LED-Based Photoacoustic Imaging for Monitoring Angiogenesis in Fibrin Scaffolds.

IMPACT STATEMENT: Noninvasive imaging techniques provide insight into physiology that is complementary to tissue morphology obtained by invasive histology. Optical imaging techniques, such as laser speckle contrast analysis, are used in vivo to longitudinally evaluate vascularization. Despite their high spatial resolution, these techniques have a limited imaging depth. In this study, we demonstrate how a dual LED-based photoacoustic (PA) and ultrasound system can delineate changes in perfusion at depth within scaffolds containing basic fibroblast growth factor. Perfusion changes detected by PA corroborated with vessel density. PA imaging could be a noninvasive and sensitive method for evaluating vascularization at depth in larger constructs.

X-ray Phase Contrast Allows Three Dimensional, Quantitative Imaging of Hydrogel Implants.

Three dimensional imaging techniques are needed for the evaluation and assessment of biomaterials used for tissue engineering and drug delivery applications. Hydrogels are a particularly popular class of materials for medical applications but are difficult to image in tissue using most available imaging modalities. Imaging techniques based on X-ray Phase Contrast (XPC) have shown promise for tissue engineering applications due to their ability to provide image contrast based on multiple X-ray properties. In this manuscript, we investigate the use of XPC for imaging a model hydrogel and soft tissue structure. Porous fibrin loaded poly(ethylene glycol) hydrogels were synthesized and implanted in a rodent subcutaneous model. Samples were explanted and imaged with an analyzer-based XPC technique and processed and stained for histology for comparison. Both hydrogel and soft tissues structures could be identified in XPC images. Structure in skeletal muscle adjacent could be visualized and invading fibrovascular tissue could be quantified. There were no differences between invading tissue measurements from XPC and the gold-standard histology. These results provide evidence of the significant potential of techniques based on XPC for 3D imaging of hydrogel structure and local tissue response.

Controlled release of fibrin matrix-conjugated platelet derived growth factor improves ischemic tissue regeneration by functional angiogenesis.

Sustained, local, low dose growth factor stimulus of target tissues/cells is believed to be of imminent importance in tissue regeneration and engineering. Recently, a technology was developed to bind growth factors to a fibrin matrix using the transglutaminase (TG) activity of factor XIIIa, thus allowing prolonged release through enzymatic cleavage. In this study we aimed to determine whether TG-PDGF.AB in fibrin could improve tissue regeneration in a standard ischemic flap model. In vitro determination of binding and release kinetics of TG-PDGF.AB allowed proof of concept of the developed binding technology. A single spray application of TG-PDGF.AB in fibrin matrix at a concentration of 10 and 100ng/ml significantly reduced ischemia-induced flap tissue necrosis in vivo on day 7 after ischemic impact compared to controls. TG-PDGF.AB at a concentration of 100ng/ml fibrin induced distinct angiogenesis as reflected by significantly improved tissue perfusion assessed by laser Doppler imaging as well as enhanced von Willebrand factor (vWF) protein expression determined by immunohistochemical means. In addition, significantly more mature microvessels were observed with 100ng/ml TG-PDGF.AB in fibrin compared to control and vehicle groups as evidenced by an improved smooth muscle actin (sma)/vWF protein ratio. In conclusion, PDGF.AB in a conjugated fibrin matrix effectively reduced ischemia-induced tissue necrosis, increased tissue perfusion and induced the growth of a mature and functional neovasculature. The sealing properties of the fibrin matrix in conjunction with the prolonged growth factor stimulus enabled by the TG-hook binding technology may present an innovative and suitable tool in tissue regeneration. STATEMENT OF SIGNIFICANCE: In our experimental study we elucidated recombinant platelet derived growth factor (PDGF) as a potential candidate in inducing angiogenesis. To avoid preterm growth factor degradation in vivo PDGF.AB was covalently linked to a fibrin scaffold using a bi-domain functionalized peptide (FXIII substrate site and plasmin cleavage site). This allowed PDGF binding to fibrin during spray application to the donor site and subsequent prolonged release via endogenous plasmin. This resulted in a mature vascular network thus enhancing tissue perfusion and consequently improved clinical outcome. With our present work we could certainly provide researchers and clinicians with an innovative versatile and reproducible technology not only to induce functional vascularity but also to improve attempts in tissue engineering in general by e.g. using different growth factors. Hence, we believe that this approach studied in the present work may provide a valuable input in an effort to drive the aim forward bringing experimental work in tissue engineering to clinic by using a clinically well characterized and used fibrin scaffold in combination with a human recombinant growth factor (fibrin scaffold linked with the specific binding technology).

