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by Keyword: Differentiation


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Ciapetti, G., Di Pompo, G., Avnet, S., Martini, D., Diez-Escudero, A., Montufar, E. B., Ginebra, M. P., Baldini, N., (2017). Osteoclast differentiation from human blood precursors on biomimetic calcium-phosphate substrates Acta Biomaterialia 50, 102-113

The design of synthetic bone grafts to foster bone formation is a challenge in regenerative medicine. Understanding the interaction of bone substitutes with osteoclasts is essential, since osteoclasts not only drive a timely resorption of the biomaterial, but also trigger osteoblast activity. In this study, the adhesion and differentiation of human blood-derived osteoclast precursors (OCP) on two different micro-nanostructured biomimetic hydroxyapatite materials consisting in coarse (HA-C) and fine HA (HA-F) crystals, in comparison with sintered stoichiometric HA (sin-HA, reference material), were investigated. Osteoclasts were induced to differentiate by RANKL-containing supernatant using cell/substrate direct and indirect contact systems, and calcium (Ca++) and phosphorus (P5+) in culture medium were measured. We observed that OCP adhered to the experimental surfaces, and that osteoclast-like cells formed at a rate influenced by the micro- and nano-structure of HA, which also modulate extracellular Ca++. Qualitative differences were found between OCP on biomimetic HA-C and HA-F and their counterparts on plastic and sin-HA. On HA-C and HA-F cells shared typical features of mature osteoclasts, i.e. podosomes, multinuclearity, tartrate acid phosphatase (TRAP)-positive staining, and TRAP5b-enzyme release. However, cells were less in number compared to those on plastic or on sin-HA, and they did not express some specific osteoclast markers. In conclusion, blood-derived OCP are able to attach to biomimetic and sintered HA substrates, but their subsequent fusion and resorptive activity are hampered by surface micro-nano-structure. Indirect cultures suggest that fusion of OCP is sensitive to topography and to extracellular calcium. Statement of Significance The novelty of the paper is the differentiation of human blood-derived osteoclast precursors, instead of mouse-derived macrophages as used in most studies, directly on biomimetic micro-nano structured HA-based surfaces, as triggered by osteoblast-produced factors (RANKL/OPG), and influenced by chemistry and topography of the substrate(s). Biomimetic HA-surfaces, like those obtained in calcium phosphate cements, are very different from the conventional calcium phosphate ceramics, both in terms of topography and ion exchange. The role of these factors in modulating precursors’ differentiation and activity is analysed. The system is closely reproducing the physiological process of attachment of host cells and further maturation to osteoclasts toward resorption of the substrate, which occurs in vivo after filling bone defects with the calcium phosphate grafts.

Keywords: Bone resorption, Differentiation, Hydroxyapatite, Ionic exchange, Osteoclasts, Topography


Zhao, M., Altankov, G., Grabiec, U., Bennett, M., Salmeron-Sanchez, M., Dehghani, F., Groth, T., (2016). Molecular composition of GAG-collagen I multilayers affects remodeling of terminal layers and osteogenic differentiation of adipose-derived stem cells Acta Biomaterialia 41, 86-99

