Fibrin Matrices Research Articles

Enhanced neurite growth from mammalian neurons in three-dimensional salmon fibrin gels

Three-dimensional fibrin matrices have been used as cellular substrates in vitro and as bridging materials for central nervous system repair. Cells can be embedded within fibrin gels since the polymerization process is non-toxic, making fibrin an attractive scaffold for transplanted cells. Most studies have utilized fibrin prepared from human or bovine blood proteins. However, fish fibrin may be well suited for neuronal growth since fish undergo remarkable central nervous system regeneration and molecules implicated in this process are present in fibrin. We assessed the growth of mammalian central nervous system neurons in bovine, human, and salmon fibrin and found that salmon fibrin gels encouraged the greatest degree of neurite (dendrite and axon) growth and were the most resistant to degradation by cellular proteases. The neurite growth-promoting effect was not due to the thrombin used to polymerize the gels nor to any copurifying plasminogen. Copurified fibronectin partially accounted for the effect on neurites, and blockade of fibrinogen/fibrin-binding integrins markedly decreased neurite growth. Anion exchange chromatography revealed different elution profiles for salmon and mammalian fibrinogens. These data demonstrate that salmon fibrin encourages the growth of neurites from mammalian neurons and suggest that salmon fibrin may be a beneficial scaffold for neuronal regrowth after CNS injury.

VEGF increases the fibrinolytic activity of endothelial cells within fibrin matrices: Involvement of VEGFR-2, tissue type plasminogen activator and matrix metalloproteinases

Proteolysis of fibrin matrices by endothelial cells plays essential roles in the migratory and morphogenic differentiation processes underlying angiogenesis. Using an in vitro fibrinolysis model consisting of human umbilical vein endothelial cells (HUVECs) embedded in a three dimensional fibrin matrix, we show that VEGF, an angiogenic cytokine that plays a crucial role in the onset of angiogenesis, is a potent activator of HUVEC-mediated fibrinolysis. This VEGF-dependent fibrin degradation was completely abrogated by inhibitors of either the plasminogen activator/plasmin or matrix metalloproteinases (MMP) proteolytic systems, suggesting the involvement of both classes of proteases in fibrin degradation. Accordingly, VEGF-induced fibrinolysis correlated with an increase in the expression of tPA and of some MMPs, such as MT2-MMP and was completely blocked by a neutralizing antibody against tPA. Overall, these results indicate that efficient proteolysis of three dimensional fibrin matrices during VEGF-mediated angiogenesis involves a complex interplay between the MMP and plasmin-mediated proteolytic systems.

Implantation of VEGF transfected preadipocytes improves vascularization of fibrin implants on the cylinder chorioallantoic membrane (CAM) model

The successful substitution or augmentation of soft tissues by implantation of three dimensional cell constructs, consisting of human preadipocytes and fibrin glue as a carrier matrix, requires a rapid and homogeneous vascularization of the whole implant in order to provide a sufficient blood supply of centrally situated cells. Previous investigations have shown that under in vivo conditions primary human preadipocytes induce vascularization of fibrin matrices by secretion of several growth factors, such as VEGF and bFGF. The current study investigates whether vascularization of implants can be improved by transplantation of preadipocytes following transfection with a VEGF‐vector. Transfection was performed by electroporation with an pCMX‐GFP and pCMX‐VEGF165 vector. Transfection efficiency (GFP expression) and VEGF expression were determined in vitro by FACS analysis and VEGF immunoassay, respectively. In vivo investigations to determine the vascularization of the implants were performed on the cylinder chorioallantoic membrane (CAM). Four million VEGF transfected cells were transferred within a fibrin matrix onto the CAM on the 7th day of incubation and after 8 days the vascularization of the implant was histologically examined and evaluated by means of a computer‐assisted image analysis program. Transfection of preadipocytes with the GFP vector by electroporation yielded transfection efficiencies between 12% and 41% of surviving cells. Results of the VEGF immunoassay demonstrated that VEGF expression was significantly higher following transfection. Investigations on the CAM outlined a significantly higher rate of vascularization in the transfected vs. control population. Our investigations demonstrate that primary human preadipocytes can be successfully transfected by electroporation with a VEGF vector. The enhanced VEGF expression on transfected cells results in an increase of vascularization of the cell constructs on the CAM.