Fibrin biomatrix-conjugated platelet-derived growth factor AB accelerates wound healing in severe thermal injury.

Controlled delivery of growth factors from biodegradable biomatrices could accelerate and improve impaired wound healing. The study aim was to determine whether platelet-derived growth factor AB (PDGF.AB) with a transglutaminase (TG) crosslinking substrate site released from a fibrin biomatrix improves wound healing in severe thermal injury. The binding and release kinetics of TG-PDGF.AB were determined in vitro. Third-degree contact burns (dorsum of Yorkshire pigs) underwent epifascial necrosectomy 24 h post-burn. Wound sites were covered with autologous meshed (3:1) split-thickness skin autografts and either secured with staples or attached with sprayed fibrin sealant (FS; n = 8/group). TG-PDGF.AB binds to the fibrin biomatrix using the TG activity of factor XIIIa, and is subsequently released through enzymatic cleavage. Three doses of TG-PDGF.AB in FS (100 ng, 1 µg and 11 µg/ml FS) were tested. TG-PDGF.AB was bound to the fibrin biomatrix as evidenced by western blot analysis and subsequently released by enzymatic cleavage. A significantly accelerated and improved wound healing was achieved using sprayed FS containing TG-PDGF.AB compared to staples alone. Low concentrations (100 ng-1 µg TG-PDGF.AB/ml final FS clot) demonstrated to be sufficient to attain a nearly complete closure of mesh interstices 14 days after grafting. TG-PDGF.AB incorporated in FS via a specific binding technology was shown to be effective in grafted third-degree burn wounds. The adhesive properties of the fibrin matrix in conjunction with the prolonged growth factor stimulus enabled by this binding technology could be favourable in many pathological situations associated with wound-healing disturbances. Copyright © 2013 John Wiley & Sons, Ltd.

Fibrin gels loaded with cisplatin and cisplatin-hyaluronate complexes tested in a subcutaneous human melanoma model.

Fibrin gels are attractive biomaterials for local delivery of a variety of agents, from drugs to proteins. Similarly, polymer-anticancer-drug conjugates and nanoparticles are emerging as potential candidates for cancer treatment. Combining these different approaches, we have studied the efficacy of fibrin gels loaded with cisplatin (DDP) and a complex of DDP with hyaluronate (DDP-HA) for tumor growth inhibition in a melanoma model. Loaded gels prepared at relatively high fibrinogen concentration (22 mg/ml) showed good in vitro antiproliferative activities, prolonged release of the anticancer drug, and a long persistence (10-15 days) in vivo when implanted subcutaneously (sc) in immunodeficient mice. Gels loaded with DDP or DDP-HA containing 1/3 or even 1/6 of their systemic dose (6 mg/kg) and positioned under the tumor mass in mice bearing a sc human SK-Mel-28 tumor showed an antitumor activity better than that of the original parent compound given intraperitoneally (ip). Moreover, in an additional experiment in vivo, fibrin gels loaded with N-trimethyl chitosan-based nanoparticles containing a DDP-HA complex were assayed, resulting in a further 8 % improvement of anticancer activity, with lesser adverse systemic toxic effects. Taken together, these results suggest that the combination of fibrin gels and drugs complexed with suitable macromolecules holds great promise for loco-regional anticancer therapy of melanoma and other surgically removable cancer types.

In vivo molecular imaging of thrombosis and thrombolysis using a fibrin-binding positron emission tomographic probe.

