Sanchez, A.; Mata, Á.; Mateos, M.; Rodriguez-Cabello , J.C.; Alonso, M.; Planell, J.; Engel, E. Biomaterials Vol. 68, p. 42-53 DOI: 10.1016/j.biomaterials.2015.07.062 Date of publication: 2015-11-01 Journal article
Bone tissue engineering demands alternatives overcoming the limitations of traditional approaches in the context of a constantly aging global population. In the present study, elastin-like recombinamers hydrogels were produced by means of carbodiimide-catalyzed crosslinking with citric acid, a molecule suggested to be essential for bone nanostructure. By systematically studying the effect of the relative abundance of reactive species on gelation and hydrogel properties such as functional groups content, degradation and structure, we were able to understand and to control the crosslinking reaction to achieve hydrogels mimicking the fibrillary nature of the extracellular matrix. By studying the effect of polymer concentration on scaffold mechanical properties, we were able to produce hydrogels with a stiffness value of 36.13 +/- 10.72 kPa, previously suggested to be osteoinductive. Microstructured and mechanically-tailored hydrogels supported the growth of human mesenchymal stem cells and led to higher osteopontin expression in comparison to their non-tailored counterparts. Additionally, tailored hydrogels were able to rapidly self-mineralize in biomimetic conditions, evidencing that citric acid was successfully used both as a crosslinker and a bioactive molecule providing polymers with calcium phosphate nucleation capacity. (C) 2015 Elsevier Ltd. All rights reserved.
Electrospinning is a method that can be used to efficiently produce scaffolds that mimic the fibrous structure of natural tissue, such as muscle structures or the extracellular matrix of bone. The technique is often used as a way of depositing composites (organic/inorganic materials) to obtain bioactive nanofibers which have the requisite mechanical properties for use in tissue engineering. However, many factors can influence the formation and collection of fibers, including experimental variables such as the parameters of the solution of the electrospun slurry. In this study, we assessed the influence of the polymer concentration, glass content and glass hydrolysis level on the morphology and thickness of fibers produced by electrospinning for a PCL-(Si-Ca-P-2) bioactive ormoglassorganically modified glassblend. Based on previous assays, this combination of materials shows good angiogenic and osteogenic properties, which gives it great potential for use in tissue engineering. The results of our study showed that blend preparation directly affected the features of the resulting fibers, and when the parameters of the blend are precisely controlled, fibers with a regular diameter could be produced fairly easily when 2,2,2-trifluoroethanol was used as a solvent instead of tetrahydrofuran. The diameter of the homogeneous fibers ranged from 360 to 620 nm depending on the experimental conditions used. This demonstrates that experimental optimization of the electrospinning process is crucial in order to obtain a deposit of hybrid nanofibers with a regular shape. (c) 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 103B: 1287-1293, 2015.
Many cell therapies rely on the ability of mesenchymal stromal cells (MSCs) to diffuse and localize throughout the target tissue-such as tumoral and ischemic tissues-, in response to specific cytokine signals, rather than being concentrated at the site of implantation. Therefore, it is fundamental to engineer biomaterial carriers as reservoirs, from which cells can migrate, possibly in a controlled manner. In this work, microcarriers (µCs) made of polylactic acid are characterized as MSC delivery vehicles capable of modulating key chemotactic pathways. The effect of different functionalization strategies on MSC migratory behavior from the µCs is studied in vitro in relation to SDF-1a/CXCR4 axis,-a major actor in MSC recruitment, chemotaxis and homing. Collagen and arginine-glycine-aspartic acid (RGD) peptides were either covalently grafted or physisorbed on µC surface. While stable covalent modifications promoted better cell adhesion and higher proliferation compared to physisorption, the functionalization method of the µCs also affected the cells migratory behavior in response to SDF-1a (CXCL12) stimulation. Less stable coatings (physisorbed) showed sensibly higher number of migrating cells than covalent collagen/RGD coatings. The combination of physic-chemical cues provided by protein/peptide functionalization and stimuli induced by 3D culture on µCs improved MSC expression of CXCR4, and exerted a control over cell migration, a condition suitable to promote cell homing after transplantation in vivo. These are key findings to highlight the impact of surface modification approaches on chemokine-triggered cell release, and allow designing biomaterials for efficient and controlled cell delivery to damaged tissues.
Kovtun, A.; goeckelmann , M.; Montufar, E.; Ginebra, M.P.; Planell, J.; Santin, M.; Ignatius, A. Acta biomaterialia Vol. 12, p. 242-249 DOI: 10.1016/j.actbio.2014.10.034 Date of publication: 2015-01-15 Journal article
Major limitations of calcium phosphate cements (CPCs) are their relatively slow degradation rate and the lack of macropores allowing the ingrowth of bone tissue. The development of self-setting cement foams has been proposed as a suitable strategy to overcome these limitations. In previous work we developed a gelatine-based hydroxyapatite foam (G-foam), which exhibited good injectability and cohesion, interconnected porosity and good biocompatibility in vitro. In the present study we evaluated the in vivo performance of the G-foam. Furthermore, we investigated whether enrichment of the foam with soybean extract (SG-foam) increased its bioactivity. G-foam, SG-foam and non-foamed CPC were implanted in a critical-size bone defect in the distal femoral condyle of New Zealand white rabbits. Bone formation and degradation of the materials were investigated after 4, 12 and 20 weeks using histological and biomechanical methods. The foams maintained their macroporosity after injection and setting in vivo. Compared to non-foamed CPC, cellular degradation of the foams was considerably increased and accompanied by new bone formation. The additional functionalization with soybean extract in the SG-foam slightly reduced the degradation rate and positively influenced bone formation in the defect. Furthermore, both foams exhibited excellent biocompatibility, implying that these novel materials may be promising for clinical application in non-loaded bone defects. (C) 2014 Acta Materialia Inc. Published by Elsevier Ltd.