The effect of molecular composition of multilayers, by pairing type I collagen (Col I) with either hyaluronic acid (HA) or chondroitin sulfate (CS) was studied regarding the osteogenic differentiation of adhering human adipose-derived stem cells (hADSCs). Polyelectrolyte multilayer (PEM) formation was based primarily on ion pairing and on additional intrinsic cross-linking through imine bond formation with Col I replacing native by oxidized HA (oHA) or CS (oCS). Significant amounts of Col I fibrils were found on both native and oxidized CS-based PEMs, resulting in higher water contact angles and surface potential under physiological condition, while much less organized Col I was detected in either HA-based multilayers, which were more hydrophilic and negatively charged. An important finding was that hADSCs remodeled Col I at the terminal layers of PEMs by mechanical reorganization and pericellular proteolytic degradation, being more pronounced on CS-based PEMs. This was in accordance with the higher quantity of Col I deposition in this system, accompanied by more cell spreading, focal adhesions (FA) formation and significant α2β1 integrin recruitment compared to HA-based PEMs. Both CS-based PEMs caused also an increased fibronectin (FN) secretion and cell growth. Furthermore, significant calcium phosphate deposition, enhanced ALP, Col I and Runx2 expression were observed in hADSCs on CS-based PEMs, particularly on oCS-containing one. Overall, multilayer composition can be used to direct cell-matrix interactions, and hence stem cell fates showing for the first time that PEMs made of biogenic polyelectrolytes undergo significant remodeling of terminal protein layers, which seems to enable cells to form a more adequate extracellular matrix-like environment. Statement of Significance: Natural polymer derived polyelectrolyte multilayers (PEMs) have been recently applied to adjust biomaterials to meet specific tissue demands. However, the effect of molecular composition of multilayers on both surface properties and cellular response, especially the fate of human adipose derived stem cells (hADSCs) upon osteogenic differentiation has not been studied extensively, yet. In addition, no studies exist that investigate a potential cell-dependent remodeling of PEMs made of extracellular matrix (ECM) components like collagens and glycosaminoglycans (GAGs). Furthermore, there is no knowledge whether the ability of cells to remodel PEM components may provide an added value regarding cell growth and differentiation. Finally, it has not been explored yet, how intrinsic cross-linking of ECM derived polyelectrolytes that improve the stability of PEMs will affect the differentiation potential of hADSCs. The current work aims to address these questions and found that the type of GAG has a strong effect on properties of multilayers and osteogenic differentiation of hADSCs. Additionally, we also show for the first time that PEMs made of biogenic polyelectrolytes undergo significant remodeling of terminal layers as completely new finding, which allows cells to form an ECM-like environment supporting differentiation upon osteogenic lineage. The finding of this work may open new avenues of application of PEM systems made by layer by layer (LbL) technique in tissue engineering and regenerative medicine.

Keywords: Collagen reorganization, Glycosaminoglycans, Layer-by-layer technique, Mesenchymal stem cells, Osteogenic differentiation


Montserrat, N., Garreta, E., Izpisua Belmonte, J. C., (2016). Regenerative strategies for kidney engineering FEBS Journal 283, (18), 3303-3324

The kidney is the most important organ for water homeostasis and waste excretion. It performs several important physiological functions for homeostasis: it filters the metabolic waste out of circulation, regulates body fluid balances, and acts as an immune regulator and modulator of cardiovascular physiology. The development of in vitro renal disease models with pluripotent stem cells (both human embryonic stem cells and induced pluripotent stem cells) and the generation of robust protocols for in vitro derivation of renal-specific-like cells from patient induced pluripotent stem cells have just emerged. Here we review major findings in the field of kidney regeneration with a major focus on the development of stepwise protocols for kidney cell production from human pluripotent stem cells and the latest advances in kidney bioengineering (i.e. decellularized kidney scaffolds and bioprinting). The possibility of generating renal-like three-dimensional structures to be recellularized with renal-derived induced pluripotent stem cells may offer new avenues to develop functional kidney grafts on-demand.

Keywords: Induced pluripotent stem cells, Kidney disease, Kidney engineering, Pluripotent stem cells, Renal differentiation


Lagunas, Anna, Martinez, Elena, Samitier, Josep, (2015). Surface-bound molecular gradients for the high throughput screening of cell responses Frontiers in Bioengineering and Biotechnology 3, Article 132

Chemical gradient surfaces are described as surfaces with a gradually varying composition along their length. Continuous chemical gradients have recently been proposed as alternative to discrete microarrays for the high throughput screening of the effects of ligand concentration in cells. Here we review some of the most recent examples in which gradients have been used to evaluate the effect of a varying ligand concentration in cell adhesion, morphology, growth and differentiation of cells, including some of our recent findings. They show the importance of the organization of ligands at the nanoscale, which is highlighted by abrupt changes in cell behavior at critical concentration thresholds.

Keywords: Cell Adhesion, Cell Differentiation, Cell growth, Cell morphology, Molecular gradient


de Oñate, L., Garreta, E., Tarantino, C., Martínez, E., Capilla, E., Navarro, I., Gutiérrez, J., Samitier, J., Campistol, J.M., Muñoz-Cánovas, P., Montserrat, N., (2015). Research on skeletal muscle diseases using pluripotent stem cells Muscle Cell and Tissue (ed. Sakuma, K.), InTech (Rijeka, Croatia) , 333-357

