonic Stem Cells

The potential of embryonic stem (ES) cells for both self-renewal and differentiation into cells of all three germ layers has generated immense interest in utilizing these cells for tissue engineering or cell-based therapies. However, the ability to culture undifferentiated ES cells without the use of feeder cells as well as means to obtain homogeneous, differentiated cell populations devoid of residual pluripotent ES cells still remain major challenges. Here we have applied murine ES cells to topographically microstructured surface libraries, BioSurface Structure Arrays (BSSA), and investigated whether these could be used to (i) identify topographically microstructured growth supports alleviating the need for feeder cells for expansion of undifferentiated ES cells and (ii) identify specific types of microstructures enforcing differentiation of ES cells. The BSSA surfaces arrays consisted of 504 different topographical microstructures each located in a tester field of 3 x 3 mm. The murine ES cell lines CJ7 and KH2 were seeded upon the BSSA libraries and specific topographical structures facilitating either undifferentiated ES cell growth or enhancing spreading indicative of differentiation of the ES cells were identified. Secondly serial passage of undifferentiated CJ7 ES cells on selected microstructures, identified in the screening of these BSSA libraries, showed that these cells had retained germ-line potential. These results indicate that one specific type of topographical surface microstructures, identified by the BSSA technology, can substitute for feeder cells and that another subset may be used to eliminate undifferentiated ES cells from a population of differentiated ES cells.

Focal Complex Maturation and Bridging on 200 nm Vitronectin but Not Fibronectin Patches Reveal Different Mechanisms of Focal Adhesion Formation

The effects of protein type and pattern size on cell adhesion, spreading, and focal adhesion development are studied. Fibronectin and vitronectin patterns from 0.1 to 3 μm produced by colloidal lithography reveal important differences in how cells adhere to and bridge focal adhesions across protein nanopatterns versus micropatterns. Vinculin and zyxin in focal adhesions but not integrins are seen to bridge ligand gaps. Differences in protein mechanical properties are implicated as important factors in focal adhesion development.

Effect of IL-4 and IL-13 on IFN-γ-induced production of nitric oxide in mouse macrophages infected with herpes simplex virus type 2

Interleukin (IL)‐4 and IL‐13 share a wide range of activities. Prominent among these is the ability to antagonize many interferon (IFN)‐γ‐induced activities. Here we demonstrate that IL‐4 and IL‐13 totally abrogate IFN‐γ‐induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) mRNA and protein synthesis in a murine macrophage cell line. IFN‐γ‐treated cells infected with herpes simplex virus type 2 (HSV‐2) or costimulated with tumor necrosis factor (TNF)‐α showed an enhanced reactivity, which was only partially reduced by IL‐4/13. The results indicate that IL‐4 and IL‐13 function by intervening with a step prior to iNOS transcription by antagonizing IFN‐γ‐induced signal(s) without counteracting synergistic virus‐ or TNF‐α‐induced signals. The beneficial effect of a sustained NO production in foci of virus infection is suggested.

Control of proliferation and osteogenic differentiation of human dental-pulp-derived stem cells by distinct surface structures

The ability to control the behavior of stem cells provides crucial benefits, for example, in tissue engineering and toxicity/drug screening, which utilize the stem cells capacity to engineer new tissues for regenerative purposes and the testing of new drugs in vitro. Recently, surface topography has been shown to influence stem cell differentiation; however, general trends are often difficult to establish due to differences in length scales, surface chemistries and detailed surface topographies. Here we apply a highly versatile screening approach to analyze the interplay of surface topographical parameters on cell attachment, morphology, proliferation and osteogenic differentiation of human mesenchymal dental-pulp-derived stem cells (DPSCs) cultured with and without osteogenic differentiation factors in the medium (ODM). Increasing the inter-pillar gap size from 1 to 6 μm for surfaces with small pillar sizes of 1 and 2 μm resulted in decreased proliferation and in more elongated cells with long pseudopodial protrusions. The same alterations of pillar topography, up to an inter-pillar gap size of 4 μm, also resulted in enhanced mineralization of DPSCs cultured without ODM, while no significant trend was observed for DPSCs cultured with ODM. Generally, cells cultured without ODM had a larger deposition of osteogenic markers on structured surfaces relative to the unstructured surfaces than what was found when culturing with ODM. We conclude that the topographical design of biomaterials can be optimized for the regulation of DPSC differentiation and speculate that the inclusion of ODM alters the ability of the cells to sense surface topographical cues. These results are essential in order to transfer the use of this highly proliferative, easily accessible stem cell into the clinic for use in cell therapy and regenerative medicine.