45 Effect of fibrin matrices on increased proliferation and spreading of pre-term trophoblasts and placental villi modulation of coagulation and angiogenesis functions

45 Effect of fibrin matrices on increased proliferation and spreading of pre-term trophoblasts and placental villi modulation of coagulation and angiogenesis functions

Regulation of Postangiogenic Neovessel Survival by β1 and β3 Integrins in Collagen and Fibrin Matrices

We used the aortic ring model of angiogenesis to investigate the role of β₁ and β₃ integrins in postangiogenic vascular survival in collagen and fibrin matrices. Confocal microscopy studies showed that both β₁ and β₃ integrins were expressed in endothelial cells and pericytes of sprouting neovessels. Antibody blocking experiments demonstrated that β₁ integrins but not β₃ integrins were required for angiogenic sprouting in collagen. Conversely, in fibrin, blockade of both integrins was needed to inhibit angiogenesis whereas treatment with either antibody alone was ineffective. Antibody-mediated blockade of β₁ but not β₃ integrins accelerated vascular regression in collagen. In contrast, both anti-β₁ and -β₃ integrin antibodies were required to promote neovessel breakdown in fibrin. These results demonstrate that angiogenic sprouting and postangiogenic neovessel survival in collagen are critically dependent on β₁ integrins. They also indicate that these processes involve a redundant repertoire of β₁ and β₃ integrins when angiogenesis occurs in fibrin. Thus, pharmacologic targeting of integrin receptors aimed at blocking neovessel formation and survival must be tailored to the specific extracellular matrix environment in which angiogenesis takes place.

Fine-tuning of a three-dimensional microcarrier-based angiogenesis assay for the analysis of endothelial-mesenchymal cell co-cultures in fibrin and collagen gels

A prerequisite for successful tissue engineering is the existence of a functional microvascular network. We hypothesized that such networks can be created and quantified in an in vitro setting by co-culturing endothelial cells (ECs) with tissue-specific 'bystander cells' in 3-D gel matrices. To test this hypothesis we adapted a previously described in vitro microcarrier-based angiogenesis assay (V. Nehls and D. Drenckhahn, 1995, Microvasc Res 50: 311-322). On optimizing this assay, we noted that the initial EC-microcarrier coverage depended on EC type and seeding technique employed to coat the microcarrier beads with the ECs. A confluent EC monolayer on the microcarrier surfaces formed only when bovine aortic endothelial cells (BAECs) were admixed to the beads under gentle agitation on an orbital shaker. After embedding BAEC-covered microcarrier beads into a sandwich-like arrangement of collagen or fibrin gels, we assessed cellular outgrowth at different serum concentrations in terms of migration distance and sprout formation. Quantifiable sprout formation was highest at 1% fetal bovine serum (FBS) in collagen matrices and at 0.1% FBS in fibrin matrices. At higher serum concentration, excess cell migration and formation of clusters prevented quantitative analysis of sprouting. Following the fine-tuning of this angiogenesis assay, we co-cultured BAECs with adipose tissue-derived fibroblasts (FBs) and vascular smooth muscle cells (SMCs). While FBs were able to increase the average migration distance of BAECs in both matrices, SMCs enhanced BAEC migration in fibrin, but not in collagen gels. By contrast, the number of newly formed sprouts in fibrin gels was increased by both cell types. We conclude that in this model bystander cells enhance EC network formation in a matrix-dependent manner. Additionally, these results stress the importance of carefully selecting the experimental parameters of a given in vitro angiogenesis model.