BACKGROUND: Fibrin is a major component of arterial and venous thrombi and represents an ideal candidate for molecular imaging of thrombosis. Here, we describe imaging properties and target uptake of a new fibrin-specific positron emission tomographic probe for thrombus detection and therapy monitoring in 2 rat thrombosis models. METHODS AND RESULTS: The fibrin-binding probe FBP7 was synthesized by conjugation of a known short cyclic peptide to a cross-bridged chelator (CB-TE2A), followed by labeling with copper-64. Adult male Wistar rats (n=26) underwent either carotid crush injury (mural thrombosis model) or embolic stroke (occlusive thrombosis model) followed by recombinant tissue-type plasminogen activator treatment (10 mg/kg, IV). FBP7 detected thrombus location in both animal models with a high positron emission tomographic target-to-background ratio that increased over time (>5-fold at 30-90 minutes, >15-fold at 240-285 minutes). In the carotid crush injury animals, biodistribution analysis confirmed high probe uptake in the thrombotic artery (≈0.5%ID/g; >5-fold greater than blood and other tissues of the head and thorax). Similar results were obtained from ex vivo autoradiography of the ipsilateral versus contralateral carotid arteries. In embolic stroke animals, positron emission tomographic-computed tomographic imaging localized the clot in the internal carotid/middle cerebral artery segment of all rats. Time-dependent reduction of activity at the level of the thrombus was detected in recombinant tissue-type plasminogen activator-treated rats but not in vehicle-injected animals. Brain autoradiography confirmed clot dissolution in recombinant tissue-type plasminogen activator-treated animals, but enduring high thrombus activity in control rats. CONCLUSIONS: We demonstrated that FBP7 is suitable for molecular imaging of thrombosis and thrombolysis in vivo and represents a promising candidate for bench-to-bedside translation.

Long-lasting fibrin matrices ensure stable and functional angiogenesis by highly tunable, sustained delivery of recombinant VEGF164.

Clinical trials of therapeutic angiogenesis by vascular endothelial growth factor (VEGF) gene delivery failed to show efficacy. Major challenges include the need to precisely control in vivo distribution of growth factor dose and duration of expression. Recombinant VEGF protein delivery could overcome these issues, but rapid in vivo clearance prevents the stabilization of induced angiogenesis. Here, we developed an optimized fibrin platform for controlled delivery of recombinant VEGF, to robustly induce normal, stable, and functional angiogenesis. Murine VEGF164 was fused to a sequence derived from α2-plasmin inhibitor (α2-PI1-8) that is a substrate for the coagulation factor fXIIIa, to allow its covalent cross-linking into fibrin hydrogels and release only by enzymatic cleavage. An α2-PI1-8-fused variant of the fibrinolysis inhibitor aprotinin was used to control the hydrogel degradation rate, which determines both the duration and effective dose of factor release. An optimized aprotinin-α2-PI1-8 concentration ensured ideal degradation over 4 wk. Under these conditions, fibrin-α2-PI1-8-VEGF164 allowed exquisitely dose-dependent angiogenesis: concentrations ≥25 µg/mL caused widespread aberrant vascular structures, but a 500-fold concentration range (0.01-5.0 µg/mL) induced exclusively normal, mature, nonleaky, and perfused capillaries, which were stable after 3 mo. Optimized delivery of fibrin-α2-PI1-8-VEGF164 was therapeutically effective both in ischemic hind limb and wound-healing models, significantly improving angiogenesis, tissue perfusion, and healing rate. In conclusion, this optimized platform ensured (i) controlled and highly tunable delivery of VEGF protein in ischemic tissue and (ii) stable and functional angiogenesis without introducing genetic material and with a limited and controllable duration of treatment. These findings suggest a strategy to improve safety and efficacy of therapeutic angiogenesis.

Fibrin nanoparticles as Possible vehicles for drug delivery.

BACKGROUND: Several issues have been raised emphasizing the harmful toxic effects of metal nanoparticles towards biological systems. Search of biological nanoparticles with excellent biocompatibility and bioavailability could address this problem. METHODS: Fibrin nanoparticles (FNP) were prepared using a novel technique and characterized for their physico-chemical properties. In vitro studies were performed to examine cytotoxicity and cellular uptake of FNP. Innate immune response to FNP was studied by (i) estimating in vitro generation of complement split products, C3a and C4d and (ii) in vivo expression of pro-inflammatory cytokines, TNF-α, IL-1 and IL-6. In vivo biodistribution study was carried out by intravenous administration of FITC-labelled FNP in mice. RESULTS: FNP were spherical with size ranging from 25 to 28nm. In vitro studies proved the biocompatibility of the nanoparticles, with their distribution across the cytoplasm and nucleus of treated cells. Complement activation studies showed insignificant increase in the level of C3a when compared with positive control. RT-PCR results revealed significant upregulation of TNF-α and downregulation of IL-6 cytokines after 6h of FNP administration. In vivo biodistribution studies showed moderate blood circulation time, with predominant distribution of nanoparticles in the liver followed by the lungs, kidney and spleen. Haematology, serum biochemistry, and histopathology analyses demonstrated that FNP were non-toxic. CONCLUSION: Owing to their small size, low cost, ease of preparation and excellent biocompatibility, FNP might be a promising novel material for drug delivery applications. GENERAL SIGNIFICANCE: Our results demonstrate the safe and promising use of FNP for biomedical applications.

