Electrical stimulation: Effective cue to direct osteogenic differentiation of mesenchymal stem cells?

Mesenchymal stem cells (MSCs) play a major role in bone tissue engineering (BTE) thanks to their capacity for osteogenic differentiation and being easily available. In vivo, MSCs are exposed to an electroactive microenvironment in the bone niche, which has piezoelectric properties. The correlation between the electrically active milieu and bone's ability to adapt to mechanical stress and self-regenerate has led to using electrical stimulation (ES) as physical cue to direct MSCs differentiation towards the osteogenic lineage in BTE. This review summarizes the different techniques to electrically stimulate MSCs to induce their osteoblastogenesis in vitro, including general electrical stimulation and substrate mediated stimulation by means of conductive or piezoelectric cell culture supports. Several aspects are covered, including stimulation parameters, treatment times and cell culture media to summarize the best conditions for inducing MSCs osteogenic commitment by electrical stimulation, from a critical point of view. Electrical stimulation activates different signaling pathways, including bone morphogenetic protein (BMP) Smad-dependent or independent, regulated by mitogen activated protein kinases (MAPK), extracellular signal-regulated kinases (ERK) and p38. The roles of voltage gate calcium channels (VGCC) and integrins are also highlighted according to their application technique and parameters, mainly converging in the expression of RUNX2, the master regulator of the osteogenic differentiation pathway. Despite the evident lack of homogeneity in the approaches used, the ever-increasing scientific evidence confirms ES potential as an osteoinductive cue, mimicking aspects of the in vivo microenvironment and moving one step forward to the translation of this approach into clinic.

Poly(vinylidene) fluoride membranes coated by heparin/collagen layer-by-layer, smart biomimetic approaches for mesenchymal stem cell culture.

The use of piezoelectric materials in tissue engineering has grown considerably since inherent bone piezoelectricity was discovered. Combinations of piezoelectric polymers with magnetostrictive nanoparticles (MNP) can be used to magnetoelectrically stimulate cells by applying an external magnetic field which deforms the magnetostrictive nanoparticles in the polymer matrix, deforming the polymer itself, which varies the surface charge due to the piezoelectric effect. Poly(vinylidene) fluoride (PVDF) is the piezoelectric polymer with the largest piezoelectric coefficients, being a perfect candidate for osteogenic differentiation. As a first approach, in this paper, we propose PVDF membranes containing magnetostrictive nanoparticles and a biomimetic heparin/collagen layer-by-layer (LbL) coating for mesenchymal stem cell culture. PVDF membranes 20% (w/v) with and without cobalt ferrite oxide (PVDF-CFO) 10% (w/w) were produced by non-solvent induced phase separation (NIPS). These membranes were found to be asymmetric, with a smooth surface, crystallinity ranging from 65% to 61%, and an electroactive ß-phase content of 51.8% and 55.6% for PVDF and PVDF-CFO, respectively. Amine groups were grafted onto the membrane surface by an alkali treatment, confirmed by ninhydrin test and X-ray photoelectron spectroscopy (XPS), providing positive charges for the assembly of heparin/collagen layers by the LbL technique. Five layers of each polyelectrolyte were deposited, ending with collagen. Human mesenchymal stem cells (hMSC) were used to test cell response in a short-term culture (1, 3 and 7 days). Nucleus cell counting showed that LbL favored cell proliferation in PVDF-CFO over non-coated membranes.

Fluctuations of conformational mobility of macromolecules around the glass transition.

The heterogeneity of local dynamics in disordered systems is behind some key features of glass transition. In order to improve our understanding of the molecular dynamics in disordered systems in the vicinity of the glass transition, different parameters have been proposed to quantitatively describe dynamical heterogeneity. In the case of polymers, free volume models relate the macromolecular mobility to the free or accessible volume. The relationship between dynamic heterogeneity and fluctuations of accessible volume seems straightforward. In the present work, the heterogeneity of local dynamics in polymeric systems is analyzed by computer simulation with the bond fluctuation model. The value of the accessible volume around each polymer chain is evaluated from a snapshot or static structure at each system state, resulting in a distribution of accessible volume that reflects system heterogeneity. The relationship between the average value and the standard deviation of free volume distributions at different temperatures fits a master curve for different systems, regardless of the specific inter- and intramolecular interaction potentials that define each material. The dynamic slowdown around the glass transition is accompanied by a clear evolution of the mean value and shape of the accessible free volume distribution. The relative fluctuation of the dynamically accessible volume has been used as a parameter to quantitatively describe heterogeneity. The fluctuation varies with temperature with remarkable differences between the liquid and glassy states of the systems studied, presenting a peak at the glass transition temperature, which can be interpreted as a reflection of the distribution of local glass transition temperatures.

