by Keyword: scaffold
Narciso M, Ulldemolins A, Júnior C, Otero J, Navajas D, Farré R, Gavara N, Almendros I, (2022). Novel Decellularization Method for Tissue Slices Frontiers In Bioengineering And Biotechnology 10,
Decellularization procedures have been developed and optimized for the entire organ or tissue blocks, by either perfusion of decellularizing agents through the tissue’s vasculature or submerging large sections in decellularizing solutions. However, some research aims require the analysis of native as well as decellularized tissue slices side by side, but an optimal protocol has not yet been established to address this need. Thus, the main goal of this work was to develop a fast and efficient decellularization method for tissue slices—with an emphasis on lung—while attached to a glass slide. To this end, different decellularizing agents were compared for their effectiveness in cellular removal while preserving the extracellular matrix. The intensity of DNA staining was taken as an indicator of remaining cells and compared to untreated sections. The presence of collagen, elastin and laminin were quantified using immunostaining and signal quantification. Scaffolds resulting from the optimized protocol were mechanically characterized using atomic force microscopy. Lung scaffolds were recellularized with mesenchymal stromal cells to assess their biocompatibility. Some decellularization agents (CHAPS, triton, and ammonia hydroxide) did not achieve sufficient cell removal. Sodium dodecyl sulfate (SDS) was effective in cell removal (1% remaining DNA signal), but its sharp reduction of elastin signal (only 6% remained) plus lower attachment ratio (32%) singled out sodium deoxycholate (SD) as the optimal treatment for this application (6.5% remaining DNA signal), due to its higher elastin retention (34%) and higher attachment ratio (60%). Laminin and collagen were fully preserved in all treatments. The SD decellularization protocol was also successful for porcine and murine (mice and rat) lungs as well as for other tissues such as the heart, kidney, and bladder. No significant mechanical differences were found before and after sample decellularization. The resulting acellular lung scaffolds were shown to be biocompatible (98% cell survival after 72 h of culture). This novel method to decellularize tissue slices opens up new methodological possibilities to better understand the role of the extracellular matrix in the context of several diseases as well as tissue engineering research and can be easily adapted for scarce samples like clinical biopsies. Copyright © 2022 Narciso, Ulldemolins, Júnior, Otero, Navajas, Farré, Gavara and Almendros.
Keywords: biocompatibility, bioscaffold recellularization, extracellular matrix, flow, impact, lung, scaffolds, tissue slices, Ammonia, Bio-scaffolds, Biocompatibility, Biological organs, Bioscaffold recellularization, Cell removal, Cells, Collagen, Cytology, Decellularization, Dna, Dna signals, Elastin, Extracellular matrices, Extracellular matrix, Extracellular-matrix, Glycoproteins, Laminin, Lung, Mammals, Recellularization, Scaffolds (biology), Sodium deoxycholate, Sulfur compounds, Tissue, Tissue slice, Tissue slices
Tejo-Otero A, Fenollosa-Artés F, Achaerandio I, Rey-Vinolas S, Buj-Corral I, Mateos-Timoneda MÁ, Engel E, (2022). Soft-Tissue-Mimicking Using Hydrogels for the Development of Phantoms Gels 8, 40
With the currently available materials and technologies it is difficult to mimic the mechanical properties of soft living tissues. Additionally, another significant problem is the lack of information about the mechanical properties of these tissues. Alternatively, the use of phantoms offers a promising solution to simulate biological bodies. For this reason, to advance in the state-of-the-art a wide range of organs (e.g., liver, heart, kidney as well as brain) and hydrogels (e.g., agarose, polyvinyl alcohol –PVA–, Phytagel –PHY– and methacrylate gelatine –GelMA–) were tested regarding their mechanical properties. For that, viscoelastic behavior, hardness, as well as a non-linear elastic mechanical response were measured. It was seen that there was a significant difference among the results for the different mentioned soft tissues. Some of them appear to be more elastic than viscous as well as being softer or harder. With all this information in mind, a correlation between the mechanical properties of the organs and the different materials was performed. The next conclusions were drawn: (1) to mimic the liver, the best material is 1% wt agarose; (2) to mimic the heart, the best material is 2% wt agarose; (3) to mimic the kidney, the best material is 4% wt GelMA; and (4) to mimic the brain, the best materials are 4% wt GelMA and 1% wt agarose. Neither PVA nor PHY was selected to mimic any of the studied tissues. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.