Interaction of human mesenchymal stem cells with osteopontin coated hydroxyapatite surfaces

In vitro studies of the initial attachment, spreading and motility of human bone mesenchymal stem cells have been carried out on bovine osteopontin (OPN) coated hydroxyapatite (HA) and gold (Au) model surfaces. The adsorption of OPN extracted from bovine milk was monitored by the quartz crystal microbalance with dissipation (QCM-D) and the ellipsometry techniques, and the OPN coated surfaces were further investigated by antigen-antibody interaction. It is shown that the OPN surface mass density is significantly lower and that the number of antibodies binding to the resulting OPN layers is significantly higher on the HA as compared to the Au surfaces. The initial attachment, spreading and motility of human mesenchymal stem cells show a larger cell area, a faster arrangement of vinculin in the basal cell membrane and more motile cells on the OPN coated HA surfaces as compared to the OPN coated Au surfaces and to the uncoated Au and HA surfaces. These in vitro results indicate that there may be great potential for OPN coated biomaterials, for instance as functional protein coatings or drug delivery systems on orthopaedic implants or scaffolds for tissue-engineering.

Osteopontin presentation affects cell adhesion—Influence of underlying surface chemistry and nanopatterning of osteopontin

Osteopontin is a promising coating material for biomaterials, being important both in remodeling and formation of mineralized tissue and in immunological responses. We have investigated cell attachment to osteopontin adsorbed at different surface chemistries (-NH(2), -COOH, -CH(3), and bare gold) and to osteopontin presented as a nanopattern of 50 nm protein patches separated by a nonadhesive background. MDA-MB-435 cells adhere well to osteopontin presented at the hydrophilic chemistries (-NH2, -COOH, and gold) suggesting that osteopontin is presented in a functional form on these surfaces. On the amine surface, the cell attachment appears partly driven by electrostatic attraction between the positively charged substrate and the negatively charged cell membrane, whereas the spreading of the cells depends on the specific interaction with osteopontin presented at the surface. Significantly, fewer cells adhere to osteopontin presented at the methyl-terminated hydrophobic surface and the cells are less spread. On the nanopatterned osteopontin, only a very low number of cells adhered and those few attached cells showed an elongated morphology with few adhesion points to the surface. This indicates that the adhesive patches are not large enough to support stable focal contacts. The good cell attachment and spreading on the hydrophilic surfaces holds promise for osteopontin as a future coating for biomaterials.

Triple basepair changes within and adjacent to the conserved YY1 motif upstream of the U3 enhancer repeats of SL3-3 murine leukemia virus cause a small but significant shortening of latency of T-lymphoma induction.

A highly conserved sequence upstream of the transcriptional enhancer in the U3 of murine leukemia viruses (MLVs) was reported to mediate negative regulation of their expression. In transient expression studies, negative regulation was reported to be conferred by coexpression of the transcription factor YY1, which binds to a motif in the upstream conserved region (UCR). To address the function of the UCR and its YY1-motif in an in vivo model of MLV-host interactions we introduced six consecutive triple basepair mutations into this region of the potent T-lymphomagenic SL3-3 MLV. We report that all mutants have retained their replication competence and that they all, like the SL3-3 wild type (wt), induce T-cell lymphomas when injected into newborn mice of the SWR strain. However, all mutants induced disease with slightly shorter latency periods than the wt SL3-3, suggesting that the YY1 motif as well as its immediate context in the UCR have a negative effect on the pathogenicity of the virus. This result may have implications for the design of retroviral vectors.