Human Mesenchymal Stem Cells Growth and Osteogenic Differentiation on Piezoelectric Poly(vinylidene fluoride) Microsphere Substrates.

The aim of this work was to determine the influence of the biomaterial environment on human mesenchymal stem cell (hMSC) fate when cultured in supports with varying topography. Poly(vinylidene fluoride) (PVDF) culture supports were prepared with structures ranging between 2D and 3D, based on PVDF films on which PVDF microspheres were deposited with varying surface density. Maintenance of multipotentiality when cultured in expansion medium was studied by flow cytometry monitoring the expression of characteristic hMSCs markers, and revealed that cells were losing their characteristic surface markers on these supports. Cell morphology was assessed by scanning electron microscopy (SEM). Alkaline phosphatase activity was also assessed after seven days of culture on expansion medium. On the other hand, osteoblastic differentiation was monitored while culturing in osteogenic medium after cells reached confluence. Osteocalcin immunocytochemistry and alizarin red assays were performed. We show that flow cytometry is a suitable technique for the study of the differentiation of hMSC seeded onto biomaterials, giving a quantitative reliable analysis of hMSC-associated markers. We also show that electrosprayed piezoelectric poly(vinylidene fluoride) is a suitable support for tissue engineering purposes, as hMSCs can proliferate, be viable and undergo osteogenic differentiation when chemically stimulated.

Kinetic study of thermal degradation of chitosan as a function of deacetylation degree.

Thermal degradation of chitosan with varying deacetylation degree (DD) ranging between 50 and 85% was analyzed by dynamic thermogravimetric analysis at different heating rates. The present study focused on the temperature range between 500 and 800K, above water evaporation. Thermal degradation showed a main degradation stage in this temperature interval with a second stage that appeared in the weight derivative curves as a shoulder in the high temperature side of the main peak with increasing intensity as the DD decreased. The Kissinger and isoconversional Ozawa-Flynn-Wall models were employed to evaluate the Ea of both thermal degradation processes. Different kinetic models were tested to computer simulate the thermogravimetric traces calculating the model parameters with a non-linear least squares fitting routine. The Sestack-Berggren model allowed reproducing accurately the overlapping of the two degradation mechanisms and calculating the mass fraction lost in each of them revealing the coupling between the two degradation mechanisms.

Surface stiffening and enhanced photoluminescence of ion implanted cellulose - polyvinyl alcohol - silica composite.

Novel Cellulose (Cel) reinforced polyvinyl alcohol (PVA)-Silica (Si) composite which has good stability and in vitro degradation was prepared by lyophilization technique and implanted using N(3+) ions of energy 24keV in the fluences of 1×10(15), 5×10(15) and 1×10(16)ions/cm(2). SEM analysis revealed the formation of microstructures, and improved the surface roughness on ion implantation. In addition to these structural changes, the implantation significantly modified the luminescent, thermal and mechanical properties of the samples. The elastic modulus of the implanted samples has increased by about 50 times compared to the pristine which confirms that the stiffness of the sample surface has increased remarkably on ion implantation. The photoluminescence of the native cellulose has improved greatly due to defect site, dangling bonds and hydrogen passivation. Electric conductivity of the ion implanted samples was improved by about 25%. Hence, low energy ion implantation tunes the mechanical property, surface roughness and further induces the formation of nano structures. MG63 cells seeded onto the scaffolds reveals that with the increase in implantation fluence, the cell attachment, viability and proliferation have improved greatly compared to pristine. The enhancement of cell growth of about 59% was observed in the implanted samples compared to pristine. These properties will enable the scaffolds to be ideal for bone tissue engineering and imaging applications.

Prediction of the "in vivo" mechanical behavior of biointegrable acrylic macroporous scaffolds.