Keywords: brain, composite hydrogel, elastography, hardness, hydrogels, in-vitro, liver, materials, mechanical-properties, mimicking, soft tissues, tissue scaffolding, viscoelasticity, warner-braztler shear test, Dynamic mechanical analysis, Hardness, Hydrogels, Materials, Mimicking, Soft tissues, Tissue scaffolding, Viscoelastic characterization, Viscoelasticity, Warner–braztler shear test
Raymond, Yago, Johansson, Linh, Thorel, Emilie, Ginebra, Maria-Pau, (2022). Translation of three-dimensional printing of ceramics in bone tissue engineering and drug delivery Mrs Bulletin 47, 59-69
Keywords: augmentation, calcium-phosphate, expansion, fabrication, hydroxyapatite, made artificial bones, osteogenesis, reconstruction, regeneration, 3-d printing, 3d printers, 3d printing, 3d-printing, Abstracting, Additives, Biomaterial, Bone, Bone regeneration, Bone substitution, Bone tissue engineering, Ceramic, Ceramics, Clinic, Controlled drug delivery, Personalized medicines, Surgical planning, Targeted drug delivery, Three-dimensional-printing, Tricalcium phosphate scaffolds
Zeinali, Reza, del Valle, Luis J., Franco, Lourdes, Yousef, Ibraheem, Rintjema, Jeroen, Alemán, Carlos, Bravo, Fernando, Kleij, Arjan W., Puiggalí, Jordi, (2022). Biobased Terpene Derivatives: Stiff and Biocompatible Compounds to Tune Biodegradability and Properties of Poly(butylene succinate) Polymers 14,
Different copolymers incorporating terpene oxide units (e.g., limonene oxide) have been evaluated considering thermal properties, degradability, and biocompatibility. Thus, polycarbonates and polyesters derived from aromatic, monocyclic and bicyclic anhydrides have been considered. Furthermore, ring substitution with myrcene terpene has been evaluated. All polymers were amorphous when evaluated directly from synthesis. However, spherulites could be observed after the slow evaporation of diluted chloroform solutions of polylimonene carbonate, with all isopropene units possessing an R configuration. This feature was surprising considering the reported information that suggested only the racemic polymer was able to crystallize. All polymers were thermally stable and showed a dependence of the maximum degradation rate temperature (from 242 °C to 342 °C) with the type of terpene oxide. The graduation of glass transition temperatures (from 44 °C to 172 °C) was also observed, being higher than those corresponding to the unsubstituted polymers. The chain stiffness of the studied polymers hindered both hydrolytic and enzymatic degradation while a higher rate was detected when an oxidative medium was assayed (e.g., weight losses around 12% after 21 days of exposure). All samples were biocompatible according to the adhesion and proliferation tests performed with fibroblast cells. Hydrophobic and mechanically consistent films (i.e., contact angles between 90° and 110°) were obtained after the evaporation of chloroform from the solutions, having different ratios of the studied biobased polyterpenes and poly(butylene succinate) (PBS). The blend films were comparable in tensile modulus and tensile strength with the pure PBS (e.g., values of 330 MPa and 7 MPa were determined for samples incorporating 30 wt.% of poly(PA-LO), the copolyester derived from limonene oxide and phthalic anhydride. Blends were degradable, biocompatible and appropriate to produce oriented-pore and random-pore scaffolds via a thermally-induced phase separation (TIPS) method and using 1,4-dioxane as solvent. The best results were attained with the blend composed of 70 wt.% PBS and 30 wt.% poly(PA-LO). In summary, the studied biobased terpene derivatives showed promising properties to be used in a blended form for biomedical applications such as scaffolds for tissue engineering.
Keywords: alternating copolymerization, biobased materials, biodegradability, composites, crystallization, cyclohexene oxide, induced phase-separation, limonene oxide, mechanical-properties, polyesters, scaffolds, spherulites, terpene derivatives, thermal properties, thermally-induced phase separation, Acetone, Bio-based, Bio-based materials, Biobased materials, Biocompatibility, Biodegradability, Butenes, Cell culture, Chlorine compounds, Degradation, Evaporation, Glass transition, Limonene oxide, Monoterpenes, Phase separation, Poly (butylenes succinate), Polybutylene succinate, Property, Ring-opening copolymerization, Scaffolds, Spheru-lites, Tensile strength, Terpene derivatives, Thermal properties, Thermally induced phase separation, Thermally-induced phase separation, Thermally?induced phase separation, Thermodynamic properties, Thermogravimetric analysis
Pérez-González, Carlos, Ceada, Gerardo, Matejcic, Marija, Trepat, Xavier, (2022). Digesting the mechanobiology of the intestinal epithelium Current Opinion In Genetics & Development 72, 82-90
The dizzying life of the homeostatic intestinal epithelium is governed by a complex interplay between fate, form, force and function. This interplay is beginning to be elucidated thanks to advances in intravital and ex vivo imaging, organoid culture, and biomechanical measurements. Recent discoveries have untangled the intricate organization of the forces that fold the monolayer into crypts and villi, compartmentalize cell types, direct cell migration, and regulate cell identity, proliferation and death. These findings revealed that the dynamic equilibrium of the healthy intestinal epithelium relies on its ability to precisely coordinate tractions and tensions in space and time. In this review, we discuss recent findings in intestinal mechanobiology, and highlight some of the many fascinating questions that remain to be addressed in this emerging field.Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.
