Development of an antibacterial nanocomposite hydrogel for human dental pulp engineering.

Hydrogel-based regenerative endodontic procedures (REPs) are considered to be very promising therapeutic strategies to reconstruct the dental pulp (DP) tissue in devitalized human teeth. However, the success of the regeneration process is limited by residual bacteria that may persist in the endodontic space after the disinfection step and contaminate the biomaterial. The aim of this work was to develop an innovative fibrin hydrogel incorporating clindamycin (CLIN)-loaded Poly (d,l) Lactic Acid (PLA) nanoparticles (NPs) to provide the hydrogel with antibacterial properties. CLIN-PLA-NPs were synthesized by a surfactant-free nanoprecipitation method and their microphysical properties were assessed by dynamic light scattering, electrophoretic mobility and scanning electron microscopy. Their antimicrobial efficacy was evaluated on Enteroccocus fæcalis by the determination of the minimal inhibitory concentration (MIC) and the minimal biofilm inhibition and eradication concentrations (MBIC and MBEC). Antibacterial properties of the nanocomposite hydrogel were verified by agar diffusion assays. NP distribution into the hydrogel and release from it were evaluated using fluorescent PLA-NPs. NP cytotoxicity was assessed on DP mesenchymal stem cells (DP-MSCs) incorporated into the hydrogel. Type I collagen synthesis was investigated after 7 days of culture by immunohistochemistry. We found that CLIN-PLA-NPs displayed a drug loading of 10 ± 2 µg per mg of PLA polymer and an entrapment efficiency of 43 ± 7%. Antibiotic loading did not affect NP size, polydispersity index and zeta potential. The MIC for Enterococcus fæcalis was 32 µg mL-1. MBIC50 and MBEC50 were 4 and 16 µg mL-1, respectively. CLIN-PLA-NPs appeared homogenously distributed throughout the hydrogel. CLIN-PLA-NP-loaded hydrogels clearly inhibited E. faecalis growth. DP-MSC viability and type I collagen synthesis within the fibrin hydrogel were not affected by CLIN-PLA-NPs. In conclusion, CLIN-PLA-NP incorporation into the fibrin hydrogel gave the latter antibacterial and antibiofilm properties without affecting cell viability and function. This formulation could help establish an aseptic environment supporting DP reconstruction and, accordingly, might be a valuable tool for REPs.

Improved mechanical properties by modifying fibrin scaffold with PCL and its biocompatibility evaluation.

Previous studies have proved that fibrin is an excellent scaffold material for tissue engineered blood vessel. However, the mechanical properties of fibrin are not enough. One way to solve the problem is to combine polymer materials with fibrin to enhance its biomechanical properties. In this study, a novel polycaprolactone (PCL)/fibrin composite scaffold was prepared by electrospinning technology. The morphological, physicochemical analysis, blood compatibility, biomechanical properties, biocompatibility and biodegradability of this vascular scaffold were evaluated. The results showed that electrospun PCL/fibrin scaffold possessed smaller aperture and larger fiber diameter than that of fibrin scaffold. The swelling ratio of the vascular PCL/fibrin scaffold at (0:100), (10:90), (20:80) and (30:70) was 112 ± 5.3, 103 ± 6.9, 94 ± 5.9 and 89 ± 3.4%, respectively. Mechanical properties of fibrin scaffolds were enhanced significantly by the addition of PCL. Furthermore, the time of plasma re-calcification, activated partial thromboplastin time and thromboplastin time in four different proportions of PCL/fibrin scaffolds were similar to that of the control group. Degradation experiments in vitro demonstrated that the degradation rate of PCL/fibrin scaffold was closely related to the content of PCL. MTT assays and immunofluorescence staining indicated that the stem cells cocultured with the PCL/fibrin scaffold had good proliferation behavior. Live/dead assay confirmed that the number of MSCs in the PCL/fibrin (10:90) group was significantly increased as compared to other groups. The tests in vivo results showed PCL/fibrin scaffold could promote cell infiltration and tissue regeneration and its degradation in vivo was faster than that of PCL scaffold. In summary, PCL/fibrin (20:80) scaffold exhibited balanced mechanical properties and degradability, as well as good cell compatibility properties; therefore, it was a promising tissue engineering material for vascular graft.

