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Biomedical subjects

Gerard Marx

Publications and source records attributed to Gerard Marx.

10 recordsLinked to original sources
PubMed2006

Simulating fibrin clotting time.

The clotting time (CT) of fibrinogen mixed with thrombin decreased, then increased with increasing fibrinogen levels. By contrast, log CT decreased monotonically with respect to the log level of activating enzyme (thrombin or reptilase). Here, the CT was determined over a large range of fibrinogen concentration (to 100 mg ml(-1)) at a fixed level of enzyme. A new parameter, [Fib]min, the minimal fibrinogen concentration required for thrombin or reptilase-instigated phase change (coagulation), was determined as [Fib]min = 0.2 +/- 0.05 microM fibrinogen. A dynamic simulation program (Stella) was employed to organize simulations based on simple and complex coagulation mechanisms, which generated CT values. The successful simulation aimed at forming [Fib]min and "recognized" the binding of unreacted fibrinogen with intermediate fibrin protofibrils. The "virtual data" mimicked the biphasic experimental CT values over a wide range of concentrations. Fibrinogen appeared to act in three modalities: as a thrombin substrate; as a precursor of fibrin; and as a competitor for fibrin protofibrils. The optimized simulation may provide a basis for predicting CT in more complex systems, such as pathological plasmas or whole blood or at high concentrations encountered with fibrin sealant.

Batroxobin↗
PubMed2006

High-yield isolation, expansion, and differentiation of rat bone marrow-derived mesenchymal stem cells with fibrin microbeads.

Fibrin microbeads (FMB), made of extensively cross-linked dense and partially denatured fibrin, were used as a matrix for efficient isolation of mesenchymal stem cells (MSC) from rat bone marrow (BM). After 2 days of incubation of FMB with whole BM in suspension, a high number of cells of mesenchymal origin attached to the FMB. On the 14th day after their transfer to plastic, the yield of the cells isolated via FMB was approximately 3-4 times higher than that obtained by currently used protocols based solely on plastic adhesion. This implies that the number of MSC in BM may be higher than previously reported. FACS analyses and immunostaining showed the mesenchymal characteristics of these cells by positive staining for fibronectin, vimentin, CD49E, and CD29. Immediately after isolation, less than 20% of the cells still expressed the hematopoietic markers CD11b and CD45. Most of these cells were eventually eliminated after further expansion of the isolated cells on plastic. Cells isolated via FMB were expanded in culture for more than 4 months and could be defined as MSC along this time period based on their ability to differentiate into precursors of mesenchymal tissues, such as osteogenic, adipogenic, and chondrogenic cells. Similar differentiation plasticity was observed in clones derived from single cells from whole MSC populations isolated via FMB. Based on our results we propose that FMB can serve as a 3-dimensional biodegradable matrix for isolation, differentiation, and possibly implantation of MSC for tissue regeneration.

Animals↗
PubMed2005

Interactions of carboplatin with fibrin(ogen), implications for local slow release chemotherapy.

The effect of carboplatin (CPt) on fibrin(ogen) clot formation and the possible use of this combination for local slow release chemotherapy were examined. CPt significantly reduced thrombin-induced fibrin clotting time (CT) and increased clot turbidity in a concentration-dependent manner. When CPt was mixed with physiological levels of fibrinogen (>1 mg/ml), electron-dense nanoparticles (3 nm) were formed, as demonstrated by both optical particle counter and transmission electron microscopy (TEM). Upon thrombin-induced coagulation, the CPt nanoparticles were trapped within the fibrin mesh. At higher fibrinogen levels (>5 mg/ml), the 3-nm CPt nanoparticles aggregated, so that approximately 2% and approximately 0.5% of the CPt on the fibrinogen appeared as larger particles of 10 and 50 nm, respectively. Dialysis experiments showed that 60-70% of the CPt was released from the fibrin clot within one hour as a non-particulate soluble form, while approximately 30% of particulate CPt were retained. Up to 5 mg/ml this portion of firmly attached CPt was dependent of the initial drug level. CPt released from the fibrin by either diffusion or by fibrinolysis exhibited cytotoxic activity towards retinoblastoma (RB) cell lines (Y-79 and Weri RB1) equivalent to free drug. Our study indicates that CPt enhances fibrin clot formation and suggests the use of fibrin with high dose CPt for slow release chemotherapy against localized tumors such as retinoblastoma.

Antineoplastic Agents↗
PubMed2004

Liposome transduction into cells enhanced by haptotactic peptides (Haptides) homologous to fibrinogen C-termini.