Keywords: crypt fission, designer matrices, differentiation, growth, gut, migration, model, scaffold, tissue mechanics, Cell migration, Cell proliferation, Ex vivo study, Human tissue, Intestine epithelium, Monolayer culture, Organoid, Review, Stem-cell, Tension, Traction therapy
Garreta, E, Nauryzgaliyeva, Z, Montserrat, N, (2021). Human induced pluripotent stem cell-derived kidney organoids toward clinical implementations Curr Opin Biomed Eng 20,
The generation of kidney organoids from human pluripotent stem cells (hPSCs) has represented a relevant scientific achievement in the organoid field. Importantly, hPSC-derived kidney organoids contain multiple nephron-like structures that exhibit some renal functional characteristics and have the capacity to respond to nephrotoxic agents. In this review, we first discuss how bioengineering approaches can help overcome current kidney organoid challenges. Next, we focus on recent works exploiting kidney organoids for drug screening and disease modeling applications. Finally, we provide a state of the art on current research toward the potential application of kidney organoids and renal cells derived from hPSCs for future renal replacement therapies.
Keywords: Bioengineering, Converting enzyme-ii, Crispr/cas9 gene editing, Disease, Disease modeling, Extracellular-matrix, Generation, Human pluripotent stem cells, Kidney organoids, Kidney regeneration, Model, Mouse, Reveals, Scaffold, Transplantation
Duro-Castano, Aroa, Rodríguez-Arco, Laura, Ruiz-Pérez, Lorena, De Pace, Cesare, Marchello, Gabriele, Noble-Jesus, Carlos, Battaglia, Giuseppe, (2021). One-Pot Synthesis of Oxidation-Sensitive Supramolecular Gels and Vesicles BIOMACROMOLECULES 22, 5052-5064
Polypeptide-based nanoparticles offer unique advantages from a nanomedicine perspective such as biocompatibility, biodegradability, and stimuli-responsive properties to (patho)physiological conditions. Conventionally, self-assembled polypeptide nanostructures are prepared by first synthesizing their constituent amphiphilic polypeptides followed by postpolymerization self-assembly. Herein, we describe the one-pot synthesis of oxidation-sensitive supramolecular micelles and vesicles. This was achieved by polymerization-induced self-assembly (PISA) of the N-carboxyanhydride (NCA) precursor of methionine using poly(ethylene oxide) as a stabilizing and hydrophilic block in dimethyl sulfoxide (DMSO). By adjusting the hydrophobic block length and concentration, we obtained a range of morphologies from spherical to wormlike micelles, to vesicles. Remarkably, the secondary structure of polypeptides greatly influenced the final morphology of the assemblies. Surprisingly, wormlike micellar morphologies were obtained for a wide range of methionine block lengths and solid contents, with spherical micelles restricted to very short hydrophobic lengths. Wormlike micelles further assembled into oxidation-sensitive, self-standing gels in the reaction pot. Both vesicles and wormlike micelles obtained using this method demonstrated to degrade under controlled oxidant conditions, which would expand their biomedical applications such as in sustained drug release or as cellular scaffolds in tissue engineering.
Keywords: alpha-amino-acid, hydrogels, leuchs anhydrides, platform, polypeptides, transformation, triggered cargo release, Amino acids, Amphiphilics, Biocompatibility, Biodegradability, Block lengths, Controlled drug delivery, Dimethyl sulfoxide, Ethylene, Gels, Hydrophobicity, Medical nanotechnology, Methionine, Micelles, Morphology, One-pot synthesis, Organic solvents, Oxidation, Physiological condition, Polyethylene oxides, Post-polymerization, Ring-opening polymerization, Scaffolds (biology), Self assembly, Stimuli-responsive properties, Supramolecular chemistry, Supramolecular gels, Supramolecular micelles, Wormlike micelle
Chausse, Victor, Schieber, Romain, Raymond, Yago, Ségry, Brian, Sabaté, Ramon, Kolandaivelu, Kumaran, Ginebra, Maria-Pau, Pegueroles, Marta, (2021). Solvent-cast direct-writing as a fabrication strategy for radiopaque stents Additive Manufacturing 48, 102392
Keywords: biocompatibility, bioresorbable stents, degradation, mechanical-properties, poly(epsilon-caprolactone), poly-l-lactic acid, polylactide, radiopacity, thermogel, x-ray imaging, Barium sulfate, Biocompatibility, Bioresorbable, Bioresorbable scaffolds, Bioresorbable stent, Bioresorbable stents, Blood vessels, Computerized tomography, Controlled drug delivery, Coronary heart disease, Direct-writing, Endothelial cells, Fabrication strategies, Injection molding, Lactic acid, Poly-l-lactic acid, Poly-l-lactic acids, Radiopacity, Scaffolds (biology), Solvent cast, Solvent-cast direct-writing, Solvents, Stents, Struts, Sulfur compounds, Targeted drug delivery, X-ray imaging
Raymond Y, Bonany M, Lehmann C, Thorel E, Benítez R, Franch J, Espanol M, Solé-Martí X, Manzanares MC, Canal C, Ginebra MP, (2021). Hydrothermal processing of 3D-printed calcium phosphate scaffolds enhances bone formation in vivo: a comparison with biomimetic treatment Acta Biomaterialia 135, 671-688
