Osteogenic differentiation of human mesenchymal stem cells on electroactive substrates.

This study investigates the effect of electroactivity and electrical charge distribution on the biological response of human bone marrow stem cells (hBMSCs) cultured in monolayer on flat poly(vinylidene fluoride), PVDF, substrates. Differences in cell behaviour, including proliferation, expression of multipotency markers CD90, CD105 and CD73, and expression of genes characteristic of different mesenchymal lineages, were observed both during expansion in basal medium before reaching confluence and in confluent cultures in osteogenic induction medium. The crystallisation of PVDF in the electrically neutral α-phase or in the electroactive phase ß, both unpoled and poled, has been found to have an important influence on the biological response. In addition, the presence of a permanent positive or negative surface electrical charge distribution in phase ß substrates has also shown a significant effect on cell behaviour.

Structure, morphology, adhesion and in vitro biological evaluation of antibacterial multi-layer HA-Ag/SiO2/TiN/Ti coatings obtained by RF magnetron sputtering for biomedical applications.

Biocompatible and antibacterial multi-layer coatings of hydroxyapatite (HA)-Ag/SiO2/TiN/Ti were obtained on the Ti-6Al-4V alloy, by means of the magnetron sputtering technique. During characterization of the coatings, the chemical composition was evaluated by energy dispersive X-ray spectroscopy and the phase analysis was carried out by X-ray diffraction. The morphology of the coatings was observed by field emission scanning electron microscopy, while transmission electron microscopy was used to appreciate their structure. The adhesion of the coatings to the substrate was evaluated by micro scratch test. The in vitro biological response was evaluated in terms of cytotoxicity, adhesion and differentiation of mouse mesenchymal stem cells, as well as adhesion and bacterial viability of Staphylococcus aureus strain. Through the compositional study carried out, the deposition of the HA phase was verified, with a Ca/P ratio close to 1.67 and the characteristic diffraction peaks of this compound. The structural study of the coatings evidenced the obtention of multi-layer architectures. The use of an intermediate SiO2/TiN/Ti trilayer was found to improve adhesion between HA-Ag and the substrate by 84%. Finally, the in vitro biological tests carried out indicated a potentially non-toxic character in the coatings. Additionally, an antibacterial effect was registered at low concentrations of Ag (<0.25 mg/L).

In vitro assessment of the biological response of Ti6Al4V implants coated with hydroxyapatite microdomains.

Dental implantology is still an expanding field of scientific study because of the number of people that receive dental therapies throughout their lives worldwide. Recovery times associated to dental surgery are still long and demand strategies to improve integration of metallic devices with hard tissues. In this work, an in vitro ceramic coating is proposed to improve and accelerate osseointegration of titanium surfaces conceived to be used as dental implants or hip or knee prosthesis, shaped either as dishes or screws. Such coating consists of hydroxyapatite microdomains on the implant surfaces obtained in vitro by immersion of titanium alloy samples (Ti6Al4V) in a simulated body fluid. This titanium alloy is highly used in implant dentistry and trauma surgery, among other fields. Once the immersion times under physiological conditions yielding to different ceramic topographies on this alloy were set, the acellular coating time of major interest so as to optimize its biological development was determined. For this purpose, dental pulp mesenchymal cells were cultured on titanium coated surfaces with different hydroxyapatite outline, and cell adhesion, proliferation and morphology were followed through histological techniques and scanning electron microscopy. It was found that 4 days of acellular hydroxyapatite coating led to a significant cell adhesion on the titanium alloys at an early stage (6 h). Cells tended although to detach from the surface of the coating over time, but those adhered on domains of intricated topography or hydroxyapatite cauliflowers proliferated on them, leading to isolated large cell clusters. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2723-2729, 2016.

Differentiation of mesenchymal stem cells for cartilage tissue engineering: Individual and synergetic effects of three-dimensional environment and mechanical loading.