Magnetically-Guided Self-Assembly of Fibrin Matrices with Ordered Nano-Scale Structure for Tissue Engineering

Magnetically-Guided Self-Assembly of Fibrin Matrices with Ordered Nano-Scale Structure for Tissue Engineering

Magnetically-Guided Self-Assembly of Fibrin Matrices with Ordered Nano-Scale Structure for Tissue Engineering

The development of effective biological scaffold materials for tissue engineering and regenerative medicine applications hinges on the ability to present precise environmental cues to specific cell populations to guide their position and function. Natural extracellular matrices have an ordered nano-scale structure that can modulate cell behaviors critical for developmental control, including directional cell motility. Here we describe a method for fabricating fibrin gels with defined architecture on the nanometer scale in which magnetic forces are used to position thrombin-coated magnetic micro-beads in a defined 2-dimensional array and thereby guide the self-assembly of fibrin fibrils through catalytic cleavage of soluble fibrinogen substrate. Time-lapse and confocal microscopy confirmed that fibrin fibrils nucleate near the surface of the thrombin-coated beads and extend out in a radial direction to form these gels. When controlled magnetic fields were used to position the beads in hexagonal arrays, the fibrin nano-fibrils that polymerized from the beads oriented preferentially along the bead--bead axes in a geodesic (minimal path) pattern. These biocompatible scaffolds supported adhesion and spreading of human microvascular endothelial cells, which exhibited co-alignment of internal actin stress fibers with underlying fibrin nano-fibrils within some membrane extensions at the cell periphery. This magnetically-guided, biologically-inspired microfabrication system is unique in that large scaffolds may be formed with little starting material, and thus it may be useful for in vivo tissue engineering applications in the future.

Mesenchymal Stem Cells Enhance Angiogenesis in Mechanically Viable Prevascularized Tissues via Early Matrix Metalloproteinase Upregulation

Angiogenesis, the sprouting of new blood vessels from existing vasculature, is a complex biological process of interest to both the treatment of numerous pathologies and the creation of thick engineered tissues. In the context of tissue engineering, one potential solution to the diffusion limitation is to create a vascular network in vitro that can subsequently anastomose with the host after implantation, allowing the implantation of thicker, more complex tissues. In this study, the ability of endothelial cells to sprout and form stable vascular networks in 3-dimensional (3D) fibrin matrices was investigated as a function of matrix density in a prevascularized tissue model. The results demonstrate that while increasing matrix density leads to a nearly 7-fold increase in compressive stiffness, vascular sprouting is virtually eliminated in the most dense matrix condition. However, the addition of human mesenchymal stem cells (HMSCs) to the denser matrices reverses this effect, resulting in an up to a 7-fold increase in network formation. Although the matrix metalloproteinases (MMPs) MMP-2, MMP-9, and MT1-MMP are all upregulated early on with the addition of HMSCs, MT1-MMP appears to play a particularly important role in the observed angiogenic response among these proteases. This study provides a means to design stiffer prevascularized tissues utilizing naturally derived substrates, and its results may yield new mechanistic insights into stem cell-based angiogenic therapies.

Rationally designed peptides for controlled release of nerve growth factor from fibrin matrices

The purpose of this research was to identify peptide sequences with varying affinity for nerve growth factor (NGF) and use them in the rational design of affinity-based drug delivery systems. A phage display library (12 amino acid random peptide sequence) was screened against NGF-conjugated chromatography resin three times and fractions containing phage of varying affinity were eluted by decreasing the pH of the eluent. These phages were isolated, amplified; then their DNA was purified and sequenced to determine the identity of the random peptide domain. Consensus peptides based on these sequences were synthesized and screened for their ability to bind NGF and release it at different rates from fibrin matrices. The ability of fibrin matrices containing these peptides and NGF to deliver to biologically active NGF was tested using a chick dorsal root ganglia model. A mathematical model was developed to further understand how the affinity of a peptide can modulate release of NGF and to aid in design optimization for the delivery system. The peptides identified in this study were determined to have varying affinities for NGF suggesting that this approach can serve as a model for tailoring the affinity of a drug delivery system for a target protein drug.