Hydrothermal (H) processes accelerate the hydrolysis reaction of α-tricalcium phosphate (α-TCP) compared to the long-established biomimetic (B) treatments. They are of special interest for patient-specific 3D-printed bone graft substitutes, where the manufacturing time represents a critical constraint. Altering the reaction conditions has implications for the physicochemical properties of the reaction product. However, the impact of the changes produced by the hydrothermal reaction on the in vivo performance was hitherto unknown. The present study compares the bone regeneration potential of 3D-printed α-TCP scaffolds hardened using these two treatments in rabbit condyle monocortical defects. Although both consolidation processes resulted in biocompatible scaffolds with osseointegrative and osteoconductive properties, the amount of newly formed bone increased by one third in the hydrothermal vs the biomimetic samples. B and H scaffolds consisted mostly of high specific surface area calcium-deficient hydroxyapatite (38 and 27 m2 g-1, respectively), with H samples containing also 10 wt.% β-tricalcium phosphate (β-TCP). The shrinkage produced during the consolidation process was shown to be very small in both cases, below 3%, and smaller for H than for B samples. The differences in the in vivo performance were mainly attributed to the distinct crystallisation nanostructures, which proved to have a major impact on permeability and protein adsorption capacity, using BSA as a model protein, with B samples being highly impermeable. Given the crucial role that soluble proteins play in osteogenesis, this is proposed to be a relevant factor behind the distinct in vivo performances observed for the two materials. Statement of significance: The possibility to accelerate the consolidation of self-setting calcium phosphate inks through hydrothermal treatments has aroused great interest due to the associated advantages for the development of 3D-printed personalised bone scaffolds. Understanding the implications of this approach on the in vivo performance of the scaffolds is of paramount importance. This study compares, for the first time, this treatment to the long-established biomimetic setting strategy in terms of osteogenic potential in vivo in a rabbit model, and relates the results obtained to the physicochemical properties of the 3D-printed scaffolds (composition, crystallinity, nanostructure, nanoporosity) and their interaction with soluble proteins.
Keywords: 3d printing, behavior, biomimetic, bone scaffolds, calcium phosphate, deficient hydroxyapatite, design, graft, hydrothermal, in vivo, morbidity, osteoinduction, porosity, standard, tricalcium phosphate, 3d printing, Biomimetic, Bone scaffolds, Calcium phosphate, Fibula free-flap, Hydrothermal, In vivo
Brennan M, Monahan DS, Brulin B, Gallinetti S, Humbert P, Tringides C, Canal C, Ginebra MP, Layrolle P, (2021). Biomimetic versus sintered macroporous calcium phosphate scaffolds enhanced bone regeneration and human mesenchymal stromal cell engraftment in calvarial defects Acta Biomaterialia 135, 689-704
In contrast to sintered calcium phosphates (CaPs) commonly employed as scaffolds to deliver mesenchymal stromal cells (MSCs) targeting bone repair, low temperature setting conditions of calcium deficient hydroxyapatite (CDHA) yield biomimetic topology with high specific surface area. In this study, the healing capacity of CDHA administering MSCs to bone defects is evaluated for the first time and compared with sintered beta-tricalcium phosphate (β-TCP) constructs sharing the same interconnected macroporosity. Xeno-free expanded human bone marrow MSCs attached to the surface of the hydrophobic β-TCP constructs, while infiltrating the pores of the hydrophilic CDHA. Implantation of MSCs on CaPs for 8 weeks in calvaria defects of nude mice exhibited complete healing, with bone formation aligned along the periphery of β-TCP, and conversely distributed within the pores of CDHA. Human monocyte-osteoclast differentiation was inhibited in vitro by direct culture on CDHA compared to β-TCP biomaterials and indirectly by administration of MSC-conditioned media generated on CDHA, while MSCs increased osteoclastogenesis in both CaPs in vivo. MSC engraftment was significantly higher in CDHA constructs, and also correlated positively with bone in-growth in scaffolds. These findings demonstrate that biomimetic CDHA are favorable carriers for MSC therapies and should be explored further towards clinical bone regeneration strategies. Statement of significance: Delivery of mesenchymal stromal cells (MSCs) on calcium phosphate (CaP) biomaterials enhances reconstruction of bone defects. Traditional CaPs are produced at high temperature, but calcium deficient hydroxyapatite (CDHA) prepared at room temperature yields a surface structure more similar to native bone mineral. The objective of this study was to compare the capacity of biomimetic CDHA scaffolds with sintered β-TCP scaffolds for bone repair mediated by MSCs for the first time. In vitro, greater cell infiltration occurred in CDHA scaffolds and following 8 weeks in vivo, MSC engraftment was higher in CDHA compared to β-TCP, as was bone in-growth. These findings demonstrate the impact of material features such as surface structure, and highlight that CDHA should be explored towards clinical bone regeneration strategies.
