Self-assembled nanofiber coatings for controlling cell responses.

Nanofibers are thought to enhance cell adhesion, growth, and function. We demonstrate that the choice of building blocks in self-assembling nanofiber systems can be used to control cell behavior. The use of 2 D-coated, self-assembled nanofibers in controlling lens epithelial cells, fibroblasts, and mesenchymal stem cells was investigated, focusing on gene and protein expression related to the fibrotic response. To this end, three nanofibers with different characteristics (morphology, topography, and wettability) were compared with two standard materials frequently used in culturing cells, TCPS, and a collagen type I coating. Cell metabolic activity, cell morphology, and gene and protein expression were analyzed. The most hydrophilic nanofiber with more compact network consisting of small fibers proved to provide a beneficial 2 D environment for cell proliferation and matrix formation while decreasing the fibrotic/stress behavior in all cell lines when compared with TCPS and the collagen type I coating. This nanofiber demonstrates the potential to be used as a biomimetic coating to study the development of fibrosis through epithelial-to-mesenchymal transition. This study also shows that nanofiber structures do not enhance cell function by definition, because the physico-chemical characteristics of the nanofibers influence cell behavior as well and actually can be used to regulate cell behavior toward suboptimal performance. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2252-2265, 2017.

Recombinant gelatin microspheres: novel formulations for tissue repair?

Microspheres (MSs) can function as multifunctional scaffolds in different approaches of tissue repair (TR), as a filler, a slow-release depot for growth factors, or a delivery vehicle for cells. Natural cell adhesion-supporting extracellular matrix components like gelatin are good materials for these purposes. Recombinant production of gelatin allows for on-demand design of gelatins, which is why we aim at developing recombinant gelatin (RG) MSs for TR. Two types of MSs (50 < Ø < 100 microm) were prepared by crosslinking two RGs, Syn-RG, and the arginine-glycine-aspartate-containing Hu-RG. The MSs were characterized, and their tissue reaction and degradation in rats was examined. Histological analysis of the explants after 14 and 28 days in vivo also showed that Syn-RG was degraded slower than Hu-RG, which correlated with the in vitro degradation assay. Hu-RG explants displayed more cellular ingrowth (60% vs. 15% for Syn-RG at day 14), which was associated with extracellular matrix deposition and vascularization. The infiltrating cells consisted of mainly macrophages, part of which fused to giant cells locally, and fibroblasts. No differences were found in matrix metalloproteinase mRNA levels, whereas gelatinase activity was clearly higher in Hu-RG explants. In conclusion, the in vitro and in vivo results of these novel formulations pave the way for cell- and/or factor-driven TR by these RG MSs.

On the role and fate of LPS-dephosphorylating activity in the rat liver.

Gut-derived lipopolysaccharide (LPS) plays a role in the pathogenesis of liver diseases like fibrosis. The enzyme alkaline phosphatase (AP) is present in, among others, the intestinal wall and liver and has been previously shown to dephosphorylate LPS. Therefore, we investigated the effect of LPS on hepatic AP expression and the effect of AP on LPS-induced hepatocyte responses. LPS-dephosphorylating activity was expressed at the hepatocyte canalicular membrane in normal and fibrotic animals. In addition to this, fibrotic animals also displayed high LPS-dephosphorylating activity around bile ducts. The enzyme was shown to dephosphorylate LPS from several bacterial species. LPS itself rapidly enhanced the intrahepatic mRNA levels for this enzyme within 2 h by a factor of seven. Furthermore, in vitro and in vivo studies showed that exogenous intestinal AP quickly bound to the asialoglycoprotein receptor on hepatocytes. This intestinal isoform significantly attenuated LPS-induced hepatic tumor necrosis factor-alpha and nitric oxide (nitrite and nitrate) responses in vitro. The enzyme also reduced LPS-induced hepatic glycogenolysis in vivo. This study shows that LPS enhances AP expression in hepatocytes and that intestinal AP is rapidly taken up by these same cells, leading to an attenuation of LPS-induced responses in vivo. Gut-derived LPS-dephosphorylating activity or enzyme upregulation within hepatocytes by LPS may therefore be a protective mechanism within the liver.

A new technique for preparing precision-cut slices from small intestine and colon for drug biotransformation studies.

INTRODUCTION: A new technique was developed to prepare precision-cut slices from small intestine and colon with the object of studying the biotransformation of drugs in these organs. METHODS: Rat intestinal slices were prepared in two different ways. In the first method, slices were punched out of the small intestine. In the second method, precision-cut slices were made from agarose-filled and -embedded intestines, using the Krumdieck tissue slicer. This method was also applied to colon tissue. Viability of the slices was determined by analysis of intracellular ATP and RNA levels and morphology. Drug metabolizing activity was studied using lidocaine, testosterone, and 7-ethoxycoumarin (7-EC) as phase I substrates, and 7-hydroxycoumarin (7-HC) as a phase II substrate. RESULTS: Precision-cut slices made from agarose-filled and -embedded intestine better preserved ATP levels than tissue that was punched out of the intestinal wall. After 24 h of incubation, morphology in precision cut-slices showed was quite well preserved while punched out tissue was almost completely autolytic after incubation. In addition, total RNA amount and quality was much better maintained in precision-cut slices, when compared to punched out tissue. Both intestinal slices and punched-out tissue showed high, and comparable, phase I and phase II biotransformation activities. DISCUSSION: It is concluded that preparing precision-cut 0.25 mm slices out of agarose-filled and -embedded intestine provides an improvement, compared with punched-out tissue, and that both intestinal and colon slices are useful preparations for in vitro biotransformation studies.