Search

A common problem in the design of tissue engineered scaffolds using electrospun scaffolds is the poor cellular infiltration into the structure. To tackle this issue, three approaches to scaffold design using electrospinning were investigated: selective leaching of a water-soluble fiber phase (poly ethylene oxide (PEO) or gelatin), the use of micron-sized fibers as the scaffold, and a combination of micron-sized fibers with codeposition of a hyaluronic acid-derivative hydrogel, Heprasil. These designs were achieved by modifying a conventional electrospinning system with two charged capillaries and a rotating mandrel collector. Three types of scaffolds were fabricated: medical grade poly(ε-caprolactone)/collagen (mPCL/Col) cospun with PEO or gelatin, mPCL/Col meshes with micron-sized fibers, and mPCL/Col microfibers cosprayed with Heprasil. All three scaffold types supported attachment and proliferation of human fetal osteoblasts. However, selective leaching only marginally improved cellular infiltration when compared to meshes obtained by conventional electrospinning. Better cell penetration was seen in mPCL/Col microfibers, and this effect was more pronounced when Heprasil regions were present in the structure. Thus, such techniques could be further exploited for the design of cell permeable fibrous meshes for tissue engineering applications. [ABSTRACT FROM AUTHOR]

Hyaluronan (HA) derivatives containing thiol-reactive electrophilic esters were prepared to react with thiol-modified macromolecules to give cross-linker-free hydrogels. Specifically, HA was converted to two haloacetate derivatives, HA bromoacetate (HABA) and HA iodoacetate (HAIA). In cytotoxicity assays, these reactive macromolecules predictably induced cell death in a dose-dependent manner. Cross-linker-free synthetic extracellular matrix (sECM) hydrogels were prepared by thiol alkylation using HAIA and HABA as polyvalent electrophiles and thiol-modified HA (CMHA-S) with or without thiol-modified gelatin (Gtn-DTPH) as polyvalent nucleophiles. When primary human fibroblasts were seeded on the surface of the sECMs containing only the electrophilic HA haloacetate and nucleophilic CMHA-S components, no significant cytoadherence was observed. Cell attachment and viability was 17% (HABA) to 30% (HAIA) lower on HA haloacetate cross-linked hydrogels than on CMHA-S that had been oxidatively cross-linked via disulfide-bonds. In contrast, sECMs that included Gtn-DTPH allowed fibroblasts to attach, spread, and proliferate. Taken together, the HA haloacetates are attractive candidates for producing cross-linker-free sECM biomaterials that can function either as anti-adhesive barriers or as cytoadhesive sECMs for cell culture in pseudo-3-D. [ABSTRACT FROM AUTHOR]

Covering: 2006 up to June 2010, but also including representative examples of prior work [ABSTRACT FROM AUTHOR]

The phosphorylated inositol diphosphates, including the diphosphoinositol pentakisphosphate regioisomers, play critical roles in signal transduction and cellular regulation. In particular, the IP7isomer 5-PP-Ins(1,2,3,4,6)P5is implicated in a nonenzymatic phosphate transfer converting a protein serine phosphate residue to a serine diphosphate. A scalable, practical new synthesis of 5-PP-Ins(1,2,3,4,6)P5is described that also allows access to a variety of IP7and IP8regioisomers. The identity of the synthetic 5-PP-Ins(1,2,3,4,6)P5was validated using IP6K1 to catalyze the conversion of IP7+ ADP to ATP + IP6. [ABSTRACT FROM AUTHOR]

Synthetic extracellular matrix hydrogels can be used for three-dimensional cell culture, wound repair, and tissue engineering. Using the bifunctional electrophile poly(ethylene glycol) diacrylate (PEGDA), thiol-modified glycosaminoglycans and polypeptides can be cross-linked into biocompatible materials in the presence of cells or tissues. However, the rate of in situ cross-linking with PEGDA under physiological conditions may occur too slowly for clinical applications requiring a fast-curing preparation. To explore a wider range of cross-linking time courses, five homo-bifunctional PEG derivatives were synthesized and examined as cross-linking agents for thiol-modified derivatives of hyaluronan (HA). Thiol reaction rate constants were measured over a pH range of 7.4 to 8.6. The order of reactivity for the functional groups used was determined to be maleimide > iodoacetate > bromoacetate > iodoacetamide > acrylate bromoacetamide, with rates increasing exponentially with increasing pH. The range of gelation times at physiological pH varied from less than 1 min to over 2 h. Addition of the cross-linkers to cell culture medium showed minimal cytotoxicity toward primary human dermal fibroblasts at concentrations anticipated during in situ cross-linking. Moreover, hydrogels prepared from thiol-modified gelatin and thiol-modified HA were biocompatible and supported attachment and proliferation of fibroblasts and hepatocytes. [ABSTRACT FROM AUTHOR]

