Kerstin Reimers

Cultivation of keratinocytes and fibroblasts in a three-dimensional bovine collagen-elastin matrix (Matriderm®) and application for full thickness wound coverage in vivo.

New skin substitutes for burn medicine or reconstructive surgery pose an important issue in plastic surgery. Matriderm® is a clinically approved three-dimensional bovine collagen-elastin matrix which is already used as a dermal substitute of full thickness burn wounds. The drawback of an avital matrix is the limited integration in full thickness skin defects, depending on the defect size. To further optimize this process, Matriderm® has also been studied as a matrix for tissue engineering of skin albeit long-term cultivation of the matrix with cells has been difficult. Cells have generally been seeded onto the matrix with high cell loss and minimal time-consuming migration. Here we developed a cell seeded skin equivalent after microtransfer of cells directly into the matrix. First, cells were cultured, and microinjected into Matriderm®. Then, cell viability in the matrix was determined by histology in vitro. As a next step, the skin substitute was applied in vivo into a full thickness rodent wound model. The wound coverage and healing was observed over a period of two weeks followed by histological examination assessing cell viability, proliferation and integration into the host. Viable and proliferating cells could be found throughout the entire matrix. The presented skin substitute resembles healthy skin in morphology and integrity. Based on this study, future investigations are planned to examine behaviour of epidermal stem cells injected into a collagen-elastin matrix under the aspects of establishment of stem cell niches and differentiation.

NoBP, a nuclear fibroblast growth factor 3 binding protein, is cell cycle regulated and promotes cell growth.

ABSTRACT Secreted and nuclear forms of fibroblast growth factor 3 (FGF3) have opposing effects on cells. The secreted form stimulates cell growth and transformation, while the nuclear form inhibits DNA synthesis and cell proliferation. By using the yeast two-hybrid system we have identified a nucleolar FGF3 binding protein (NoBP) which coimmunoprecipitated and colocalized with FGF3 in transfected COS-1 cells. Characterization of the NoBP binding domain of FGF3 exactly matched the sequence requirements of FGF3 for its translocation into the nucleoli, suggesting that NoBP might be the nucleolar binding partner of FGF3 essential for its nucleolus localization. Carboxyl-terminal domains of NoBP contain linear nuclear and nucleolar targeting motifs which are capable of directing a heterologous protein β-galactosidase to the nucleus and the nucleoli. While NoBP expression was detected in all analyzed proliferating established cell lines, NoBP transcription was rapidly downregulated in the promyelocytic leukemia cell line HL60 when induced to differentiate. Analysis on the expression pattern of NoBP mRNA throughout the cell cycle in HeLa cells synchronized by lovastatin demonstrated a substantial upregulation during the late G1/early S phase. NoBP overexpression conferred a proliferating effect onto NIH 3T3 cells and can counteract the inhibitory effect of nuclear FGF3, suggesting a role of NoBP in controlling proliferation in cells. We propose that NoBP is the functional target of nuclear FGF3 action.

The anti-apoptotic protein lifeguard is expressed in breast cancer cells and tissues

Lifeguard (LFG) is an anti-apoptotic protein that inhibits Fas-mediated death in tumour cells. However, the molecular function of human LFG in the carcinogenesis of human breast cells is uncertain. We studied the expression and function of endogenous LFG in four breast cancer cell lines (MCF-7, MDA-MB-231, T-47D and HS 578T), a human breast epithelial cell line (HS 578Bst), and in healthy and cancerous breast tissues. Molecular (Western blot and RT-PCR) and immunohistochemical techniques were used to investigate the LFG expression. To investigate the breast cancer cell proliferation in the presence of Fas, we performed fluorescent cell viability assays. The possible association of Fas with LFG was analyzed by immunofluorescence microscopy. In this paper, we provide convincing evidence that LFG is overexpressed in several human breast cancer cell lines. More importantly, we found that the LFG expression correlates with high tumour grades in primary breast tumours. Finally, we demonstrated that Fas sensitivity is reduced in breast cancer cell lines expressing LFG. Our results indicated that LFG is strongly expressed in breast cancer epithelial cells. Moreover, the overexpression of LFG correlated with tumour grade and reduced Fas sensitivity. Our findings support the idea that LFG may have a role in the downregulation of apoptosis in breast cancer cells.

Biomechanics and biocompatibility of woven spider silk meshes during remodeling in a rodent fascia replacement model.

