Gerard Marx

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.

VIRUCIDAL TREATMENT OF BLOOD PROTEIN PRODUCTS WITH UVC RADIATION

The virus safety of blood derivatives continues to be of concern, especially with respect to nonenveloped and/or heat-stable viruses. Previously, we demonstrated that treatment of whole plasma, AHF concentrate or fibrinogen with short wavelength ultraviolet light (UVC) results in the inactivation of > or = 10(6) infectious doses (ID) of encephalomyocarditis virus (EMCV), hepatitis A virus (HAV) and porcine parvovirus (PPV), each of which is nonenveloped. Protein recovery was enhanced greatly by inclusion of the flavonoid, rutin, added prior to UVC exposure to quench reactive oxygen species. We now report on the treatment of albumin and intravenous immune globulin (IVIG) isolated by a previously described, integrated chromatographic method. Albumin was treated with either 0.1 or 0.2 J/cm2 UVC in the presence of 0.8 or 1.6 mM rutin; IVIG was treated with either 0.05 or 0.1 J/cm2 UVC in the presence of 0.5 or 1.0 mM rutin. Our results show that > or = 10(6.9) ID of EMCV and PPV were inactivated under each of the conditions studied except the treatment of albumin with 0.1 J/cm2 UVC in the presence of 1.6 mM rutin where 10(4.3) ID of EMCV and > or = 10(6.9) ID of PPV were killed. It appears that the sensitivity of PPV to UVC exceeds that of EMCV and that virus kill with UVC is higher in IVIG than in albumin. In the absence of rutin, UVC increased the extent of aggregation of both albumin and IVIG by two- to three-fold. With rutin present, the increase in albumin aggregation was reduced, and it was virtually eliminated by subsequent processing on Sephacryl S-200, a step in the existing procedure designed to remove aggregates. The increase in aggregation of IVIG appeared to be eliminated on inclusion of either 0.5 mM or 1 mM rutin. We conclude that both albumin and IVIG can be treated with UVC to inactivate > or = 10(6) ID of nonenveloped viruses. The inclusion of rutin during treatment helps protect against protein aggregation.

Protecting Fibrinogen with Rutin during UVC Irradiation for Viral Inactivation

Abstract— Fibrinogen solutions were irradiated with UVC (254 nm) to inactivate contaminating viruses. In order to protect fibrinogen during UVC irradiation, 0.5 mM rutin was added prior to UVC exposure and subsequently removed during processing. Viral kill by 0.1 J/cm2 UVC resulted in the following inactivation values (log 10): non‐lipid‐enveloped viruses: Parvo 5.5; encephalomyocarditis virus 6.5; hepatitis A virus 6.5: lipid‐enveloped viruses: human immunodeficiency virus 5.7; vesicular stomatitis virus 5.7. Fibrinogen irradiated with 0.5 mM rutin did not significantly differ from unirradiated material in terms of clot time and breaking strength. In the absence of rutin, UVC irradiation of fibrinogen at similar fluence led to loss of solubility, increased clot time and the cleavage of fibrino‐peptides that reacted with dinitro‐phenyl hydrazine as a test for ketonic carbonyl groups. High‐performance liquid chromatography and mass spectrometry data showed that rutin exposed to UVC formed numerous breakdown, oxidation and combinational products. Experiments with 3H‐rutin showed that after UVC irradiation, subsequent processing by a C18 resin and alcohol precipitation removed >99% rutin, representing <10 ppm rutin in the final fibrinogen preparations. Residual 3H‐rutin was not covalently bonded to the fibrinogen. Immunochemical studies with rabbit antisera to UVC irradiated (with rutin) fibrinogen showed the absence of neoimmungens. By all measures, rutin prevents fibrinogen degradation during virucidal UVC irradiation.

High-Yield Isolation, Expansion, and Differentiation of Murine Bone Marrow-Derived Mesenchymal Stem Cells Using Fibrin Microbeads (FMB)

Transplantation of adult mesenchymal stem cells (MSCs) could provide a basis for tissue regeneration. MSCs are typically isolated from bone marrow (BM) based on their preferential adherence to plastic, although with low efficiency in terms of yield and purity. Extensive expansion is needed to reach a significant number of MSCs for any application. Fibrin microbeads (FMB) were designed to attach mesenchymal cells and to provide a matrix for their expansion. The current study was aimed at isolating a high yield of purified BM-derived mouse MSCs based on their preferential adherence and proliferation on FMB in suspension cultures. MSCs could be downloaded to plastics or further expanded on FMB. The yield of MSCs obtained by the FMB isolation technique was about one order of magnitude higher than that achieved by plastic adherence, suggesting that these cells are more abundant than previously reported. FMB-isolated cells were classified as MSCs by their fibroblastic morphology, self-renewal ability, and expression profile of their surface antigens, as examined by flow cytometry and immunostaining. In cell culture, the isolated MSCs could be induced to differentiate into three different mesodermal lineages, as demonstrated by histochemical stains and by RT-PCR analyses of tissue-specific genes. MSCs were also able to differentiate into osteocytes while still cultured on FMB. Our results suggest that FMB might serve as an efficient platform for the isolation, expansion, and differentiation of mouse BM-derived MSCs to be subsequently implanted for tissue regeneration.

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.

Heparin as inhibitor of mammalian protein synthesis II. Degree of sulfation; Related sulfated mucopolysaccharides

Abstract Heparin is a potent inhibitor of initiation of translation of rabbit globin mRNA in a Krebs ascites cell-free system, as well as being a potent anticoagulant. Progressive desulfation of heparin in the presence of acid for 1 h decreases heparins anticoagulation activity by 50%, while incubation of heparin for longer than 8 h is necessary to cause 50% loss of heparins activity as a protein-synthesis inhibitor. Partial resulfation of amino groups of inactive desulfated heparin restores heparins protein-synthesis inhibitory ability almost completely, while negligible anticoagulant activity is restored. Proteoheparin is active as both a protein-synthesis inhibitor and an anticoagulant, while heparan sulfate lacks both activities.