The generation of induced pluripotent stem cells (iPSCs), especially the generation of patient-derived pluripotent stem cells (PSCs) suitable for disease modelling in vitro, opens the door for the potential translation of stem-cell related studies into the clinic. Successful replacement, or augmentation, of the function of damaged cells by patient-derived differentiated stem cells would provide a novel cell-based therapy for skeletal muscle-related diseases. Since iPSCs resemble human embryonic stem cells (hESCs) in their ability to generate cells of the three germ layers, patient-specific iPSCs offer definitive solutions for the ethical and histo-incompatibility issues related to hESCs. Indeed human iPSC (hiPSC)-based autologous transplantation is heralded as the future of regenerative medicine. Interestingly, during the last years intense research has been published on disease-specific hiPSCs derivation and differentiation into relevant tissues/organs providing a unique scenario for modelling disease progression, to screen patient-specific drugs and enabling immunosupression-free cell replacement therapies. Here, we revise the most relevant findings in skeletal muscle differentiation using mouse and human PSCs. Finally and in an effort to bring iPSC technology to the daily routine of the laboratory, we provide two different protocols for the generation of patient-derived iPSCs.

Keywords: Pluripotent stem cells, Myogenic differentiation, Disease modelling, Patient-specific induced pluripotent stem cells, Muscular dystrophy


Vila, Olaia F., Bagó, Juli R., Navarro, Melba, Alieva, Maria, Aguilar, Elisabeth, Engel, Elisabeth, Planell, Josep, Rubio, Nuria, Blanco, Jerónimo, (2013). Calcium phosphate glass improves angiogenesis capacity of poly(lactic acid) scaffolds and stimulates differentiation of adipose tissue-derived mesenchymal stromal cells to the endothelial lineage Journal of Biomedical Materials Research - Part A 101A, (4), 932-941

The angiogenic capacity of a new biomaterial composite of poly(lactic acid) and calcium phosphate glass (PLA/CaP) was analyzed by noninvasive bioluminescence imaging (BLI) and histological procedures. Human adipose tissue-derived mesenchymal stromal cells expressing cytomegalovirus (CMV) promoter regulated Photinus pyralis luciferase (hAMSC-PLuc) grew up to 30 times the initial cell load, in vitro, when seeded in PLA/CaP scaffolds, but suffered an initial growth crisis followed by recovery when the scaffolds were subcutaneously implanted in SCID mice. To analyze changes in gene expression, hAMSC-PLuc cells were double labeled with a CMV promoter regulated Renilla reniformis luciferase and a Photinus pyralis luciferase reporter regulated by either the PECAM promoter or a hypoxia response element (HRE) artificial promoter and seeded in PLA/CaP and PLA scaffolds implanted in SCID mice. Analysis by BLI showed that hAMSCs in scaffolds were induced to differentiate to the endothelial lineage and did this faster in PLA/CaP than in PLA scaffolds. Endothelial differentiation correlated with a decrease in the activity of HRE regulated luciferase expression, indicative of a reduction of hypoxia. Histological analysis showed that PLA/CaP scaffolds were colonized by a functional host vascular system. Moreover, colonization by isolectin B4 positive host cells was more effective in PLA/CaP than in PLA scaffolds, corroborating BLI results.

Keywords: Scaffold, Bioluminescence imaging, Cell differentiation, Angiogenesis, Mesenchymal stromal cells


Gustavsson, J., Ginebra, M. P., Planell, J., Engel, E., (2012). Osteoblast-like cellular response to dynamic changes in the ionic extracellular environment produced by calcium-deficient hydroxyapatite Journal of Materials Science-Materials in Medicine 23, (10), 2509-2520

Solution-mediated reactions due to ionic substitutions are increasingly explored as a strategy to improve the biological performance of calcium phosphate-based materials. Yet, cellular response to well-defined dynamic changes of the ionic extracellular environment has so far not been carefully studied in a biomaterials context. In this work, we present kinetic data on how osteoblast-like SAOS-2 cellular activity and calcium-deficient hydroxyapatite (CDHA) influenced extracellular pH as well as extracellular concentrations of calcium and phosphate in standard in vitro conditions. Since cells were grown on membranes permeable to ions and proteins, they could share the same aqueous environment with CDHA, but still be physically separated from the material. In such culture conditions, it was observed that gradual material-induced adsorption of calcium and phosphate from the medium had only minor influence on cellular proliferation and alkaline phosphatase activity, but that competition for calcium and phosphate between cells and the biomaterial delayed and reduced significantly the cellular capacity to deposit calcium in the extracellular matrix. The presented work thus gives insights into how and to what extent solution-mediated reactions can influence cellular response, and this will be necessary to take into account when interpreting CDHA performance both in vitro and in vivo.

