Dunia M. García Cruz

Chitosan microparticles as injectable scaffolds for tissue engineering

The use of chitosan microparticles as injectable carriers for cell transplantation represents a promising alternative to avoid the drawbacks of the implantation of other forms of three‐dimensional (3D) scaffolds seeded with cells. In this study, a 3D construct is obtained in vitro by combining chitosan microparticles crosslinked with genipin and goat bone marrow stromal cells (GBMCs). Cell viability and the morphology of GBMCs were evaluated after culture for 7 and 14 days. Our results show the feasibility of chitosan microparticles as potential injectable scaffolds for tissue engineering and regenerative medicine. Copyright

Differentiation of mesenchymal stem cells in chitosan scaffolds with double micro and macroporosity.

Bone Marrow mesenchymal stem cells can be induced to differentiate into osteoblasts to regenerate damaged bone tissue using tissue engineering techniques. In this study, we examine the use of chitosan scaffolds with double pore structure prepared by an innovative method that combines freeze gelation (that produces micropores) and particle leaching out technique (that produces interconnected spherical macropores) seeking to enhance the osteogenic differentiation of goat bone marrow stromal cells (GBMSCs). The double pore architecture of the scaffold was characterized by scanning electron microscopy (SEM), microcomputed tomography and confocal laser scanning microscopy. The obtained hierarchical pore structure allowed very efficient seeding of GBMSCs that are able to occupy the whole volume of the scaffold, showing good adhesion and proliferation. GBMSCs were differentiated into osteoblasts as indicated by alkaline phosphatase activity and osteocalcin expression. The results of this study demonstrate that chitosan scaffold may be promising biomaterial for bone regeneration.

Blending polysaccharides with biodegradable polymers. II. Structure and biological response of chitosan/polycaprolactone blends

Blends of polycaprolactone (PCL) and chitosan (CHT) were prepared by casting from the mixture of solutions of both components in suitable solvents. PCL, and CHT, form phase separated blends with improved mechanical properties and increased water sorption ability with respect to pure PCL. The morphology of the system was investigated by scanning electron microscopy (SEM), atomic force microscopy (AFM) and confocal microscopy. Dispersed domains of CHT in the semicrystalline PCL matrix were found in samples with less than 20% CHT but cocontinuous phase morphologies are found in blends with 20% or more CHT. This feature was corroborated by the temperature dependence of the elastic modulus measured by dynamic mechanical properties as a function of temperature. It was observed that for those blends above 20 wt% CHT, the mechanical stability of the system was kept even after melting of the PCL phase. Primary human chondrocytes were cultured on the different substrates. Cell morphology was studied by SEM and the viability and proliferation was investigated by the colorimetric MTT assay. Different protein conformations were found by AFM on CHT and PCL samples which were related to the biological performance of the substrates. Hydrophilicty of the material is not directly related to the biological response and the sample with 20 wt% CHT shows better results than the other blends with respect to chondrocyte viability and proliferation. However, the results obtained in the blends are worse than in pure PCL. It seems to be correlated with the surface energy of the different blends rather than hydrophilicity.

Stirred flow bioreactor modulates chondrocyte growth and extracellular matrix biosynthesis in chitosan scaffolds.

The aim of this study is to show the favorable effect of simple dynamic culture conditions on chondrogenesis of previously expanded human chondrocytes seeded in a macroporous scaffold with week cell-pore walls adhesion. We obtained enhanced chondrogenesis by the combination of chitosan porous supports with a double micro- and macro-pore structure and cell culture in a stirring bioreactor. Cell-scaffold constructs were cultured under static or mechanically stimulated conditions using an intermittent stirred flow bioreactor during 28 days. In static culture, the chondrocytes were homogeneously distributed throughout the scaffold pores; cells adhered to the scaffold pore walls, showed extended morphology and were able to proliferate. Immunofluorescense and biochemical assays showed abundant type I collagen deposition at day 28. However, the behavior of chondrocytes submitted to mechanical stimuli in the bioreactor was completely different. Mechanical loading influenced cell morphology and extracellular matrix composition. Under dynamic conditions, chondrocytes kept their characteristic phenotype and tended to form cell aggregates surrounded by a layer of the main components of the hyaline cartilage extracellular matrix, type II collagen, and aggrecan. An enhanced aggrecan and collagen type II production was observed in engineered cartilage constructs cultured under stirred flow compared with those cultured under static conditions.

