Stefano Agnello

In situ forming hydrogels of hyaluronic acid and inulin derivatives for cartilage regeneration.

An in situ forming hydrogel obtained by crosslinking of amino functionalized hyaluronic acid derivatives with divinylsulfone functionalized inulin (INU-DV) has been here designed and characterized. In particular two hyaluronic acid derivatives bearing respectively a pendant ethylenediamino (EDA) portion (HA-EDA) and both EDA and octadecyl pendant groups (HA-EDA-C18) were crosslinked through an azo-Michael reaction with INU-DV. Gelation time and consumption of DV portions have been evaluated on hydrogel obtained using HA-EDA and HA-EDA-C18 derivatives with a concentration of 3% w/v and a ratio 80/20 w/w respect to the crosslinker INU-DV. The presence of pendant C18 chains improves mechanical performances of hydrogels and decreases the susceptibility to hyaluronidase hydrolysis. Bovine chondrocytes, encapsulated during crosslinking, sufficiently survive and efficiently proliferate until 28 days of analysis.

Injectable in situ forming hydrogels based on natural and synthetic polymers for potential application in cartilage repair

In this work we prepared two new hyaluronic acid (HA) based in situ forming hydrogels for the potential treatment of articular cartilage damages. In particular the amino derivative of HA (HA-EDA) and its graft copolymer with α-elastin (HA-EDA-g-α-elastin) were crosslinked, in mild physiological conditions via Michael-type addition, with α,β-poly(N-2-hydroxyethyl)-DL-aspartamide (PHEA) derivatized with divinylsulfone (DV). The swelling and degradation profile of the obtained hydrogels as well as the metabolic activity of incorporated bovine articular chondrocytes were investigated. Histological analysis and scanning electron microscopy (SEM) were performed to analyze the morphology of cells after scheduled times of incubation. Moreover the influence of the α-elastin on the elastic modulus of cell free and cell containing hydrogels has been studied through atomic force microscopy (AFM).

Synthesis, mechanical and thermal rheological properties of new gellan gum derivatives

New derivatives of gellan gum (GG) were prepared by covalent attachment of octadecylamine (C18-NH2) to polysaccharide backbone via amide linkage by using bis(4-nitrophenyl) carbonate (4-NPBC) as a coupling agent. The effect of the alkyl chain grafted onto hydrophilic backbone of high molecular weight GG was investigated in terms of physicochemical properties and ability of new derivatives to form hydrogels. A series of hydrogels was obtained in solutions with different kind and concentration of ions and their stability and mechanical properties were evaluated. The obtained derivatives resulted soluble at temperature lower than starting GG and physicochemical properties of obtained hydrogels suggested their potential use in biomedical field.

Interpenetrated 3D porous scaffolds of silk fibroin with an amino and octadecyl functionalized hyaluronic acid

An ethylenediamine (EDA) and octadecylamine (–C18) hyaluronic acid (HA) derivative, named HA–EDA–C18, has been used for the production of interpenetrated composite biomaterials with silk fibroin. The peculiar ionic strength sensibility of this HA derivative allows the production of porous matrices without the need for chemical crosslinking. Scaffolds have been produced through a salt leaching procedure by exploiting the properties of silk fibroin and HA–EDA–C18 to physically crosslink when forced through a syringe loaded with NaCl. The porosity of the sponges, comprised between 70–80%, was dependent on the amount of each polymer and NaCl size distribution. Moreover, through FT-IR analysis, it has been evaluated how the blend composition influences the conformational changes of fibroin. This study demonstrates that HA–EDA–C18 induces the transition of silk fibroin to its β-sheet conformation. Moreover, the HA–EDA–C18 weight fraction in composite sponges regulates the scaffold susceptibility to protease and hyaluronidase. FT-IR analysis and enzymatic hydrolysis studies suggest that the homogeneous interpenetration of polymers occurs. In vitro studies indicate that the presence of fibroin improves the viability and attachment of bovine chondrocytes.