Sachot, N.; Mateos, M.; Planell, J.; Velders, A.; lewandowska, M.; Engel, E.; Castaño, O. Nanoscale Vol. 7, num. 37, p. 15349-15361 DOI: 10.1039/c5nr04275e Date of publication: 2015-01-01 Journal article
Hybrid materials are being extensively investigated with the aim of mimicking the ECM microenvironment to develop effective solutions for bone tissue engineering. However, the common drawbacks of a hybrid material are the lack of interactions between the scaffold's constituents and the masking of its bioactive phase. Conventional hybrids often degrade in a non-homogeneous manner and the biological response is far from optimal. We have developed a novel material with strong interactions between constituents. The bioactive phase is directly exposed on its surface mimicking the structure of the ECM of bone. Here, polylactic acid electrospun fibers have been successfully and reproducibly coated with a bioactive organically modified glass (ormoglass, Si-Ca-P-2 system) covalently. In comparison with the pure polymeric mats, the fibers obtained showed improved hydrophilicity and mechanical properties, bioactive ion release, exhibited a nanoroughness and enabled good cell adhesion and spreading after just one day of culture (rMSCs and rEPCs). The fibers were coated with different ormoglass compositions to tailor their surface properties (roughness, stiffness, and morphology) by modifying the experimental parameters. Knowing that cells modulate their behavior according to the exposed physical and chemical signals, the development of this instructive material is a valuable advance in the design of functional regenerative biomaterials.
Rajzer, I.; Menaszek, E.; Kwiatkowski, R.; Castaño, O.; Planell, J. Materials science and engineering C. Biomimetic and supramolecular systems Vol. 44, p. 183-190 DOI: 10.1016/j.msec.2014.08.017 Date of publication: 2014-11-01 Journal article
In this study gelatin (Gel) modified with calcium phosphate nanoparticles (SG5) and polycaprolactone (PCL) were used to prepare a 3D bi-layer scaffold by collecting electrospun PCL and gelatin/SG5 fibers separately in the same collector. The objective of this study was to combine the desired properties of PCL and Gel/SG5 in the same scaffold in order to enhance mineralization, thus improving the ability of the scaffold to bond to the bone tissue. The scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and the wide angle X-ray diffraction (WAXD) measurements confirmed that SG5 nanoparticles were successfully incorporated into the fibrous gelatin matrix. The composite Gel/SG5/PCL scaffold exhibited more enhanced mechanical properties than individual Gel and Gel/SG5 scaffolds. The presence of SG5 nanoparticles accelerated the nucleation and growth of apatite crystals on the surface of the composite Gel/SG5/PCL scaffold in simulated body fluid (SBF). The osteoblast response in vitro to developed electrospun scaffolds (PCL and Gel/SG5/PCL) was investigated by using normal human primary NHOst cell lines. NHOst cell culture studies showed that higher alkaline phosphatase (ALP) activity and better mineralization were obtained in the case of composite materials than in pure PCL scaffolds. The mechanically strong PCL scaffold served as a skeleton, while the Gel/SG5 fibers facilitated cell spreading and mineralization of the scaffold. (C) 2014 Elsevier B.V. All rights reserved.
Bioprinting allows the fabrication of living constructs with custom-made architectures by spatially controlled deposition of multiple bioinks. This is important for the generation of tissue, such as osteochondral tissue, which displays a zonal composition in the cartilage domain supported by the underlying subchondral bone. Challenges in fabricating functional grafts of clinically relevant size include the incorporation of cues to guide specific cell differentiation and the generation of sufficient cells, which is hard to obtain with conventional cell culture techniques. A novel strategy to address these demands is to combine bioprinting with microcarrier technology. This technology allows for the extensive expansion of cells, while they form multi-cellular aggregates, and their phenotype can be controlled. In this work, living constructs were fabricated via bioprinting of cell-laden microcarriers. Mesenchymal stromal cell (MSC)-laden polylactic acid microcarriers, obtained via static culture or spinner flask expansion, were encapsulated in gelatin methacrylamide-gellan gum bioinks, and the printability of the composite material was studied. This bioprinting approach allowed for the fabrication of constructs with high cell concentration and viability. Microcarrier encapsulation improved the compressive modulus of the hydrogel constructs, facilitated cell adhesion, and supported osteogenic differentiation and bone matrix deposition by MSCs. Bilayered osteochondral models were fabricated using microcarrier-laden bioink for the bone compartment. These findings underscore the potential of this new microcarrier-based biofabrication approach for bone and osteochondral constructs.
Mateos, M.; Castaño, O.; Planell, J.; Engel, E. Journal of materials science. Materials in medicine Vol. 25, num. 7, p. 1781-1787 DOI: 10.1007/s10856-014-5199-z Date of publication: 2014-07 Journal article
Surface biofunctionalisation of many biodegradable polymers is one of the used strategies to improve the biological activity of such materials. In this work, the introduction of collagen type I over the surface of a biodegradable polymer (poly lactic acid) processed in the forms of films and fibers leads to an enhancing of the cellular adhesion of human dermal fibroblast when compared to unmodified polymer and biomolecule-physisorbed polymer surface. The change of topography of the material does not affect the cellular adhesion but results in a higher proliferation of the fibroblast cultured over the fibers. Moreover, the difference of topography governs the cellular morphology, i.e. cells adopt a more stretched conformation where cultured over the films while a more elongated with lower area morphology are obtained for the cells grown over the fibers. This study is relevant for designing and modifying different biodegradable polymers for their use as scaffolds for different applications in the field of Tissue Engineering and Regenerative Medicine.
