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.

Epitope mapping of neutralizing TSST-1 specific antibodies induced by immunization with toxin or toxoids.

Toxic shock syndrome toxin-1 (TSST-1), a superantigen produced by Staphylococcus aureus, is a potent stimulator of the immune system. T-cells are activated by crosslinking of MHC class II molecules on antigen presenting cells with T-cell receptors (TCR). TSST-1 is associated with the majority of the cases of menstrual staphylococcal toxic shock, a severe and life-threatening multisystem disorder. Even though antibody mediated protection has been studied, information on antibody specificity directed to individual antigenic determinants of the protein is incomplete. To obtain immunogens with low toxicity, we generated a double-site mutant (dmTSST-1), modified at solvent-exposed residues predicted to be important for both MHC class II and TCR binding, and detoxified recombinantly expressed TSST-1 (rTSST-1) as well as native TSST-1 (nTSST-1) isolated from Staphylococcus aureus by treatment with formaldehyde. Rabbits were immunized with rTSST-1, nTSST-1, dmTSST-1, and formaldehyde inactivated toxoids. The sera obtained were used to map the antigen-reactive regions of the molecule and to identify specificities of antibodies induced by immunization with the different antigens. To detect linear antigenic epitopes of TSST-1 the reactivity of the sera with 11-meric peptides having an overhang of four residues, covering the entire molecule of TSST-1, have been studied. We found that sera of TSST-1 immunized rabbits predominantly reacted with N-terminal residues 1-15, while sera generated with formaldehyde inactivated toxoid recognized a total of 7 regions located at the N- and C-terminus and internal sites of TSST-1. Despite different specificities all sera were able to inhibit TSST-1 induced proliferation of human mononuclear cells.

Double mutant and formaldehyde inactivated TSST-1 as vaccine candidates for TSST-1-induced toxic shock syndrome.

Up to now there is no treatment for staphylococcal toxic shock syndrome, a disease mainly induced by toxic shock syndrome toxin-1(TSST-1). There is great demand in finding means to control the disease, one of them is the development of an effective and safe vaccine against TSST-1. In this study we constructed a series of vaccine candidates and investigated their biological activity, toxicity, and potential to invoke an immune response. TSST-1 was isolated from Stahylococcus aureus supernatants and recombinantly expressed as a N-terminal 6x histidine-tagged protein in Escherichia coli. In order to obtain molecules with minimal toxicity we constructed single mutants (G31R and H135A) and one double mutant (G31R/H135A) with both residues exchanged. We also detoxified native TSST-1 isolated from S. aureus, and recombinantly expressed TSST-1 by treatment with formaldehyde. Functional activity of native and recombinant TSST-1 and grade of inocuity of mutants and toxoids was determined by investigating mitogenity, T-cell activation, and cytokine release upon stimulation of human mononuclear cells with the vaccine candidates. All substances were tested in a rabbit immunization study. After primary immunization and three additional boosts all vaccinated animals developed antibody titers against TSST-1 and were protected against challenge with a lethal doses of superantigen potentiated with lipopolysaccharide.