Bio-/Nanoimmobilization Platform Based on Bioinspired Fibrin-Bone@Polydopamine-Shell Adhesive Composites for Biosensing.

Inspired by blood coagulation and mussel adhesion, we report novel adhesive fibrin-bone@polydopamine (PDA)-shell composite matrix as highly efficient immobilization platform for biomacromolecules and nanomaterials. Fibrin, as a bioglue, and PDA, as a chemical adhesive, are integrated in a one-pot simultaneous polymerization consisting of biopolymerization of fibrinogen and chemical polymerization of dopamine. Fibrin fibers act as adhesive bones to construct scaffold, while PDA coat on the scaffold to form adhesive shell, generating 3D porous composite matrix with unique bone@shell structure. Two types of enzymes (glucose oxidase and acetylcholinesterase) and Au nanoparticles were adopted as respective model biomolecules and nanomaterials to investigate the immobilization capability of the matrix. The bionanocomposites showed high efficiency in capturing nanoparticles and enzymes, as well as significant mass-transfer and biocatalysis efficiencies. Therefore, the bionanocomposites exhibited significant potential in biosensing of glucose and paraoxon with limits of detection down to 5.2 µM and 4 ppt, respectively. The biological-chemical-combined polymerization strategy and composite platform with high immobilization capacity and mass-transfer efficiency open up a novel way for the preparation of high-performance bionanocomposites for various applications, in particular, biosensing.

Biofabrication of injectable fibrin microtissues for minimally-invasive therapies: application of surfactants.

Microtissues created from the protein fibrin and containing embedded cells can be used in modular tissue engineering approaches to create larger, hierarchical and complex tissue structures. In this paper we demonstrate an emulsification-based method for the production of such fibrin microtissues containing fibroblasts (FB) and endothelial cells (EC) and designed to promote tissue vascularization. Surfactants can be beneficial in the microtissue fabrication process to reduce aggregation and to facilitate recovery of microtissues from the emulsion, thereby increasing yield. The nonionic surfactants Pluronic L101® and Tween 20® both increased microtissue yield in a dose-dependent fashion. Cell viability of both human FB and human EC remained high after exposure to low surfactant concentrations but decreased with increasing surfactant concentration. L101 was markedly less cytotoxic than Tween, and therefore was the surfactant of choice in this application. The yield of cell-laden microtissues increased with increasing L101 concentration, though microtissues were slightly larger at low concentrations. The total metabolic activity of cells in retrieved microtissues was bimodal and was highest at an L101 concentration of 0.10% wt/vol. Network formation by EC in microtissues embedded in surrounding 3D fibrin hydrogels was also most extensive in microtissues made using an L101 concentration of 0.10% wt/vol. Minimally-invasive delivery of microtissue populations was demonstrated by injection through a standard 18 G needle, and the ability to form robust endothelial networks was maintained in injected microtissue populations. Taken together, these data demonstrate a facile emulsification-based method to create modular, cell-laden hydrogel microtissues that can be delivered by injection to promote tissue regeneration. Appropriate selection of the type and concentration of surfactant used in the process can be used to maximize viability and specialized function of the embedded cells. Such biomaterial-based microtissues may have broad applicability in cell-based therapies and tissue engineering.

A comprehensive mechanism of fibrin network formation involving early branching and delayed single- to double-strand transition from coupled time-resolved X-ray/light-scattering detection.

The formation of a fibrin network following fibrinogen enzymatic activation is the central event in blood coagulation and has important biomedical and biotechnological implications. A non-covalent polymerization reaction between macromolecular monomers, it consists basically of two complementary processes: elongation/branching generates an interconnected 3D scaffold of relatively thin fibrils, and cooperative lateral aggregation thickens them more than 10-fold. We have studied the early stages up to the gel point by fast fibrinogen:enzyme mixing experiments using simultaneous small-angle X-ray scattering and wide-angle, multi-angle light scattering detection. The coupled evolutions of the average molecular weight, size, and cross section of the solutes during the fibrils growth phase were thus recovered. They reveal that extended structures, thinner than those predicted by the classic half-staggered, double-stranded mechanism, must quickly form. Following extensive modeling, an initial phase is proposed in which single-bonded "Y-ladder" polymers rapidly elongate before undergoing a delayed transition to the double-stranded fibrils. Consistent with the data, this alternative mechanism can intrinsically generate frequent, random branching points in each growing fibril. The model predicts that, as a consequence, some branches in these expanding "lumps" eventually interconnect, forming the pervasive 3D network. While still growing, other branches will then undergo a Ca(2+)/length-dependent cooperative collapse on the resulting network scaffolding filaments, explaining their sudden thickening, low final density, and basic mechanical properties.