Objective:The aim of this study was to investigate biomechanical and immunogenic properties of spider silk meshes implanted as fascia replacement in a rat in vivo model. Background:Meshes for hernia repair require optimal characteristics with regard to strength, elasticity, and cytocompatibility. Spider silk as a biomaterial with outstanding mechanical properties is potentially suitable for this application. Methods:Commercially available meshes used for hernia repair (Surgisis and Ultrapro) were compared with handwoven meshes manufactured from native dragline silk of Nephila spp. All meshes were tied onto the paravertebral fascia, whereas sham-operated rats were sutured without mesh implantation. After 4 or 14 days, 4 weeks, and 4 or 8 months, tissue samples were analyzed concerning inflammation and biointegration both by histological and biochemical methods and by biomechanical stability tests. Results:Histological sections revealed rapid cell migration into the spider silk meshes with increased numbers of giant cells compared with controls with initial decomposition of silk fibers after 4 weeks. Four months postoperatively, spider silk was completely degraded with the formation of a stable scar verified by constant tensile strength values. Surgisis elicited excessive stability loss from day 4 to day 14 (P < 0.001), with distinct inflammatory reaction demonstrated by lymphocyte and neutrophil invasion. Ultrapro also showed decreasing strength and poor elongation behavior, whereas spider silk samples had the highest relative elongation (P < 0.05). Conclusions:Hand-manufactured spider silk meshes with good biocompatibility and beneficial mechanical properties seem superior to standard biological and synthetic meshes, implying an innovative alternative to currently used meshes for hernia repair.

Applying amphibian limb regeneration to human wound healing: a review.

In contrast to the limited regenerative ability found in human wound healing, which often results in unsatisfying and deficient scar formation, urodele amphibians, with the Mexican axolotl as a prime example, expose an extraordinary regenerative capacity. This regeneration leads to a perfect restoration of tissue architecture, function, and aesthetics with the axolotl being actually able to reclaim complete limbs. Evolutionary considerations suggest that regeneration might be a biologic principle which also underlies human wound healing. Experimental findings, such as comparative studies on transforming growth factor-&bgr; and fibroblast growth factor accentuate this assumption. Regeneration, as recent data indicate, might be a question of adaptive immunity. The loss of regenerative potency correlates with the decrease of regeneration in most species, whereas the Mexican axolotl lacks adaptive immunity throughout its life. The characterization of molecular pathways as a prerequisite for any control of regenerative processes sets an increasing indication toward the transfer into human beings. Some regenerative techniques, eg, recombinant transforming growth factor-&bgr; have already emerged. Molecular findings suggest that there is an intrinsic regenerative capacity in humans which might be initiated under appropriate circumstances. The Mexican axolotl is liable to diverse surgical and molecular approaches. Though well-known among developmental biologists, its exploitation for experimental Plastic Surgery still has to be established. We therefore intend to give an introduction to amphibian regeneration and the common evolutionary roots of regeneration and human wound healing, as we believe that Plastic Surgery takes a unique advantage of performing basic research on amphibian regeneration.

AmbLOXe--an epidermal lipoxygenase of the Mexican axolotl in the context of amphibian regeneration and its impact on human wound closure in vitro.

OBJECTIVEnThe Mexican axolotl (Ambystoma mexicanum) is a well-characterized example for intrinsic regeneration. As lipoxygenase signaling is of crucial importance to scarless mammalian wound healing, we postulated that lipoxygenases might be expressed during amphibian regeneration and they might also influence human cells under appropriate conditions. In this study we identified an amphibian lipoxygenase and evaluated its impact on human cells in an in vitro wound model.nnnMETHODSncDNA encoding for amphibian epidermal lipoxygenase (AmbLOXe) was polymerase chain reaction amplified and sequenced followed by phylogenic classification based on T-coffee alignment. Distribution of AmbLOXe was examined in various Ambystoma tissues, using polymerase chain reaction and in situ hybridization. Lipoxgenase influence was investigated using an outgrowth model of amphibian epidermal cells. Human osteosarcoma, as well as keratinocyte cell lines expressing AmbLOXe, were tested concerning in vitro wound closure in a monolayer scratch model.nnnRESULTSnWe isolated AmbLOXe from Ambystoma limb bud blastema identified as a homologue of human epidermal lipoxygenase. Amphibian epidermal lipoxygenase is expressed in Axolotl limb blastema and in epidermal cells which show decreased cell migration and proliferation rates when treated with LOX inhibitors. Furthermore, human osteosarcoma and keratinocyte cells showed increased rates of cell migration if transfected with AmbLOXe.nnnCONCLUSIONnIn this study, AmbLOXe, a new effector of amphibian regeneration is described. In consideration of the presented data, AmbLOXe is important for amphibian epidermal cell proliferation and migration. As AmbLOXe expressing human osteosarcoma and keratinocyte cell lines showed increased rates of in vitro wound closure, an influence of amphibian mediators on human cells could be described for the first time.

