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.

Capacitively coupled electrical stimulation of rat chondroepiphysis explants: A histomorphometric analysis.

The growth plate is a cartilaginous layer present from the gestation period until the end of puberty where it ossifies joining diaphysis and epiphysis. During this period several endocrine, autocrine, and paracrine processes within the growth plate are carried out by chondrocytes; therefore, a disruption in cellular functions may lead to pathologies affecting bone development. It is known that electric fields impact the growth plate; however, parameters such as stimulation time and electric field intensity are not well documented. Accordingly, this study presents a histomorphometrical framework to assess the effect of electric fields on chondroepiphysis explants. Bones were stimulated with 3.5 and 7 mV/cm, and for each electric field two exposure times were tested for 30 days (30 min and 1 h). Results evidenced that electric fields increased the hypertrophic zones compared with controls. In addition, a stimulation of 3.5 mV/cm applied for 1 h preserved the columnar cell density and its orientation. Moreover, a pre-hypertrophy differentiation in the center of the chondroepiphysis was observed when explants were stimulated during 1 h with both electric fields. These findings allow the understanding of the effect of electrical stimulation over growth plate organization and how the stimulation modifies chondrocytes morphophysiology.

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.

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.

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.

Computational methodology to determine fluid related parameters of non regular three-dimensional scaffolds.

The application of three-dimensional (3D) biomaterials to facilitate the adhesion, proliferation, and differentiation of cells has been widely studied for tissue engineering purposes. The fabrication methods used to improve the mechanical response of the scaffold produce complex and non regular structures. Apart from the mechanical aspect, the fluid behavior in the inner part of the scaffold should also be considered. Parameters such as permeability (k) or wall shear stress (WSS) are important aspects in the provision of nutrients, the removal of metabolic waste products or the mechanically-induced differentiation of cells attached in the trabecular network of the scaffolds. Experimental measurements of these parameters are not available in all labs. However, fluid parameters should be known prior to other types of experiments. The present work compares an experimental study with a computational fluid dynamics (CFD) methodology to determine the related fluid parameters (k and WSS) of complex non regular poly(L-lactic acid) scaffolds based only on the treatment of microphotographic images obtained with a microCT (µCT). The CFD analysis shows similar tendencies and results with low relative difference compared to those of the experimental study, for high flow rates. For low flow rates the accuracy of this prediction reduces. The correlation between the computational and experimental results validates the robustness of the proposed methodology.

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.

Preparation of highly porous hydroxyapatite from cuttlefish bone.

Hydroxyapatite structures for tissue engineering applications have been produced by hydrothermal (HT) treatment of aragonite in the form of cuttlefish bone at 200 degrees C. Aragonite (CaCO(3)) monoliths were completely transformed into hydroxyapatite after 48 h of HT treatment. The substitution of CO(3) (2-) groups predominantly into the PO(4) (3-) sites of the Ca(10)(PO(4))(6)(OH)(2) structure was suggested by FT-IR spectroscopy and Rietveld structure refinement. The intensity of the nu(3)PO(4) (3-) bands increase, while the intensity of the nu(2)CO(3) (2-) bands decrease with the duration of HT treatment resulting in the formation of carbonate incorporating hydroxyapatite. The SEM micrographs have shown that the interconnected hollow structure with pillars connecting parallel lamellae in cuttlefish bone is maintained after conversion. Specific surface area (S (BET)) and total pore volume increased and mean pore size decreased by HT treatment.