In vitro/ex vivo evaluation of multifunctional collagen/chitosan/hyaluronic acid hydrogel-based alendronate delivery systems.

Injectable hydrogel-based materials have emerged as promising alendronate (ALN) delivery systems for the treatment of osteoporosis. However, their intrinsic permeability limits the sustained delivery of small-molecule drugs. In response to this challenge, we present the multifunctional hybrids composed of mesoporous silica particles decorated with hydroxyapatite and loaded with alendronate (MSP-NH2-HAp-ALN), which are immobilized in collagen/chitosan/hyaluronic acid-based hydrogel. We have mainly focused on the biological in vitro/ex vivo evaluation of developed composites. It was found that the extracts released from tested systems do not exhibit hemolytic properties and are safe for blood elements and the human liver cell model. The resulting materials create an environment conducive to differentiating human bone marrow mesenchymal stem cells and reduce the viability of osteoclast precursors (RAW 264.7). Importantly, even the system with the lowest concentration of ALN caused a substantial cytotoxic effect on RAW 264.7 cells; their viability decreased to 20 % and 10 % of control on 3 and 7 day of culture. Additionally, prolonged ALN release (up to 20 days) with minimized burst release was observed, while material features (wettability, swellability, degradation, mechanical properties) depended on MSP-NH2-HAp-ALN content. The obtained data indicate that developed composites establish a high-potential formulation for safe and effective osteoporosis therapy.

Investigation the Effect of MR Fluid Composition on Properties at Low Strain Ranges.

The paper presents the results of eight magnetorheological (MR) fluids of different compositions. Magnetite and carbonyl iron were used as magnetic particles. MR fluids based on glycerin and OKS 352 oil were produced using stabilizers in the form of oleic acid and Aerosil 200 (Evonik Resource Efficiency GmbH, Hanau, Germany) silica; additives such as graphite and yellow dextrin were also used. The aim of the study was to determine the properties of various combinations of components on the dynamic properties of MR fluids, i.e., properties characterizing the fluid within the range of low deformations, as well as to investigate the effect of different compositions on structural yield stress and flow stress prepared MR fluids at different magnetic field induction values.

Towards Controlling the Local Bone Tissue Remodeling-Multifunctional Injectable Composites for Osteoporosis Treatment.

Alendronate (ALN) is the most commonly prescribed oral nitrogen-containing bisphosphonate for osteoporosis therapy. However, its administration is associated with serious side effects. Therefore, the drug delivery systems (DDS) enabling local administration and localized action of that drug are still of great importance. Herein, a novel multifunctional DDS system based on the hydroxyapatite-decorated mesoporous silica particles (MSP-NH2-HAp-ALN) embedded into collagen/chitosan/chondroitin sulfate hydrogel for simultaneous osteoporosis treatment and bone regeneration is proposed. In such a system, the hydrogel serves as a carrier for the controlled delivery of ALN at the site of implantation, thus limiting potential adverse effects. The involvement of MSP-NH2-HAp-ALN in the crosslinking process was established, as well as the ability of hybrids to be used as injectable systems. We have shown that the attachment of MSP-NH2-HAp-ALN to the polymeric matrix provides a prolonged ALN release (up to 20 days) and minimizes the initial burst effect. It was revealed that obtained composites are effective osteoconductive materials capable of supporting the osteoblast-like cell (MG-63) functions and inhibiting osteoclast-like cell (J7741.A) proliferation in vitro. The purposely selected biomimetic composition of these materials (biopolymer hydrogel enriched with the mineral phase) allows their biointegration (in vitro study in the simulated body fluid) and delivers the desired physicochemical features (mechanical, wettability, swellability). Furthermore, the antibacterial activity of the composites in in vitro experiments was also demonstrated.

Lysine-functionalized chondroitin sulfate improves the biological properties of collagen/chitosan-based injectable hydrogels.