Keywords: beta-tricalcium phosphate, bone regeneration, calcium deficient hydroxyapatite, differentiation, engraftment, human bone marrow mesenchymal stromal cells, hydroxyapatite scaffolds, in-vitro, inhibition, osteogenesis, osteoinduction, stem-cells, surface-topography, tissue, Beta-tricalcium phosphate, Bone regeneration, Calcium deficient hydroxyapatite, Engraftment, Human bone marrow mesenchymal stromal cells
Rubí-Sans G, Nyga A, Rebollo E, Pérez-Amodio S, Otero J, Navajas D, Mateos-Timoneda MA, Engel E, (2021). Development of Cell-Derived Matrices for Three-Dimensional in Vitro Cancer Cell Models Acs Applied Materials & Interfaces 13, 44108-44123
Most morphogenetic and pathological processes are driven by cells responding to the surrounding matrix, such as its composition, architecture, and mechanical properties. Despite increasing evidence for the role of extracellular matrix (ECM) in tissue and disease development, many in vitro substitutes still fail to effectively mimic the native microenvironment. We established a novel method to produce macroscale (>1 cm) mesenchymal cell-derived matrices (CDMs) aimed to mimic the fibrotic tumor microenvironment surrounding epithelial cancer cells. CDMs are produced by human adipose mesenchymal stem cells cultured in sacrificial 3D scaffold templates of fibronectin-coated poly-lactic acid microcarriers (MCs) in the presence of macromolecular crowders. We showed that decellularized CDMs closely mimic the fibrillar protein composition, architecture, and mechanical properties of human fibrotic ECM from cancer masses. CDMs had highly reproducible composition made of collagen types I and III and fibronectin ECM with tunable mechanical properties. Moreover, decellularized and MC-free CDMs were successfully repopulated with cancer cells throughout their 3D structure, and following chemotherapeutic treatment, cancer cells showed greater doxorubicin resistance compared to 3D culture in collagen hydrogels. Collectively, these results support the use of CDMs as a reproducible and tunable tool for developing 3D in vitro cancer models.
Keywords: 3d cell-derived matrices, adipose mesenchymal stem cells, collagen matrix, colorectal adenocarcinoma, cytotoxicity assay, deposition, expansion, extracellular microenvironment, extracellular-matrix, fibronectin, growth, macromolecular crowders, microcarriers, scaffolds, tissue, 3d cell-derived matrices, Adipose mesenchymal stem cells, Cytotoxicity assay, Extracellular microenvironment, Macromolecular crowders, Mesenchymal stem-cells, Microcarriers
del-Mazo-Barbara L, Ginebra MP, (2021). Rheological characterisation of ceramic inks for 3D direct ink writing: A review JOURNAL OF THE EUROPEAN CERAMIC SOCIETY 41, 18-33
3D printing is a competitive manufacturing technology, which has opened up new possibilities for the fabrication of complex ceramic structures and customised parts. Extrusion-based technologies, also known as direct ink writing (DIW) or robocasting, are amongst the most used for ceramic materials. In them, the rheological properties of the ink play a crucial role, determining both the extrudability of the paste and the shape fidelity of the printed parts. However, comprehensive rheological studies of printable ceramic inks are scarce and may be difficult to understand for non-specialists. The aim of this review is to provide an overview of the main types of ceramic ink formulations developed for DIW and a detailed description of the more relevant rheological tests for assessing the printability of ceramic pastes. Moreover, the key rheological parameters are identified and linked to printability aspects, including the values reported in the literature for different ink compositions.
Keywords: 3-dimensional structures, behavior, deposition, direct ink writing, freeform fabrication, gelation, glass scaffolds, mechanical-properties, printability, rheology, robocasting, suspensions, 3d printing, Direct ink writing, Phosphate scaffolds, Printability, Rheology, Robocasting
Konka J, Buxadera-Palomero J, Espanol M, Ginebra M-P, (2021). 3D printing of hierarchical porous biomimetic hydroxyapatite scaffolds: Adding concavities to the convex filaments Acta Biomaterialia 134, 744-759
Porosity plays a key role on the osteogenic performance of bone scaffolds. Direct Ink Writing (DIW) allows the design of customized synthetic bone grafts with patient-specific architecture and controlled macroporosity. Being an extrusion-based technique, the scaffolds obtained are formed by arrays of cylindrical filaments, and therefore have convex surfaces. This may represent a serious limitation, as the role of surface curvature and more specifically the stimulating role of concave surfaces in osteoinduction and bone growth has been recently highlighted. Hence the need to design strategies that allow the introduction of concave pores in DIW scaffolds. In the current study, we propose to add gelatin microspheres as a sacrificial material in a self-setting calcium phosphate ink. Neither the phase transformation responsible for the hardening of the scaffold nor the formation of characteristic network of needle-like hydroxyapatite crystals was affected by the addition of gelatin microspheres. The partial dissolution of the gelatin resulted in the creation of spherical pores throughout the filaments and exposed on the surface, increasing filament porosity from 0.2 % to 67.9 %. Moreover, the presence of retained gelatin proved to have a significant effect on the mechanical properties, reducing the strength but simultaneously giving the scaffolds an elastic behavior, despite the high