TIMP Independence of Matrix Metalloproteinase (MMP)-2 Activation by Membrane Type 2 (MT2)-MMP Is Determined by Contributions of Both the MT2-MMP Catalytic and Hemopexin C Domains

The important and distinct contribution that membrane type 2 (MT2)-matrix metalloproteinase (MMP) makes to physiological and pathological processes is now being recognized. This contribution may be mediated in part through MMP-2 activation by MT2-MMP. Using Timp2-/- cells, we previously demonstrated that MT2-MMP activates MMP-2 to the fully active form in a pathway that is TIMP-2-independent but MMP-2 hemopexin carboxyl (C) domain-dependent. In this study cells expressing MT2-MMP as well as chimera proteins in which the C-terminal half of MT2-MMP and MT1-MMP were exchanged showed that the MT2-MMP catalytic domain has a higher propensity than that of MT1-MMP to initiate cleavage of the MMP-2 prodomain in the absence of TIMP-2. Although we demonstrate that MT2-MMP is a weak collagenase, this first activation cleavage was enhanced by growing the cells in type I collagen gels. The second activation cleavage to generate fully active MMP-2 was specifically enhanced by a soluble factor expressed by Timp2-/- cells and was MT2-MMP hemopexin C domain-dependent; however, the RGD sequence within this domain was not involved. Interestingly, in the presence of TIMP-2, a MT2-MMP.MMP-2 trimolecular complex formed, but activation was not enhanced. Similarly, TIMP-3 did not promote MT2-MMP-mediated MMP-2 activation but inhibited activation at higher concentrations. This study demonstrates the influence that both the catalytic and hemopexin C domains of MT2-MMP exert in determining TIMP independence in MMP-2 activation. In tissues or pathologies characterized by low TIMP-2 expression, this pathway may represent an alternative means of rapidly generating low levels of active MMP-2.

A new approach to tissue engineering of vascularized skeletal muscle

Tissue Engineering of skeletal muscle tissue still remains a major challenge. Every neo-tissue construct of clinically relevant dimensions is highly dependent on an intrinsic vascularisation overcoming the limitations of diffusion conditioned survival. Approaches incorporating the arteriovenous-loop model might bring further advances to the generation of vascularised skeletal muscle tissue. In this study 12 syngeneic rats received transplantation of carboxy-fluorescine diacetate-succinimidyl ester (CFDA)-labelled, expanded primary myoblasts into a previously vascularised fibrin matrix, containing a microsurgically created AV loop. As control cells were injected into fibrin-matrices without AV-loops. Intra-arterial ink injection followed by explantation was performed 2, 4 and 8 weeks after cell implantation. Specimens were evaluated for CFDA, MyoD and DAPI staining, as well as for mRNA expression of muscle specific genes. Results showed enhanced fibrin resorption in dependence of AV loop presence. Transplanted myoblasts could be detected in the AV loop group even after 8 weeks by CFDA-fluorescence, still showing positive MyoD staining. RT-PCR revealed gene expression of MEF-2 and desmin after 4 weeks on the AV loop side, whereas expression analysis of myogenin and MHCembryo was negative. So far myoblast injection in the microsurgical rat AV loop model enhances survival of the cells, keeping their myogenic phenotype, within pre-vascularised fibrin matrices. Probably due to the lack of potent myogenic stimuli and additionally the rapid resorption of the fibrin matrix, no formation of skeletal muscle-like tissue could be observed. Thus further studies focussing on long term stability of the matrix and the incorporation of neural stimuli will be necessary for generation of vascularised skeletal muscle tissue.

Molecular weight fibrinogen variants determine angiogenesis rate in a fibrin matrix in vitro and in vivo.

During wound repair, fibrin acts both as a barrier to prevent blood loss and as a temporary matrix for the invasion and ingrowth of endothelial and tissue cells. A well-controlled angiogenesis process in the fibrinous exudate matrix is crucial for optimal wound healing. The composition and structure of the fibrin matrix are important determinants of the invasion of endothelial cells and capillary-tube formation into the matrix. Fibrinogen circulates in a high and low molecular weight form (HMW and LMW, respectively) and the purpose of this study was to investigate how fibrin matrices from these naturally occurring fibrinogen variants influence angiogenesis. Angiogenesis was studied using an in vitro model in which human microvascular endothelial cells (hMVEC) were cultured on three-dimensional fibrin matrices from different fibrinogen forms, and using two in vivo mouse models. The in vitro angiogenesis in an HMW-fibrin matrix shows increased cell and tubular structure ingrowth compared with unfractionated fibrin matrix (median increase 58%, range 46-234%). The ingrowth of tubular structures in an LMW-fibrin matrices is decreased when compared with unfractionated fibrin (median decrease 70%, range 67-100%). Similar results were observed for in vivo angiogenesis. The naturally occurring fibrinogen variants HMW- and LMW-fibrin modulate the angiogenic capacity of endothelial cells in fibrin matrices. The different effects of the molecular weight fibrinogen variants provide further insight in the matrix characteristics in angiogenesis and could possibly be applied in the context of tissue engineering and wound healing.