Novel bioactive collagen/chitosan/lysine-functionalized chondroitin sulfate (CSmod) injectable hydrogels are presented. The modification of CS with amine groups introduced with lysine moieties (the degree of substitution about 21%) guarantees its covalent binding with the hydrogel network while genipin crosslinking. Both the physicochemical and biological features of developed hydrogels might be adjusted by playing with CSmod and crosslinking agent concentrations. It was revealed that materials became more hydrophobic with increased CSmod content, while crosslinking degree and enzymatic degradation studies established the influence of CSmod concentration and Ch:CSmod ratio on the crosslinking process. In situ rheological experiments verified the injectability of resulted systems. The biological in vitro evaluation demonstrated that all designed materials are biocompatible as they supported proliferation and adhesion of MG-63 cell line. In vitro biomineralization study employing simulated body fluid model revealed CSmod-content dependent bioactivity of obtained hydrogels. Importantly for pristine collagen/chitosan materials, the formation of apatite-like structures was not observed. Our findings demonstrate that developed injectable ColChCSmod hydrogels particularly system with the greatest CSmod concentration exhibits high bioactive potential, without the need of applying additional inducers what renders them promising materials within tissue engineering applications.

Bioactive hydrogel scaffolds reinforced with alkaline-phosphatase containing halloysite nanotubes for bone repair applications.

Alkaline phosphatase (ALP), biomineralization promoting enzyme, was immobilized in halloysite (HAL) nanotubes and used as a bioactive component of the chitosan (CH) and chitosan-collagen (C-CH) hydrogel scaffolds for bone regeneration. The influence of HAL-ALP and collagen on the properties of the obtained materials was studied. 30 wt% of HAL-ALP increased significantly the swelling ratio of chitosan-based scaffolds. The presence of both: collagen and HAL-ALP had positive effect on the scaffolds' porosity, which was considerably higher for C-CH. The presence of HAL has improved the mechanical properties of both types of scaffolds, while the addition of 20% of collagen to the chitosan hydrogels have considerably decreased their storage modulus. Biomineralization tests showed that although mineral was formed in both CH and C-CH scaffolds with HAL-ALP, the process was more effective for collagen-containing hydrogels. Biological studies, performed using MG-63 osteoblast-like cell line showed that C-CH scaffolds, especially those after biomineralization, were a better material for cell adhesion and growth. Both types of scaffolds degraded slowly in physiological pH. C-CH scaffolds containing 30% of HAL-ALP have the highest potential as bioactive material for bone regeneration.

Bioactive yet antimicrobial structurally stable collagen/chitosan/lysine functionalized hyaluronic acid - based injectable hydrogels for potential bone tissue engineering applications.

Novel, biocompatible, multifunctional, injectable genipin crosslinked collagen/chitosan/lysine-modified hyaluronic acid based hydrogels (ColChHAmod) were prepared in a facile, one-step procedure. The novelty of the current approach lies in the functionalization of hyaluronic acid (HA) with primary amine groups by lysine attachment, and its further use as a component of the injectable sol. The obtained derivative, HAmod, could form, upon crosslinking with genipin, covalent bonds with other components of the hydrogel network, resulting in structurally stable, better-defined hydrogels. We have demonstrated that, by adjusting HAmod content and genipin concentration, hydrogels with tunable physicochemical characteristics (swelling, wettability, tendency for enzymatic degradation) and properties adequate for the potential bone tissue regeneration can be prepared. Storage modulus measurements indicated that HAmod has positive effect on mechanical characteristics of hydrogels prepared. It was also revealed that the ColChHAmod-based hydrogels are characterized by a high porosity (85-95%). The in situ rheological measurements confirmed the injectability of the obtained hydrogels. The in vitro cell culture studies showed that the surface of all materials prepared was biocompatible, as they supported proliferation and adhesion of osteoblast-like cells followed by ALP expression. The intrinsic antibacterial activity of the hydrogels against Escherichia coli was also demonstrated in in vitro experiment.

Genipin crosslinked bioactive collagen/chitosan/hyaluronic acid injectable hydrogels structurally amended via covalent attachment of surface-modified silica particles.