This study examines a biocompatible scaffold series of random copolymer networks P(EA-HEA) made of Ethyl Acrylate, EA, and 2-Hydroxyl Ethyl Acrylate, HEA. The P(EA-HEA) scaffolds have been synthesized with varying crosslinking density and filled with a Poly(Vinyl Alcohol), PVA, to mimic the growing cartilaginous tissue during tissue repair. In cartilage regeneration the scaffold needs to have sufficient mechanical properties to sustain the compression in the joint and, at the same time, transmit mechanical signals to the cells for chondrogenic differentiation. Mechanical tests show that the elastic modulus increases with increasing crosslinking density of P(EA-HEA) scaffolds. The water plays an important role in the mechanical behavior of the scaffold, but highly depends on the crosslinking density of the proper polymer. Furthermore, when the scaffold with hydrogel is tested it can be seen that the modulus increases with increasing hydrogel density. Even so, the mechanical properties are inferior than those of the scaffolds with water filling the pores. The hydrogel inside the pores of the scaffolds facilitates the expulsion of water during compression and lowers the mechanical modulus of the scaffold. The P(EA-HEA) with PVA shows to be a good artificial cartilage model with mechanical properties close to native articular cartilage.

MC3T3-E1 Cell Response to Ti1-xAgx and Ag-TiNx Electrodes Deposited on Piezoelectric Poly(vinylidene fluoride) Substrates for Sensor Applications.

In the sensors field, titanium based coatings are being used for the acquisition/application of electrical signals from/to piezoelectric materials. In this particular case, sensors are used to detect dynamic mechanical loads at early stages after intervention of problems associated with prostheses implantation. The aim of this work is to select an adequate electrode for sensor applications capable, in an initial stage to avoid bone cell adhesion, but at a long stage, permit osteointegration and osteoinduction. This work reports on the evaluation of osteoblast MC3T3-E1 cells behavior in terms of proliferation, adhesion and long-term differentiation of two different systems used as sensor electrodes: Ti1-xAgx and Ag-TiNx deposited by d.c. and pulsed magnetron sputtering at room temperature on poly(vinylidene fluoride) (PVDF). The results indicated an improved effect of Ag-TiNx electrodes compared with Ti1-xAgx and TiN, in terms of diminished cell adhesion and proliferation at an initial cell culture stage. Nevertheless, when cell culture time is longer, cells grown onto Ag-TiNx electrodes are capable to proliferate and also differentiate at proper rates, indicating the suitability of this coating for sensor application in prostheses devices. Thus, the Ag-TiNx system was considered the most promising electrode for tissue engineering applications in the design of sensors for prostheses to detect dynamic mechanical loads.

Design and validation of a biomechanical bioreactor for cartilage tissue culture.

Specific tissues, such as cartilage, undergo mechanical solicitation under their normal performance in human body. In this sense, it seems necessary that proper tissue engineering strategies of these tissues should incorporate mechanical solicitations during cell culture, in order to properly evaluate the influence of the mechanical stimulus. This work reports on a user-friendly bioreactor suitable for applying controlled mechanical stimulation--amplitude and frequency--to three-dimensional scaffolds. Its design and main components are described, as well as its operation characteristics. The modular design allows easy cleaning and operating under laminar hood. Different protocols for the sterilization of the hermetic enclosure are tested and ensure lack of observable contaminations, complying with the requirements to be used for cell culture. The cell viability study was performed with KUM5 cells.

Macroporous thin membranes for cell transplant in regenerative medicine.

The aim of this paper is to present a method to produce macroporous thin membranes made of poly (ethyl acrylate-co-hydroxyethyl acrylate) copolymer network with varying cross-linking density for cell transplantation and prosthesis fabrication. The manufacture process is based on template techniques and anisotropic pore collapse. Pore collapse was produced by swelling the membrane in acetone and subsequently drying and changing the solvent by water to produce 100 microns thick porous membranes. These very thin membranes are porous enough to hold cells to be transplanted to the organism or to be colonized by ingrowth from neighboring tissues in the organism, and they present sufficient tearing stress to be sutured with surgical thread. The obtained pore morphology was observed by Scanning Electron Microscope, and confocal laser microscopy. Mechanical properties were characterized by stress-strain experiments in tension and tearing strength measurements. Morphology and mechanical properties were related to the different initial thickness of the scaffold and the cross-linking density of the polymer network. Seeding efficiency and proliferation of mesenchymal stem cells inside the pore structure were determined at 2 h, 1, 7, 14 and 21 days from seeding.