Transactivation of lifeguard (LFG) by Akt-/LEF-1 pathway in MCF-7 and MDA-MB 231 human breast cancer cells

Lifeguard (LFG) has been identified as a molecule that uniquely inhibits death mediated by Fas. The molecular function of human LFG and its regulation in carcinogenesis is uncertain. In our study, we investigated the potential regulation of LFG expression by Akt/LEF-1 pathway. The Glycogen synthase kinase-3 (GSK3) can be regulated by different signaling pathways including those mediated by protein kinase Akt. Inhibition of GSK3β subunits activity results in the stabilisation of the β-catenin protein and its accumulation in the nucleus, where it associates with members of the TCF/LEF-1 family of transcription factors to mediate gene transcription. In Western blots, RT–PCR and by small interfering RNA directed against LEF-1, we demonstrated that LFG expression correlates with GSK3β and LEF-1 activation. Moreover, we showed that LFG mRNA was down-regulated after transfection with siRNA against LEF-1 in MDA-MB-231 cells. Our results therefore identify LFG as a target of the Akt/LEF-1 pathway in MDA-MB-231 breast tumour cells, a regulation which could play a key role in breast tumour progression.

In vitro wound healing assays - State of the art

Abstract Wound healing is essential for the restoration of the barrier function of the skin. During this process, cells at the wound edges proliferate and migrate, leading to re-epithelialization of the wound surface. Wound healing assays are used to study the molecular mechanisms of wound repair, as well as in the investigation of potential therapeutics and treatments for improved healing. Numerous models of wound healing have been developed in recent years. In this review, we focus on in vitro assays, as they allow a fast, cost-efficient and ethical alternative to animal models. This paper gives a general overview of 2-dimensional (2D) cell monolayer assays by providing a description of injury methods, as well as an evaluation of each assay’s strengths and limitations. We include a section reviewing assays performed in 3-dimensional (3D) culture, which employ bioengineered skin models to capture complex wound healing mechanics like cell-matrix interactions and the interplay of different cell types in the healing process. Finally, we discuss in detail available software tools and algorithms for data analysis.

In Vitro Evaluation of Spider Silk Meshes as a Potential Biomaterial for Bladder Reconstruction

Reconstruction of the bladder by means of both natural and synthetic materials remains a challenge due to severe adverse effects such as mechanical failure. Here we investigate the application of spider major ampullate gland-derived dragline silk from the Nephila edulis spider, a natural biomaterial with outstanding mechanical properties and a slow degradation rate, as a potential scaffold for bladder reconstruction by studying the cellular response of primary bladder cells to this biomaterial. We demonstrate that spider silk without any additional biological coating supports adhesion and growth of primary human urothelial cells (HUCs), which are multipotent bladder cells able to differentiate into the various epithelial layers of the bladder. HUCs cultured on spider silk did not show significant changes in the expression of various epithelial-to-mesenchymal transition and fibrosis associated genes, and demonstrated only slight reduction in the expression of adhesion and cellular differentiation genes. Furthermore, flow cytometric analysis showed that most of the silk-exposed HUCs maintain an undifferentiated immunophenotype. These results demonstrate that spider silk from the Nephila edulis spider supports adhesion, survival and growth of HUCs without significantly altering their cellular properties making this type of material a suitable candidate for being tested in pre-clinical models for bladder reconstruction.

Paracrine loop of keratinocyte proliferation and directed neuritic outgrowth in a neuroepithelial coculture.

AbstractIn the absence of skin innervation, wound healing is delayed and chronic nonhealing wounds may occur. Keratinocytes produce neurotrophic factors, such as nerve growth factor (NGF), which has been suggested to attract primary cutaneous afferent axons and exert mitogenic effects on keratinocytes. The present study was performed to examine the interaction of primary human keratinocytes (hKTs) and rat cutaneous primary afferent dorsal root ganglion (DRG) neurons with regard to neuritic outgrowth and keratinocyte proliferation. Neuritic outgrowth was assessed with neurofilament immunostaining where cell bodies and fine neuritic processes were identified. Neuritic outgrowth of neurons alone in culture is spatially random and radial. Neurites in cocultures of DRG neurons insinuated between the hKTs and grew to “clumps” of hKTs within the cultures. Immunostaining with anti-NGF antibody indicates that hKTs expressed the neurotrophin NGF. Proliferation of keratinocytes was significantly enhanced in coculture with DRG and hKT, and NGF levels were increased as compared to DRG or hKT culture alone. These results indicate a dynamic interaction between DRG neurons and hKTs whereby the DRG neurons issue neurites in association with hKTs and the hKTs up-regulate NGF and increase their proliferation rate. These findings support the hypothesis that nerve-skin interactions play a significant role in wound healing.