Fibrin chain cross-linking, fibrinolysis, and in vivo sealing efficacy of differently structured fibrin sealants.

In this study, we compared the sealing characteristics and efficacy of a fibrin sealant with reduced plasminogen (FS-rplg) and a fibrin sealant with aprotinin as a fibrinolysis inhibitor (FS-apr). The relevant sealing characteristics including clot structure, fibrin chain cross-linking, and clot lysis were tested in the laboratory. The sealing efficacy was then investigated in a follow-up animal model to determine differences in the in vivo sealing properties. A total of 46 animals were available for the final analysis with 23 animals in each treatment arm. In conclusion, we saw differences in vitro between FS-rplg and FS-apr in ultrastructure and α-chain cross-linking rates as well as in the rate of fibrinolysis. These differences may explain the significantly enhanced sealing efficacy in FS-apr compared to FS-rplg shown in vivo in a rabbit intestinal model.

BMP-2 but not VEGF or PDGF in fibrin matrix supports bone healing in a delayed-union rat model.

Treatment of delayed bone healing and non-unions after fractures, osteotomies or arthrodesis still is a relevant clinical challenge. Artificially applied growth factors can increase bone healing and progressively gain importance in clinical routine. The aim of this study was to determine the effects of rhPDGF-BB, rhVEGF-165, and rhBMP-2 in fibrin matrix on bone healing in a delayed-union rat model. Thirty-seven rats underwent a first operation where a standardized femoral critical size defect was created. A silicone spacer was implanted to impair vascularization within the defect. At 4 weeks the spacer was removed in a second operation and rhPDGF-BB, rhVEGF-165, or rhBMP-2 were applied in a fibrin clot. Animals in a fourth group received a fibrin clot without growth factors. At 8 weeks fibrin bound rhBMP-2 treated animals showed a significantly increased union rate and bone volume within the defect compared to the other groups. Single application of fibrin bound rhPDGF-BB and rhVEGF-165 failed to increase bone healing in our atrophic non-union model.

Enamel matrix protein adsorption to root surfaces in the presence or absence of human blood.

BACKGROUND: The clinical use of an enamel matrix derivative (EMD) has been shown to promote formation of new cementum, periodontal ligament (PDL), and bone and to significantly enhance the clinical outcomes after regenerative periodontal surgery. It is currently unknown to what extent the bleeding during periodontal surgery may compete with EMD adsorption to root surfaces. The aim of this study is to evaluate the effect of blood interactions on EMD adsorption to root surfaces mimicking various clinical settings and to test their ability to influence human PDL cell attachment and proliferation. METHODS: Teeth extracted for orthodontic reasons were subjected to ex vivo scaling and root planing and treated with 24% EDTA, EMD, and/or human blood in six clinically related settings to determine the ability of EMD to adsorb to root surfaces. Surfaces were analyzed for protein adsorption via scanning electron microscopy and immunohistochemical staining with an anti-EMD antibody. Primary human PDL cells were seeded on root surfaces and quantified for cell attachment and cell proliferation. RESULTS: Plasma proteins from blood samples altered the ability of EMD to adsorb to root surfaces on human teeth. Samples coated with EMD lacking blood demonstrated a consistent even layer of EMD adsorption to the root surface. In vitro experiments with PDL cells demonstrated improved cell attachment and proliferation in all samples coated with EMD (irrespective of EDTA) when compared to samples containing human blood. CONCLUSION: Based on these findings, it is advised to minimize blood interactions during periodontal surgeries to allow better adsorption of EMD to root surfaces.

