3d collagen hydrogel promotes in vitro langerhans islets vascularization through ad-mvfs angiogenic activity

Adipose derived microvascular fragments (ad-MVFs) consist of effective vascularization units able to reassemble into efficient microvascular networks. Because of their content in stem cells and related angiogenic activity, ad-MVFs represent an interesting tool for applications in regenerative medicine. Here we show that gentle dissociation of rat adipose tissue provides a mixture of ad-MVFs with a length distribution ranging from 33–955 µm that are able to maintain their original morphology. The isolated units of ad-MVFs that resulted were able to activate transcriptional switching toward angiogenesis, forming tubes, branches, and entire capillary networks when cultured in 3D collagen type-I hydrogel. The proper involvement of metalloproteases (MMP2/MMP9) and serine proteases in basal lamina and extracellular matrix ECM degradation during the angiogenesis were concurrently assessed by the evaluation of alpha-smooth muscle actin (αSMA) expression. These results suggest that collagen type-I hydrogel provides an adequate 3D environment supporting the activation of the vascularization process. As a proof of concept, we exploited 3D collagen hydrogel for the setting of ad-MVF–islet of Langerhans coculture to improve the islets vascularization. Our results suggest potential employment of the proposed in vitro system for regenerative medicine applications, such as the improving of the islet of Langerhans engraftment before transplantation

Neural Crest-Derived Chondrocytes Isolation for Tissue Engineering in Regenerative Medicine

Chondrocyte transplantation has been successfully tested and proposed as a clinical procedure aiming to repair articular cartilage defects. However, the isolation of chondrocytes and the optimization of the enzymatic digestion process, as well as their successful in vitro expansion, remain the main challenges in cartilage tissue engineering. In order to address these issues, we investigated the performance of recombinant collagenases in tissue dissociation assays with the aim of isolating chondrocytes from bovine nasal cartilage in order to establish the optimal enzyme blend to ensure the best outcomes of the overall procedure. We show, for the first time, that collagenase H activity alone is required for effective cartilage digestion, resulting in an improvement in the yield of viable cells. The extracted chondrocytes proved able to grow and activate differentiation/dedifferentiation programs, as assessed by morphological and gene expression analyses

Nanoformulations based on collagenases loaded into halloysite/Veegum® clay minerals for potential pharmaceutical applications

The design and development of nanomaterials capable of penetrate cancer cells is fundamental when anticancer therapy is involved. The use of collagenase (Col) is useful since this enzyme can degrade collagen, mainly present in the tumor extracellular matrix. However, its use is often limited since collagenase suffers from inactivation and short half-life. Use of recombinant ultrapure collagenase or carrier systems for their delivery are among the strategies adopted to increase the enzyme stability. Herein, based on the more stability showed by recombinant enzymes and the possibility to use them in anticancer therapy, we propose a novel strategy to further increase their stability by using halloysite nanotubes (HNTs) as carrier. ColG and ColH were supramolecularly loaded onto HNTs and used as fillers for Veegum gels. The systems could be used for potential local administration of collagenases for solid tumor treatment. All techniques adopted for characterization showed that halloysite interacts with collagenases in different ways depending with the Col considered. Furthermore, the hydrogels showed a very slow release of the collagenases within 24 h. Finally, biological assays were performed by studying the digestion of a type-I collagen matrix highlighting that once released the Col still possessed some activity. Thus we developed carrier systems that could further increase the high recombinant collagenases stability, preventing their inactivation in future in vivo applications for potential local tumor treatment

A Fibrillar Biodegradable Scaffold for Blood Vessels Tissue Engineering

In recent years there has been a growing interest for the development of tubular scaffolds employed to assist the replacement of small blood vessels. Materials designed for this purpose need to be biodegradable, have good mechanical properties and improve cell adhesion, proliferation and differentiation. To obtain biomaterials with these properties, electrospinning seems to be one of the most useful technique. Several biodegradable synthetic polymers or constituents of the extracellular matrix (ECM) have been electrospun showing optimal mechanical properties and biodegradability. However, such polymers are lacking in versatile chemical structure affordable to immobilize growth factors or chemokines. The glycosaminoglycan heparin is able to bind several growth factors like vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) and, when grafted onto the scaffold surface it is able to attract cells thus improving their proliferation and differentiation. Aim of this research was the production and the preliminary in vitro biological characterization of a new biodegradable material, obtained by electrospun a polyaminoacid-graft-polyester copolymer. The electrospun biomaterial has been successfully grafted with heparin exploiting the better chemical reactivity of the polyaminoacid portions of the graft copolymer. Then its morphology has been investigated by scanning electron microscopy (SEM) and the potential biodegradability of the material has been studied until 60 days. Preliminary biological data in vitro, on human endothelial cells, show a good compatibility of the scaffold obtained by electrospinning, with regard to cell adhesion and proliferation. Experiments are in progress to evaluate the effects of heparin on cell differentiation

