Nora E. Paul

Topographical control of human macrophages by a regularly microstructured polyvinylidene fluoride surface

In this study we investigated the influence of surface topography on the inflammatory response of human macrophages. We generated different polyvinylidene fluoride (PVDF) surfaces including (i) a smooth surface of PVDF spherulites as a control, (ii) a randomly nanotextured surface with alumina particles, and (iii) a microstructure using laser ablation. The identical chemistry of all PVDF surfaces was demonstrated by X-ray photoelectron spectroscopy. The topography was evaluated by white light interferometry and X-profile analysis. Macrophages were cultured on the different surfaces including lipopolysaccharide (LPS) treatment as an inflammatory activator. Our results demonstrate that the microstructured surface but not the nanotexured significantly affects the activation of primary human macrophages by inducing a specific cytokine and gene expression pattern. This activation resulted in a subtype of macrophages with pro- but also anti-inflammatory properties. Interestingly, the response on the topography differed from that triggered by LPS, pointing to a different activation state of the cells. Our data clearly show that a particular topography induces an inflammatory response. This suggests that the modification of topography could influence the inflammatory potency of a biomaterial and hence could affect the biocompatibility of implants.

Induction of specific macrophage subtypes by defined micro-patterned structures.

In this study, we investigated the influence of different perfluoropolyether (PFPE) microstructures on the inflammatory response of human macrophages. We generated four different microstructured PFPE surfaces by replica molding from silicon masters. The function-associated surface markers 27E10 and CD163 were monitored using flow cytometry to measure the pro- and anti-inflammatory reactions. Inflammatory mediator expression was measured at the protein and mRNA level. Lipopolysaccharide treatment served as positive control for pro-inflammatory activation. We observed that each micropattern induced a specific morphology, phenotype and mediator profile. A microstructure of regular grooves induced a pro-inflammatory phenotype (M1) which was not accompanied by release of pro-inflammatory mediators. However, the larger cylindrical posts induced an anti-inflammatory phenotype (M2) with a remarkable down-regulation of CXCL10. Smaller posts with a shorter distance exhibited a stronger pro-inflammatory response than those with a longer distance, on the levels of both phenotype and mediator release. Regression analysis suggests that the geometrical parameters of the microstructures, specifically the period of structures, may play an important role in macrophage response. Optimization of such microstructures may provide a method to invoke a predictable response of macrophages to implants and control the mediator release.

Differential expression of influx and efflux transport proteins in human antigen presenting cells

Abstract:  Human macrophages (MΦ) express cytochrome P450 enzymes verifying their capacity to metabolize a variety of endogenous and exogenous substances. Here we analysed the mRNA and protein expression of transport proteins involved in the uptake or export of drugs, hormones and arachidonic acid metabolites in dendritic cells (DC) and MΦ compared to their precursors – blood monocytes – using cDNA microarray, RT‐PCR, Western‐blot and immunostaining techniques. The transport proteins studied included members of the solute carrier organic anion transporter family (SLCO) and the multidrug resistance associated proteins (MRP) 1–6 belonging to the ATP‐binding cassette subfamily C (ABCC). We found that only mRNA for SLCO‐2B1, ‐3A1, and ‐4A1 were present in monocytes, MΦ and DC. Most interestingly the expression of SLCO‐2B1 was markedly enhanced in MΦ as compared to monocytes and DC. The presence of mRNA for ABCC1, 3, 4, 5 and 6 in all three cell types was demonstrated. On protein level ABCC1/MRP1 which has been identified as leukotriene C4 transporter was found to be the most abundant transporter in MΦ and DC. Blocking the ABCC1/MRP1 activity with the specific inhibitor MK571 resulted in a phenotypic change in DC but not in MΦ. Our data show that human blood monocytes and monocyte derived MΦ as well as DC express a specific profile of transporters involved in uptake and export of exogenous molecules like allergens or drugs, but also of endogenous substances in particular of inflammatory lipid mediators like leukotrienes and prostaglandins.

Chondrogenic Differentiation of Human Adipose-Derived Stem Cells: A New Path in Articular Cartilage Defect Management?

According to data published by the Centers for Disease Control and Prevention, over 6 million people undergo a variety of medical procedures for the repair of articular cartilage defects in the U.S. each year. Trauma, tumor, and age-related degeneration can cause major defects in articular cartilage, which has a poor intrinsic capacity for healing. Therefore, there is substantial interest in the development of novel cartilage tissue engineering strategies to restore articular cartilage defects to a normal or prediseased state. Special attention has been paid to the expansion of chondrocytes, which produce and maintain the cartilaginous matrix in healthy cartilage. This review summarizes the current efforts to generate chondrocytes from adipose-derived stem cells (ASCs) and provides an outlook on promising future strategies.