Regenerative medicine strategies to promote recovery following traumatic brain injuries are currently focused on the use of biomaterials as delivery systems for cells or bioactive molecules. This study shows that cell-free biomimetic scaffolds consisting of radially aligned electrospun poly-L/DL. lactic acid (PLA70/30) nanofibers release L-lactate and reproduce the 3D organization and supportive function of radial glia embryonic neural stem cells. The topology of PLA nanofibers supports neuronal migration while L-lactate released during PLA degradation acts as an alternative fuel for neurons and is required for progenitor maintenance. Radial scaffolds implanted into cavities made in the postnatal mouse brain fostered complete implant vascularization, sustained neurogenesis, and allowed the long-term survival and integration of the newly generated neurons. Our results suggest that the endogenous central nervous system is capable of regeneration through the in vivo dedifferentiation induced by biophysical and metabolic cues, with no need for exogenous cells, growth factors, or genetic manipulation. (C) 2014 Elsevier Ltd. All rights reserved.
Regenerative medicine strategies to promote recovery following traumatic brain injuries are currently focused on the use of biomaterials as delivery systems for cells or bioactive molecules. This study shows that cell-free biomimetic scaffolds consisting of radially aligned electrospun poly-l/dl lactic acid (PLA70/30) nanofibers release l-lactate and reproduce the 3D organization and supportive function of radial glia embryonic neural stem cells. The topology of PLA nanofibers supports neuronal migration while l-lactate released during PLA degradation acts as an alternative fuel for neurons and is required for progenitor maintenance. Radial scaffolds implanted into cavities made in the postnatal mouse brain fostered complete implant vascularization, sustained neurogenesis, and allowed the long-term survival and integration of the newly generated neurons. Our results suggest that the endogenous central nervous system is capable of regeneration through the invivo dedifferentiation induced by biophysical and metabolic cues, with no need for exogenous cells, growth factors, or genetic manipulation.
Sanzana, E.; Navarro, M.; Ginebra, M.P.; Planell, J.; Ojeda, A.; Montecinos, H. Journal of biomedical materials research. Part A Vol. 102, num. 6, p. 1767-1773 DOI: 10.1002/jbm.a.34845 Date of publication: 2014-06-01 Journal article
The aim of this work is to shed light on the role of porosity and pore architecture in the in vivo bone regeneration capacity of biodegradable glass scaffolds. A calcium phosphate glass in the system P2O5-CaO-Na2O-TiO2 was foamed using two different porogens, namely albumen and hydrogen peroxide (H2O2); the resulting three-dimensional porous structures were characterized and implanted in New Zealand rabbits to study their in vivo behavior. Scaffolds foamed with albumen displayed a monomodal pore size distribution centered around 150 m and a porosity of 82%, whereas scaffolds foamed with H2O2 showed lower porosity (37%), with larger elongated pores, and multimodal size distribution. After 12 weeks of implantation, histology results revealed a good osteointegration for both types of scaffolds. The quantitative morphometric analysis showed the substitution of the biomaterial by new bone in the case of glasses foamed with albumen. In contrast, bone neoformation and material resorption were significantly lower in the defects filled with the scaffolds foamed with H2O2. The results obtained in this study showed that both calcium phosphate glass scaffolds were osteoconductive, biocompatible, and biodegradable materials. However, differences in porosity, pore architecture, and microstructure led to substantially different in vivo response. (c) 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 1767-1773, 2014.
Bone is the main store of calcium and progenitor cells in the body. During the resorption process, the local calcium concentration reaches 8-40 mM, and the surrounding cells are exposed to these fluctuations in calcium. This stimulus is a signal that is detected through the calcium sensing receptor (CaSR), which modulates chemotactic and proliferative G protein-dependent signaling pathways. The objective of the present work is to evaluate the roles of extracellular calcium ([Ca2+](o)) and the CaSR in osteoinduction. Rat bone marrow mesenchymal stromal cells (rBMSCs) were stimulated with 10 mM of Ca2+. Several experiments were conducted to demonstrate the effect of [Ca2+](o) on chemotaxis, proliferation and differentiation on the osteoblastic lineage. It was found that [Ca2+](o) induces rBMSCs to migrate and proliferate in a concentration-dependent manner. Real-time polymerase chain reaction and immunofluorescence also revealed that 10 mM Ca2+ stimulates overexpression of osteogenic markers in rBMSCs, including alkaline phosphatase (ALP), bone sialoprotein, collagen Ia1 and osteocalcin. Functional assays determining ALP activity and mineralization tests both corroborate the increased expression of these markers in rBMSCs stimulated with Ca2+. Moreover, CaSR blockage inhibited the cellular response to stimulation with high concentrations of [Ca2+](o), revealing that the CaSR is a key modulator of these cellular responses. (C) 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Castaño, O.; Sachot, N.; Xuriguera, E.; Engel, E.; Planell, J.; Park, J.; Jin, G.; Kim, T.H; Kim, J.; Kim, H-W. ACS applied materials and interfaces Vol. 6, num. 10, p. 7512-7522 DOI: 10.1021/am500885v Date of publication: 2014-05-28 Journal article
In bone regeneration, silicon-based calcium phosphate glasses (Bioglasses) have been widely used since the 1970s. However, they dissolve very slowly because of their high amount of Si (SiO2 > 45%). Recently, our group has found that calcium ions released by the degradation of glasses in which the job of silicon is done by just 5% of TiO2 are effective angiogenic promoters, because of their stimulation of a cell-membrane calcium sensing receptor (CaSR). Based on this, other focused tests on angiogenesis have found that Bioglasses also have the potential to be angiogenic promoters even with high contents of silicon (80%); however, their slow degradation is still a problem, as the levels of silicon cannot be decreased any lower than 45%. In this work, we propose a new generation of hybrid organically modified glasses, ormoglasses, that enable the levels of silicon to be reduced, therefore speeding up the degradation process. Using electrospinning as a faithful way to mimic the extracellular matrix (ECM), we successfully produced hybrid fibrous mats with three different contents of Si (40, 52, and 70%), and thus three different calcium ion release rates, using an ormoglass-polycaprolactone blend approach. These mats offered a good platform to evaluate different calcium release rates as osteogenic promoters in an in vivo subcutaneous environment. Complementary data were collected to complement Ca2+ release analysis, such as stiffness evaluation by AFM, zeta-potential, morphology evaluation by FESEM, proliferation and differentiation analysis, as well as in vivo subcutaneous implantations. Material and biological characterization suggested that compositions of organic/inorganic hybrid materials with a Si content equivalent to 40%, which were also those that released more calcium, were osteogenic. They also showed a greater ability to form blood vessels. These results suggest that Si-based ormoglasses can be considered an efficient tool for calcium release modulation, which could play a key role in the angiogenic promoting process.