Inyección de fibrina epidural como tratamiento conservador de la fuga persistente de líquido cefalorraquídeo tras cateterización intratecal / No disponible

No disponible

Elution of ciprofloxacin from acrylic bone cement and fibrin clot: an in vitro study.

The purpose of this study was to investigate the release of ciprofloxacin from acrylic bone cement and fibrin clot. Under sterile conditions, bone cement and fibrin clot were individually mixed with ciprofloxacin. Ten specimens of each complex were placed in 1 ml of nutrient broth and incubated at 37 degrees C. The nutrient broth was changed daily, and the removed samples were stored at -70 degrees C until the antibiotic concentration in each sample was determined by a microbiological method. The maximum level in bone cement specimens was obtained at the second day (80.80 microg/ml) and its diffusion was rapid at first, decreasing gradually over a period of 365 days. Fibrin clot biodegradable specimens released high concentrations of ciprofloxacin (1.52-49.91 microg/ml) in vitro for the period of time needed to treat bone infections (i.e. 65 days). We conclude that the high release of ciprofloxacin in vitro from acrylic bone cement and fibrin clot is very promising since the obtained levels are much higher than the required minimal inhibitory concentration (MIC) against the implicated pathogens in soft tissue and bone infections. The in vivo relevance of the obtained results requires carefully performed studies in animal models.

Interaction of fibrin(ogen) with apolipoprotein(a): further characterization and identification of a novel lysine-dependent apolipoprotein(a)-binding site within the gamma chain 287-411 region.

Interaction of lipoprotein(a) with fibrin associated with atherosclerotic lesions promotes its accumulation in the lesions, thereby contributing to the development of atherothrombosis. Numerous studies revealed that this interaction occurs through the apolipoprotein(a) [apo(a)] component of lipoprotein(a) and COOH-terminal Lys residues generated by partial degradation of fibrin with plasmin (a COOH-Lys-dependent mechanism). At the same time, the mechanism of the interaction of apo(a) with intact fibrin(ogen) remained unclear. Our recent study identified the Lys-independent apo(a)-binding sites within the fibrin(ogen) alphaC domains which contribute to an alternative Lys-independent mechanism. In this study, we performed direct measurements of the interaction between apo(a) and various fibrin(ogen) fragments representing the whole fibrin(ogen) molecule except the alphaC regions. The experiments revealed that the apo(a)-binding site, identified previously within fibrinogen gamma chain residues 207-235 [Klose, R., et al. (2000) J. Biol. Chem. 275, 38206-38212], is a high-affinity site and mainly Lys-independent, suggesting that it should also contribute to the Lys-independent mechanism. The experiments also identified a novel Lys-dependent high-affinity apo(a)-binding site within the sequence of gamma chain residues 287-411. This site may provide interaction of apo(a) with intact fibrin(ogen) through another alternative mechanism, which depends on internal Lys residues. Thus, apo(a) may interact with intact fibrin through the Lys-independent and Lys-dependent mechanisms, while the COOH-Lys-dependent mechanism may prevail in the presence of fibrinolytic activity.

Bleomycin-induced pulmonary fibrosis in fibrinogen-null mice.

Mice deleted for the plasminogen activator inhibitor-1 (PAI-1) gene are relatively protected from developing pulmonary fibrosis induced by bleomycin. We hypothesized that PAI-1 deficiency reduces fibrosis by promoting plasminogen activation and accelerating the clearance of fibrin matrices that accumulate within the damaged lung. In support of this hypothesis, we found that the lungs of PAI-1(-/-) mice accumulated less fibrin after injury than wild-type mice, due in part to enhanced fibrinolytic activity. To further substantiate the importance of fibrin removal as the mechanism by which PAI-1 deficiency limited bleomycin-induced fibrosis, bleomycin was administered to mice deficient in the gene for the Aalpha-chain of fibrinogen (fib). Contrary to our expectation, fib(-/-) mice developed pulmonary fibrosis to a degree similar to fib(+/-) littermate controls, which have a plasma fibrinogen level that is 70% of that of wild-type mice. Although elimination of fibrin from the lung was not in itself protective, the beneficial effect of PAI-1 deficiency was still associated with proteolytic activity of the plasminogen activation system. In particular, inhibition of plasmin activation and/or activity by tranexamic acid reversed both the accelerated fibrin clearance and the protective effect of PAI-1 deficiency. We conclude that protection from fibrosis by PAI-1 deficiency is dependent upon increased proteolytic activity of the plasminogen activation system; however, complete removal of fibrin is not sufficient to protect the lung.