Bioactive organic inorganic poly(CLMA-co-HEA)/silica nanocomposites

A series of novel poly(CLMA-co-HEA)/silica nanocomposites is synthesized from caprolactone 2-(methacryloyloxy)ethyl ester (CLMA) and 2-hydroxyethyl acrylate (HEA) as organic comonomers and the simultaneous sol-gel polymerization of tetraethyloxysilane (TEOS) as silica precursor, in different mass ratios up to a 30 wt% of silica. The nanocomposites are characterized as to their mechanical and thermal properties, water sorption, bioactivity and biocompatibility, reflecting the effect on the organic matrix provided by the silica network formation. The nanocomposites nucleate the growth of hydroxyapatite (HAp) on their surfaces when immersed in the simulated body fluid of the composition used in this work. Proliferation of the MC3T3 osteoblast-like cells on the materials was assessed with the MTS assay showing their biocompatibility. Immunocytochemistry reveals osteocalcin and type I collagen production, indicating that osteoblast differentiation was promoted by the materials, and calcium deposition was confirmed by von Kossa staining. The results indicate that these poly(CLMA-co-HEA)/silica nanocomposites could be a promising biomaterial for bone tissue engineering.

Cytotoxic effect of 4-hydroxytamoxifen conjugate material on human Schwann cells: Synthesis and characterization

In this study, the toxicity of 4-hydroxytamoxifen (4-OHT) on human Schwann cells (HSCs) was evaluated. Substantial alterations in the cell morphology and viability were observed at 4-OHT concentrations higher than 3 µg/mL. Therefore, we designed and synthesized a drug–polymer conjugate, based on N-(2-hydroxypropyl)methacrylamide (HPMA) and ethyl acrylate (EA) for delivering 4-OHT to the target tissue without the detrimental consequences of the systemic therapy currently used. The macromer carrier of 4-OHT (MATX), with a functionalization degree of 80%, was synthesized in two steps and verified by 1H-NMR and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy. MATX was conjugated to the poly(HPMA-co-EA) copolymer network via radical polymerization. The influence of MATX on the physical, chemical, and mechanical properties of poly(HPMA-co-EA-co-MATX) with a ratio of 69/29/2 wt% was compared to those of poly(HPMA-co-EA) networks with a similar feed mixture. The in vitro release of 4-OHT within 1 month was 6 wt% of the total amount of drug linked to the copolymer backbone.

Polymer chains incorporating caprolactone and arginine–glycine–aspartic acid functionalities: Synthesis, characterization and biological response in vitro of the Schwann cell

This study describes a strategy for the covalent immobilization of active adhesion peptide moieties onto polymers through the intermediacy of itaconic acid. The arginine–glycine–aspartic acid peptide was grafted to a novel poly(caprolactone 2-(methacryloyloxy) ethyl ester)-co-itaconic acid bulk biomaterial, in order to improve the cell adhesion of the polymer. First, the arginine–glycine–aspartic acid sequence was grafted onto itaconic acid via an amidation reaction using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide as activation complex. The itaconic acid–arginine–glycine–aspartic acid macromer was characterized by Fourier transform infrared spectroscopy and 1H-NMR, yielding a functionalization degree of 85%. In a second step, poly(caprolactone 2-(methacryloyloxy) ethyl ester-co-itaconic acid–arginine–glycine–aspartic acid) (with a feed mixture of 90 wt% of caprolactone 2-(methacryloyloxy) ethyl ester and 10 wt% of itaconic acid–arginine–glycine–aspartic acid macromer) and a series of copolymers of caprolactone 2-(methacryloyloxy) ethyl ester and itaconic acid with different compositions (weight fractions of itaconic acid up to 20 wt%) were synthesized by radical copolymerization. The microstructure and network architecture of the new polymer systems were investigated. Mechanical moduli of poly(caprolactone 2-(methacryloyloxy) ethyl ester-co-itaconic acid), evaluated by dynamic–mechanical analysis, increase with the itaconic acid content. In poly(caprolactone 2-(methacryloyloxy) ethyl ester-co-itaconic acid–arginine–glycine–aspartic acid), the glass transition temperature and the mechanical moduli of the system are smaller than in the nonfunctionalized poly(caprolactone 2-(methacryloyloxy) ethyl ester-co-itaconic acid) copolymers, and the polymer is less hydrophilic. The results indicate that arginine–glycine–aspartic acid grafting of poly(caprolactone 2-(methacryloyloxy) ethyl ester-co-itaconic acid) copolymer networks can be useful for tissue engineering applications, because regenerative processes in the nervous system can be promoted and accelerated, thus, opening a possibility to generate materials with a high potential for clinical applicability.