Enrica Briganti

Fibrin-based scaffold incorporating VEGF- and bFGF-loaded nanoparticles stimulates wound healing in diabetic mice

Diabetic skin ulcers are difficult to heal spontaneously due to the reduced levels and activity of endogenous growth factors. Recombinant human vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are known to stimulate cell proliferation and accelerate wound healing. Direct delivery of VEGF and bFGF at the wound site in a sustained and controllable way without loss of bioactivity would enhance their biological effects. The aim of this study was to develop a poly(ether)urethane-polydimethylsiloxane/fibrin-based scaffold containing poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with VEGF and bFGF (scaffold/GF-loaded NPs) and to evaluate its wound healing properties in genetically diabetic mice (db/db). The scaffold application on full-thickness dorsal skin wounds significantly accelerated wound closure at day 15 compared to scaffolds without growth factors (control scaffold) or containing unloaded PLGA nanoparticles (scaffold/unloaded NPs). However, the closure rate was similar to that observed in mice treated with scaffolds containing free VEGF and bFGF (scaffold/GFs). Both scaffolds containing growth factors induced complete re-epithelialization, with enhanced granulation tissue formation/maturity and collagen deposition compared to the other groups, as revealed by histological analysis. The ability of the scaffold/GF-loaded NPs to promote wound healing in a diabetic mouse model suggests its potential use as a dressing in patients with diabetic foot ulcers.

Long term performance of small-diameter vascular grafts made of a poly(ether)urethane-polydimethylsiloxane semi-interpenetrating polymeric network.

In the past years considerable research efforts have been directed at developing more suitable synthetic vascular grafts, but small-diameter vascular grafts (SDVGs) perform less well than autogenous arterial or venous grafts. Grafts such as Dacron and ePTFE have often been used as alternatives to autologous grafts, but they have shown poor patency rates when used in small-diameter sizes or low-flow locations. Nevertheless, despite these efforts no alternative concepts have emerged yet that promises to replace the current generation of synthetic grafts soon. The purpose of this preliminary in vivo study was to assess the blood and tissue compatibility behaviors of a novel compliant SDVGs, fabricated with a poly(ether)urethane-polydimethylsiloxane (PEtU-PDMS) semi-interpenetrating polymeric network (semi-IPN) and featuring two different porous layers in the wall thickness. Grafts were implanted according to anastomotic techniques which emulate the flow conditions clinically adopted for peripheral or aorto-coronary bypass procedures. Relatively long grafts were implanted in the common carotid artery of adult sheep and compared to standard ePTFE grafts of the same size and length implanted controlaterally. The animal experimentation showed superior handling and compliance characteristics, and patency rates of PEtU-PDMS grafts in comparison with a standard ePTFE graft, and the ability of remodelling in vivo while being gradually replaced by a natural tissue with no sign of calcification.

Glubran2 Surgical Glue: In Vitro Evaluation of Adhesive and Mechanical Properties

BACKGROUND In surgical and endoscopic procedures, tissue adhesives are commonly used as reinforcement of sutures or as bonding and hemostatic agents. Fibrin glues do not guarantee adequate properties for many clinical applications; on the contrary, cyanoacrylate glues guarantee high bonding strength between biologic tissues. The aim of this study was to provide evidence regarding adhesive and strength properties of a widely used cyanoacrylate glue, Glubran2, GEM s.r.l., Viareggio, Italy. Comparative tests were also carried out on a commercial fibrin glue. MATERIAL AND METHODS Glubran2 is a modified n-butyl-2-cyanoacrylate glue approved for internal and external use, in Europe. The glue, on contact with living tissues polymerizes rapidly, generating a film that guarantees firm adherence of tissues. In this study, adhesive properties on biologic substrates, both of Glubran2 and of fibrin glue, were investigated according to American Society for Testing and Materials (ASTM) standards, while their strength, after polymerization on an inert substrate, was investigated according to Deutsches Institut Für Normung (DIN) standards. RESULTS All tests evidenced a strong bonding capability of Glubran2 on biologic tissues and high tensile strength of polymerized film; high breaking strength of polymerized glue was highlighted by tensile tests. CONCLUSION The present study fills the gap concerning Glubran2 adhesive and tensile properties. All tests showed the intrinsic tensile strength of polymerized Glubran2 and its capability to realize a higher-resistance bonding among biologic tissues, in comparison with fibrin glue, giving strong indication of its usefulness in surgical and endoscopic practice, especially in a wet environment.

