Processing methods for human amniotic membrane as scaffold for tissue engineering with mesenchymal stromal human cells.

Tissue engineering is an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function. The aims of this work were to compare chemically and physically processed human Amniotic Membranes (hAM) and analyze the cytocompatibility and proliferation rate (PR) of two primary human mesenchymal stromal cell lines, from different sources and donor conditions seeded over these scaffolds. The evaluated hAM processes were: cold shock to obtain a frozen amniotic membrane (FEAM) with remaining dead epithelial cells, denudation of hAM with trypsin for 20/10 min (DEAM20/10) or treatment with sodium dodecyl sulfate to decellularized hAM (DAM). All samples were sterilized with gamma radiation. The selection of the treated hAM to then generate composites was performed by scanning and transmission electron microscopy and characterization by X-ray diffraction, selecting DEAM10 and FEAM as scaffolds for cell seeding. Two sources of primary human stromal cells were used, both developed by our researchers, human Dental Pulp Stem Cells (hDPSC) from living donors and human Mesenchymal Stromal Cells (hMSC) from bone marrow isolated from brain dead donors. This last line of cells conveys a novel source of human cells that, to our knowledge, have not been tested as part of this type of construct. We developed four in vitro constructs without cytotoxicity signs and with different PR depending on the scaffolds and cells. hDPSC and hMSC grew over both FEAM and DEAM10, but DEAM10 allowed higher PR.

Synthesis and characterization of a bovine collagen: GAG scaffold with Uruguayan raw material for tissue engineering.

Tissue engineering (TE) and regenerative medicine offer strategies to improve damaged tissues by using scaffolds and cells. The use of collagen-based biomaterials in the field of TE has been intensively growing over the past decades. Mesenchymal stromal cells (MSCs) and dental pulp stem cells (DPSCs) are promising cell candidates for development of clinical composites. In this study, we proposed the development of a bovine collagen type I: chondroitin-6-sulphate (CG) scaffold, obtained from Uruguayan raw material (certified as free bovine spongiform encephalopathy), with CG crosslinking enhancement using different gamma radiation doses. Structural, biomechanical and chemical characteristics of the scaffolds were assessed by Scanning Electron Microscopy, axial tensile tests, FT-IR and Raman Spectroscopy, respectively. Once we selected the most appropriate scaffold for future use as a TE product, we studied the behavior of MSCs and DPSCs cultured on the scaffold by cytotoxicity, proliferation and differentiation assays. Among the diverse porous scaffolds obtained, the one with the most adequate properties was the one exposed to 15 kGy of gamma radiation. This radiation dose contributed to the crosslinking of molecules, to the formation of new bonds and/or to the reorganization of the collagen fibers. The selected scaffold was non-cytotoxic for the tested cells and a suitable substrate for cell proliferation. Furthermore, the scaffold allowed MSCs differentiation to osteogenic, chondrogenic, and adipogenic lineages. Thus, this work shows a promising approach to the synthesis of a collagen-scaffold suitable for TE.

Ablation of frequent premature ventricular complex in an athlete.

Premature ventricular complex are common findings in the exam of many athletes. There is no extensive scientific evidence in the management of this situation particularly when associated with borderline contractile function of the left ventricle. In this case report, we present a 35-year-old asymptomatic healthy athlete with high incidence (over 10,000 beats in 24 h) of premature ventricular complex and left ventricular dilatation with dysfunction, which persisted after a resting period of 6 months without training. We performed radiofrequency ablation of the premature ventricular complex focus. After 1-year follow-up, he was asymptomatic without arrhythmia and the left ventricle normalized its size and function as shown by echocardiogram and cardiac magnetic resonance.

Biodritin microencapsulated human islets of Langerhans and their potential for type 1 diabetes mellitus therapy.