Fabrication of fibrin based electrospun multiscale composite scaffold for tissue engineering applications.

Fabricating scaffolds mimicking the native extracellular matrix (ECM) in both structure and function is a key challenge in the field of tissue engineering. Previously we have demonstrated a novel electrospinnig method for the fabrication of fibrin nanofibers using Poly(vinyl alcohol) (PVA) as an 'electrospinning-driving' polymer. Here we demonstrate the fabrication and characterization of a multiscale fibrin based composite scaffold with polycaprolactone (PCL) by sequential electrospinning of PCL microfibers and fibrin nanofibers. This multiscale scaffold has great potential for tissue engineering applications due to the combined benefits of biological nanofibers such as cell attachment and proliferation and that of microfibers such as open structure, larger pore size and adequate mechanical strength. Physico chemical characterization of the electrospun scaffold was done by Scanning Electron Microscopy (SEM), Contact angle analysis, fibrin specific Phosphotungstic acid haematoxyllin (PTAH) staining and evaluation of mechanical properties. SEM data revealed the formation of bead free nanofibers of fibrin with a fiber diameter ranging from 50-500 nm and microfibers of PCL in the size range of 1 microns to 2.5 microns. These dimensions mimic the hierarchical structure of ECM found in native tissues. Cell attachment and viability studies using human mesenchymal stem cells (hMSC) revealed that the scaffold is non toxic and supports cell attachment, spreading and proliferation. In addition, we examined the inflammatory potential of the scaffold to demonstrate its usefulness in tissue engineering applications.

Recombinant human fibrinogen that produces thick fibrin fibers with increased wound adhesion and clot density.

Human fibrinogen is a biomaterial used in surgical tissue sealants, scaffolding for tissue engineering, and wound healing. Here we report on the post-translational structure and functionality of recombinant human FI (rFI) made at commodity levels in the milk of transgenic dairy cows. Relative to plasma-derived fibrinogen (pdFI), rFI predominantly contained a simplified, neutral carbohydrate structure and >4-fold higher levels of the γ'-chain transcriptional variant that has been reported to bind thrombin and Factor XIII. In spite of these differences, rFI and pdFI were kinetically similar with respect to the thrombin-catalyzed formation of protofibrils and Factor XIIIa-mediated formation of cross-linked fibrin polymer. However, electron microscopy showed rFI produced fibrin with much thicker fibers with less branching than pdFI. In vivo studies in a swine liver transection model showed that, relative to pdFI, rFI made a denser, more strongly wound-adherent fibrin clot that more rapidly established hemostasis.

Oligonucleotide-peptide conjugates: solid-phase synthesis under acidic conditions and use in ELISA assays.

Here we used solid-phase methods to prepare oligonucleotides carrying fibrin/ filaggrin citrullinated peptides. Post-synthetic conjugation protocols were successfully applied for the synthesis of oligonucleotides carrying small peptides. A stepwise protocol using acid treatment for the final deprotection allowed the preparation of polypyrimidine oligonucleotides carrying longer and arginine-rich peptides. An ELISA-based test using the oligonucleotide-citrullinated peptide conjugates was developed for the detection of anti-citrullinated protein/peptide antibodies in human serum from rheumatoid arthritis patients.

Control of anti-thrombogenic properties: surface-induced self-assembly of fibrinogen fibers.

Wound healing is a complex process initiated by the formation of fibrin fibers and endothelialization. Normally, this process is triggered in a wound by thrombin cleavage of fibrinopeptides on fibrinogen molecules, which allows them to self spontaneously-assemble into large fibers that provide the support structure of the clot and promote healing. We have found that the fibrous structures can also form without thrombin on most polymer or metal surfaces, including those commonly used for stents. We show that the relatively hydrophobic E and D regions of the fibrinogen molecule are adsorbed on these surfaces, exposing the αC domains, which in turn results in the formation of large fiber structures that promote endothelial cell adhesion. We show that the entire process can be suppressed when stents or other substrates are coated with polymers that are functionalized to bind the αC domains, leading to the development of potentially nonthrombogenic implant materials.

Perspectives and challenges in regenerative medicine using plasma rich in growth factors.

Plasma rich in growth factors (PRGF-Endoret) is an endogenous therapeutic technology that is gaining interest in regenerative medicine due to its potential to stimulate and accelerate tissue healing and bone regeneration. This autologous biotechnology is designed for the in situ delivery of multiple cellular modulators and the formation of a fibrin scaffold, thereby providing different formulations that can be widely used in numerous medical and scientific fields including dentistry, oral implantology, orthopedics, ulcer treatment and tissue engineering among others. Here we discuss the important progress that has been accomplished in this field. Furthermore, a comprehensive outlook of the intriguing therapeutic applications of this technology is presented.