Collagen, chitosan and hyaluronic acid based multicomponent injectable and in situ gellating biomimetic hybrid materials for bone tissue engineering applications were prepared in one-step procedure. The bioactive phase in the form of surface-modified silica particles was introduced to the solutions of biopolymers and simultaneously crosslinked with genipin both the biopolymer matrix and dispersed particles at 37 °C. The novel approach presented here involved the use of silica particles which surfaces were priory functionalized with amino groups. That modification makes possible the covalent attachment of silica particles to the polymeric hydrogel network on crosslinking with genipin. That methodology is especially important as it makes possible to obtain the hybrid materials (biopolymer-silica particles) in which the problems related to the potential phase separation of mineral particles, hindering their in vivo application can be eliminated. The hybrids of various compositions were obtained and their physicochemical and biological properties were determined. The in vitro experiments performed under simulated body fluid conditions revealed that the amino-functionalized silica particles covalently attached to the biopolymeric network are still bioactive. Finally, the in vitro cell culture studies shown that the materials developed are biocompatible as they supported MG-63 cells adhesion, proliferation as well as Alkaline phosphatase (ALP) expression.

Collagen/chitosan/hyaluronic acid - based injectable hydrogels for tissue engineering applications - design, physicochemical and biological characterization.

Studies on synthesis, physico-chemical and biological properties of novel biomimetic materials, potentially useful as injectable hydrogels are presented. These materials are in situ prepared chemically crosslinked collagen/chitosan/hyaluronic acid-based hydrogels exhibiting potential for tissue regeneration. Optimization of hydrogels involved testing the effect of various concentration of crosslinking agent (genipin) as well as different ratios of biopolymers used on their properties. The changes in the content of hyaluronic acid and in the genipin concentration used have been shown to be crucial. Employing the highest concentration of crosslinking agent studied (20 mM) the hydrogels of compact structure, characterized by good mechanical properties and prolonged degradation profile can be obtained. Changing the HA content in sol mixture the hydrogel of various wettability; more or less hydrophilic when compared to pure collagen/chitosan hydrogels can be fabricated. The in vitro cell culture study has shown that the surface of the prepared materials ensures suitable biocompatibility. These hydrogels can support the proliferation and adhesion of MG-63 cell line as it was demonstrated using Alamar Blue assay and SEM observations. It is believed that the collagen/chitosan/hyaluronic acid hydrogels crosslinked with genipin are particularly promising materials for bone regeneration procedures, especially attractive for regeneration of small bone losses. This is the first paper in the litearature presenting results of studies on that type of biopolymeric injectable hydrogels chemically crosslinked with genipin.

Tuning of elasticity and surface properties of hydrogel cell culture substrates by simple chemical approach.

When designing materials for tissue engineering applications various parameters characterizing both materials and tissue have to be taken into account. The characteristics such as chemistry, elasticity, wettability, roughness and morphology of the substrate's surface have significant impact on cell behavior. The paper presents biopolymer (collagen/chitosan) based hydrogel materials with tunable elasticity and surface properties useful for fabrication of substrates for cell culture. Using simple chemical approach involving the change in concentration of crosslinking agent (genipin) and composition of the reaction mixture the hydrogels characterized with various features were obtained. Detailed analysis of morphology, topography, roughness and elasticity of surface performed using Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM) and rheological measurements has shown that the topographical aspects and roughness parameter can be modulated in nanoscale regime (13-47 nm). Substrate's elasticity could be modified in a wide range (0.2-270 kPa). Biological in vitro studies on fibroblasts behavior revealed that the materials prepared provide satisfactory conditions for cell culture, ensuring their high viability, good adhesion and normal morphology. The genipin crosslinked collagen-chitosan hydrogels characterized by denser fiber structure, higher elasticity and lower surface roughness are the most attractive supports for fibroblasts cultivation. The results obtained indicate that the properties of the materials developed can be easily tailored to the needs of the given type of cells.