Keywords: Alkaline-phosphatase activity, Saos-2 cells, In-vitro, bone mineralization, Biological basis, Differentiation, Culture, Matrix, Proliferation, Topography


Woods, N. B., Parker, A. S., Moraghebi, R., Lutz, M. K., Firth, A. L., Brennand, K. J., Berggren, W. T., Raya, A., Belmonte, J. C. I., Gage, F. H., Verma, I. M., (2011). Brief report: Efficient generation of hematopoietic precursors and progenitors from human pluripotent stem cell lines Stem Cells 29, (7), 1158-1164

By mimicking embryonic development of the hematopoietic system, we have developed an optimized in vitro differentiation protocol for the generation of precursors of hematopoietic lineages and primitive hematopoietic cells from human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSCs). Factors such as cytokines, extra cellular matrix components, and small molecules as well as the temporal association and concentration of these factors were tested on seven different human ESC and iPSC lines. We report the differentiation of up to 84% human CD45+ cells (average 41% +/- 16%, from seven pluripotent lines) from the differentiation culture, including significant numbers of primitive CD45+/CD34+ and CD45+/CD34+/CD38- hematopoietic progenitors. Moreover, the numbers of hematopoietic progenitor cells generated, as measured by colony forming unit assays, were comparable to numbers obtained from fresh umbilical cord blood mononuclear cell isolates on a per CD45+ cell basis. Our approach demonstrates highly efficient generation of multipotent hematopoietic progenitors with among the highest efficiencies reported to date (CD45+/CD34+) using a single standardized differentiation protocol on several human ESC and iPSC lines. Our data add to the cumulating evidence for the existence of an in vitro derived precursor to the hematopoietic stem cell (HSC) with limited engrafting ability in transplanted mice but with multipotent hematopoietic potential. Because this protocol efficiently expands the preblood precursors and hematopoietic progenitors, it is ideal for testing novel factors for the generation and expansion of definitive HSCs with long-term repopulating ability.

Keywords: Differentiation, Hematopoiesis, Hematopoietic progenitors, Pluripotent stem cells


Perán, M., Sánchez-Ferrero, A., Tosh, D., Marchal, J. A., Lopez, E., Alvarez, P., Boulaiz, H., Rodríguez-Serrano, F., Aranega, A., (2011). Ultrastructural and molecular analyzes of insulin-producing cells induced from human hepatoma cells Cytotherapy 13, (2), 193-200

Background aims. Diabetes type I is an autoimmune disease characterized by the destruction of pancreatic insulin-producing (beta-) cells and resulting in external insulin dependence for life. Islet transplantation represents a potential treatment for diabetes but there is currently a shortage of suitable organs donors. To augment the supply of donors, different strategies are required to provide a potential source of beta-cells. These sources include embryonic and adult stem cells as well as differentiated cell types. The main goal of this study was to induce the transdifferentiation (or conversion of one type cell to another) of human hepatoma cells (HepG2 cells) to insulin-expressing cells based on the exposure of HepG2 cells to an extract of rat insulinoma cells (RIN). Methods. HepG2 cells were first transiently permeabilized with Streptolysin O and then exposed to a cell extract obtained from RIN cells. Following transient exposure to the RIN extract, the HepG2 cells were cultured for 3 weeks. Results. Acquisition of the insulin-producing cell phenotype was determined on the basis of (i) morphologic and (ii) ultrastructural observations, (iii) immunologic detection and (iv) reverse transcription (RT)-polymerase chain reaction (PCR) analysis. Conclusions. This study supports the use of cell extract as a feasible method for achieve transdifferentiation of hepatic cells to insulin-producing cells.

Keywords: Beta-cells, Diabetes, Insulin-producing cells, Transdifferentiation


Byrne, Damien P., Lacroix, Damien, Prendergast, Patrick J., (2011). Simulation of fracture healing in the tibia: Mechanoregulation of cell activity using a lattice modeling approach Journal of Orthopaedic Research 29, (10), 1496-1503

In this study, a three-dimensional (3D) computational simulation of bone regeneration was performed in a human tibia under realistic muscle loading. The simulation was achieved using a discrete lattice modeling approach combined with a mechanoregulation algorithm to describe the cellular processes involved in the healing process namely proliferation, migration, apoptosis, and differentiation of cells. The main phases of fracture healing were predicted by the simulation, including the bone resorption phase, and there was a qualitative agreement between the temporal changes in interfragmentary strain and bending stiffness by comparison to experimental data and clinical results. Bone healing was simulated beyond the reparative phase by modeling the transition of woven bone into lamellar bone. Because the simulation has been shown to work with realistic anatomical 3D geometry and muscle loading, it demonstrates the potential of simulation tools for patient-specific pre-operative treatment planning.