Minimalism in Radiation Synthesis of Biomedical Functional Nanogels

A scalable, single-step, synthetic approach for the manufacture of biocompatible, functionalized micro- and nanogels is presented. In particular, poly(N-vinyl pyrrolidone)-grafted-(aminopropyl)methacrylamide microgels and nanogels were generated through e-beam irradiation of PVP aqueous solutions in the presence of a primary amino-group-carrying monomer. Particles with different hydrodynamic diameters and surface charge densities were obtained at the variance of the irradiation conditions. Chemical structure was investigated by different spectroscopic techniques. Fluorescent variants were generated through fluorescein isothiocyanate attachment to the primary amino groups grafted to PVP, to both quantify the available functional groups for bioconjugation and follow nanogels localization in cell cultures. Finally, a model protein, bovine serum albumin, was conjugated to the nanogels to demonstrate the attachment of biologically relevant molecules for targeting purposes in drug delivery. The described approach provides a novel strategy to fabricate biohybrid nanogels with a very promising potential in nanomedicine

Mouse Sphingosine Kinase 1a Is Negatively Regulated through Conventional PKC-Dependent Phosphorylation at S373 Residue

Sphingosine kinase is a lipid kinase that converts sphingosine into sphingosine-1-phosphate, an important signaling molecule with intracellular and extracellular functions. Although diverse extracellular stimuli influence cellular sphingosine kinase activity, the molecular mechanisms underlying its regulation remain to be clarified. In this study, we investigated the phosphorylation-dependent regulation of mouse sphingosine kinase (mSK) isoforms 1 and 2. mSK1a was robustly phosphorylated in response to extracellular stimuli such as phorbol ester, whereas mSK2 exhibited a high basal level of phosphorylation in quiescent cells regardless of agonist stimulation. Interestingly, phorbol ester-induced phosphorylation of mSK1a correlated with suppression of its activity. Chemical inhibition of conventional PKCs (cPKCs) abolished mSK1a phosphorylation, while overexpression of PKC alpha, a cPKC isoform, potentiated the phosphorylation, in response to phorbol ester. Furthermore, an in vitro kinase assay showed that PKC alpha directly phosphorylated mSK1a. In addition, phosphopeptide mapping analysis determined that the S373 residue of mSK1a was the only site phosphorylated by cPKC. Interestingly, alanine substitution of S373 made mSK1a refractory to the inhibitory effect of phorbol esters, whereas glutamate substitution of the same residue resulted in a significant reduction in mSK1a activity, suggesting the significant role of this phosphorylation event. Taken together, we propose that mSK1a is negatively regulated through cPKC-dependent phosphorylation at S373 residueopen

Recovery of Bioactive Compounds from Marine Organisms: Focus on the Future Perspectives for Pharmacological, Biomedical and Regenerative Medicine Applications of Marine Collagen

Marine environments cover more than 70% of the Earth's surface and are among the richest and most complex ecosystems. In terms of biodiversity, the ocean represents an important source, still not widely exploited, of bioactive products derived from species of bacteria, plants, and animals. However, global warming, in combination with multiple anthropogenic practices, represents a serious environmental problem that has led to an increase in gelatinous zooplankton, a phenomenon referred to as jellyfish bloom. In recent years, the idea of "sustainable development" has emerged as one of the essential elements of green-economy initiatives; therefore, the marine environment has been re-evaluated and considered an important biological resource. Several bioactive compounds of marine origin are being studied, and among these, marine collagen represents one of the most attractive bio-resources, given its use in various disciplines, such as clinical applications, cosmetics, the food sector, and many other industrial applications. This review aims to provide a current overview of marine collagen applications in the pharmacological and biomedical fields, regenerative medicine, and cell therapy

nCDase and SphK-1 localization in vesicles shed by tumour cells and their biological roles.