The effect of mechanical stress on the proliferation, adipogenic differentiation and gene expression of human adipose-derived stem cells.

To allow for a better implementation of external volume expansion to clinical applications for soft tissue regeneration, it is necessary to comprehensively understand the underlying mechanisms. As human adipose‐derived stem cells (hASCs) play a crucial role in soft tissue enlargement, we investigated the impact of cyclic stretch on gene expression, proliferation rate and adipogenic differentiation of these cells. After cyclic stretching, RNA was extracted and subjected to DNA microarray analysis and reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR). Also, the expression of FABP4 mRNA was analysed by RT‐qPCR to test whether mechanical stretch affected adipogenic differentiation of hASCs. The proliferation rate was assessed using the alamarBlue assay and Ki‐67 staining. A cell cycle analysis was performed with flow cytometry and Western blot. We found that cyclic stretch significantly induced the expression of CYP1B1 mRNA. Furthermore, the adipogenic differentiation of hASCs was impaired, as was the proliferation. This was partly due to a decrease in extracellular signal‐regulated kinase (ERK) 1/2 and histone H3 phosphorylation, suggesting a growth arrest in the G2/M phase of the cell cycle. Enrichment analyses demonstrated that stretch‐regulated genes were over‐represented in pathways and biological processes involved in extracellular matrix organization, vascular remodelling and responses to cell stress. Taken together, mechanical stress impaired both proliferation and adipogenic differentiation, but led to a tissue‐remodelling phenotype of hASCs. These data suggest that extracellular matrix remodelling and neoangiogenesis may play a more important role in external volume expansion than proliferation and adipogenesis of hASCs. Copyright

RNA Isolation from Adipose Tissue: An Optimized Procedure for High RNA Yield and Integrity

Adipose tissue physiology strongly differs between body regions. Reasonable amounts of intact RNA are required to analyze the local differences on a molecular level. Therefore, we developed an optimized RNA isolation procedure from adipose tissue samples. Excised human subcutaneous adipose tissue was obtained from elective operations, and the RNA Stabilization Reagent (RNAlater) was tested for its effect on RNA integrity. Additionally, 3 different RNA isolation kits were evaluated for efficacy in isolating RNA from tissue samples. The samples displayed a significant loss in recoverable RNA and signs of RNA degradation 30 to 60 minutes after tissue excision. The application of RNAlater significantly delayed this degradation. Using the RNeasy Lipid Tissue Kit resulted in a significantly higher RNA yield compared to the RNeasy Mini Kit. Our results demonstrate that combining the RNAlater and RNeasy Lipid Tissue Kit results in a higher yield of RNA even from relatively small tissue samples with guaranteed RNA integrity.

The RGD Coupling Strategy Determines the Inflammatory Response of Human Primary Macrophages In Vitro and Angiogenesis In Vivo

Surface modifications of implants are frequently done using bioactive peptides. However, immune cells such as macrophages might evoke a rejection of an implant due to an undesired activation by the materials. Here, the influence of different strategies for peptide immobilization onto (poly)-vinylidene fluoride (PVDF) on inflammation and angiogenesis is studied. The inflammatory response of human primary macrophages is investigated by analyzing inflammatory cytokine expression. Surface roughness and adsorptive coupling have only minor effects on macrophage activation. Acrylic acid (AAc)-based covalent RGD-coupling leads to the most favorable cellular reaction, indicated by increased VEGF release. Chemical vapor deposition treated surfaces are inert, but additional covalent coupling of RGD induces a pronounced proinflammatory reaction. An in vivo angiogenesis study reveals that covalent coupling of RGD results in delayed but increased angiogenesis. It is concluded that for implant decoration with peptides, the substrate material has to be selected carefully to prevent inflammatory immune responses.