Carbon nanotube composite peptide-based biosensors as putative diagnostic tools for rheumatoid arthritis.

This work reports on the fabrication and performance of a simple amperometric immunosensor device to be potentially used for the detection of serum anti-citrullinated peptide antibodies (ACPAs), which are specific for rheumatoid arthritis (RA) autoimmune disease. Sera of RA patients contain antibodies to different citrullinated peptides and proteins such as fibrin or filaggrin. Herein, a chimeric fibrin-filaggrin synthetic peptide (CFFCP1) was used as a recognition element anchored to the surface of a multiwalled carbon nanotube-polystyrene (MWCNT-PS) based electrochemical transducer. The transducer fabrication process is described in detail together with its successful electrochemical performance in terms of repeatability and reproducibility of the corresponding amperometric response. The resulting immunosensor approach was initially tested in sera of rabbits previously inoculated with the synthetic peptide and eventually applied to the detection of ACPAs in human sera. A comparative study was carried out using control serum from a blood donor, which demonstrated the selectivity of the immunosensor response and its sensitivity for the detection of anti-CFFCP1 antibodies present in RA patients.

Coronary arteries angiogenesis in ischemic myocardium: biocompatibility and biodegradability of various hydrogels.

To evaluate the biocompatibility and biodegradability of various hydrogels and choose suitable hydrogels for the coronary arteries angiogenesis in ischemic myocardium, we synthesized six kinds of hyaluronan hydrogels, fibrin hydrogel, poly(vinyl alcohol)-chitosan hydrogel, and obtained elastin hydrogels. We examined their degradation rates and cytotoxicity in vitro. Then, hydrogels were implanted into rat adductor muscles for 1, 2, or 4 weeks. Hydrogels and surrounding tissues were resected, followed by hematoxylin and eosin staining, Masson's trichrome staining, and immunohistochemical staining for measurements of degradation, immune response, and angiogenesis. 2-Iminothiolane grafted hyaluronan hydrogel and periodate oxidated hyaluronan hydrogel presented rapid degradation rates, low quantity of inflammation-mediating cells (12 +/- 3 and 12 +/- 4 per 2.5 x 10(-3) mm(2), respectively, at week 2), thin fibrous capsules (scores were 3.8 +/- 0.1 and 4.0 +/- 0.3 per 0.33 mm(2), respectively, at week 2) with dense blood vessels in the areas surrounding the implanted hydrogels. 2-Iminothiolane grafted hyaluronan and periodate oxidated hyaluronan hydrogels with appropriate degradation rates and low immune responses were suitable for coronary arteries angiogenesis in ischemic myocardium.

Development of a composite degradable/nondegradable tissue-engineered vascular graft.

The present study aimed to determine the feasibility of constructing a reinforced autologous vascular graft by combining the advantages of fibrin gel as an autologous cell carrier material with the inherent mechanical strength of an integrated mesh structure. It was hypothesized that the mesh and dynamic culture conditions could be combined to generate mechanically stable and implantable vascular grafts within a shorter cultivation period than traditional methods. A two-step moulding technique was developed to integrate a polyvinylidene fluoride (PVDF) mesh (pore size: 1-2 mm) in the wall of a fibrin-based vascular graft (I.D. 5 mm) seeded with carotid myofibroblasts. The graft was cultured under increasing physiological flow conditions for 2 weeks. Histology, burst strength, and suture retention strength were evaluated. Cell growth and tissue development was excellent within the fibrin gel matrix surrounding the PVDF fibers, and tissue structure demonstrated remarkable similarity to native tissue. The grafts were successfully subjected to physiological flow rates and pressure gradients from the outset, and mechanical properties were enhanced by the mesh structure. Mean suture retention strength of the graft tissue was 6.3 N and the burst strength was 236 mm Hg. Using the vascular composite graft technique, the production of tissue engineered, small-caliber vascular grafts with good mechanical properties within a conditioning period of 14 days is feasible.

Synthesis of overlapping fibrin citrullinated peptides and their use for diagnosing rheumatoid arthritis.