Chondrogenesis of dedifferentiated chondrocytes and mesenchymal stem cells is influenced not only by soluble molecules like growth factors, but also by the cell environment itself. The latter is achieved through both mechanical cues - which act as stimulation factor and influences nutrient transport - and adhesion to extracellular matrix cues - which determine cell shape. Although the effects of soluble molecules and cell environment have been intensively addressed, few observations and conclusions about the interaction between the two have been achieved. In this work, we review the state of the art on the single effects between mechanical and biochemical cues, as well as on the combination of the two. Furthermore, we provide a discussion on the techniques currently used to determine the mechanical properties of materials and tissues generated in vitro, their limitations and the future research needs to properly address the identified problems. STATEMENT OF SIGNIFICANCE: The importance of biomechanical cues in chondrogenesis is well known. This paper reviews the existing literature on the effect of mechanical stimulation on chondrogenic differentiation of mesenchymal stem cells in order to regenerate hyaline cartilage. Contradictory results found with respect to the effect of different modes of external loading can be explained by the different properties of the scaffolding system that holds the cells, which determine cell adhesion and morphology and spatial distribution of cells, as well as the stress transmission to the cells. Thus, this review seeks to provide an insight into the interplay between external loading program and scaffold properties during chondrogenic differentiation. The review of the literature reveals an important gap in the knowledge in this field and encourages new experimental studies. The main issue is that in each of the few cases in which the interplay is investigated, just two groups of scaffolds are compared, leaving intermediate adhesion conditions out of study. The authors propose broader studies implementing new high-throughput techniques for mechanical characterization of tissue engineering constructs and the inclusion of fatigue analysis as support methodology to more exhaustive mechanical characterization.

Determining the influence of N-acetylation on water sorption in chitosan films.

Water absorption in chitosan rapidly increases when the deacetylation degree decreases between 85 and 45%. This seems to contradict the fact that water absorption in chitin is much lower than that of chitosan. The aim of this paper is to understand this feature by measuring the main parameters affecting equilibrium water content. Since swelling capacity depends on the water-polymer interaction, the Flory Huggins interaction parameter was evaluated, finding small or null dependence on the deacetylation degree. Other factor influencing elastic energy is chain stiffness related to the elastic modulus that was measured as a function of deacetylation degree. Besides, crystalline structure was measured by X-ray diffraction patterns as a characteristic of cross-linking density. These observations led us to conclude that the instability of crystals during the swelling process increases with decreasing deacetylation degree, explaining the high equilibrium water content of low deacetylation chitosans.

Cell-free cartilage engineering approach using hyaluronic acid-polycaprolactone scaffolds: a study in vivo.

Polycaprolactone scaffolds modified with cross-linked hyaluronic acid were prepared in order to establish whether a more hydrophilic and biomimetic microenvironment benefits the progenitor cells arriving from bone marrow in a cell-free tissue-engineering approach. The polycaprolactone and polycaprolactone/hyaluronic acid scaffolds were characterized in terms of morphology and water absorption capacity. The polycaprolactone and polycaprolactone/hyaluronic acid samples were implanted in a chondral defect in rabbits; bleeding of the subchondral bone was provoked to generate a spontaneous healing response. Repair at 1, 4, 12, and 24 weeks was assessed macroscopically using the International Cartilage Repair Society score and the Oswestry Arthroscopy Score and microscopically using immunohistological staining for collagen type I and type II, and for Ki-67. The presence of hyaluronic acid improves scaffold performance, which supports a good repair response without biomaterial pre-seeding.

Silica coating of the pore walls of a microporous polycaprolactone membrane to be used in bone tissue engineering.

Polycaprolactone/silica microporous hybrid membranes were produced in two steps: A microporous polycaprolactone membrane with an interconnected porosity of 80% was obtained via a freeze extraction procedure, then silica was formed by a sol-gel reaction inside the micropores using tetraethyl orthosilicate, TEOS, as silica precursor. It is shown that silica forms a thin coating layer homogeneously distributed over the pore walls when sol-gel reaction is catalyzed by hydrochloric acid, while it forms submicron spherical particles when using basic catalyzer. Some physical properties and the viability and osteoblastic differentiation of bone marrow rat mesenchymal stem cells cultured on pure and hybrid membranes were studied.

Fibronectin fixation on poly(ethyl acrylate)-based copolymers.

The aim of this paper is to quantify the adhered fibronectin (FN; by adsorption and/or grafting) and the exposure of its cell adhesive motifs (RGD and FNIII7-10) on poly(ethyl acrylate) (PEA) copolymers whose chemical composition has been designed to increase wettability and to introduce acid functional groups. FN was adsorbed to PEA, poly(ethyl acrylate-co-hydroxyethyl acrylate), poly(ethyl acrylate-co-acrylic acid), and poly(ethyl acrylate-co-methacrylic acid) copolymers, and covalently cross-linked to poly(ethyl acrylate-co-acrylic acid) and poly(ethyl acrylate-co-methacrylic acid) copolymers. Amount of adhered FN and exhibition of RGD and FNIII7- 10 fragments involved in cell adhesion were quantified with enzyme-linked immunosorbent assay tests. Even copolymers with a lower content of the hydrophilic component showed a decrease in water contact angle. In addition, FN was successfully fixed on all surfaces, especially on the hydrophobic surfaces. However, it was demonstrated that exposure of its cell adhesion sequences, which is the key factor in cell adhesion and proliferation, was higher for hydrophilic surfaces.