A composite fibrin-based scaffold for controlled delivery of bioactive pro-angiogenetic growth factors

The aim of this study was to fabricate and characterize in vitro a novel composite scaffold that, combining good mechanical properties with a controlled and sustained release of bioactive pro-angiogenetic growth factors, should be useful for angiogenesis induction in organs/tissues in which is also necessary to give resistance and mechanical strength. Composite scaffolds, constituted by a synthetic biocompatible material, a poly(ether)urethane-polydimethylsiloxane blend, and a biological polymer, the fibrin, were manufactured by spray, phase-inversion technique. During the manufacturing process heparin and heparin-binding growth factors, such as VEGF(165) and bFGF, were incorporated into the fibrin layer. Microscopical examinations showed a homogeneous fibrin layer firmly adherent on top of the synthetic material. Tensile tests highlighted the high elasticity of the composite scaffold and its capability to maintain integrity up to high deformation. VEGF(165) and bFGF release were controlled by fibrinogen concentration, whereas it was not affected by heparin concentration, as revealed by ELISA assay. The biological activity of the released growth factors was maintained as demonstrated by HUVEC proliferation. Finally, scaffolds induced a low monocyte mRNA expression of inflammatory markers (IL-8, L-SEL, LFA-1 and iNOS). In conclusion, the new composite scaffolds, once implanted, providing a co-localization and temporal distribution of bioactive VEGF and bFGF in addition to good mechanical properties, may be useful to stimulate new vessels formation in ischemic tissues.

Tissue response to poly(ether)urethane-polydimethylsiloxane-fibrin composite scaffolds for controlled delivery of pro-angiogenic growth factors

The development of a scaffold able to mimic the mechanical properties of elastic tissues and to induce local angiogenesis by controlled release of angiogenic growth factors could be applied in the treatment of several ischemic diseases. For this purpose a composite scaffold made of a poly(ether)urethane-polydimethylsiloxane (PEtU-PDMS) semi-interpenetrating polymeric network (semi-IPN) and fibrin loaded growth factors (GFs), such as VEGF and bFGF, was manufactured using spray, phase-inversion technique. To evaluate the contribution of each scaffold component with respect to tissue response and in particular to blood vessel formation, three different scaffold formulations were developed as follows: 1) bare PEtU-PDMS; 2) PEtU-PDMS/Fibrin; and 3) PEtU-PDMS/Fibrin + GFs. Scaffolds were characterized in vitro respect to their morphology, VEGF and bFGF release kinetics and bioactivity. The induction of in vivo angiogenesis after subcutaneous and ischemic hind limb scaffold implantation in adult Wistar rats was evaluated at 7 and 14 days by immunohistological analysis (IHA), while Laser Doppler Perfusion Imaging (LDPI) was performed in the hind limbs at 0, 3, 7, 10 and 14 days. IHA of subcutaneously implanted samples showed that at 7 and 14 days the PEtU-PDMS/Fibrin + GFs scaffold induced a statistically significant increase in number of capillaries compared to bare PEtU-PDMS scaffold. IHA of ischemic hind limb showed that at 14 days the capillary number induced by PEtU-PDMS/Fibrin + GFs scaffolds was higher than that of PEtU-PDMS/Fibrin scaffolds. Moreover, at both time-points PEtU-PDMS/Fibrin scaffolds induced a significant increase in number of capillaries compared to bare PEtU-PDMS scaffolds. LDPI showed that at 10 and 14 days the ischemic/non-ischemic blood perfusion ratio was significantly greater in the PEtU-PDMS/Fibrin + GFs than in the other scaffolds. In conclusion, this study showed that the semi-IPN composite scaffold acting as a pro-angiogenic GFs delivery system has therapeutic potential for the local treatment of ischemic tissue and wound healing.