content of ceramic as a continuous phase. Notwithstanding the inherent difficulty of in vitro cultures with this highly reactive material an enhancement of MG-63 cell proliferation, as well as better spreading of hMSCs was recorded on the developed scaffolds. Statement of significance: Recent studies have stressed the role that concave surfaces play in tissue regeneration and, more specifically, in osteoinduction and osteogenesis. Direct ink writing enables the production of patient-specific bone grafts with controlled architecture. However, besides many advantages, it has the serious limitation that the surfaces obtained are convex. In this article, for the first time we develop a strategy to introduce concave pores in the printed filaments of biomimetic hydroxyapatite by incorporation and partial dissolution of gelatin microspheres. The retention of part of the gelatin results in a more elastic behavior compared to the brittleness of hydroxyapatite scaffolds, while the needle-shaped nanostructure of biomimetic hydroxyapatite is maintained and gelatin-coated concave pores on the surface of the filaments enhance cell spreading. © 2021 The Authors
Keywords: 3d printing, bioceramics, biomimetic, bone, bone regeneration, concavity, concavity, bone regeneration, gelatin, hydrogel, hydroxyapatite, microspheres, osteoinduction, porosity, porous filament, substitutes, tissue-growth, 3d printing, Biomimetic, Calcium-phosphate scaffolds, Concavity, bone regeneration, Gelatin, Hydroxyapatite, Porous filament
Velasco-Mallorqui, F, Rodriguez-Comas, J, Ramon-Azcon, J, (2021). Cellulose-based scaffolds enhance pseudoislets formation and functionality Biofabrication 13,
In vitro research for the study of type 2 diabetes (T2D) is frequently limited by the availability of a functional model for islets of Langerhans. To overcome the limitations of obtaining pancreatic islets from different sources, such as animal models or human donors, immortalized cell lines as the insulin-producing INS1E beta-cells have appeared as a valid alternative to model insulin-related diseases. However, immortalized cell lines are mainly used in flat surfaces or monolayer distributions, not resembling the spheroid-like architecture of the pancreatic islets. To generate islet-like structures, the use of scaffolds appeared as a valid tool to promote cell aggregations. Traditionally-used hydrogel encapsulation methods do not accomplish all the requisites for pancreatic tissue engineering, as its poor nutrient and oxygen diffusion induces cell death. Here, we use cryogelation technology to develop a more resemblance scaffold with the mechanical and physical properties needed to engineer pancreatic tissue. This study shows that carboxymethyl cellulose (CMC) cryogels prompted cells to generate beta-cell clusters in comparison to gelatin-based scaffolds, that did not induce this cell organization. Moreover, the high porosity achieved with CMC cryogels allowed us to create specific range pseudoislets. Pseudoislets formed within CMC-scaffolds showed cell viability for up to 7 d and a better response to glucose over conventional monolayer cultures. Overall, our results demonstrate that CMC-scaffolds can be used to control the organization and function of insulin-producing beta-cells, representing a suitable technique to generate beta-cell clusters to study pancreatic islet function.
Keywords: biomaterial, cryogel, pancreatic islets, scaffold, tissue engineering, ?-cell, Architecture, Beta-cell, Beta-cell heterogeneity, Biomaterial, Carboxymethyl cellulose, Cell culture, Cell death, Cell engineering, Cell organization, Cells, Cellulose, Cryogel, Cryogels, Cytoarchitecture, Delivery, Encapsulation methods, Gelation, Gene-expression, Immortalized cells, Insulin, Insulin secretory responses, Islets of langerhans, Mechanical and physical properties, Monolayer culture, Monolayers, Pancreatic islets, Pancreatic tissue, Pancreatic-islets, Proliferation, Scaffold, Scaffolds, Scaffolds (biology), Size, Tissue, Tissue engineering
Raymond Y, Thorel E, Liversain M, Riveiro A, Pou J, Ginebra MP, (2021). 3D printing non-cylindrical strands: Morphological and structural implications Additive Manufacturing 46,
Conventional direct ink writing uses circular nozzles and, therefore, results in cylindrical strands. 3D printing with non-circular nozzles adds new degrees of freedom to this versatile technology, and allows obtaining structures with higher specific surface area or even introducing concave surfaces in the printed architecture. This is an enticing prospect for countless applications, including tissue engineering, chemical reaction catalysts, water evaporators and electrochemical energy storage devices. Despite this, it has been hardly explored by the 3D-printing community. Herein, we develop for the first time 3D printed structures with complex filament section morphologies using a custom-made modular nozzle and a self-setting ceramic ink. The fast elastic recovery of the ink allows obtaining good shape fidelity in the printed filaments, permitting the creation of intricate surfaces with up to 30% concavity and increasing up to 2.5 times the specific surface area compared to cylindrical strands. The use of non-circular nozzles introduces some specific constraints in the printing process. The geometry of the nozzle determines the stable printing directions, and nozzle orientation becomes a critical parameter to achieve a stable printing. Strand torsion, a phenomenon that remains unnoticed with circular nozzles, may result in relevant changes in the geometrical features of the printed structures.