Enhancement of adipose tissue formation by implantation of adipogenic-differentiated preadipocytes

Engineered adipose tissue could be used for the reconstruction or augmentation of soft tissues lost due to mastectomy or lumpectomy in plastic and reconstructive surgery. Preadipocytes are a feasible cell source for adipose tissue regeneration. However, the enhancement of the in vivo adipogenic conversion of preadipocytes remains a major task. In vitro, the adipogenic differentiation of preadipocytes prior to implantation might enhance the adipose tissue regeneration. In the present study, we investigated whether implantation of adipogenic-differentiated preadipocytes enhances the adipose tissue formation compared with implantation of undifferentiated preadipocytes. We also investigated whether basic fibroblast growth factor (bFGF) further enhances the adipose tissue formation mediated by the implantation of adipogenic-differentiated preadipocytes. A fibrin matrix containing human preadipocytes cultured in adipogenic differentiation-inducing conditions with (group 1) or without (group 2) bFGF was injected into the subcutaneous spaces of athymic mice. Fibrin matrices containing undifferentiated human preadipocytes with (group 3) or without (group 4) bFGF were also implanted. Six weeks after implantation, the implanted cells formed new tissues in all groups. Importantly, the implantation of adipogenic-differentiated preadipocytes resulted in more extensive adipogenesis than the implantation of undifferentiated preadipocytes, as evaluated by adipose tissue area and human adipocyte-specific gene expression in the newly formed tissues. In addition, bFGF enhanced neovascularization in the newly formed tissues and further enhanced the adipogenesis mediated by the adipogenic-differentiated preadipocytes. The present study demonstrates that the implantation of adipogenic-differentiated preadipocytes enhances adipose tissue regeneration, as compared with the implantation of undifferentiated preadipocytes, and that cell transplantation-mediated adipogenesis can be further enhanced by the delivery of bFGF.

Novel Transgenic Rabbit Model Sheds Light on the Puzzling Role of Matrix Metalloproteinase-12 in Atherosclerosis

Matrix metalloproteinases (MMPs), also termed matrixins, are known to affect atherosclerosis in many diverse ways.1–3 Apart from a role in the progression of atherosclerotic lesions, MMPs have also been involved in plaque destabilization and rupture and in the development of aneurysms. Despite intense research efforts during the last decade, it has been challenging to formulate a unifying model on the specific role of MMPs in atherosclerosis. In fact, mouse genetic studies have yielded controversial insights, highly dependent on genetic background, dietary regimen, and arterial site of analysis, whereas human genetic association studies do not prove causality of MMPs in atherosclerosis. Moreover, the lack of specific inhibitors precluded pharmacological dissection of the role of MMPs in vivo. There is thus a need for alternative strategies to decipher the MMP puzzle in atherosclerosis. In this issue of Circulation , Liang et al4 used a novel approach: They generated transgenic rabbits to study the role of MMP-12, also known as macrophage elastase, in atherosclerosis. Their results indicate that MMP-12 causes media destruction and pseudoaneurysm formation and, more surprisingly, accelerates plaque growth in an animal model that more closely resembles atherosclerosis in humans. Article p 1993 MMPs belong to one of the most ancient families of enzymes that arose in evolution soon after the first signs of life to regulate, in general, interactions of cells with the extracellular matrix (ECM). To cope with this formidable challenge, more than 65 structurally related MMPs developed in bacteria, plants, nematodes, fruit flies, sea urchins, and vertebrates. One of their key activities is breaking down ECM substrates in the basement membrane and interstitial matrix.1,2 As such, MMPs facilitate migration of macrophages and smooth muscle cells (SMCs) in atherosclerotic plaques by degrading ECM barriers, exposing novel ECM adhesion sites, and relieving the binding between basement membrane …