Effect of Platelet Lysate on Human Cells Involved in Different Phases of Wound Healing

Background Platelets are rich in mediators able to positively affect cell activity in wound healing. Aim of this study was to characterize the effect of different concentrations of human pooled allogeneic platelet lysate on human cells involved in the different phases of wound healing (inflammatory phase, angiogenesis, extracellular matrix secretion and epithelialization). Methodology/Principal Findings Platelet lysate effect was studied on endothelial cells, monocytes, fibroblasts and keratinocytes, in terms of viability and proliferation, migration, angiogenesis, tissue repair pathway activation (ERK1/2) and inflammatory response evaluation (NFκB). Results were compared both with basal medium and with a positive control containing serum and growth factors. Platelet lysate induced viability and proliferation at the highest concentrations tested (10% and 20% v/v). Whereas both platelet lysate concentrations increased cell migration, only 20% platelet lysate was able to significantly promote angiogenic activity (p<0.05 vs. control), comparably to the positive control. Both platelet lysate concentrations activated important inflammatory pathways such as ERK1/2 and NFκB with the same early kinetics, whereas the effect was different for later time-points. Conclusion/Significance These data suggest the possibility of using allogeneic platelet lysate as both an alternative to growth factors commonly used for cell culture and as a tool for clinical regenerative application for wound healing.

A combined synthetic-fibrin scaffold supports growth and cardiomyogenic commitment of human placental derived stem cells.

Aims A potential therapy for myocardial infarction is to deliver isolated stem cells to the infarcted site. A key issue with this therapy is to have at ones disposal a suitable cell delivery system which, besides being able to support cell proliferation and differentiation, may also provide handling and elastic properties which do not affect cardiac contractile function. In this study an elastic scaffold, obtained combining a poly(ether)urethane-polydimethylsiloxane (PEtU-PDMS) semi-interpenetrating polymeric network (s-IPN) with fibrin, was used as a substrate for in vitro studies of human amniotic mesenchymal stromal cells (hAMSC) growth and differentiation. Methodology/Principal Findings After hAMSC seeding on the fibrin side of the scaffold, cell metabolic activity and proliferation were evaluated by WST-1 and bromodeoxyuridine assays. Morphological changes and mRNAs expression for cardiac differentiation markers in the hAMSCs were examined using immunofluorescence and RT-PCR analysis. The beginning of cardiomyogenic commitment of hAMSCs grown on the scaffold was induced, for the first time in this cell population, by a nitric oxide (NO) treatment. Following NO treatment hAMSCs show morphological changes, an increase of the messenger cardiac differentiation markers [troponin I (TnI) and NK2 transcription factor related locus 5 (Nkx2.5)] and a modulation of the endothelial markers [vascular endothelial growth factor (VEGF) and kinase insert domain receptor (KDR)]. Conclusions/Significance The results of this study suggest that the s-IPN PEtU-PDMS/fibrin combined scaffold allows a better proliferation and metabolic activity of hAMSCs cultured up to 14 days, compared to the ones grown on plastic dishes. In addition, the combined scaffold sustains the beginning of hAMSCs differentiation process towards a cardiomyogenic lineage.

The effect of gamma irradiation on physical–mechanical properties and cytotoxicity of polyurethane–polydimethylsiloxane microfibrillar vascular grafts

Poly(ether) urethane (PEtU)–polydimethylsiloxane (PDMS) based materials have been processed by a spray, phase-inversion technique to produce microfibrillar small-diameter vascular grafts; however the effect of sterilization upon these grafts is still unknown. This study investigated the effect of gamma irradiation on grafts made of PEtU–PDMS materials containing different PDMS concentrations. Sterilisation-induced changes in surface chemical structure and morphology were assessed by infrared spectroscopy, light and scanning electron microscopy. Tensile tests were used to examine changes in mechanical properties and the cytotoxicity evaluation was performed on L929 fibroblasts. The study demonstrated that physical–chemical and mechanical properties of PEtU–PDMS grafts, at each PDMS concentration, were not significantly affected by the exposure to gamma irradiation, moreover no sign of cytotoxicity was observed after sterilisation. Although in vitro experiments have been promising, further in vivo studies are necessary to evaluate the biodegradation behaviour of PEtU–PDMS graft after gamma irradiation, before any clinical application.