Sphingolipid metabolism is a dynamic process resulting in the formation of a number of bioactive metabolites including ceramide, ceramide-1-phosphate, sphingosine e sphingosine-1-phosphate (S1P). (Pyne and Pyne; Biochem. J. 2000; 349:385-402). Following sphingomyelinase activation, sphingomyelin is hydrolyzed to ceramide, which is considered to be an inducer of cell growth arrest, differentiation and apoptosis. (Hannun et. al 1996; Science: 274:1855-1859). Ceramidase catalyzes the deacylation of ceramide to produce a free fatty acid and sphingosine. The enzyme sphingosine kinase (SphK) catalyzes the formation of S1P from sphingosine and ATP (Olivera et al. J.Biol.Chem. 1998; 273:12576- 12583). Two distinct SphK isoforms, SphK1 and SphK2, have been cloned and characterized. (Liu et.al. J.Biol.Chem. 2000; 275: 19513-19520) and recently, alternatively spliced variants of human SphK1 and SphK2, differing in their amino terminal portions, have also been described (Billich et. al J.Biol.Chem. 2003; 278; 47408-47415). SphK1 and SphK2 differ in their relative tissue distribution, sub cellular localization and biochemical activities, consistent with distinct biologic functions for these two enzymes (Saba et al. Circ. Res. 2004; 94:724-734). SphK2 presents a nuclear localization signal sequence and is localized in the cell nucleus (Igarashi et. al. J.Biol.Chem 2003; 278: 46832-46839). SphK1 is primarily localized in the cytosol. PMA and TNF\u3b1 induce the phosphorylation of SphK1 Ser 225, through the activation of MAPK and ERK1/2. Phosphorylation of SphK1results in its plasma membrane localization and in its activation. (Pitson et al. Embo J. 2003; 22: 5491-550). SphK1 is a cell surface-active kinase and an extracellular protein. As several secreted proteins, like for instance FGF-1 and FGF-2, SphK1 molecule lacks a conventional leader secretion signal sequence. The mechanism of its release from the cell occurs via a non classical pathway independent of the endoplasmic reticulum/Golgi system but requiring functional actin dynamics (Ancellin et al J.Biol.Chem. 2002; 277: 6667-6675). SphK1 activity, and therefore production of S1P at the cell periphery and/or in the extracellular medium, was shown to regulate a wide variety of cellular processes, including promotion of cell proliferation, survival and motility (Olivera et al. J.Biol.Chem. 2003; 278: 46452-46460). S1P is an important proangiogenic factor and its ability to promote capillary morphogenesis in endothelial cell is significantly enhanced when S1P is associated with FGF-2 (Harvey et al. J Lab. Clin. Med 2002; 140: 188-198). Since we already reported that FGF-2 release occurs by vesicle shedding (Taverna et. al. J.Biol.Chem. 2003; 278: 51911-51919), we hypothesized and tried to demonstrate the possibility that S1P is produced in shed vesicles and that it exerts a synergic role with vesicle associated FGF-2, in the induction of endothelial cell differentiation. We also considered the hypothesis that enzymes involved in sphingolipid degradation could play a role in vesicle shedding. Our experimental dates indicate: \ub7 nCDase and SphK1 are both present in shed vesicles in biologically active forms, together with their lipidic substrates. \ub7 The enzymes of sphingolipid metabolism are not involved in the process of vesicle shedding. \ub7 In SK Hep-1 hepatocarcinoma cells, which we used in most of our experiments, FGF-2 and both nCDase and SphK1 are simultaneously released in shed vesicles. \ub7 Shed vesicles exert chemiotactic effects on endothelial cells and have the ability to promote their morphogenesis in capillary-like structures. \ub7 Since these effects are typical of both FGF-2 and S1P, to neutralize effects of vesicle-associated FGF-2, we denatured the protein components of vesicles by a 10 minutes treatment at 100 C\ub0. This treatment is known todenature FGF-2 (Vemuri et al. 1994; J Pharm Pharmacol. 46: 481-486) while S1P is reported to be stable at temperatures below 120 C\ub0 (Harvey et al. J Lab. Clin. Med 2002; 140: 188-198). Heat treatment substantially decreased the chemiotactic effect of vesicles but only had a small effect on their capability to induce formation of capillary-like structures. \ub7 Promotion of endothelial cell morphogenesis was increased in vesicles shed by cells overexpressing SphK1 and it was decreased in cells expressing a dominant negative SphK1 isoform. In summary our dates show that shed vesicles are vehicles for both FGF-2 and SphK1 secretion, and that FGF-2 and S1P, produced in the vesicle membrane by the joined catalytic action of nCDase and SphK1, exert a synergic action on endothelial cells inducing angiogenesis

Development of injectable and durable kefiran hydro-alcoholic gels

Injectable, in-situ forming kefiran gels have been developed for potential applications as implantable drug delivery devices or scaffolds for tissue regeneration. Concentrated solutions (4, 5 and 6%w) of kefiran, extracted from kefir grains, have been assessed in term of viscosity and injectability through G26 syringe needles, and for their ability to undergo gelation upon mixing with different alcohols. Propylene glycol (PG) has been selected as gelling agent because it ensures homogenous gelation in relatively short times (from few minutes up to 6 h). The investigation of the rheological behavior of kefiran/PG gels varying polymer concentration and temperature (25 °C and 37 °C) has provided interesting hints to support a possible gelation mechanism that accounts also for the observed influence of the alcohol type. Finally, the study of kefiran/PG gels has been complemented with the investigation on selected formulations of the swelling/degradation behavior upon immersion in isotonic buffer solution for up to 40 days at 37 °C; of the ability of the gels to retain and/or release two model molecules; and within vitro cell viability and cytotoxicity tests, to support the absence of toxic effects on cells induced by direct contact with the gels or by leached components from these gels