Pharmacokinetic study of fibrin clot-ciprofloxacin complex: an in vitro and in vivo experimental investigation.

We prepared a composite of fibrin clot and ciprofloxacin for use as a biodegradable antibiotic delivery system with sustained effect for the treatment of chronic osteomyelitis. In vitro, ten experiments were performed in which 10 mg of ciprofloxacin were incorporated into 4 ml of fibrin clot. The clots were preserved in nutrient broth and incubated at 37 degrees C for a total of 60 days. Every 24 h a broth specimen was obtained, and the ciprofloxacin concentration was determined by microbiological assay. The maximum level of antibiotic was noted on the first day (49.9 +/- 5.1 mg/l). The ciprofloxacin-fibrin clot complexes usually disintegrated after 60 days. In vivo, the fibrin-ciprofloxacin clots were made as previously described. The composite was implanted in the medullary canal of rabbit tibiae, and the antibiotic concentration was measured in bone, muscle, skin and serum. In all tissues around the implant, the concentration of antibiotic exceeded the minimum inhibitory concentration against the common causative organisms of osteomyelitis for 10 days. The implant caused no systemic side-effects, and it is likely to prove clinically useful as a drug delivery system for treating chronic osteomyelitis.

Effect of local hemostatics on bone induction in rats: a comparative study of bone wax, fibrin-collagen paste, and bioerodible polyorthoester with and without gentamicin.

Local hemostatics for osseous tissue should preferably be absorbable and biocompatible and should not inhibit osteogenesis. The tissue response and effect on demineralized bone-induced heterotopic osteogenesis in the abdominal muscle of 120 male Wistar rats by different local hemostatics were evaluated by light microscopy and 85Sr uptake analyses. Non-absorbable bone wax of 88% beeswax and absorbable bovine fibrin-collagen paste both significantly inhibited osteoinduction, whereas a bioerodible polyorthoester drug delivery system with or without 4% gentamicin did not. Bone wax was not absorbed and induced a chronic foreign body reaction. Fibrin-collagen paste induced less inflammation with numerous monocytes and macrophages with engulfed material. Bioerodible polyorthoester caused a very moderate tissue reaction and was mostly resorbed at week 4.

Localization and visualization of pulmonary emboli with radiolabeled fibrin-specific monoclonal antibody.

Indium-111-labeled monoclonal antibody 64C5 specific for the beta-chain of fibrin monomer was used to image canine (n = 6) experimental pulmonary emboli (at least one barium-thrombin and one copper-coil induced clot per dog). Uptake of 111In-64C5 and 125I-control-DIG26-11 were compared in 10 clots (7 barium-thrombin and 3 copper-coil) identified in the lungs. There was no difference in the blood clearance of 111In-64C5 and 125I-DIG26-11. Uptake of 111In-64C5 (0.183 +/- 0.105, mean %ID/g) was greater than 125I-DIG26-11 (0.024 +/- 0.025) in pulmonary clots (p less than 0.001). Mean thrombus to blood ratios at 24 hr were 6.78:1 for 64C5 and 0.57:1 for DIG26-11. The clots visualized in vivo were larger (0.315 +/- 0.381 g) than clots not visualized (0.089 +/- 0.098). Negative images were recorded in three dogs with pulmonary emboli, injected with 111In-labeled control monoclonal antibody 3H3. These data suggest that 111In-labeled antifibrin can detect large pulmonary emboli in vivo.

Resorption of fibrin tissue adhesive sealant by isolated osteoclasts in culture.

The tissue tolerance of fibrin tissue adhesive sealants has been investigated. These materials are increasingly being used to close wounds and to stabilize spongy bone transplants. An in vitro model consisting of isolated osteoclasts in primary cell culture was used. The 2 commercially available tissue adhesive sealant protein solutions differ substantially with regard to the spreading and resorption capacity of the cultivated osteoclasts.