Relationship between micro-porosity, water permeability and mechanical behavior in scaffolds for cartilage engineering.

In tissue engineering the design and optimization of biodegradable polymeric scaffolds with a 3D-structure is an important field. The porous scaffold provide the cells with an adequate biomechanical environment that allows mechanotransduction signals for cell differentiation and the scaffolds also protect the cells from initial compressive loading. The scaffold have interconnected macro-pores that host the cells and newly formed tissue, while the pore walls should be micro-porous to transport nutrients and waste products. Polycaprolactone (PCL) scaffolds with a double micro- and macro-pore architecture have been proposed for cartilage regeneration. This work explores the influence of the micro-porosity of the pore walls on water permeability and scaffold compliance. A Poly(Vinyl Alcohol) with tailored mechanical properties has been used to simulate the growing cartilage tissue inside the scaffold pores. Unconfined and confined compression tests were performed to characterize both the water permeability and the mechanical response of scaffolds with varying size of micro-porosity while volume fraction of the macro-pores remains constant. The stress relaxation tests show that the stress response of the scaffold/hydrogel construct is a synergic effect determined by the performance of the both components. This is interesting since it suggests that the in vivo outcome of the scaffold is not only dependent upon the material architecture but also the growing tissue inside the scaffold׳s pores. On the other hand, confined compression results show that compliance of the scaffold is mainly controlled by the micro-porosity of the scaffold and less by hydrogel density in the scaffold pores. These conclusions bring together valuable information for customizing the optimal scaffold and to predict the in vivo mechanical behavior.

An experimental fatigue study of a porous scaffold for the regeneration of articular cartilage.

The aim of this experimental study is to predict the long-term mechanical behavior of a porous scaffold implanted in a cartilage defect for tissue engineering purpose. Fatigue studies were performed by up to 100,000 unconfined compression cycles in a polycaprolactone (PCL) scaffold with highly interconnected pores architecture. The scaffold compliance, stress-strain response and hysteresis energy have been measured after different number of fatigue cycles, while the morphology has been observed by scanning electron microscopy at the same fatigue times. To simulate the growing tissue in the scaffold/tissue construct, the scaffold was filled with an aqueous solution of polyvinyl alcohol (PVA) and subjected to repeating cycles of freezing and thawing that increase the hydrogel stiffness. Fatigue studies show that the mechanical loading provokes failure of the dry scaffold at a smaller number of deformation cycles than when it is immersed in water, and also that 100,000 compressive dynamic cycles do not affect the scaffold/gel construct. This shows the stability of the scaffold implanted in a chondral defect and gives a realistic simulation of the mechanical performance from implantation of the empty scaffold to regeneration of the new tissue inside the scaffold's pores.

In vitro mechanical fatigue behavior of poly-ɛ-caprolactone macroporous scaffolds for cartilage tissue engineering: Influence of pore filling by a poly(vinyl alcohol) gel.

Polymeric scaffolds used in regenerative therapies are implanted in the damaged tissue and submitted to repeated loading cycles. In the case of articular cartilage engineering, an implanted scaffold is typically subjected to long-term dynamic compression. The evolution of the mechanical properties of the scaffold during bioresorption has been deeply studied in the past, but the possibility of failure due to mechanical fatigue has not been properly addressed. Nevertheless, the macroporous scaffold is susceptible to failure after repeated loading-unloading cycles. In this work fatigue studies of polycaprolactone scaffolds were carried by subjecting the scaffold to repeated compression cycles in conditions simulating the scaffold implanted in the articular cartilage. The behavior of the polycaprolactone sponge with the pores filled with a poly(vinyl alcohol) gel simulating the new formed tissue within the pores was compared with that of the material immersed in water. Results were analyzed with Morrow's criteria for failure and accurate fittings are obtained just up to 200 loading cycles. It is also shown that the presence of poly(vinyl alcohol) increases the elastic modulus of the scaffolds, the effect being more pronounced with increasing the number of freeze/thawing cycles.

An "in vitro" experimental model to predict the mechanical behavior of macroporous scaffolds implanted in articular cartilage.