Fibrin-chitosan composite substrate for in vitro culture of chondrocytes.

The aim of this study was to develop a biocompatible monolayer substrate based on fibrin and chitosan for in vitro culture of chondrocytes. Fibrin-chitosan composite substrates combined the proved cell adhesion properties of fibrin with the hydrophilicity and poor adhesion capacity of chitosan. Chitosan microspheres were produced by coacervation method, agglomerated within a fibrin network and subsequently crosslinked with genipin. The composite substrate was stable for 28 days of culture due to the high crosslinking density. Human chondrocytes cultured on the composite substrate were viable during the culture period. At the end of culture time (28 days) the composite substrate showed low cellular proliferation, 41% more collagen type II and 13% more production of sulfated glycosaminoglycans with respect to the amounts found at 14 days. The study revealed that dedifferentiated chondrocytes cultured in monolayer on the composite substrate can re-acquire characteristics of differentiated cells without using three-dimensional substrates or chondrogenic media.

Proliferation and differentiation of goat bone marrow stromal cells in 3D scaffolds with tunable hydrophilicity.

We have synthesized methacrylate-endcapped caprolactone networks with tailored water sorption ability, poly(CLMA-co-HEA), in the form of three-dimensional (3D) scaffolds with the same architecture but exhibiting different hydrophilicity character (x(HEA)=0, 0.3, 0.5), and we investigated the interaction of goat bone marrow stromal cells (GBMSCs) with such structures. For this purpose, GBMSCs were seeded and cultured for 3, 7, 14, 21, and 28 days onto the developed scaffolds. Cells have proliferated throughout the whole scaffold volume. Cell adhesion and morphology were analyzed by SEM, whereas cell viability and proliferation was assessed by MTS test and DNA quantification concluding that numbers of cells increased as a function of the culturing time (until day 14) and also with the hydrophobic content in the samples (from 50 to 100% of CLMA). No significant difference between samples with 100% and 70% of CLMA were detected in some cases. Osteoblastic differentiation was followed by assessing the alkaline phosphatase activity of cells, as well as type I collagen and osteocalcin expressions levels until day 21. The three markers were positive at days 14 and 21 when cells were cultured in 100% CLMA substrates which suggests osteoblastic differentiation of mesenchymal stem cells within these scaffolds. On the other hand, when the CLMA content decreases (until 50%), type I collagen and osteocalcin were positive but ALP was negative indicating that the differentiation process is affected by hydrophilic content. We suggest that such system may be useful to extract information on the effect of materials' wettability on the corresponding biological performance in a 3D environment. Such general insights may be relevant in the context of biomaterials selection for tissue engineering strategies.

Structure and properties of methacrylate-endcapped caprolactone networks with modulated water uptake for biomedical applications.

Methacrylate-endcapped caprolactone (CLMA) networks were synthesized and copolymerized with 2-hydroxyethyl acrylate (HEA) seeking to tailor the hydrophilicity of the system. The resulting structure of the copolymer network is investigated by differential scanning calorimetry, thermogravimetry, and Fourier transform infrared spectroscopy. The dynamic swelling behavior and the equilibrium water sorption is measured and correlated with the microstructure. The experimental results allow one to conclude that the new material is a random copolymer of both components, HEA and CLMA. The effect of cell attachment and proliferation on the new copolymer networks was observed by in vitro culture of human chondrocytes up to 8 days. Enhanced cellular adhesion, similar to that obtained with tissue culture polystyrene (TCPS), was obtained in the hydrophilized systems. The new copolymers are appropriate for the fabrication of scaffolds with controlled porosity for tissue engineering.

Acrylic scaffolds with interconnected spherical pores and controlled hydrophilicity for tissue engineering.

Polymer scaffolds are obtained in which the geometric characteristics (pore size, connectivity, porosity) and the physico-chemical properties of the resulting material can be controlled in an independent way. The interconnected porous structure was obtained using a template of sintered PMMA microspheres of controlled size. Copolymerization of hydrophobic ethyl acrylate and hydrophilic hydroxyethyl methacrylate comonomers took place in the free space of the template, different comonomer ratio gave rise to different hydrophilicity degrees of the material keeping the same pore architecture. The morphology of the resulting scaffolds was investigated by scanning electron microscopy (SEM), the porosity of the material calculated, and the mechanical properties compared with those of the bulk (non porous) material of the same composition.