Serra, T.; Ortiz, M.; Engel, E.; Planell, J.; Navarro, M. Materials science and engineering C. Biomimetic and supramolecular systems Vol. 38, p. 55-62 DOI: 10.1016/j.msec.2014.01.003 Date of publication: 2014-05-01 Journal article
Achieving high quality 3D-printed structures requires establishing the right printing conditions. Finding processing conditions that satisfy both the fabrication process and the final required scaffold properties is crucial. This work stresses the importance of studying the outcome of the plasticizing effect of PEG on PLA-based blends used for the fabrication of 3D-direct-printed scaffolds for tissue engineering applications. For this, PLA/PEG blends with 5, 10 and 20% (w/w) of PEG and PLA/PEG/bioactive CaP glass composites were processed in the form of 3D rapid prototyping scaffolds. Surface analysis and differential scanning calorimetry revealed a rearrangement of polymer chains and a topography, wettability and elastic modulus increase of the studied surfaces as PEG was incorporated. Moreover, addition of 10 and 20% PEG led to non-uniform 3D structures with lower mechanical properties. In vitro degradation studies showed that the inclusion of PEG significantly accelerated the degradation rate of the material. Results indicated that the presence of PEG not only improves PLA processing but also leads to relevant surface, geometrical and structural changes including modulation of the degradation rate of PLA-based 3D printed scaffolds. (C) 2014 Elsevier B.V. All rights reserved.
Dessi, M.; Alvarez, M.; De Santis, R.; Ginebra, M.P.; Planell, J.; Ambrosio, L. Journal of materials science. Materials in medicine Vol. 25, num. 2, p. 283-295 DOI: 10.1007/s10856-013-5071-6 Date of publication: 2014-02 Journal article
An alternative approach to bone repair for less invasive surgical techniques, involves the development of biomaterials directly injectable into the injury sites and able to replicate a spatially organized platform with features of bone tissue. Here, the preparation and characterization of an innovative injectable bone analogue made of calcium deficient hydroxyapatite and foamed gelatin is presented. The biopolymer features and the cement self-setting reaction were investigated by rheological analysis. The porous architecture, the evolution of surface morphology and the grains dimension were analyzed with electron microscopy (SEM/ESEM/TEM). The physico-chemical properties were characterized by X-ray diffraction and FTIR analysis. Moreover, an injection test was carried out to prove the positive effect of gelatin on the flow ensuing that cement is fully injectable. The cement mechanical properties are adequate to function as temporary substrate for bone tissue regeneration. Furthermore, MG63 cells and bone marrow-derived human mesenchymal stem cells (hMSCs) were able to migrate and proliferate inside the pores, and hMSCs differentiated to the osteoblastic phenotype. The results are paving the way for an injectable bone substitute with properties that mimic natural bone tissue allowing the successful use as bone filler for craniofacial and orthopedic reconstructions in regenerative medicine.
We present the immobilization on synthetic substrates of elastin-like recombinamers (ELR) that combine a bioactive motif for cell adhesion with protein antifouling properties. Physical adsorption of the recombinamers and covalent-grafting through organosilane chemistry were investigated. The biochemically-modified surfaces were thoroughly characterized and tested for protein absorption in serum by fluorescence-labelling, XPS, Ellipsometry, and OWLS. The ELR were successfully grafted and stable, even upon mechanical stresses; being the covalent bonding favourable over physical adsorption. The coated metal surfaces exhibited excellent reduction of serum protein adsorption (9 ng/cm2) compared to the bare metal surface (310 ng/cm2). Non-specific protein adsorption may mask the introduced bioactive motifs; therefore, the bioactivated surfaces should display serum-protein antifouling properties. Finally, improved hMSCs response was assessed on the bioactivated substrates. In summary, the coatings simultaneously displayed anti-fouling and bioactive properties. These studies investigated key factors to enhance tissue material interactions fundamental for the design of bioactive devices and future biomedical applications
We present the immobilization on synthetic substrates of elastin-like recombinamers (ELR) that combine a bioactive motif for cell adhesion with protein antifouling properties. Physical adsorption of the recombinamers and covalent-grafting through organosilane chemistry were investigated. The biochemically-modified surfaces were thoroughly characterized and tested for protein absorption in serum by fluorescence-labelling, XPS, Ellipsometry, and OWLS. The ELR were successfully grafted and stable, even upon mechanical stresses; being the covalent bonding favourable over physical adsorption. The coated metal surfaces exhibited excellent reduction of serum protein adsorption (9 ng/cm2) compared to the bare metal surface (310 ng/cm2). Non-specific protein adsorption may mask the introduced bioactive motifs; therefore, the bioactivated surfaces should display serum-protein antifouling properties. Finally, improved hMSCs response was assessed on the bioactivated substrates. In summary, the coatings simultaneously displayed anti-fouling and bioactive properties. These studies investigated key factors to enhance tissue material interactions fundamental for the design of bioactive devices and future biomedical applications.