A model is proposed to assess mechanical behavior of tissue engineering scaffolds and predict their performance "in vivo" during tissue regeneration. To simulate the growth of tissue inside the pores of the scaffold, the scaffold is swollen with a Poly (Vinyl alcohol) solution and subjected to repeated freezing and thawing cycles. In this way the Poly (Vinyl alcohol) becomes a gel whose stiffness increases with the number of freezing and thawing cycles. Mechanical properties of the construct immersed in water are shown to be determined, in large extent, by the water mobility constraints imposed by the gel filling the pores. This is similar to the way that water mobility determines mechanical properties of highly hydrated tissues, such as articular cartilage. As a consequence, the apparent elastic modulus of the scaffold in compression tests is much higher than those of the empty scaffold or the gel. Thus this experimental model allows assessing fatigue behavior of the scaffolds under long-term dynamic loading in a realistic way, without recourse to animal experimentation.

Fatigue prediction in fibrin poly-ε-caprolactone macroporous scaffolds.

Tissue engineering applications rely on scaffolds that during its service life, either for in-vivo or in vitro applications, are under loading. The variation of the mechanical condition of the scaffold is strongly relevant for cell culture and has scarcely been addressed. The fatigue life cycle of poly-ε-caprolactone, PCL, scaffolds with and without fibrin as filler of the pore structure were characterized both dry and immersed in liquid water. It is observed that the there is a strong increase from 100 to 500 in the number of loading cycles before collapse in the samples tested in immersed conditions due to the more uniform stress distributions within the samples, the fibrin loading playing a minor role in the mechanical performance of the scaffolds.

Biomimetic hydroxyapatite coating on pore walls improves osteointegration of poly(L-lactic acid) scaffolds.

Polymer-ceramic composites obtained as the result of a mineralization process hold great promise for the future of tissue engineering. Simulated body fluids (SBFs) are widely used for the mineralization of polymer scaffolds. In this work an exhaustive study with the aim of optimizing the mineralization process on a poly(L-lactic acid) (PLLA) macroporous scaffold has been performed. We observed that when an air plasma treatment is applied to the PLLA scaffold its hydroxyapatite nucleation ability is considerably improved. However, plasma treatment only allows apatite deposition on the surface of the scaffold but not in its interior. When a 5 wt % of synthetic hydroxyapatite (HAp) nanoparticles is mixed with PLLA a more abundant biomimetic hydroxyapatite layer grows inside the scaffold in SBF. The morphology, amount, and composition of the generated biomimetic hydroxyapatite layer on the pores' surface have been analyzed. Large mineralization times are harmful to pure PLLA as it rapidly degrades and its elastic compression modulus significantly decreases. Degradation is retarded in the composite scaffolds because of the faster and extensive biomimetic apatite deposition and the role of HAp to control the pH. Mineralized scaffolds, covered by an apatite layer in SBF, were implanted in osteochondral lesions performed in the medial femoral condyle of healthy sheep. We observed that the presence of biomimetic hydroxyapatite on the pore's surface of the composite scaffold produces a better integration in the subchondral bone, in comparison to bare PLLA scaffolds.

Gelatin microparticles aggregates as three-dimensional scaffolding system in cartilage engineering.

A three-dimensional (3D) scaffolding system for chondrocytes culture has been produced by agglomeration of cells and gelatin microparticles with a mild centrifuging process. The diameter of the microparticles, around 10 µ, was selected to be in the order of magnitude of the chondrocytes. No gel was used to stabilize the construct that maintained consistency just because of cell and extracellular matrix (ECM) adhesion to the substrate. In one series of samples the microparticles were charged with transforming growth factor, TGF-ß1. The kinetics of growth factor delivery was assessed. The initial delivery was approximately 48 % of the total amount delivered up to day 14. Chondrocytes that had been previously expanded in monolayer culture, and thus dedifferentiated, adopted in this 3D environment a round morphology, both with presence or absence of growth factor delivery, with production of ECM that intermingles with gelatin particles. The pellet was stable from the first day of culture. Cell viability was assessed by MTS assay, showing higher absorption values in the cell/unloaded gelatin microparticle pellets than in cell pellets up to day 7. Nevertheless the absorption drops in the following culture times. On the contrary the cell viability of cell/TGF-ß1 loaded gelatin microparticle pellets was constant during the 21 days of culture. The formation of actin stress fibres in the cytoskeleton and type I collagen expression was significantly reduced in both cell/gelatin microparticle pellets (with and without TGF-ß1) with respect to cell pellet controls. Total type II collagen and sulphated glycosaminoglycans quantification show an enhancement of the production of ECM when TGF-ß1 is delivered, as expected because this growth factor stimulate the chondrocyte proliferation and improve the functionality of the tissue.