Fibrin gels entrapment of a doxorubicin-containing targeted polycyclodextrin: Evaluation of in vivo antitumor activity in orthotopic models of human neuroblastoma.

In vivo local antitumor activity of fibrin gels (FBGs) loaded with the poly-cyclodextrin oCD-NH2/Dox, compared to free Dox, was evaluated in two mouse orthotopic neuroblastoma (NB) models, after positioning of the releasing devices in the visceral space. FBGs were prepared at the fibrinogen (FG) concentrations of 22 and 40 mg/ml clotted in the presence of 0.81 mM/mg FG Ca2+ and 1.32 U/mg FG thrombin. Our results indicate that FBGs loaded with oCD-NH2/Dox and applied as neoadjuvant loco-regional treatment, show an antitumor activity significantly greater than that displayed by the same FBGs loaded with identical dose of Dox or after free Dox administered intra venous (iv). In particular, FBGs prepared at 40 mg/ml showed a slightly lower antitumor activity, although after their positioning we observed a significant initial reduction of tumor burden lasting for several days after gel implantation. FBGs at 22 mg/ml loaded with oCD-NH2/Dox and applied after tumor removal (adjuvant treatment model) showed a significantly better antitumor activity than the iv administration of free Dox, with 90% tumor regrowth reduction compared to untreated controls. In all cases the weight loss post-treatment was limited after gel application, although in the adjuvant treatment the loss of body weight lasted longer than in the other treatment modality. In accordance with our recent published data on the low local toxic effects of FBGs, the present findings also underline an increase of the therapeutic index of Dox when locally administered through FBGs loaded with the oCD-NH2/Dox complex.

An Enzyme from Aristolochia indica Destabilizes Fibrin-ß Amyloid Co-Aggregate: Implication in Cerebrovascular Diseases.

Fibrinogen and ß-amyloid (Aß) peptide independently form ordered aggregates but in combination, they form disordered structures which are resistant to fibrinolytic enzymes like plasmin and cause severity in cerebral amyloid angiopathy (CAA). A novel enzyme of 31.3 kDa has been isolated from the root of the medicinal plant Aristolochia indica that showed fibrinolytic as well as fibrin-Aß co-aggregate destabilizing properties. This enzyme is functionally distinct from plasmin. Thrombolytic action of the enzyme was demonstrated in rat model. The potency of the plant enzyme in degrading fibrin and fibrin-plasma protein (Aß, human serum albumin, lysozyme, transthyretin and fibronectin) co-aggregates was demonstrated by atomic force microscopy, scanning electron microscopy and confocal microscopy that showed better potency of the plant enzyme as compared to plasmin. Moreover, the plant enzyme inhibited localization of the co-aggregate inside SH-SY5Y human neuroblastoma cells and also co-aggregate induced cytotoxicity. Plasmin was inefficient in this respect. In the background of limited options for fragmentation of these co-aggregates, the plant enzyme may appear as a potential proteolytic enzyme.

Physiologically clotted fibrin--preparation and characterization for tissue engineering and drug delivery applications.

Fibrin used for biomedical applications is prepared by mixing concentrated solutions of fibrinogen and thrombin in presence of cross-linking agents such as Factor XIII or glutaraldehyde. The main drawbacks associated with this procedure include cost, complexity and time required for fibrin preparation. Hence, present study deals with the characterization of physiologically clotted fibrin (PF) for bone tissue engineering and drug delivery applications. For this the physico-chemical properties of PF were compared with those of the conventionally prepared fibrin (CF). Further MTT and haemolytic assays were performed for both PF and CF to compare their biocompatibility. The amount of alkaline phosphatase produced and calcium secreted by MG-63 cells in the presence of PF and CF were used to relate the osteogenic potency of PF with that of CF. Gallic acid, an anti-cancer drug was loaded within PF and CF and their role in drug delivery was compared.

A role for L-glutamate ionotropic receptors in the development of rat neurogenic pulmonary edema.