Haptides are 19-21mer cell-binding peptides equivalent to sequences on the C-termini of fibrinogen beta chain (Cbeta), gamma chain (preCgamma) and the extended alphaE chain of fibrinogen (CalphaE). In solution, Haptides accumulated in cells by non-saturable kinetics [Exp. Cell Res. 287 (2003) 116]. This study describes Haptide interactions with liposomes and Haptide-mediated liposome uptake by cells. Haptides became incorporated into negatively charged liposomes, changing their zeta potential. Atomic force microscopy and particle sizing by light scattering showed that the liposomes dissolved Haptide nanoparticles and absorbed them from solution. Pre-mixing fluorescent rhodamine-containing liposomes or "stealth" doxorubicin (DOX)-containing liposomes (Doxil) with Cbeta, preCgamma or to a lesser degree CalphaE, significantly enhanced their uptake by fibroblasts and endothelial cells. Confocal microscopy showed Haptide-induced liposome uptake saturated above approximately 40 microM Haptide. Cytotoxicity tests with lower concentrations of Doxil liposomes indicated that premixing with approximately 40 microM Cbeta or preCgamma increased their toxicity by one order of magnitude. It was evident that the liposomes complexed with an amphiphilic Haptide are transduced through cell membranes, probably by a non-receptor-mediated process. These results suggest that Cbeta or pre-Cgamma could be employed to augment the cellular uptake of drugs in liposomal formulations.

Animals↗
PubMed2004

Fibrinogen C-terminal peptidic sequences (Haptides) modulate fibrin polymerization.

We previously described synthetic peptides of 19-21 amino acid residues, homologous to the C-termini of fibrinogen Fib(340) and Fib(420), from the beta-chain (Cbeta), the extended alphaE chain (CalphaE) and near the end of the gamma-chain (preCgamma) which elicited attachment (haptotactic) responses from mesenchymal cells. We named these haptotactic peptides -Haptides. The effects of Haptides on fibrin clot formation was evaluated and their possible effects on platelet aggregation was examined. The Haptides Cbeta,CalphaE and preCgamma, (2-10 micro M) increased fibrin clot turbidity and also decreased thrombin-induced clotting time. Higher concentrations (>120 micro M of Cbeta or preCgamma) induced fibrinogen precipitation even without thrombin. These precipi-tates exhibited different ultrastructure from thrombin-induced fibrin by scanning and transmission microscopy. C-terminal peptides of the other fibrinogen chains exerted no such effects. Sepharose beads covalently coated with either whole fibrinogen or Haptides (SB-Fib or SB-Haptide) highly adsorbed free (FITC) Haptides. In aqueous solution, Haptides formed nano-par-ticles with average size of approximately 150 nm in diameter. We suggest that such positively charged aggregates could serve to nucleate and accelerate fibrin gel formation. These results also indicate that Cbeta and preCgamma sequences within fibrin(ogen) participate in the docking and condensation of fibrin(ogen) during its assembly into a fibrin clot. By contrast, Haptides up to 100 micro M did not bind to platelets, and had no effect on platelet aggregation. Our findings highlight the roles of the C-terminal sequences of the beta and gamma chains in fibrin(ogen) polymerization as well as in cell attachment.

Blood Coagulation↗
PubMed2003

New cell attachment peptide sequences from conserved epitopes in the carboxy termini of fibrinogen.

Fibrinogen seems to contribute significantly to cell binding and recruitment into wounds besides its major role in clot formation. We describe 19- to 21-mer cell-binding (haptotactic) peptides from the C-termini of fibrinogen beta-chain (Cbeta), the extended alphaE chain, and near the C-terminal of the gamma-chain. When these peptides were covalently bound to a biologically inert matrix such as Sepharose beads (SB), they elicited beads attachment to cells, mostly of mesenchymal origin (including fibroblasts, endothelial cells, and smooth muscle cells) as well as some transformed cell lines. Based on such haptotactic activity, these peptides were termed "haptides." By contrast, peptides homologous to fibrinogen C-termini alpha- and gamma-chains elicited no such activity. The haptide Cbeta could not block the interaction of fibroblasts with antibodies directed against integrins beta(1), alpha(v), alpha(v)beta(1), alpha(v)beta(3), and alphaIIbeta(3). Moreover, GRGDS peptide could not inhibit enhanced cell binding to SB-Cbeta, as expected from an integrin-mediated process. In soluble form the haptides were accumulated in cells with nonsaturable kinetics without any toxic or proproliferative effects in concentrations up to 80 microM. These findings suggest that the conserved haptidic sequences within fibrin(ogen) can be associated with the adhesion and migration of cells into fibrin clots and may have a significant role in normal wound healing and in various pathological conditions.