Keywords: Tissue differentiation, Computational analysis, Mechanical conditions, Bone regeneration, Weight-bearing, Proliferation, Osteoblast, Stiffness, Ingrowth, Scaffold


Milan, J. L., Planell, J. A., Lacroix, D., (2010). Simulation of bone tissue formation within a porous scaffold under dynamic compression Biomechanics and Modeling in Mechanobiology 9, (5), 583-596

A computational model of mechanoregulation is proposed to predict bone tissue formation stimulated mechanically by overall dynamical compression within a porous polymeric scaffold rendered by micro-CT. Dynamic compressions of 0.5-5% at 0.0025-0.025 s(-1) were simulated. A force-controlled dynamic compression was also performed by imposing a ramp of force from 1 to 70 N. The model predicts homogeneous mature bone tissue formation under strain levels of 0.5-1% at strain rates of 0.0025-0.005 s(-1). Under higher levels of strain and strain rates, the scaffold shows heterogeneous mechanical behaviour which leads to the formation of a heterogeneous tissue with a mixture of mature bone and fibrous tissue. A fibrous tissue layer was also predicted under the force-controlled dynamic compression, although the same force magnitude was found promoting only mature bone during a strain-controlled compression. The model shows that the mechanical stimulation of bone tissue formation within a porous scaffold closely depends on the loading history and on the mechanical behaviour of the scaffold at local and global scales.

Keywords: Bone tissue engineering, Scaffold, Tissue differentiation, Mechanoregulation, Finite element analysis


Morgenstern, C., Schwaibold, M., Randerath, W. J., Bolz, A., Jané, R., (2010). An invasive and a noninvasive approach for the automatic differentiation of obstructive and central hypopneas IEEE Transactions on Biomedical Engineering 57, (8), 1927-1936

The automatic differentiation of obstructive and central respiratory events is a major challenge in the diagnosis of sleep-disordered breathing. Esophageal pressure (Pes) measurement is the gold-standard method to identify these events. This study presents a new classifier that automatically differentiates obstructive and central hypopneas with the Pes signal and a new approach for an automatic noninvasive classifier with nasal airflow. An overall of 28 patients underwent night polysomnography with Pes recording, and a total of 769 hypopneas were manually scored by human experts to create a gold-standard annotation set. Features were automatically extracted from the Pes signal to train and test the classifiers (discriminant analysis, support vector machines, and adaboost). After a significantly (p < 0.01) higher incidence of inspiratory flow limitation episodes in obstructive hypopneas was objectively, invasively assessed compared to central hypopneas, the feasibility of an automatic noninvasive classifier with features extracted from the airflow signal was demonstrated. The automatic invasive classifier achieved a mean sensitivity, specificity, and accuracy of 0.90 after a 100-fold cross validation. The automatic noninvasive feasibility study obtained similar hypopnea differentiation results as a manual noninvasive classification algorithm. Hence, both systems seem promising for the automatic differentiation of obstructive and central hypopneas.

Keywords: Automatic differentiation, Central hypopnea, Esophageal pressure (Pes), Inspiratory flow limitation (IFL), Noninvasive classification, Obstructive hypopnea


Morgenstern, C., Schwaibold, M., Randerath, W., Bolz, A., Jané, R., (2010). Automatic non-invasive differentiation of obstructive and central hypopneas with nasal airflow compared to esophageal pressure Engineering in Medicine and Biology Society (EMBC) 32nd Annual International Conference of the IEEE , IEEE (Buenos Aires, Argentina) , 6142-6145

The differentiation of obstructive and central respiratory events is a major challenge in the diagnosis of sleep disordered breathing. Esophageal pressure (Pes) measurement is the gold-standard method to identify these events but its invasiveness deters its usage in clinical routine. Flattening patterns appear in the airflow signal during episodes of inspiratory flow limitation (IFL) and have been shown with invasive techniques to be useful to differentiate between central and obstructive hypopneas. In this study we present a new method for the automatic non-invasive differentiation of obstructive and central hypopneas solely with nasal airflow. An overall of 36 patients underwent full night polysomnography with systematic Pes recording and a total of 1069 hypopneas were manually scored by human experts to create a gold-standard annotation set. Features were automatically extracted from the nasal airflow signal to train and test our automatic classifier (Discriminant Analysis). Flattening patterns were non-invasively assessed in the airflow signal using spectral and time analysis. The automatic non-invasive classifier obtained a sensitivity of 0.71 and an accuracy of 0.69, similar to the results obtained with a manual non-invasive classification algorithm. Hence, flattening airflow patterns seem promising for the non-invasive differentiation of obstructive and central hypopneas.