BACKGROUND: Microencapsulation of pancreatic islets with polymeric compounds constitutes an attractive alternative therapy for type 1 diabetes mellitus. The major limiting factor is the availability of a biocompatible and mechanically stable polymer. We investigated the potential of Biodritin, a novel polymer constituted of alginate and chondroitin sulfate, for islet microencapsulation. METHODS: Biodritin microcapsules were obtained using an air jet droplet generator and gelated with barium or calcium chloride. Microencapsulated rat insulinoma RINm5F cells were tested for viability using the [3-(4,5-dimetyl-thiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide] [MTT] colorimetric assay. Microencapsulated rat pancreatic islets were coincubated with macrophages derived from mouse peritoneal liquid to assess the immunomodulatory potential of the microcapsules, using quantitative real time-PCR (qPCR). Biodritin biocompatibility was demonstrated by subcutaneous injection of empty microcapsules into immunocompetent Wistar rats. Insulin secretion by microencapsulated human pancreatic islets was evaluated using an electrochemoluminescent assay. Microencapsulated human islets transplanted into chemically induced diabetic mice were monitored for reversal of hyperglycemia. RESULTS: The metabolic activity of microencapsulated RINm5F cells persisted for at least 15 days. Interleukin-1beta expression by macrophages was observed during coculture with islets microencapsulated with Biodritin-CaCl2, but not with Biodritin-BaCl2. No statistical difference in glucose-stimulated insulin secretion was observed between nonencapsulated and microencapsulated islets. Upon microencapsulated islet transplantation, the blood glucose level of diabetic mice normalized; they remained euglycemic for at least 60 days, displaying normal oral glucose tolerance tests. CONCLUSION: This study demonstrated that Biodritin can be used for islet microencapsulation and reversal of diabetes; however, further investigations are required to assess its potential for long-term transplantation.

Heparin-like compounds prepared by chemical modification of capsular polysaccharide from E. coli K5.

O-Sulfation of sulfaminoheparosan SAH, a glycosaminoglucuronan with the structure-->4)-beta-D-GlcA(1-->4)-beta-D-GlcNSO3(-)-(1-->, obtained by N-deacetylation and N-sulfation of the capsular polysaccharide from E. coli K5, was investigated in order to characterize the sulfation pattern eliciting heparin-like activities. SAH was reacted (as the tributylammonium salt in N,N-dimethylformamide) with pyridine-sulfur trioxide under systematically different experimental conditions. The structure of O-sulfated products (SAHS), as determined by mono- and two-dimensional 1H and 13C NMR, varied with variation of reaction parameters. Sulfation of SAH preferentially occurred at O-6 of the GlcNSO3- residues. Further sulfation occurred either at O-3 or at O-2 of the GlcA residues, depending on the experimental conditions. Products with significantly high affinity for antithrombin and antifactor Xa activity were obtained under well-defined conditions. These products contained the trisulfated aminosugar GlcNSO3-3,6SO3-, which is a marker component of the pentasaccharide sequence through which heparin binds to antithrombin.

Different effects of mucosal, bovine lung and chemically modified heparin on selected biological properties of basic fibroblast growth factor.