Animals↗
PubMed2003

Evolution of fibrin glue applicators.

Fibrin glue (FG) is used worldwide as a potent surgical tool, which establishes hemostasis in wounds and also bonds tissue. The standard FG applicator is based on a dual-syringe system. This review, based mainly on the patent literature, describes the development of the quasi-standard dual syringe system as well as the rise of other FG applicator designs based on mechanical force (ratchet systems), Bernoulli gas flow, positive gas pressure, or electro-servo devices. The packaging of commercial FG components is reviewed within the context of "loading" the FG applicators and the need to minimize the number of needles required to access the packaged (vials) components. Parameters such as internal clogging, homogeneity of spray, the requirement for gas or vacuum house lines, the number of parts that must be handled, and the time required to assemble the applicator, load it, and have it ready for use are also discussed. A rating system is proposed that permits one to use such parameters to rank the various applicator designs, relative to the dual-syringe system. Hopefully, this review will stimulate the design of better FG applicators and packaging required for elective surgery, emergency treatments, and tissue engineering in the 21st century.

Equipment Design↗
PubMed2002

Characterizing fibrin glue performance as modulated by heparin, aprotinin, and factor XIII.

We describe the performance of fibrin glue (FG) as modulated by heparin, aprotinin, or factor XIII levels. In vitro tests and a rat kidney excision model demonstrated that the hemostatic efficacy of fibrin was not modulated by aprotinin. Overlapping rat skin sections demonstrated that adhesion strength (AS) was proportional to the area of overlap as well as to fibrinogen levels. AS was not modulated by exogenous heparin or aprotinin and was independent of the endogenous factor XIII in fibrinogen. SDS-PAGE developed by Coomassie or Western blots with anti-gamma chain antibody confirmed that normal skin sections contain adequate trans-glutaminase to maximally cross-link normal, as well as XIII-depleted, fibrin. Fibrin glue (FG) sprayed onto rat skin incision wounds with a dual channel spray applicator acted in 2 phases: initially (day 1), compared to wounds stapled without or treated with only thrombin, FG significantly increased breaking strength. In the second phase of wound healing (after day 3), all groups achieved increased but equivalent breaking strength. FG containing aprotinin (to 3000 U/m; Immuno, Behringwerke, Germany) exhibited initial tissue bonding strength equivalent to fibrin without aprotinin, but histological examination showed delayed fibrinolysis and a concomitant slower regeneration of granulation tissue. Thus, our data indicated that aprotinin was not particularly beneficial to wound healing and that the endogenous factor XIII level in the fibrinogen did not contribute significantly to skin bonding. Rather, the tissue supplied adequate trans-glutaminase activity required to crosslink fibrin to itself and to the tissue.

Animals↗
PubMed2002

Fibrin microbeads for isolating and growing bone marrow-derived progenitor cells capable of forming bone tissue.

It has been demonstrated that bone marrow (BM)-derived pluripotent stem cells can be incorporated into muscle, bone, nerve, lung, stomach, intestine, and skin. Fibrin-based biodegradable microbeads (FMB) were developed for culturing, in suspension, a high density of cells, mostly of mesenchymal origin. In the current study, FMB were used to isolate and expand mesenchymal progenitor cells from BM of mice and rats. Cells from BM isolated on FMB (FMB-BM cells) were visualized by fluorescent confocal microscopy and quantified by a modified MTS colorimetric assay. Downloading the BM cells from FMB onto plastic induced their differentiation into islets of cells with osteogenic phenotype that secreted mineralized extracellular matrix. This was augmented by inducers of osteogenesis, such as ascorbic acid, beta-glycerophosphate, and dexamethasone, or osteoblast-growth peptides (OGP). Implanting FMB-BM cells under the kidney capsule in mouse tested the osteogenic potential of these cells in vivo. Thirty days after implantation, bone structures with typical BM elements were seen in 8/53 kidneys in 6-Gy-irradiated mice and in 1/10 kidneys in nonirradiated recipients; bone formation was verified by soft x-ray imaging and elemental analysis that showed elevated Ca and Fe in the implant region. FMB-BM cells - downloaded onto plastic flasks, cultured for 2 weeks, mechanically harvested and then implanted - induced 100% bone formation in both irradiated (6/6) and nonirradiated (3/3) mice. Histology revealed well-organized bone structures under the kidney capsule, including osteoblasts and typical elements of BM. Our findings demonstrate that FMB are capable of isolating and expanding progenitor cells from BM for osteogenesis and possibly for regenerating other mesenchymal tissues.

Animals↗