Keywords: Practical, Experimental/ biomedical measurement, Feature extraction, Flow measurement, Medical disorders, Medical signal processing, Patient diagnosis, Pneumodynamics, Pressure measurement, Signal classification, Sleep, Spectral analysis/ automatic noninvasive differentiation, Obstructive hypopnea, Central hypopnea, Inspiratory flow limitation, Nasal airflow, Esophageal pressure, Polysomnography, Feature extraction, Discriminant analysis, Spectral analysis


Prendergast, P. J., Checa, S., Lacroix, D., (2010). Computational models of tissue differentiation Computational Modeling in Biomechanics (ed. Suvranu De, Farshid Guilak, Mohammad R. K. Mofrad), Springer-Verlag Berlin (Berlin) 3, 353-372

Readers of this chapter will learn about our approach to computer simulation of tissue differentiation in response to mechanical forces. It involves defining algorithms for mechanoregulation of each of following cell activities: proliferation, apoptosis, migration, and differentiation using a stimulus based on a combination of strain and fluid flow (Prendergast et al., J. Biomech., 1997) - algorithms are based on a lattice-modelling which also facilitates building algorithms for complex processes such as angiogenesis. The algorithms are designed to be collaboratable individually. They can be combined to create a computational simulation method for tissue differentiation, using finite element analysis to compute the mechanical stimuli in even quite complex biomechanical environments. Examples are presented of the simulation method in use.

Keywords: Mechanobiology, Lattice modeling, Differentiation, Tissue engineering, Finite element modeling, Scaffolds


Nicolas, O., Gavin, R., Del Rio, J. A., (2009). New insights into cellular prion protein (PrPc) functions: The "ying and yang" of a relevant protein Brain Research Reviews 61, (2), 170-184

The conversion of cellular prion protein (PrPc) a GPI-anchored protein, into a protease-K-resistant and infective form (generally termed PrPsc) is mainly responsible for Transmissible Spongiform Encephalopathies (TSEs), characterized by neuronal degeneration and progressive loss of basic brain functions. Although PrPc is expressed by a wide range of tissues throughout the body, the complete repertoire of its functions has not been fully deter-mined. Recent studies have confirmed its participation in basic physiological processes such as cell proliferation and the regulation of cellular homeostasis. Other studies indicate that PrPc interacts with several molecules to activate signaling cascades with a high number of cellular effects. To deter-mine PrPc functions, transgenic mouse models have been generated in the last decade. In particular, mice lacking specific domains of the PrPc protein have revealed the contribution of these domains to neurodegenerative processes. A dual role of PrPc has been shown, since most authors report protective roles for this protein while others describe pro-apoptotic functions. in this review, we summarize new findings on PrPc functions, especially those related to neural degeneration and cell signaling.

Keywords: Prion, Doppel, Shadoo, Cell death, Cell proliferation, Cell differentiation


Martinez, E., Lagunas, A., Mills, C. A., Rodriguez-Segui, S., Estevez, M., Oberhansl, S., Comelles, J., Samitier, J., (2009). Stem cell differentiation by functionalized micro- and nanostructured surfaces Nanomedicine 4, (1), 65-82

New fabrication technologies and, in particular, new nanotechnologies have provided biomaterial and biomedical scientists with enormous possibilities when designing customized supports and scaffolds with controlled nanoscale topography and chemistry. The main issue now is how to effectively design these components and choose the appropriate combination of structure and chemistry to tailor towards applications as challenging and complex as stem cell differentiation. Occasionally, an incomplete knowledge of the fundamentals of biological differentiation process has hampered this issue. However, the recent technological advances in creating controlled cellular microenvironments can be seen as a powerful tool for furthering fundamental biology studies. This article reviews the main strategies followed to achieve solutions to this challenge, particularly emphasizing the working hypothesis followed by the authors to elucidate the mechanisms behind the observed effects of structured surfaces on cell behavior.