The present study was undertaken to evaluate possible roles of L-glutamate ionotropic receptors in neurogenic pulmonary edema. Perfusion of L-glutamate into the fourth ventricles of rats increased nitric oxide (NO) signals in the efflux solution concentration-dependently, significantly reducing both the occurrence and severity of neurogenic pulmonary edema. This effect was completely reversed by prior intracisternal injection of an NO synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME), or an N-methyl-D-aspartate (NMDA) receptor antagonist, dizocilpine maleate (MK-801), and partially by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a 2-amino-3-hydroxy-5-methyl-4-isoxazol propionic acid (AMPA)/kainic acid receptor antagonist. Administration of MK-801 or CNQX alone, without L-glutamate, almost completely prevented neurogenic pulmonary edema development. These results suggest that endogenous L-glutamate may facilitate underlining disease process, whereas L-glutamate exogenously applied into the fourth ventricle may have an inhibitory action via release of NO, through ionotropic receptors.

New experimental models for the in vitro reconstitution of human bladder mucosa.

This study describes two experimental models for the in vitro reconstitution of the human bladder mucosa (neo-bladder): human urothelial stabilized cell lines were cultured on three-dimensional matrices, collagen or platelet-fibrin gels, containing murine fibroblast 3T3-J2 cells. Low-density seeding (2x10(4) cells/ml) of both normal (TCA-48) and neoplastic cell lines (TCA-47) on collagen matrix gave rise to isolated papillar colonies, while high-density seeding (3.75x10(6) cells/ml) led to the formation of wide pluristratified epithelial sheets, resembling the normal transitional epithelium. In contrast, high-density seeding (5x10(5) cells/ml) on platelet-fibrin matrix did not allow the formation of epithelial sheets: only isolated voluminous colonies of normal TCA-48 cells, and sparse and small colonies of neoplastic TCA-47 could be observed. Growth assays and cytotoxicity reduction tests showed that the growth inhibitory effect of platelet-fibrin gel on urothelial cells was probably due to the aspecific activation of the complement contained in the plasmatic fraction, whose precipitation forms fibrin-glue. Collectively, these findings allow us to draw the following conclusions: i) neobladders obtained by culturing urothelial cells on collagen matrix reproduce normal bladder mucosa and could be utilized in pharmacological studies; and ii) platelet-fibrin gels, that specifically inhibit neoplastic urothelial cell growth, could be used as scaffolds in surgical bladder reconstitution.

Topical application of fibrin adhesive in the rat brain: effects on different cellular elements of the wound.

Although fibrin adhesives are popular in the field of neurosurgery, the medical literature is devoid of study elucidating their effects on the brain tissue. To study the safety of applying fibrin glue to the brain and to explore new surgical potentialities, we implanted soft pellets made of fibrin glue into the brains of Wistar rat. Following 6 h and 3, 7 and 14 days post-implantation survival, the brains were removed and paraffin sections were processed for hematoxylin-eosin staining, as well as immunohistochemistry for microtubule-associated protein (MAP-1A) and glial fibrillary acidic protein. The changes in the neuronal and glial elements and also the numbers of inflammatory and endothelial cells in the vicinity of implanted fibrin glue pellets were compared with those of gelfilm pellets. The results demonstrated that topical application of fibrin glue to the brain causes significantly enhanced local accumulation of mononuclear cells and promoted angiogenesis close to the wound while not affecting the neuronal and glial elements. These findings suggest that fibrin glue can be considered as a safe supportive material for intradural procedures directly involving the brain tissue.

Mediastinal fibrin glue: hemostatic effect and tissue response in calves.

There is continued controversy regarding the effectiveness and potential adverse effects of fibrin glue. Thus, we chose to evaluate it in a model of experimental calf aortic valve replacement that has been previously well established. Concentrated fibrinogen and topical thrombin were sprayed to form a thin layer of fibrin glue over the mediastinal tissues of 20 consecutive calves undergoing aortic valve replacement. Chest tube outputs of these animals were compared with those of the preceding 20 consecutive calves undergoing aortic valve replacement without fibrin glue. All procedures were performed by the same surgeon, and no other technical changes were made between the two series. Total postoperative chest tube output (mean +/- standard error) was 553 +/- 50 mL for the calves treated with fibrin glue and 1,155 +/- 103 mL for the control calves (p less than 0.001). On histological examination of mediastinal tissues from 5 treated calves killed 6 weeks after operation, there was no evidence of inflammation, fibrosis, or residual fibrin. To our knowledge, this is the first controlled laboratory study to show that fibrin glue spray is an effective hemostatic agent and that it produces no long-term tissue reaction.