Current cancer xenograft models used to evaluate new anticancer therapies are limited to “good take” cell lines, fail to mimic normal human disease, and poorly predict clinical outcomes. We now describe the use of an injectable, in situcross-linkable synthetic extracellular matrix (sECM) to deliver and grow cancer cells in vivo. The hyaluronan (HA)-derived sECMs were seeded with breast, colon, and ovarian cancer cells prior to gelation, and then injected subcutaneously into mammary fat pads, subserosally in colons, and intracapsularly in ovaries, respectively. Two cell lines were used for each type of cancer, and results were compared with orthotopic injection of cells in serum-free medium. At 4 weeks postinjection, four parameters were measured: (i) incidence and size of cancer at the injection site, (ii) vascularization or necrosis of new cancer tissue, (iii) cancer seeding in adjacent tissues, and (iv) metastasis to lymph nodes and other vital organs. In addition, the activation of the phosphoinositide 3-kinase (PI 3-K) signaling pathway was analyzed immunohistochemically. Overall, orthotopic delivery of cancer cells in sECM hydrogels showed clear advantages: (i) increased incidence of cancer formation and reduced variability in tumor size, (ii) enhanced growth of organ-specific cancers with good tumor-tissue integration, (iii) improved vascularization and reduced necrosis within the tumor, (iv) reduced cancer seeding on adjacent tissues, and (v) better general health of animals. Thus, engineered tumors represent an improved approach to traditional tumor xenografts, and facilitate studies in cancer biology, invasion and metastasis, as well as the investigation of new therapeutic and diagnostic protocols. [ABSTRACT FROM AUTHOR]

An efficient enantioselective synthesis of sn-2-aminooxy (AO) analogues of lysophosphatidic acid (LPA) that possess palmitoyl and oleoyl acyl chains is presented. Both sn-2-AO LPA analogues are agonists for the LPA1, LPA2, and LPA4G-protein-coupled receptors, but antagonists for the LPA3receptor and inhibitors of autotaxin (ATX). Moreover, both analogues stimulate migration of intestinal epithelial cells in a scratch wound assay. [ABSTRACT FROM AUTHOR]

Insulin stimulates glucose uptake into muscle and fat cells by translocating glucose transporter 4 (GLUT4) to the cell surface, with input from phosphatidylinositol (PI) 3-kinase and its downstream effector Akt/protein kinase B. Whether PI 3,4,5-trisphosphate (PI(3,4,5)P3) suffices to produce GLUT4 translocation is unknown. We used two strategies to deliver PI(3,4,5)P3 intracellularly and two insulin-sensitive cell lines to examine Akt activation and GLUT4 translocation. In 3T3-L1 adipocytes, the acetoxymethyl ester of PI(3,4,5)P3 caused GLUT4 migration to the cell periphery and increased the amount of plasma membrane-associated phosphoAkt and GLUT4. Intracellular delivery of PI(3,4,5)P3 using polyamine carriers also induced translocation of myc-tagged GLUT4 to the surface of intact L6 myoblasts, demonstrating membrane insertion of the transporter. GLUT4 translocation caused by carrier-delivered PI(3,4,5)P3 was not reproduced by carrier-PI 4,5bisphosphate or carrier alone. Like insulin, carrier-mediated delivery of PI(3,4,5)P3 elicited redistribution of perinuclear GLUT4 and Akt phosphorylation at the cell periphery. In contrast to its effect on GLUT4 mobilization, delivered PI(3,4,5)P3 did not increase 2-deoxyglucose uptake in either L6GLUT4myc myoblasts or 3T3-L1 adipocytes. The ability of exogenously delivered PI(3,4,5)P3 to augment plasma membrane GLUT4 content without increasing glucose uptake suggests that input at the level of PI 3-kinase suffices for GLUT4 translocation but is insufficient to stimulate glucose transport. [ABSTRACT FROM AUTHOR]