Surface properties of biomaterials play a major role in the governing of cell
functionalities. It is well known that mechanical, chemical and nanotopo-
graphic cues, for example, influence cell proliferation and differentiation.
Here, we present a novel coating protocol to produce hierarchicallyengineered
fibrous scaffolds with tailorable surface characteristics, which mimic bone
extracellular matrix. Based on the sol–gel method and a succession of surface
treatments, hollow electrospun polylactic acid fibres were coated with a
silicon–calcium–phosphate bioactive organic–inorganic glass. Compared
with pure polymeric fibres that showed a completely smooth surface, the
coated fibres exhibited a nanostructured topography and greater roughness.
They also showed improved hydrophilic properties and a Young’s modulus
sixfold higher than non-coated ones, while remaining fully flexible and easy
to handle. Rat mesenchymal stem cells cultured on these fibres showed
great cellular spreading and interactions with the material. This protocol can
be transferred to other structures and glasses, allowing the fabrication of var-
ious materials with well-defined features. This novel approach represents
therefore a valuable improvement in the production of artificial matrices
able to direct stem cell fate through physical and chemical interactions
Research on surface modification of polymeric materials to guide the cellular activity in biomaterials designed for tissue engineering applications has mostly focused on the use of natural extracellular matrix (ECM) proteins and short peptides, such as RGD. However, the use of engineered proteins can gather the advantages of these strategies and avoid the main drawbacks. In this study, recombinant engineered proteins called elastin-like recombinamers (ELRs) have been used to functionalize poly(lactic) acid (PLA) model surfaces. The structure of the ELRs has been designed to include the integrin ligand RGDS and the cross-linking module VPGKG. Surface functionalization has been characterized and optimized by means of ELISA and atomic force microscopy (AFM). The results suggest that ELR functionalization creates a nonfouling canvas able to restrict unspecific adsorption of proteins. Moreover, AFM analysis reveals the conformation and disposition of ELRs on the surface. Biological performance of PLA surfaces functionalized with ELRs has been studied and compared with the use of short peptides. Cell response has been assessed for different functionalization conditions in the presence and absence of the bovine serum albumin (BSA) protein, which could interfere with the surface-cell interaction by adsorbing on the interface. Studies have shown that ELRs are able to elicit higher rates of cell attachment, stronger cell anchorages and faster levels of proliferation than peptides. This work has demonstrated that the use of engineered proteins is a more efficient strategy to guide the cellular activity than the use of short peptides, because they not only allow for better cell attachment and proliferation, but also can provide more complex properties such as the creation of nonfouling surfaces.
Research on surface modi
cation of polymeric materials to
guide the cellular activity in biomaterials designed for tissue engineering
applications has mostly focused on the use of natural extracellular matrix
(ECM) proteins and short peptides, such as RGD. However, the use of
engineered proteins can gather the advantages of these strategies and avoid the
main drawbacks. In this study, recombinant engineered proteins called elastin-
like recombinamers (ELRs) have been used to functionalize poly(lactic) acid
(PLA) model surfaces. The structure of the ELRs has been designed to
include the integrin ligand RGDS and the cross-linking module VPGKG. Surface functionalization has been characterized and
optimized by means of ELISA and atomic force microscopy (AFM). The results suggest that ELR functionalization creates a
nonfouling canvas able to restrict unspeci
c adsorption of proteins. Moreover, AFM analysis reveals the conformation and
disposition of ELRs on the surface. Biological performance of PLA surfaces functionalized with ELRs has been studied and
compared with the use of short peptides. Cell response has been assessed for di
erent functionalization conditions in the
presence and absence of the bovine serum albumin (BSA) protein, which could interfere with the surface
cell interaction by
adsorbing on the interface. Studies have shown that ELRs are able to elicit higher rates of cell attachment, stronger cell
anchorages and faster levels of proliferation than peptides. This work has demonstrated that the use of engineered proteins is a
cient strategy to guide the cellular activity than the use of short peptides, because they not only allow for better cell
attachment and proliferation, but also can provide more complex properties such as the creation of nonfouling surfaces
In bone tissue engineering, the composition of the ionic extracellular environment (IEE) can determine
both cellular fate and a biomaterial
s development and performance. Therefore, precise control of the
IEE and a perfect understanding of the dynamic changes that it can be subject to due to cellular activity
the extracellular concentrations of calcium and phosphate during long-term cultures. It was observed
that cellular in
uence onthe IEE varied greatly between the two models and could be linked to the capac-
ity of cells to deposit calcium in the extracellular matri
x. Miniaturized ion-selective electrodes that could
allow for real-time monitoring ofcalcium in aminimally invasive waywere then constructed.The electro-
des were characterized in standard
cell culture environments, prior to being successfully applied
for periods of 24h, to record the dynamics of cell-induced deposition of calcium in the extracellular
matrix, while using osteogenic media of either high or low concentrations of phosphate. As a result, this
study provides the background and technological means for the non-destructive evaluation of the IEE
and allows for the optimization and development of better models of bone tissue construction
To develop tissue engineering strategies useful for repairing damage in the central nervous system (CNS) it is essential to design scaffolds that emulate the NSC niche and its tight control of neural cell genesis, growth, and differentiation. In this study we tested two types of poly l/dl lactic acid (PLA95/5 and PLA70/30), a biodegradable material permissive for neural cell adhesion and growth, as materials for nerve regeneration. Both PLA were slightly hydrophobic and negatively charged but differed in crystallinity, stiffness and degradation rate. PLA95/5 films were highly crystalline, stiff (GPa), and did not degrade significantly in the one-month period analyzed in culture. In contrast, PLA70/30 films were more amorphous, softer (MPa) and degraded faster, releasing significant amounts of lactate into the culture medium. PLA70/30 performs better than PLA95/5 for primary cortical neural cell adhesion, proliferation and differentiation, maintaining the pools of neuronal and glial progenitor cells in vitro.