Heparins from bovine mucosa and lung, and chemically modified heparins were assayed for their capacity to: (i) protect human recombinant basic fibroblast growth factor (bFGF) from tryptic cleavage; (ii) prevent 125I-bFGF binding to heparan sulphate proteoglycans present in the extracellular matrix and on the cell surface of fetal bovine aortic endothelial GM 7373 cell cultures; (iii) affect 125I-bFGF binding to high-affinity tyrosine kinase FGF receptors present on the cell membrane of GM 7373 cells; (iv) inhibit the mitogenic activity exerted by bFGF in the same cells. The results demonstrate that the potency shown by mucosal heparins in the different assays is a direct function of size, very-low-molecular-mass heparin (2.0 kDa) being significantly less effective on a molar basis than unfractionated heparin (13.6 kDa). Increased flexibility of the backbone structure, as observed in reduced/oxidized heparins of different size, does not affect the capacity of the polysaccharide to interact with bFGF. In contrast, selective 2-O-desulphation, but not 6-O-desulphation, drastically reduced the capacity of heparin to protect bFGF from proteolytic cleavage, to affect its interaction with low- and high-affinity sites, and to inhibit its mitogenic activity. Two preparations of bovine lung heparin, differing in molecular mass, were as effective as mucosal heparin in the bFGF-tryptic-digestion assay and the endothelial-cell proteoglycan-binding assay, but they were highly inefficient at inhibiting the capacity of bFGF to interact with its tyrosine kinase receptors. Bovine lung heparins were also less effective than mucosal heparin as bFGF antagonists in GM 7373-cell-proliferation assays. N-Desulphated/N-acetylated bovine lung heparin retained only a significant capacity to protect bFGF from tryptic cleavage. The results demonstrate that different chemical features of the heparin molecule, including decrease in molecular mass, selective desulphation, disaccharide composition and clustering, affect differently the capacity of the glycosaminoglycan to interact with bFGF and to influence its biological behaviour in different assays in vitro and in endothelial cell cultures. Our findings should aid the design of synthetic oligosaccharides aimed at improving the bioavailability of bFGF when administered in vivo as a therapeutic agent.

An examination of heart proteins by two-dimensional electrophoresis.

We examined specimens from explanted human hearts by two-dimensional electrophoresis. The protocol selected includes: (a) solubilization of the sample in a urea-detergent mix; (b) charge fractionation in the presence of urea and nonionic detergent on a pH 4-10 immobilized pH gradient; (c) size fractionation on a polyacrylamide concentration gradient in the presence of sodium dodecyl sulfate; and (d) staining with silver nitrate. The method is sensitive enough for analysis of biopsies in the 1-3 mg range (wet tissue). We saw, for explanted hearts, variations in the protein pattern with the site of sample dissection. Results are presented for 11 explanted human hearts: one control organ and 10 pathological samples. The recorded pathologies included dilatative cardiomyopathy (six cases), valvulopathy (one case), ischemic cardiopathy (two cases), and graft rejection (one case). The patterns for whole extracts as well as for cytoplasmic proteins and myofibril components are compared. Extensive individual variability was observed both between control and pathological cases and among the abnormal samples.

Additives for immobilized pH gradient two-dimensional separation of particulate material: comparison between commercial and new synthetic detergents.

We describe the synthesis of two detergents, L and A15, whose performances as solubilizing agents and as additives in the first-dimension step of a two-dimensional separation are compared with those of some commercial compounds, i.e., Nonidet P-40, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate(Chaps), and sulfobetaine, on three membrane protein preparations: rat RBC ghosts, beef kidney microvilli, and spinach thylakoids. L is 3-]3-dodecylamidoprophylcbdimethylammonio propane-1-sulfonate; owing to the substitution of a dodecylamido for the dodecyl residue of SB 3-12, the concentration of urea compatible with 2% detergent increases from 4.5 M for the parent molecule up to 7 M. With all three biological samples on which the panel of different detergents has been tested in parallel, L + urea scores as the most effective solubilization medium. On red blood cells a notable qualitative difference is observed with the selective extraction by L as well as by N-dodecyl-N,N-dimethylammonio-3-propanesulfonate of a major protein (pI = ca. 5.5, Mr = ca. 100,000). A15 is derived from a tertiary amine, with one alkylic substituent (either C11 or C13) and two poly(ethylene oxide) tails (totaling 15 ethoxy residues), which is reacted with propane sultone. Approximately 30% of the product corresponds to the N-adduct and is a truly zwitterionic detergent, while 60% is an O-derivative and still contains a titratable amino group (with a pK of 7.2). A15 can thus be used for isoelectric focusing on immobilized pH gradients, as in this work, but would not be compatible with carrier ampholyte isoelectric focusing.(ABSTRACT TRUNCATED AT 250 WORDS)