Keywords: Cell pattering, Differentiation, Microcontact printing, Micropatterning, Microstructure, Nanoimprinting, Nanostructure, Stem cells


Carreras, A., Almendros, I., Acerbi, I., Montserrat, J. M., Navajas, D., Farre, R., (2009). Obstructive apneas induce early release of mesenchymal stem cells into circulating blood Sleep 32, (1), 117-119

STUDY OBJECTIVES: To investigate whether noninvasive application of recurrent airway obstructions induces early release of mesenchymal stem cells into the circulating blood in a rat model of obstructive sleep apnea. DESIGN: Prospective controlled animal study. SETTING: University laboratory. PATIENTS OR PARTICIPANTS: Twenty male Sprague-Dawley rats (250-300 g). INTERVENTIONS: A specially designed nasal mask was applied to the anesthetized rats. Ten rats were subjected to a pattern of recurrent obstructive apneas (60 per hour, lasting 15 seconds each) for 5 hours. Ten anesthetized rats were used as controls. MEASUREMENTS AND RESULTS: Mesenchymal stem cells from the blood and bone marrow samples were isolated and cultured to count the total number of colony-forming unit fibroblasts (CFU-F) of adherent cells after 9 days in culture. The number of CFU-F from circulating blood was significantly (P = 0.02) higher in the rats subjected to recurrent obstructive apneas (5.00 +/- 1.16; mean +/- SEM) than in controls (1.70 +/- 0.72). No significant (P = 0.54) differences were observed in CFU-F from bone marrow. CONCLUSIONS: Application of a pattern of airway obstructions similar to those experienced by patients with sleep apnea induced an early mobilization of mesenchymal stem cells into circulating blood.

Keywords: Adipocytes/cytology, Animals, Blood Cell Count, Bone Marrow Cells/ cytology, Cell Adhesion/physiology, Cell Count, Cell Differentiation/physiology, Cell Division/physiology, Disease Models, Animal, Fibroblasts/cytology, Male, Mesenchymal Stem Cells/ cytology, Osteocytes/cytology, Rats, Rats, Sprague-Dawley, Sleep Apnea, Obstructive/ blood, Stem Cells/cytology


Rodriguez-Segui, S. A., Pla, M., Engel, E., Planell, J. A., Martinez, E., Samitier, J., (2009). Influence of fabrication parameters in cellular microarrays for stem cell studies Journal of Materials Science: Materials in Medicine 20, (7), 1525-1533

Lately there has been an increasing interest in the development of tools that enable the high throughput analysis of combinations of surface-immobilized signaling factors and which examine their effect on stem cell biology and differentiation. These surface-immobilized factors function as artificial microenvironments that can be ordered in a microarray format. These microarrays could be useful for applications such as the study of stem cell biology to get a deeper understanding of their differentiation process. Here, the evaluation of several key process parameters affecting the cellular microarray fabrication is reported in terms of its effects on the mesenchymal stem cell culture time on these microarrays. Substrate and protein solution requirements, passivation strategies and cell culture conditions are investigated. The results described in this article serve as a basis for the future development of cellular microarrays aiming to provide a deeper understanding of the stem cell differentiation process.

Keywords: Bone-marrow, Protein microarrays, Progenitor cells, Differentiation, Surfaces, Growth, Biomaterials, Commitment, Pathways, Culture media


Engel, E., Del Valle, S., Aparicio, C., Altankov, G., Asin, L., Planell, J. A., Ginebra, M. P., (2008). Discerning the role of topography and ion exchange in cell response of bioactive tissue engineering scaffolds Tissue Engineering Part A 14, (8), 1341-1351

Surface topography is known to have an influence on osteoblast activity. However, in the case of bioactive materials, topographical changes can affect also ion exchange properties. This makes the problem more complex, since it is often difficult to separate the strictly topographical effects from the effects of ionic fluctuations in the medium. The scope of this paper is to analyze the simultaneous effect of topography and topography-mediated ion exchange on the initial cellular behavior of osteoblastic-like cells cultured on bioactive tissue engineering substrates. Two apatitic substrates with identical chemical composition but different micro/nanostructural features were obtained by low-temperature setting of a calcium phosphate cement. MG63 osteoblastic-like cells were cultured either in direct contact with the substrates or with their extracts. A strong and permanent decrease of calcium concentration in the culture medium, dependent on substrate topography, was detected. A major effect of the substrate microstructure on cell proliferation was observed, explained in part by the topography-mediated ion exchange, but not specifically by the ionic Ca(2+) fluctuations. Cell differentiation was strongly enhanced when cells were cultured on the finer substrate. This effect was not explained by the chemical modification of the medium, but rather suggested a strictly topographical effect.