To develop tissue engineering strategies useful for repairing damage in the central nervous system (CNS) it is essential to design scaffolds that emulate the NSC niche and its tight control of neural cell genesis, growth, and differentiation. In this study we tested two types of poly l/dl lactic acid (PLA95/5 and PLA70/30), a biodegradable material permissive for neural cell adhesion and growth, as materials for nerve regeneration. Both PLA were slightly hydrophobic and negatively charged but differed in crystallinity, stiffness and degradation rate. PLA95/5 films were highly crystalline, stiff (GPa), and did not degrade significantly in the one-month period analyzed in culture. In contrast, PLA70/30 films were more amorphous, softer (MPa) and degraded faster, releasing significant amounts of lactate into the culture medium. PLA70/30 performs better than PLA95/5 for primary cortical neural cell adhesion, proliferation and differentiation, maintaining the pools of neuronal and glial progenitor cells in vitro. l-lactate in the medium recapitulated PLA70/30's maintenance of neuronal restricted progenitors but did not sustain bipotential or glial restricted progenitors in the cultures, as occurred when neural cells were grown on PLA70/30. Our results suggest that PLA70/30 may mimic some of the physical and biochemical characteristics of the NSC niche. Its mechanical and surface properties may act synergistically in the modulation of bipotential and glial restricted progenitor phenotypes, while it is l-lactate, either added to the medium or released by the film that drives the maintenance of neuronal restricted progenitor cell phenotypes.
Fabrication of new biodegradable scaffolds that guide and stimulate tissue regeneration is still a major issue in tissue engineering approaches. Scaffolds that possess adequate biodegradability, pore size, interconnectivity, bioactivity and mechanical properties in accordance with the injured tissue are required. This work aimed to develop and characterize three-dimensional (3-D) scaffolds that fulfill the aforementioned requirements. For this, a nozzle-based rapid prototyping system was used to combine polylactic acid and a bioactive CaP glass to fabricate 3-D biodegradable scaffolds with two patterns (orthogonal and displaced double layer). Scanning electron microscopy and micro-computer tomography showed that 3-D scaffolds had completely interconnected porosity, uniform distribution of the glass particles, and a controlled and repetitive architecture. Surface properties were also assessed, showing that the incorporation of glass particles increased both the roughness and the hydrophilicity of the scaffolds. Mechanical tests indicated that compression strength is dependent on the scaffold geometry and the presence of glass. Preliminary cell response was studied with primary mesenchymal stem cells (MSC) and revealed that CaP glass improved cell adhesion. Overall, the results showed the suitability of the technique/materials combination to develop 3-D porous scaffolds and their initial biocompatibility, both being valuable characteristics for tissue engineering applications.
Pegueroles, Marta; Tonda-Turo, C.; Planell, J.; Gil, F.J.; Aparicio, C. Journal of the Royal Society Interface Vol. 7, num. 1-4, p. 1-13 DOI: 10.1007/s13758-012-0048-4 Date of publication: 2012-12 Journal article
Gustavsson, J.; Ginebra, M.P.; Planell, J.; Engel, E. Journal of materials science. Materials in medicine Vol. 23, num. 10, p. 2509-2520 DOI: 10.1007/s10856-012-4705-4 Date of publication: 2012-10 Journal article
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.
Smart biomaterials play a key role when aiming at successful tissue repair by means of regenerative medicine approaches, and are expected to contain chemical as well as mechanical cues that will guide the regenerative process. Recent advances in the understanding of stem cell biology and mechanosensing have shed new light onto the importance of the local microenvironment in determining cell fate. Herein we report the biological properties of a bioactive, biodegradable calcium phosphate glass/polylactic acid composite biomaterial that promotes bone marrowderived endothelial progenitor cell (EPC) mobilisation, differentiation and angiogenesis through the creation of a controlled bone healing-like microenvironment. The angiogenic response is triggered by biochemical and mechanical cues provided by the composite, which activate two synergistic cell signalling pathways: a biochemical one mediated by the calcium-sensing receptor and a mechanosensitive one regulated by non-muscle myosin II contraction. Together, these signals promote a synergistic response by activating EPCs-mediated VEGF and VEGFR-2 synthesis, which in turn promote progenitor cell homing, differentiation and tubulogenesis. These findings highlight the importance of controlling microenvironmental cues for stem/progenitor cell tissue engineering and offer exciting new therapeutical opportunities for biomaterialbased vascularisation approaches and clinical applications
Salerno, A.; Levato, R.; Mateos, M.; Engel, E.; Netti, P.; Planell, J. Journal of biomedical materials research. Part A Vol. 101, num. 3, p. 720-732 DOI: 10.1002/jbm.a.34374 Date of publication: 2012-08-31 Journal article
The present study reports a novel approach for the
design and fabrication of polylactic acid (PLA) microparticle-
based scaffolds with microstructural properties suitable for
bone and cartilage regeneration. Macroporous PLA scaffolds
with controlled shape were fabricated by means of a semicon-
tinuous process involving (1) microfluidic emulsification of a
PLA/ethyl lactate solution (5% w/v) in a span 80/paraffin oil so-
lution (3% v/v) followed by (2) particles coagulation/assembly
in an acetone/water solution for the development of a continu-
ous matrix. Porous scaffolds prepared from particles with
monomodal or bimodal size distribution, overall porosity
ranges from 93 to 96%, interparticles porosity from 41 to 54%,
and static compression moduli from 0.3 to 1.4 MPa were man-
ufactured by means of flow rate modulation of of the continu-
ous phase during emulsion. The biological response of the
scaffolds was assessed
by using bone marrow-derived
rat mesenchymal stem cells (MSCs). The results demonstrated
the ability of the scaffolds to support the extensive and uni-
form three-dimensional adhesion, colonization, and prolifera-
tion of MSCs within the entire construct
When the intervertebral disc is removed to
relieve chronic pain, subsequent segment stabilization
should restore the functional mechanics of the native disc.
