Paola Losi

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.

Evaluation of the Effect of a Gamma Irradiated DBM-Pluronic F127 Composite on Bone Regeneration in Wistar Rat

Demineralized bone matrix (DBM) is widely used for bone regeneration. Since DBM is prepared in powder form its handling properties are not optimal and limit the clinical use of this material. Various synthetic and biological carriers have been used to enhance the DBM handling. In this study we evaluated the effect of gamma irradiation on the physical-chemical properties of Pluronic and on bone morphogenetic proteins (BMPs) amount in DBM samples. In vivo studies were carried out to investigate the effect on bone regeneration of a gamma irradiated DBM-Pluronic F127 (DBM-PF127) composite implanted in the femur of rats. Gamma irradiation effects (25 kGy) on physical-chemical properties of Pluronic F127 were investigated by rheological and infrared analysis. The BMP-2/BMP-7 amount after DBM irradiation was evaluated by ELISA. Bone regeneration capacity of DBM-PF127 containing 40% (w/w) of DBM was investigated in transcortical holes created in the femoral diaphysis of Wistar rat. Bone porosity, repaired bone volume and tissue organization were evaluated at 15, 30 and 90 days by Micro-CT and histological analysis. The results showed that gamma irradiation did not induce significant modification on physical-chemical properties of Pluronic, while a decrease in BMP-2/BMP-7 amount was evidenced in sterilized DBM. Micro-CT and histological evaluation at day 15 post-implantation revealed an interconnected trabeculae network in medullar cavity and cellular infiltration and vascularization of DBM-PF127 residue. In contrast a large rate of not connected trabeculae was observed in Pluronic filled and unfilled defects. At 30 and 90 days the DBM-PF127 samples shown comparable results in term of density and thickness of the new formed tissue respect to unfilled defect. In conclusion a gamma irradiated DBM-PF127 composite, although it may have undergone a significant decrease in the concentration of BMPs, was able to maintains bone regeneration capability.

Chemical-physical and in vivo evaluations of a self-assembling amphiphilic peptide as an injectable hydrogel scaffold for biomedical applications

The self-aggregation and gelation of an amphiphilic peptide (C17H35CONH–A4G3ERGD, peptide amphiphile) were studied by light scattering, viscometry, nuclear magnetic resonance diffusometry, and atomic force microscopy. The peptide amphiphile critical aggregation concentration was evaluated to be 16 and 60 µM by light scattering and viscometry, respectively. The observed difference was attributed to the larger sensitivity of the latter technique to the presence of long fibrils. The addition of one equivalent or more of divalent cations (Ca2+ and Mg2+) to peptide amphiphile formed dense incoherent hydrogels. Based on the atomic force microscopy and nanoindentation data, both the hydrogel morphology and stiffness were independent of the cation type and peptide amphiphile concentration. However, gel stiffness increased on increasing Ca2+/peptide amphiphile molar ratio while a parallel decrease in the apparent water diffusion rate was observed by nuclear magnetic resonance diffusometry. The dispersions of endothelial progenitor cells in the peptide amphiphile hydrogels were evaluated in vivo on a rat tissue hypoxia model. Significant capillary formation at the injection site was observed by tissue appearance and histological examination, which indicated endothelial progenitor cell/peptide amphiphile hydrogel-enhanced angiogenesis in ischemic tissue.

Design and characterization of a composite material based on Sr(II)-loaded clay nanotubes included within a biopolymer matrix

This paper reports on the preparation, characterization, and cytotoxicity of a hybrid nanocomposite material made of Sr(II)-loaded Halloysite nanotubes included within a biopolymer (3-polyhydroxybutyrate-co-3-hydroxyvalerate) matrix. The Sr(II)-loaded inorganic scaffold is intended to provide mechanical resistance, multi-scale porosity, and to favor the in-situ regeneration of bone tissue thanks to its biocompatibility and bioactivity. The interaction of the hybrid system with the physiological environment is mediated by the biopolymer coating, which acts as a binder, as well as a diffusional barrier to the Sr(II) release. The degradation of the polymer progressively leads to the exposure of the Sr(II)-loaded Halloysite scaffold, tuning its interaction with osteogenic cells. The in vitro biocompatibility of the composite was demonstrated by cytotoxicity tests on L929 fibroblast cells. The results indicate that this composite material could be of interest for multiple strategies in the field of bone tissue engineering.