Hyaluronic Acid Derivative with Improved Versatility for Processing and Biological Functionalization

A hydrophobic/amino functionalized derivative of hyaluronic acid (HA-EDA-C18 ) has been processed by salt leaching technique as porous scaffold without need of chemical crosslinking. Aim of this work is to demonstrate the improved versatility of HA-EDA-C18 in terms of processing and biological functionalization. In particular, the chemical procedure to tether thiol bearing RGD peptide has been described. Moreover, the possibility to load and to control the release of slightly water soluble effectors has been demonstrated by using dexamethasone. First, the swelling and degradation profiles of the scaffolds have been investigated, then the evaluation of metabolic activity of bovine chondrocytes, the histological analysis, and microscope observations has been performed to evaluate cellular adhesion and proliferation as well as the production of collagen type II.

Spray dried hyaluronic acid microparticles for adhesion controlled aggregation and potential stimulation of stem cells

Spray-dried microparticles of a derivative of hyaluronic acid (HA) have been engineered to obtain a controlled aggregation with Human Mesenchymal Stem Cells (hMSCs) into 3D constructs. We demonstrated the utility of chemical functionalization of a native constituent of the extracellular matrix to improve processing performances and to control on stem cell adhesion and differentiation. Native hyaluronic acid (HA), cell adhesive peptides (RGD), transforming growth factor β3, dexamethasone are biological agents potentially suitable for chondrogenic stimulation of hMSCS. However unmodified HA suffers of drawbacks in terms of stability and versatility of processing. Functionalization strategies are needed to overcome these drawbacks. In this paper microparticles were produced by spray-drying of an aliphatic and amino functionalized HA derivative. Hydrophobic derivatization of HA allowed the production of microparticles stabilized by physical crosslinking and to load and to control dexamethasone release. The presence of pendant amino groups was exploited to tether cyclic RGD and transforming growth factor β3via maleimide chemistry; cyRGDC functionalization controlled hMSCs/microparticles aggregation. Chondrogenic potential was preliminary assayed by qualitative immunohistological detection.

Synthesis and evaluation of thermo-rheological behaviour and ionotropic crosslinking of new gellan gum-alkyl derivatives

This paper reports the synthesis and the physicochemical characterization of two series of gellan gum (GG) derivatives functionalized with alkyl chains with different number of carbon, from 8 to 18. In particular, low molecular weight gellan gum samples with 52.6 or 96.7 kDa, respectively, were functionalized with octylamine (C8), dodecylamine (C12) and octadecylamine (C18) by using bis(4-nitrophenyl) carbonate (4-NPBC) as a coupling agent. Thermo-rheological and ionotropic crosslinking properties of these gellan gum-alkyl derivatives were evaluated and related to the degree of derivatization in alkyl chains. Results suggested as length and degree of derivatization differently influenced coil-to-helix gelation mechanism of GG derivatives, ionotropic crosslinking, and strength of crosslinked hydrogels obtained in CaCl2 0.102 M and NaCl 0.15 M. Statement of hypothesis: The insertion of alkyl chains on the gellan gum backbone interferes with coil-to-helix transition mechanism and allows the production of hydrophobically assembled hydrogels.

Can vaccines for contagious agalactia reduce disease progression in infected animals: a preliminary study?

A flock of sheep in Central Sicily was affected by a severe outbreak of contagious agalactia (CA) caused by Mycoplasma agalactiae, affecting nearly 30% resulting in a large drop in milk production. Many ewes had warm and swollen udders that often became sclerotic and was accompanied by keratoconjunctivitis and arthritis in both adults and young. As antibiotic treatment appeared ineffective, the whole flock was given two doses of an inactivated vaccine against M agalactiae two weeks apart. A small group was selected for close examination and additional tests. A fortnight after the last vaccination mycoplasma excretion fell in all but one of the selected ewes and was undetectable in all but two animals two weeks after that. In nearly all the selected ewes, there was an improvement in milk quality and udder condition. This work provided preliminary evidence for the continued use of CA vaccines to slow or prevent disease progression.