Because of partially constrained motions and the lack of
intrinsic rotational stiffness ball-on-socket implants present
many disadvantages. Composite disc substitutes mimicking
healthy disc structures should be able to assume the role
expected for a disc substitute with fewer restrictions than
ball-on-socket implants. A biomimetic composite disc
prototype including artificial nucleus fibre-reinforced
annulus and endplates was modelled as an L4–L5 disc
substitute within a L3–L5 lumbar spine finite element
model. Different device updates, i.e. changes of material
properties fibre distributions and volume fractions and
nucleus placements were proposed. Load- and displace-
ment-controlled rotations were simulated with and without
body weight applied. The original prototype reduced
greatly the flexibility of the treated segment with signifi-
cant adjacent level effects under displacement-controlled
or hybrid rotations. Device updates allowed restoring large
part of the global axial and sagittal rotational flexibility
predicted with the intact model. Material properties played
a major role, but some other updates were identified to
potentially tune the device behaviour against specific
motions. All device versions altered the coupled interseg-
mental shear deformations affecting facet joint contact
through contact area displacements. Loads in the bony
endplates adjacent to the implants increased as the implant
stiffness decreased but did not appear to be a strong limi-
tation for the implant biomechanical and mechanobiolog-
ical functionality. In conclusion, numerical results given by
biomimetic composite disc substitutes were encouraging
with greater potential than that offered by ball-on-socket
PLA MPs are prepared via a novel and toxic-chemical-free fabrication route using ethyl lactate,
a green solvent and FDA-approved aroma. MPs are obtained by a solution jet break-up and
solvent displacement method. Adjusting flow parameters allows the tuning of MPs size
between 60 and 180 mm, with reduced polydispersity.
Morphological analysis shows microporous particles
with Janus-like surface. A fluorophore is successfully
loaded into the MPs during their formation step. This
versatile green solvent-based procedure is proven to be
suitable for drug encapsulation and delivery applications.
The method may be extended to different
droplet generation techniques.
Radial glia cells (RGC) are multipotent progenitors that generate neurons and glia during CNS development,
and which also served as substrate for neuronal migration. After a lesion, reactive glia are the
main contributor to CNS regenerative blockage, although some reactive astrocytes are also able to dedifferentiate
in situ into radial glia-like cells (RGLC), providing beneficial effects in terms of CNS
recovery. Thus, the identification of substrate properties that potentiate the ability of astrocytes to
transform into RGLC in response to a lesion might help in the development of implantable devices that
improve endogenous CNS regeneration. Here we demonstrate that functional RGLC can be induced from
in vitro matured astrocytes by using a precisely-sized micropatterned PMMA grooved scaffold, without
added soluble or substrate adsorbed biochemical factors. RGLC were extremely organized and aligned on
2 mm line patterned PMMA and, like their embryonic counterparts, express nestin, the neuron-glial
progenitor marker Pax6, and also proliferate, generate different intermediate progenitors and support
and direct axonal growth and neuronal migration. Our results suggest that the introduction of line
patterns in the size range of the RGC processes in implantable scaffolds might mimic the topography of
the embryonic neural stem cell niche, driving endogenous astrocytes into an RGLC phenotype, and thus
favoring the regenerative response in situ.
Pegueroles, Marta; García, J.; Fernández, M.; Engel, E.; Palacio, I.; Mascaraque, A.; de la Fuente, O.; Planell, J.; Castaño, O. Symposium and Annual Meeting of the International Society for Ceramics in Medicine Presentation's date: 2011-11 Presentation of work at congresses
Montufar, E.; Traykova, T.; Planell, J.; Ginebra, M.P. Materials science and engineering C. Biomimetic and supramolecular systems Vol. 31, num. 7, p. 1498-1504 DOI: 10.1016/j.msec.2011.06.008 Date of publication: 2011-10-10 Journal article
Hydroxyapatite foams are potential synthetic bone grafting materials or scaffolds for bone tissue engineering.
A novel method to obtain injectable hydroxyapatite foams consists in foaming the liquid phase of a calcium
phosphate cement. In this process, the cement powder is incorporated into a liquid foam, which acts as a
template for macroporosity. After setting, the cement hardens maintaining the macroporous structure of the
foam. In this study a low molecular weight surfactant, Polysorbate 80, and a protein, gelatine, were compared
as foaming agents of a calcium phosphate cement. The foamability of Polysorbate 80 was greater than that of
gelatine, resulting in higher macroporosity in the set hydroxyapatite foam and higher macropore
interconnectivity. Gelatine produced less interconnected foams, especially at high concentrations, due to a
higher liquid foam stability. However it increased the injectability and cohesion of the foamed paste, and
enhanced osteoblastic-like cell adhesion, all of them important properties for bone grafting materials.
Calcium phosphate compounds can potentially influence cellular fate through ionic substitutions. However,
to be able to turn such solution-mediated processes into successful directors of cellular response, a
perfect understanding of the material-induced chemical reactions in situ is required. We therefore report
on the application of home-made electrochemical microelectrodes, tested as pH and chloride sensors, for
precise spatial and temporal characterization of different aqueous environments around calcium phosphate-
based biomaterials prepared from a-tricalcium phosphate using clinically relevant liquid to powder
ratios. The small size of the electrodes allowed for online measurements in traditionally inaccessible
in vitro environments, such as the immediate material–liquid interface and the interior of curing bone
cement. The kinetic data obtained has been compared to theoretical sorption models, confirming that
the proposed setup can provide key information for improved understanding of the biochemical environment
imposed by chemically reactive biomaterials.