With the aim of developing a new enzyme-linked immunosorbent assay test to detect autoantibodies in the sera of rheumatoid arthritis patients with a high sensitivity and specificity using synthetic citrullinated peptides of fibrin (which is abundant in rheumatoid synovium) as antigenic substract, peptides belonging to alpha- and beta-fibrin chains were selected by computer-aided prediction of antigenicity and epitope mapping and synthesized in solid phase. We analysed by enzyme-linked immunosorbent assay 133 sera from patients with well-characterized rheumatic diseases, including 67 patients with rheumatoid arthritis. The results of the immunoassays reported highlight the usefulness of fibrin-related peptides in rheumatoid arthritis diagnosis and, especially, the ability and specificity of the [Cit(621,627,630)]alpha-fibrin(617-631) (alpha fib617) peptide sequence to recognize the autoantibodies that are present in rheumatoid arthritis patients.

Polymerization of rod-like macromolecular monomers studied by stopped-flow, multiangle light scattering: set-up, data processing, and application to fibrin formation.

Many biological supramolecular structures are formed by polymerization of macromolecular monomers. Light scattering techniques can provide structural information from such systems, if suitable procedures are used to collect the data and then to extract the relevant parameters. We present an experimental set-up in which a commercial multiangle laser light scattering photometer is linked to a stopped-flow mixer, allowing, in principle, the time-resolved extrapolation of the weight-average molecular weight M(w) and of the z-average square radius of gyration (z) of the polymers from Zimm-like plots. However, if elongated structures are formed as the polymerization proceeds, curved plots rapidly arise, from which M(w) and (z) cannot be recovered by linear fitting. To verify the correctness of a polynomial fitting procedure, polydisperse collections of rod-like or worm-like particles of different lengths, generated at various stages during bifunctional polycondensations of rod-like macromolecular monomers, were considered. Then, the angular dependence of their time-averaged scattered intensity was calculated in the Rayleigh-Gans-Debye approximation, with random and systematic noise also added to the data. For relatively narrow size distributions, a third-degree polynomial fitting gave satisfactory results across a broad range of conversion degrees, yielding M(w) and (z) values within 2% and no greater than 10-20%, respectively, of the calculated values. When more broad size distributions were analyzed, the procedure still performed well for semiflexible polymers, but started to seriously underestimate both M(w) and (z) when rigid rod-like particles were analyzed, even at relatively low conversion degrees. The data were also analyzed in the framework of the Casassa approximation, from which the mass per unit length of the polymers can be derived. These procedures were applied to a set of data taken on the early stages of the thrombin-catalyzed polymerization of fibrinogen, a rod-like macromolecule approximately 50 nm long. The polymers, grown in the absence of Ca(2+) by rate-limiting amounts of thrombin, appeared to be characterized by a much broader size distribution than the one expected for a classical Flory bifunctional polycondensation, and they seem to behave as relatively flexible worm-like double-stranded chains. Evidence for the formation of fibrinogen-fibrin monomer complexes is also inferred from the time dependence of the mass/length ratio. However, our data are also compatible with the presence of limited amounts of single-stranded structures in the very early stages, either as a secondary, less populated pathway, or as transient intermediates to the classical double-stranded fibrils.

Amino acids and peptides. XVIII. Synthetic peptides related to N-terminal portion of fibrin alpha-chain and their inhibitory effect on fibrinogen/thrombin clotting.

Peptide analogs of the N-terminal portion of fibrin alpha-chain were prepared and their inhibitory effects on fibrinogen/thrombin clotting were examined. Of the synthetic peptides, H-Gly-Pro-Arg-Pro-Pro-NH2 exhibited the most potent inhibitory effect.

Amino acids and peptides. XIII. Synthetic studies on N-terminal tripeptide amide analogs of fibrin alpha-chain.

N-Terminal tripeptide analogs of fibrin alpha-chain were synthesized and their inhibitory effect on fibrinogen/thrombin clotting was examined. A new water-soluble active ester, 3-pyridinium ester, was used for the synthesis. Among the synthetic peptides, H-Gly-Pro-Arg-hexamethyleneimine exhibited the highest inhibitory effect on fibrinogen-thrombin clotting.

Resorption of 125I-labeled fibrin bioplast from the dorsal muscle of rats.

Fibrin bioplast is an implant material made from plasticized fibrin. The resorption and route of elimenation of 125I-tagged bioplast plates implanted into the back muscle of rats have been studied in 18 experiments. Variation in radioactivity of the implant, appearance of the activity in the urine and fecal output, supplemented with information on the radioactivity of the implant site and that of isolated organs, reveal that the elimination of bioplast begins on the 12th day and proceeds at an even rate without pathological tissue reactions. By the 42nd day 10 to 12% of the original material is present. The elimination of the implant material is complete and occurs mainly with urine through the kidneys.