Fabrication of poly(lactic acid)-poly(ethylene oxide) electrospun membranes with controlled micro to nanofiber sizes.

Biodegradable poly(L-lactide acid) (PLLA) nanofiber membranes were prepared by electrospinning of PLLA and poly(ethylene oxide) (PEO). The selective removal of PEO by water allows to obtain smaller fiber diameters and to increase the porosity of the membranes in comparison to PLLA membranes obtained under the same electrospinning conditions. After removal of PEO membranes with fiber sizes of 260 nm and average porosity close to 80% are obtained. Thermal and infrared results confirm the poor miscibility of PLLA and PEO, with the PEO randomly distributed along the PLLA fibers. On the other, PLLA and PEO mixing strongly affect their respective degradation temperatures. The influence of the PEO in the electrospinning process is discussed and the results are correlated to the evolution of the PLLA fiber diameter.

Relaxation dynamics of poly(vinylidene fluoride) studied by dynamical mechanical measurements and dielectric spectroscopy.

The aim of this study is to analyze the mobility of polymer chains in semicrystalline poly(vinylidene fluoride) (PVDF). PVDF crystallizes from the melt in the α crystalline phase. The transformation from the α phase to the electroactive ß phase can be induced by stretching at temperatures in the range between 80 and 140 °C. The spherulitic structure of the crystalline phase is deformed during stretching to form fibrils oriented in the direction of the strain. The amorphous phase confined among the crystalline lamellae is distorted as well and some degree of orientation of the polymer chains is expected. Dynamic-mechanical and dielectric spectroscopy measurements were performed in PVDF films stretched to strain ratios up to 5 at temperatures between 80 and 140 °C. Dynamic-mechanical measurements were conducted between -60 °C and melting and in this temperature range the relaxation spectra show the main relaxation of the amorphous phase (called ß-relaxation) and at higher temperatures a relaxation related to crystallites motions (α (c)-relaxation). Although the mean relaxation times of the ß-relaxation are nearly equal in PVDF before and after crystal phase transformation, a significant change of shape of the relaxation spectrum proves the effect of chain distortion due to crystal reorganization. In stretched PVDF the elastic modulus of the polymer in the direction of deformation is significantly higher than in the transversal one, as expected by chain and crystals fibril orientation. The recovery of the deformation when the sample is heated is related with the appearance of the α (c)-relaxation. Dielectric spectroscopy spectrum shows the main relaxation of the amorphous phase and a secondary process (γ-relaxation) at lower temperatures. Stretching produces significant changes in the relaxation processes, mainly in the strength and shape of the main relaxation ß. The Havriliak-Negami function has been applied to analyze the dielectric response.

Fibronectin adsorption and cell response on electroactive poly(vinylidene fluoride) films.

Due to the large potential of electroactive materials in novel tissue engineering strategies, the aim of this work is to determine if the crystalline phase and/or the surface electrical charge of electroactive poly(vinylidene fluoride), PVDF, have influence on the biological response in monolayer cell culture. Non-polar α-PVDF and electroactive ß-PVDF were prepared. The ß-PVDF films were poled by corona discharge to show negative or positive electrical surface charge density. It has been concluded that hydrophilicity of the PVDF substrates depends significantly on crystalline phase and polarity. Furthermore, by means of atomic force microscopy and an enzyme-linked immunosorbent assay test, it has been shown that positive or negative poling strongly influences the behavior of ß-PVDF supports with respect to fibronectin (FN) adsorption, varying the exhibition of adhesion ligands of adsorbed FN. Culture of MC3T3-E1 pre-osteoeblasts proved that cell proliferation depends on surface polarity as well. These results open the viability of cell culture stimulation by mechanical deformation of a piezoelectric substrate that results in varying electrical charge densities on the substrate surface.