VEGF withdrawal induces vascular remodelling and pruning via intussusception

Once the capillary plexuses have been formed, they undergo a process of intensive remodelling including formation of feeding vessels and adaptation of the vascular branching morphology. We used the chicken chorioallantoic membrane (CAM) and the chick kidney demonstrating the effects of VEGF withdrawal. We observed emergence of remodelling and adaptation by formation of transvascular tissue pillars i.e. intussusception at vascular bifurcations (intussusceptive branching remodelling = IBR). IBR occurred by formation of pillars close to a bifurcation angle. The pillars increased in girth until they merged with the connective tissue in the bifurcation angle. Morphometric investigations demonstrated that IBR resulted in optimisation of the branching geometry such that optimality was augmented by minimization of pumping energy and building material. The formation of multiple eccentric pillars and their subsequent successive fusions meant that IBR was also extensively involved in vascular pruning, a process that results in reduction of the number of vascular branches. Putatively, sub-maintenance doses of VEGF cause vascular remodelling. Application of fibrin matrices containing VEGF121 and 165 on CAM caused a pronounced angiogenic response. Subsequent removal of the matrices resulted in extensive intussusceptive vascular pruning, with a following reduction in the number of small arteries and veins. Local application of VEGF inhibitors, induced similar changes in the CAM. Relation between VEGF withdrawal and intussusceptive pruning was also found in the developing kidney. Disappearance of the pro- and mesonephrons is related to the VEGF down regulation followed by massive intussusceptive pruning. We conclude that intussusception plays a crucial role in vascular tree remodelling and pruning in response to angiogenic factors and changing hemodynamic conditions. Funded by SNF 3100A0-104000/1

Confocal reflection imaging of 3D fibrin polymers

The reconstruction of extracellular matrix (ECM) by confocal reflection microscopy is a physical approach for monitoring the ECM structure, cell–matrix interactions and changes of ECM structure over time. We here show that confocal reflection imaging is useful in reconstructing the fibrillar architecture of 3D fibrin lattices and fibroblasts embedded therein at high resolution up to 250 nm. Together with confocal fluorescence microscopy of collagen deposited by fibroblasts, this technique allows the monitoring of fibrin remodeling by stromal cells. In conclusion, confocal reflection microscopy is a valuable technique for real-time monitoring of the remodeling of fibrin matrices, such as during thrombus formation, wound healing, and tumor formation.

Peptide-matrix-mediated gene transfer of an oxygen-insensitive hypoxia-inducible factor-1alpha variant for local induction of angiogenesis.

Hypoxia-inducible factor (HIF) constitutes a target in therapeutic angiogenesis. HIF-1alpha functions as a sensor of hypoxia and induces expression of vascular endothelial growth factor (VEGF), which then induces angiogenesis. To explore the potential of HIF-1alpha gene therapy in stimulating wound healing, we delivered a gene encoding a stabilized form of HIF-1alpha, lacking the oxygen-sensitive degradation domain, namely HIF-1alpha deltaODD, by using a previously characterized peptide-based gene delivery vector in fibrin as a surgical matrix. The peptide vector consisted of multiple domains: (i) A cysteine-flanked lysine hexamer provided DNA interactions that were stable extracellularly but destabilized intracellularly after reduction of the formed disulfide bonds. This DNA-binding domain was fused to either (ii) a fibrin-binding peptide for entrapment within the matrix or (iii) a nuclear localization sequence for efficient nuclear targeting. The HIF-1alpha deltaODD gene was expressed and translocated to the nucleus under normoxic conditions, leading to up-regulation of vascular endothelial growth factor (VEGF)-A165 mRNA and protein levels in vitro. When the peptide-DNA nanoparticles entrapped in fibrin matrices were applied to full-thickness dermal wounds in the mouse (10 microg per wound in 30 microl of fibrin), angiogenesis was increased comparably strongly to that induced by VEGF-A165 protein (1.25 microg per wound in 30 microl of fibrin). However, the maturity of the vessels induced by HIF-1alpha deltaODD was significantly higher than that induced by VEGF-A165 protein, as shown by stabilization of the neovessels with smooth muscle. Nonviral, local administration of this potent angiogenesis-inducing gene by using this peptide vector represents a powerful approach in tissue engineering and therapeutic angiogenesis.