Keywords: calcium phosphate, ceramic, ceramics, flow, geometry, microextrusion, robocasting, Calcium phosphate, Ceramic, Direct ink writing, Microextrusion, Robocasting, Scaffolds
López-Canosa A, Perez-Amodio S, Yanac-Huertas E, Ordoño J, Rodriguez-Trujillo R, Samitier J, Castaño O, Engel E, (2021). A microphysiological system combining electrospun fibers and electrical stimulation for the maturation of highly anisotropic cardiac tissue Biofabrication 13,
The creation of cardiac tissue models for preclinical testing is still a non-solved problem in drug discovery, due to the limitations related to thein vitroreplication of cardiac tissue complexity. Among these limitations, the difficulty of mimicking the functional properties of the myocardium due to the immaturity of the used cells hampers the obtention of reliable results that could be translated into human patients.In vivomodels are the current gold standard to test new treatments, although it is widely acknowledged that the used animals are unable to fully recapitulate human physiology, which often leads to failures during clinical trials. In the present work, we present a microfluidic platform that aims to provide a range of signaling cues to immature cardiac cells to drive them towards an adult phenotype. The device combines topographical electrospun nanofibers with electrical stimulation in a microfabricated system. We validated our platform using a co-culture of neonatal mouse cardiomyocytes and cardiac fibroblasts, showing that it allows us to control the degree of anisotropy of the cardiac tissue inside the microdevice in a cost-effective way. Moreover, a 3D computational model of the electrical field was created and validated to demonstrate that our platform is able to closely match the distribution obtained with the gold standard (planar electrode technology) using inexpensive rod-shaped biocompatible stainless-steel electrodes. The functionality of the electrical stimulation was shown to induce a higher expression of the tight junction protein Cx-43, as well as the upregulation of several key genes involved in conductive and structural cardiac properties. These results validate our platform as a powerful tool for the tissue engineering community due to its low cost, high imaging compatibility, versatility, and high-throughput configuration capabilities.
Keywords: bioreactor, cardiac tissue engineering, cardiomyocytes, electrospinning, fabrication, fibroblasts, heart-on-a-chip, heart-tissue, in vitro models, myocardium, orientation, platform, scaffolds, Cardiac tissue engineering, Electrospinning, Field stimulation, Heart-on-a-chip, In vitro models, Microphysiological system
García-Mintegui C, Córdoba LC, Buxadera-Palomero J, Marquina A, Jiménez-Piqué E, Ginebra MP, Cortina JL, Pegueroles M, (2021). Zn-Mg and Zn-Cu alloys for stenting applications: From nanoscale mechanical characterization to in vitro degradation and biocompatibility Bioactive Materials 6, 4430-4446
In the recent decades, zinc (Zn) and its alloys have been drawing attention as promising candidates for bioresorbable cardiovascular stents due to its degradation rate more suitable than magnesium (Mg) and iron (Fe) alloys. However, its mechanical properties need to be improved in order to meet the criteria for vascular stents. This work investigates the mechanical properties, biodegradability and biocompatibility of Zn-Mg and Zn-Cu alloys in order to determine a proper alloy composition for optimal stent performance. Nanoindentation measurements are performed to characterize the mechanical properties at the nanoscale as a function of the Zn microstructure variations induced by alloying. The biodegradation mechanisms are discussed and correlated to microstructure, mechanical performance and bacterial/cell response. Addition of Mg or Cu alloying elements refined the microstructure of Zn and enhanced yield strength (YS) and ultimate tensile strength (UTS) proportional to the volume fraction of secondary phases. Zn-1Mg showed the higher YS and UTS and better performance in terms of degradation stability in Hanks’ solution. Zn-Cu alloys presented an antibacterial effect for S. aureus controlled by diffusion mechanisms and by contact. Biocompatibility was dependent on the degradation rate and the nature of the corrosion products.
Keywords: behavior, biocompatibility, biodegradable metals, bioresorbable metals, bioresorbable scaffold, copper, corrosion properties, elastic-modulus, galvanic corrosion, microstructure, nanoindentation, redox homeostasis, zinc, Biocompatibility, Bioresorbable metals, Galvanic corrosion, Nanoindentation, Room-temperature superplasticity, Zinc alloys
Ortega MA, Rodríguez-Comas J, Yavas O, Velasco-Mallorquí F, Balaguer-Trias J, Parra V, Novials A, Servitja JM, Quidant R, Ramón-Azcón J, (2021). In Situ LSPR Sensing of Secreted Insulin in Organ-on-Chip Biosensors 11,
Organ-on-a-chip (OOC) devices offer new approaches for metabolic disease modeling and drug discovery by providing biologically relevant models of tissues and organs in vitro with a high degree of control over experimental variables for high-content screening applications. Yet, to fully exploit the potential of these platforms, there is a need to interface them with integrated non-labeled sensing modules, capable of monitoring, in situ, their biochemical response to external stimuli, such as stress or drugs. In order to meet this need, we aim here to develop an integrated technology based on coupling a localized surface plasmon resonance (LSPR) sensing module to an OOC device to monitor the insulin in situ secretion in pancreatic islets, a key physiological event that is usually perturbed in metabolic diseases such as type 2 diabetes (T2D). As a proof of concept, we developed a biomimetic islet-on-a-chip (IOC) device composed of mouse pancreatic islets hosted in a cellulose-based scaffold as a novel approach. The IOC was interfaced with a state-of-the-art on-chip LSPR sensing platform to monitor the in situ insulin secretion. The developed platform offers a powerful tool to enable the in situ response study of microtissues to external stimuli for applications such as a drug-screening platform for human models, bypassing animal testing.
