Christine Pauken

Regulation of cell adhesion during embryonic compaction of mammalian embryos: Roles for PKC and β‐catenin

Beta‐catenin has a number of roles in early development including involvement in cell adhesion, cell signaling, and developmental fate specification. This study investigates the mechanisms that regulate embryonic compaction, the first cell adhesion event in early mammalian development. Mammalian embryos can be induced to compact at an earlier developmental stage than normal by treatment with agonists that activate protein kinase C (PKC), and this treatment is used to identify and analyze the minimum essential changes required for embryonic compaction. It was predicted that: (1) since activation of PKC can induce compaction prematurely in mouse embryos, phosphorylation of the protein components of the adherens complex would occur during induced compaction and that these components would be required for the cell adhesive event; (2) these same proteins should be phosphorylated during compaction in normal development; (3) new, highly‐specific inhibitors of PKC activity would inhibit compaction during normal development and induced compaction; and (4) some PKC isotypes would become localized to the junctional membranes during the process of compaction. In agreement with these predictionst, β‐catenin became phosphorylated on serine/threonine residues both during induced compaction and normal development. Inhibitors to PKC, but not inhibitors to other kinases, blocked compaction. Furthermore, the alpha isotype of PKC is recruited to the membranes of the apposing blastomeres both during induced compaction and during normal development immediately before compaction begins and before β‐catenin becomes part of the detergent‐resistant cytoskeleton at the junction. Mol. Reprod. Dev. 54:135–144, 1999.

Degradation, cytotoxicity, and biocompatibility of NIPAAm-based thermosensitive, injectable, and bioresorbable polymer hydrogels

A thermosensitive, injectable, and bioresorbable polymer hydrogel, poly(N-isopropylacrylamide-co-dimethyl-γ-butyrolactone acrylate-co-acrylic acid) [poly(NDBA)], was synthesized by radical copolymerization with 7.00 mol % dimethyl-γ-butyrolactone acrylate in tetrahydrofuran. The chemical composition was determined by acid titration in conjunction with (1) H NMR quantification. The molecular weight and polydispersity were determined by gel permeation chromatography in conjunction with static light scattering. The degradation properties of the polymer hydrogel were characterized using differential scanning calorimetry, percentage mass loss, cloud point test, and swelling ratio over time. It was found that the initial lower critical solution temperature (LCST) of the polymer is between room temperature and body temperature and that it takes about 2 weeks for the LCST to surpass body temperature under physiological conditions. An indirect cytotoxicity test indicated that this copolymer has relatively low cytotoxicity as seen with 3T3 fibroblast cells. The in vivo-gelation and degradation study showed good agreement with in vitro-degradation findings, and no detrimental effects to adjacent tissues were observed after the complete dissolution of the polymer.

Liposomal Formulation Increases Local Delivery of Amphotericin from Bone Cement: A Pilot Study

BackgroundAmphotericin is a highly toxic hydrophobic antifungal. Delivery of amphotericin from antifungal-loaded bone cement (ALBC) is much lower than would be expected for an equivalent load of water-soluble antibacterials. Lipid formulations have been developed to decrease amphotericin toxicity. It is unknown how lipid formulations affect amphotericin release and compressive strength of amphotericin ALBC.Questions/purposesWe asked if amphotericin release from liposomal amphotericin ALBC (1) changed with amphotericin load; (2) differed from release from amphotericin deoxycholate ALBC; (3) was an active drug; and (4) if liposomal amphotericin affected the bone cement strength.MethodsForty-five standardized test cylinders were fabricated from three formulations of ALBC: Simplex™ P bone cement with 200 mg liposomal amphotericin, 800 mg liposomal amphotericin, or 800 mg amphotericin deoxycholate per batch. For each ALBC formulation, cumulative released amphotericin was determined from five cylinders, and compressive strength was measured for 10 cylinders, five before elution and five after. Activity of released amphotericin was determined by growth inhibition assay.ResultsAmphotericin release was greater for increased load of liposomal amphotericin: 770 μg for 800 mg versus 118 μg for 200 mg. Amphotericin release was greater from liposomal ALBC than from deoxycholate ALBC: 770 μg versus 23 μg over 7 days for 800 mg amphotericin. Released amphotericin was active. Compressive strength of liposomal ALBC is decreased, 67 MPa and 34 MPa by Day 7 in elution for the 200-mg and 800-mg formulations, respectively.ConclusionsLiposomal amphotericin has greater amphotericin release from ALBC than amphotericin deoxycholate. Compressive strength of liposomal amphotericin ALBC decreases to less than recommended for implant fixation. Local toxicity data are needed before liposomal amphotericin ALBC can be used clinically.

Manipulating Degradation Time in a N-isopropylacrylamide-Based Co-polymer with Hydrolysis-Dependent LCST

A thermosensitive, bioresorbable and in situ gelling co-polymer, poly(N-isopropylacrylamide-co-dimethyl-γ-butyrolactone acrylate-co-acrylic acid), was synthesized by radical co-polymerization with varying dimethyl-γ-butyrolactone acrylate (DBA) content. The materials properties were characterized using differential scanning calorimetry, gel-permeation chromatography in conjunction with static light scattering, Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR) and acid titration. The initial lower critical solution temperature (LCST) of the synthesized co-polymer is between room temperature and body temperature. With the increase of DBA content, the LCST decreases, but then increases after the ring-opening hydrolysis of the DBA side-group. The FT-IR and NMR spectra show the co-polymerization of three monomers, as well as the hydrolysis-dependent ring-opening of the DBA side-group. The addition of acrylic acid increases the initial LCST and accelerates the degradation rate of the co-polymer. An indirect cytotoxicity test indicated that this co-polymer has relatively low cytotoxicity as seen with 3T3 fibroblast cells.

Temporal differences in Erk1/2 activity distinguish among combinations of extracellular matrix components.

Rational design of biomaterials requires understanding how cells interrogate their microenvironment. In this study, human umbilical vein endothelial cells are cultured on combinations of extracellular matrix (ECM) components (collagen I, collagen IV, vitronectin, fibronectin, laminin, heparan sulfate proteoglycan, chondroitin sulfate proteoglycan), and the phosphorylation of four intracellular signaling kinases (Erk1/2, JNK, Akt1, and NFκB) is quantified. These combinations of ECM components elicit different temporal patterns of Erk1/2 phosphorylation. Collagen I-containing substrates cause Erk1/2 phosphorylation to reach maximal levels at 30 min and remain near maximal levels until 90 min. Collagen IV/laminin substrates elicit maximal phosphorylation at 30-45 min, and then phosphorylation decreases substantially at 60-90 min. All other combinations studied (collagen IV and vitronectin-based combinations) cause an increase in phosphorylation at 30-45 min, but not to maximal levels; maximal phosphorylation is reached by 60-90 min. These temporal patterns of phosphorylation may explain how a limited number of intracellular signaling pathways can distinguish among thousands of possible combinations of microenvironmental cues by adding to the information contained in each cell signaling pathway.