Keywords: Alkaline Phosphatase/metabolism, Bone Cements/pharmacology, Calcium/metabolism, Calcium Phosphates/pharmacology, Cell Adhesion/drug effects, Cell Differentiation/drug effects, Cell Proliferation/drug effects, Cell Shape/drug effects, Cells, Cultured, Culture Media, Durapatite/pharmacology, Humans, Interferometry, Ion Exchange, Materials Testing, Osteoblasts/ cytology/drug effects/enzymology/ultrastructure, Phosphorus/metabolism, Powders, Tissue Engineering, Tissue Scaffolds


Charles-Harris, M., Koch, M. A., Navarro, M., Lacroix, D., Engel, E., Planell, J. A., (2008). A PLA/calcium phosphate degradable composite material for bone tissue engineering: an in vitro study Journal of Materials Science-Materials in Medicine 19, (4), 1503-1513

Biodegradable polymers reinforced with an inorganic phase such as calcium phosphate glasses may be a promising approach to fulfil the challenging requirements presented by 3D porous scaffolds for tissue engineering. Scaffolds' success depends mainly on their biological behaviour. This work is aimed to the in vitro study of polylactic acid (PLA)/CaP glass 3D porous constructs for bone regeneration. The scaffolds were elaborated using two different techniques, namely solvent-casting and phase-separation. The effect of scaffolds' micro and macrostructure on the biological response of these scaffolds was assayed. Cell proliferation, differentiation and morphology within the scaffolds were studied. Furthermore, polymer/glass scaffolds were seeded under dynamic conditions in a custom-made perfusion bioreactor. Results indicate that the final architecture of the solvent-cast or phase separated scaffolds have a significant effect on cells' behaviour. Solvent-cast scaffolds seem to be the best candidates for bone tissue engineering. Besides, dynamic seeding yielded a higher seeding efficiency in comparison with the static method.

Keywords: Biocompatible Materials/ chemistry, Bone and Bones/ metabolism, Calcium Phosphates/ chemistry, Cell Differentiation, Cell Proliferation, Humans, Lactic Acid/ chemistry, Microscopy, Confocal, Microscopy, Electron, Scanning, Osteoblasts/metabolism, Permeability, Polymers/ chemistry, Porosity, Solvents/chemistry, Tissue Engineering/ methods


Gustavsson, J., Altankov, G., Errachid, A., Samitier, J., Planell, J. A., Engel, E., (2008). Surface modifications of silicon nitride for cellular biosensor applications Journal of Materials Science-Materials in Medicine 19, (4), 1839-1850

Thin films of silicon nitride (Si3N4) can be used in several kinds of micro-sized biosensors as a material to monitor fine environmental changes related to the process of bone formation in vitro. We found however that Si3N4 does not provide optimal conditions for osseointegration as osteoblast-like MG-63 cells tend to detach from the surface when cultured over confluence. Therefore Si3N4 was modified with self-assembled monolayers bearing functional end groups of primary amine (NH2) and carboxyl (COOH) respectively. Both these modifications enhanced the interaction with confluent cell layers and thus improve osseointegration over Si3N4. Furthermore it was observed that the NH2 functionality increased the adsorption of fibronectin (FN), promoted cell proliferation, but delayed the differentiation. We also studied the fate of pre-adsorbed and secreted FN from cells to learn more about the impact of above functionalities for the development of provisional extracellular matrix on materials interface. Taken together our data supports that Si3N4 has low tissue integration but good cellular biocompatibility and thus is appropriate in cellular biosensor applications such as the ion-sensitive field effect transistor (ISFET). COOH and NH2 chemistries generally improve the interfacial tissue interaction with the sensor and they are therefore suitable substrates for monitoring cellular growth or matrix deposition using electrical impedance spectroscopy.

Keywords: Adsorption, Amines/chemistry, Biocompatible Materials/ chemistry, Biosensing Techniques, Cell Differentiation, Cell Line, Cell Proliferation, Electric Impedance, Extracellular Matrix/metabolism, Fibronectins/chemistry, Humans, Materials Testing, Osteoblasts/ cytology, Silicon Compounds/ chemistry, Surface Properties


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