Autologous fibrin matrices: A potential source of biological mediators that modulate tendon cell activities

The use of autologous fibrin matrices has been proposed as a therapeutic strategy for the local and physiological delivery of growth factors in the treatment of several clinical conditions requiring tendon healing or tendon graft remodelling. In the present work, we investigated the proliferation, synthesis of type-I collagen and angiogenic factors by tendon cells seeded on platelet-rich (PR) and platelet-poor (PP) matrices. Furthermore, in vivo cellular and vascular effects of each treatment were examined after infiltration in Achilles tendon in sheep. Results showed that the presence of platelets within the fibrin matrices increased significantly the proliferation of tendon cells. Additionally, cultured tendon cells synthesised type I collagen and angiogenic factors such as VEGF and HGF. The synthesis of VEGF, but not of HGF, was significantly higher when platelets were present within the matrix. In the sheep model, the injection of pre-clotted plasma within tendons increased cellular density and promoted neovascularization. These results indicate that administration of fibrin matrices is a safe and easy strategy that may open new avenues for enhancing tissue healing and remodelling and influences the process of regeneration in clinical situations characterised by a poor healing outcome.

Hemostatic Factors Regulate Immune Surveillance and Contribute to the Rapid Clearance of Bacterial Pathogens In Vivo.

The local activation of coagulation system proteases and secondary deposition of fibrin may constitute important “danger” or “damage” signals that promote leukocyte activation events at sites of infection or injury. Consistent with this general hypothesis, we previously showed that mice expressing a mutant form of fibrinogen lacking the leukocyte integrin receptor αMβ2-binding motif displayed compromised clearance of bacteria in a model of S. aureus-induced peritonitis. Here, we show that mice with a complete genetic deficiency in fibrinogen (Fib−/−) show an even more profound sensitivity to S. aureus peritonitis. Fib−/− mice were found to succumb to bacterial doses that were rarely fatal in wildtype mice following intraperitoneal injection of wildtype S. aureus. This profound difference in survival profile was also seen when mice were challenged with S. aureus lacking either the bacterial fibrinogen-receptor (ClfA) or the bacterial prothrombin activator, coagulase. These results indicate that efficient bacterial clearance in control animals is related to host leukocyte engagement of fibrin(ogen) as opposed to bacterial engagement of this hemostatic factor. Following intraperitoneal inoculation with one billion CFU of S. aureus, control mice successful cleared >99% of the bacteria within 1 hour, whereas the number of bacteria retrieved within peritoneal lavage fluid of Fib−/− mice remained similar to the original input CFU. Microscopy studies and bacterial fate studies revealed that the rapid loss of viable bacteria in control animals was not due to either fibrinogen-dependent dissemination to other tissues nor the simple association of live bacteria with peritoneal surfaces. Neutrophils and macrophages appear to participate in early bacterial clearance in animals of both genotypes, but these cells appear to be overwhelmed and engorged with microbes in Fib-/− mice. Leukocyte composition and trafficking into the peritoneal cavity was not appreciably altered in fibrin(ogen)-deficient mice. Furthermore, B cells, T cells, mast cells and complement factors C3 and C5 were not found to be major factors in the fibrinogen-dependent bacterial clearance. Taken together, the available data indicate that a failure to fully implement leukocyte-mediated antimicrobial mechanisms is the primary impediment to bacterial clearance in Fib−/− mice. The findings are consistent with the hypothesis that leukocyte engagement of provisional fibrin matrices within challenged tissues is a critical parameter in leukocyte “target recognition” in vivo.