Serra, T.; Navarro, M.; Planell, J. International Conference on Advanced Research in Virtual and Rapid Prototyping p. 67-72 DOI: 10.1201/b11341-12 Presentation's date: 2011-09 Presentation of work at congresses
In the human lumbar spine, annulus fibrosus
fibres largely contribute to intervertebral disc stability.
Detailed annulus models are therefore necessary to obtain reliable predictions of lumbar spine mechanics by finite element
modelling. However, different definitions of collagen orientations coexist in the literature for healthy human lumbar annuli. Therefore, four annulus fibre-induced anisotropy models were built from reported anatomical descriptions, and inserted in a L3–L5 lumbar bi-segment finite element
model. Annulus models were, respectively, characterized by radial, tangential, radial and tangential, and no fibre orientation
gradients. The effect of rotational and axial compressive loadings was simulated and first, predictions were compared to experimental data. Then, intervertebral disc
local biomechanics was studied under axial rotation and axial compression. A new parameter, i.e. the fibre contribution
quality parameter, was computed in the anterior, lateral, postero-lateral, and posterior annuli of each model, in function
of fibre stresses, radial load distributions, and matrix shear strains. Locally, each annulus model behaved differently, affecting intervertebral disc biomechanics and segmentalmotions.
The fibre contribution quality parameter allowed establishing direct links between local annulus fibre organization and local annulus loadings, while other kinematical
and biomechanical data did not. It was concluded that functional relations should exist between local annulus fibre orientations and overall segment morphology. The proposed fibre contribution quality parameter could be used to examine
such relations and calibrate lumbar spine finite element models by locally adjusting the annulus bundle criss-cross angles. Conclusions of this study are particularly relevant to patient-specific models or artificial disc designs.
Biomaterial surface properties, via alterations
in the adsorbed protein layer, and the presence of specific
functional groups can influence integrin binding specificity,
thereby modulating cell adhesion and differentiation processes.
The adsorption of fibronectin, a protein directly
involved in osteoblast adhesion to the extracellular matrix,
has been related to different physical and chemical properties
of biomaterial surfaces. This study used blasting
particles of different sizes and chemical compositions to
evaluate the response of MG63 osteoblast-like cells on
smooth and blasted titanium surfaces, with and without
fibronectin coatings, by means of real-time reverse
transcription-polymerase chain reaction (qRT-PCR) assays.
This response included (a) expression of the a5, av and a3
integrin subunits, which can bind to fibronectin through the
RGD binding site, and (b) expression of alkaline phosphatase
(ALP) and osteocalcin (OC) as cell-differentiation
markers. ALP activity and synthesis of OC were also tested.
Cells on SiC-blasted Ti surfaces expressed higher
amounts of the a5 mRNA gene than cells on Al2O3-blasted
Ti surfaces. This may be related to the fact that SiC-blasted
surfaces adsorbed higher amounts of fibronectin due to
their higher surface free energy and therefore provided a
higher number of specific cell-binding sites. Fn-coated Tisurfaces decreased a5 mRNA gene expression, by favoring
the formation of other integrins involved in adhesion over
a5b1. The changes in a5 mRNA expression induced by the
presence of fibronectin coatings may moreover influence
the osteoblast differentiation pathway, as fibronectin coatings
on Ti surfaces also decreased both ALP mRNA
expression and ALP activity after 14 and 21 days of cell
Koch, M.A.; Vrij, E.; Engel, E.; Planell, J.; Lacroix, D. Journal of biomedical materials research. Part A Vol. 95A, num. 4, p. 1011-1018 DOI: 10.1002/jbm.a.32927 Date of publication: 2010-12-15 Journal article
A promising approach to bone tissue engineering lies
in the use of perfusion bioreactors where cells are seeded and
cultured on scaffolds under conditions of enhanced nutrient
supply and removal of metabolic products. Fluid flow alterations
can stimulate cell activity, making the engineering of tissue
more efficient. Most bioreactor systems are used to culture
cells on thin scaffold discs. In clinical use, however, bone substitutes
of large dimensions are needed. In this study, MG63
osteoblast-like cells were seeded on large porous PLA/glass
scaffolds with a custom developed perfusion bioreactor system.
Cells were seeded by oscillating perfusion of cell suspension
through the scaffolds. Applicable perfusion parameters for successful
cell seeding were determined by varying fluid flow velocity
and perfusion cycle number. After perfusion, cell seeding,
the cell distribution, and cell seeding efficiency were determined.
A fluid flow velocity of 5 mm/s had to be exceeded to
achieve a uniform cell distribution throughout the scaffold interior.
Cell seeding efficiencies of up to 50% were achieved.
Results suggested that perfusion cycle number influenced cell
seeding efficiency rather than fluid flow velocities. The cell
seeding conducted is a promising basis for further long term
cell culture studies in large porous scaffolds.
Despite their known osteoconductivity, clinical use of calcium phosphate cements is limited both by their relatively slow rate of resorption and by rheological properties incompatible with injectability. Bone in-growth and material resorption have been improved by the development of porous calcium phosphate cements. However, injectable formulations have so far only been obtained through the addition of relatively toxic surfactants. The present work describes the response of osteoblasts to a novel injectable foamed bone cement based on a composite formulation including the bioactive foaming agents soybean and gelatine. The foaming properties of both defatted soybean and gelatine gels were exploited to develop a self-hardening soy/gelatine/hydroxyapatite composite foam able to retain porosity upon injection.
After setting, the foamed paste produced a calcium-deficient hydroxyapatite scaffold, showing good injectability and cohesion as well as interconnected porosity after injection. The intrinsic bioactivity of soybean and gelatine was shown to favour osteoblast adhesion and growth. These findings suggest that injectable, porous and bioactive calcium phosphate cements can be produced for bone regeneration through minimally invasive surgery.