The Effect of Antiseptics on Adipose-Derived Stem Cells

Background: Although chemical antiseptics are the most basic measure to control wound infection and frequently come into contact with subcutaneous adipose tissue, no studies have evaluated their toxicity on adipose tissue and its cell fractions. In the present study, the effects of five different antiseptics on adipose-derived stem cells were evaluated. Methods: Human adipose-derived stem cells were harvested from healthy donors. Adipose-derived stem cell viability was measured after treatment with different concentrations of antiseptics over 5 days. Furthermore, the effect on the proliferation, adipogenic differentiation, and apoptosis/necrosis of adipose-derived stem cells was analyzed. Finally, the mRNA expression of the stem cell markers CD29, CD34, CD73, CD90, and CD105 was detected. Results: Octenisept and Betaisodona significantly reduced cell proliferation and differentiation and led to considerable adipose-derived stem cell necrosis. Octenisept decreased stem cell viability at the lowest concentrations tested, and all stem cell markers were down-regulated by Octeniseptr and Betaisodona. Lavasept and Prontosan both led to reduced stem cell viability, proliferation, and differentiation, and increased apoptosis/necrosis, although the effects were less pronounced compared with Octenisept and Betaisodona. Adipose-derived stem cells survived treatment with mafenide acetate even at high concentrations, and mafenide acetate showed minimal negative effects on their proliferation, adipogenic differentiation, cell death, and stem cell marker expression. Conclusions: Mafenide acetate may be regarded as a feasible antiseptic for the treatment of wounds with exposed adipose tissue because of its low adipose-derived stem cell toxicity. Lavasept and Prontosan are possible alternatives to mafenide acetate. Octenisept and Betaisodona, by contrast, may be used only in highly diluted solutions. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Adipose Tissue Increases the Proliferation of Melanoma Cell Lines In Vitro.

Introduction:Autologous fat grafting has been used for reconstructive and aesthetic surgery for more than a century. Although initial criticism involved fat resorption and later the possible radiological interference in mammographies, the most recent debate focuses on the carcinogenicity of fat grafts. Among malignant skin tumors, melanomas show the highest lethality and lead to significant soft tissue defects after resection with fat grafting representing a feasible therapy option for tissue reconstruction. In contrast to breast carcinoma, little data is available regarding the carcinogenicity of fat grafts in melanoma patients. The present study was designed to investigate the influence of whole adipose tissue on the growth of melanoma cell lines compared to breast cancer cell lines. Methods:Human subcutaneous adipose tissue was obtained from elective surgeries and cut into pieces ranging from 20 to 500 mg. Adipose tissue samples were co-cultivated with the human breast cancer cell line MCF-7 and the melanoma cell lines MEL-JUSO and IGR-37, and proliferation was measured using the alamarBlue proliferation assay. Additionally, the same experiment was conducted under nutrient-limited conditions. Results:We found that adipose tissue significantly increased the proliferation of the human breast cancer cell line MCF-7 and importantly also of the melanoma cell lines MEL-JUSO and IGR-37 under optimal nutrient supply. Although the impact of adipose tissue on the growth of MCF-7 cells under nutrient deprivation was comparatively weak, the proliferation of MEL-JUSO and IGR-37 cells was clearly stimulated by adipose tissue. Discussion:Our results show that adipose tissue enhances not only the growth of breast cancer but also melanoma cells even under conditions of nutrient limitation by soluble factors secreted by adipose tissue. With respect to our results and the current literature, reconstruction of soft tissue defects after malignancies including breast cancer and melanoma should be conducted with great care and only after complete tumor resection.

The Effect of Lipoaspirates on Human Keratinocytes

BACKGROUND One increasingly important trend in plastic, reconstructive, and aesthetic surgery is the use of fat grafts to improve cutaneous wound healing. In clinical practice, lipoaspirates (adipose tissue harvested by liposuction) are re-injected in a procedure called lipofilling. Previous studies, however, mainly evaluated the regenerative effect of isolated adipocytes, adipose-derived stem cells, and excised en bloc adipose tissue on keratinocytes, whereas no study to date has examined the effect of lipoaspirates. OBJECTIVES The authors aimed to investigate differences in the regenerative property of en bloc adipose tissue and lipoaspirates on keratinocytes. METHODS Human keratinocytes, lipoaspirates, and en bloc adipose tissue from 36 healthy donors were isolated. In vitro proliferation, differentiation, migration, stratification, and wound healing of keratinocyte monolayers were measured. Furthermore, secreted levels of VEGF, bFGF, IGF-1, MMP-9, and MIF were detected by ELISA. RESULTS Migration, proliferation, and wound healing of keratinocytes were increased by lipoaspirates. Interestingly, the effect of lipoaspirates on keratinocyte proliferation was significantly higher than by en bloc adipose tissue after 5 days. The differentiation of keratinocytes was equally attenuated by lipoaspirates and en bloc adipose tissue. Stratification of keratinocyte layers was enhanced by lipoaspirates and en bloc fat when compared to controls. Lipoaspirates secrete higher levels of bFGF, whereas higher levels of VEGF and IGF-1 are released by en bloc adipose tissue. CONCLUSION We show that lipoaspirates and en bloc adipose tissue have a regenerative effect on keratinocytes. One reason for the higher effect of lipoaspirates on keratinocyte proliferation may be the secretion of different cytokines.