Oligonucleotide biofunctionalization enhances endothelial progenitor cell adhesion on cobalt/chromium stents.

As the endothelium still represents the ideal surface for cardiovascular devices, different endothelialization strategies have been attempted for biocompatibility and nonthrombogenicity enhancement. Since endothelial progenitor cells (EPCs) could accelerate endothelialization, preventing thrombosis and restenosis, the aim of this study was to use oligonucleotides (ONs) to biofunctionalize stents for EPC binding. In order to optimize the functionalization procedure before its application to cobalt-chromium (Co/Cr) stents, discs of the same material were preliminarily used. Surface aminosilanization was assessed by infrared spectroscopy and scanning electron microscopy. A fluorescent endothelial-specific ON was immobilized on aminosilanized surfaces and its presence was visualized by confocal microscopy. Fluorescent ON binding to porcine blood EPCs was assessed by flow cytometry. Viability assay was performed on EPCs cultured on unmodified, nontargeting ON or specific ON-coated discs; fluorescent staining of nuclei and F-actin was then performed on EPCs cultured on unmodified or specific ON-coated discs and stents. Disc biofunctionalization significantly increased EPC viability as compared to both unmodified and nontargeting ON-coated surfaces; cell adhesion was also significantly increased. Stents were successfully functionalized with the specific ON, and EPC binding was confirmed by confocal microscopy. In conclusion, stent biofunctionalization for EPC binding was successfully achieved in vitro, suggesting its use to obtain in vivo endothelialization, exploiting the natural regenerative potential of the human body.

Healing effect of a fibrin-based scaffold loaded with platelet lysate in full-thickness skin wounds:

Chronic skin lesions are difficult to heal due to reduced levels and activity of endogenous growth factors. The platelet lysate, obtained by repeated freeze–thawing of platelet-enriched blood samples, is an easily attainable source of a wide range of growth factors and bioactive mediators involved in tissue repair. In this study, a bio-synthetic scaffold composed of poly(ether)urethane–polydimethylsiloxane material and fibrin was developed for platelet lysate delivery to chronic skin wounds. The kinetics release and the bioactivity of growth factors released from platelet lysate–loaded poly(ether)urethane–polydimethylsiloxane–fibrin scaffold were investigated, respectively, by enzyme-linked immunosorbent assay and a cell proliferation test using human fibroblasts. The in vitro experiments demonstrated that the platelet lysate–loaded poly(ether)urethane–polydimethylsiloxane–fibrin scaffold provides a sustained release of platelet derived growth factors. The cell growth in the presence of scaffold was comparable to those observed for the platelet lysate added to culture medium in free form, showing that the scaffold preparation process did not affect biological activity of growth factors. The effect of platelet lysate–loaded poly(ether)urethane–polydimethylsiloxane–fibrin scaffold on wound healing in genetically diabetic mouse (db/db) was also investigated. The application of the scaffold on full-thickness skin wounds significantly accelerated wound closure at day 15 post-surgery compared with control poly(ether)urethane–polydimethylsiloxane–fibrin scaffold (without platelet lysate) or a commercially available polyurethane film dressing. Histological analysis demonstrated an increased re-epithelialization, granulation tissue formation, and collagen deposition. The ability of the platelet lysate–loaded poly(ether)urethane–polydimethylsiloxane–fibrin scaffold to promote wound healing in vivo through simultaneous delivery of multiple active substances suggests its potential use for the treatment of diabetic foot ulcers.