Keywords: biosensor, cytoarchitecture, dna hybridization, gelatin, in situ insulin monitoring, langerhans, lspr sensors, microfluidic device, organ-on-a-chip, parallel, platform, scaffold, Human pancreatic-islets, In situ insulin monitoring, Lspr sensors, Organ-on-a-chip
Zeinali R, Del Valle LJ, Torras J, Puiggalí J, (2021). Recent progress on biodegradable tissue engineering scaffolds prepared by thermally-induced phase separation (Tips) INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 22, 3504
Porous biodegradable scaffolds provide a physical substrate for cells allowing them to attach, proliferate and guide the formation of new tissues. A variety of techniques have been developed to fabricate tissue engineering (TE) scaffolds, among them the most relevant is the thermally-induced phase separation (TIPS). This technique has been widely used in recent years to fabricate three-dimensional (3D) TE scaffolds. Low production cost, simple experimental procedure and easy processability together with the capability to produce highly porous scaffolds with controllable architecture justify the popularity of TIPS. This paper provides a general overview of the TIPS methodology applied for the preparation of 3D porous TE scaffolds. The recent advances in the fabrication of porous scaffolds through this technique, in terms of technology and material selection, have been reviewed. In addition, how properties can be effectively modified to serve as ideal substrates for specific target cells has been specifically addressed. Additionally, examples are offered with re-spect to changes of TIPS procedure parameters, the combination of TIPS with other techniques and innovations in polymer or filler selection.
Keywords: biodegradable polymer, composites, morphology, pore structure, porosity, processing parameters, thermally induced phase separation, Biodegradable polymer, Composites, Morphology, Pore structure, Porosity, Processing parameters, Thermally induced phase separation, Tissue engineering scaffold
Olmo C, Franco L, Vidal A, Del Valle LJ, Puiggalí J, (2021). Ultrasound micromolding of porous polylactide/hydroxyapatite scaffolds Express Polymer Letters 15, 389-403
© BME-PT. Ultrasound micromolding (USM) preparation of hybrid scaffolds based on polylactide (PLA) and hydroxyapatite (HAp) particles has been evaluated. PLA was stable under the applied ultrasound source since a minimum degradation was detected. Porous materials were achieved using polyethylene glycol (PEG) and NaCl salts to the initial PLA and the subsequent leaching of the micromolded specimens. To avoid cavitation and decomposition problems during micromolding, it was necessary to use HAp free of typical synthesis impurities like carbonate and nitrate compounds. Compact PLA/HAp pieces allowed a maximum HAp load of 60 wt%, while porous specimens could be obtained with a maximum load of 38 wt%. Physical characterization of new scaffolds was performed by X-ray diffraction, spectroscopic and calorimetric techniques, stress-strain tests and contact angle measurements. Results indicated that a degree of porosity of 35% and relatively good mechanical properties could be achieved (i.e., 580 MPa, 4%, and 15.6 MPa for the Young modulus, elongation at break, and tensile strength, respectively). Scaffolds showed the positive effect of HAp and porosity on cell proliferation; this latter was 40% higher than that detected for non-porous PLA specimens.
Keywords: apatite, conformation, fabrication, hydroxyapatite, micropieces, polymers, porous scaffolds, proliferation, tissue, ultrasound micromolding, vibration, Composite scaffolds, Hydroxyapatite, Micropieces, Porous scaffolds, Processing technologies, Ultrasound micromolding
Vidal, E, Guillem-Marti, J, Ginebra, MP, Combes, C, Ruperez, E, Rodriguez, D, (2021). Multifunctional homogeneous calcium phosphate coatings: Toward antibacterial and cell adhesive titanium scaffolds SURFACE & COATINGS TECHNOLOGY 405,
Implants for orthopedic applications need to be biocompatible and bioactive, with mechanical properties similar to those of surrounding natural bone. Given this scenario titanium (Ti) scaffolds obtained by Direct Ink Writing technique offer the opportunity to manufacture customized structures with controlled porosity and mechanical properties. Considering that 3D Ti scaffolds have a significant surface area, it is necessary to develop strategies against the initial bacterial adhesion in order to prevent infection in the early stages of the implantation, while promoting cell adhesion to the scaffold. The challenge is not only achieving a balance between antibacterial activity and osseointegration, it is also to develop a homogeneous coating on the inner and outer surface of the scaffold. The purpose of this work was the development of a single-step electrodeposition process in order to uniformly cover Ti scaffolds with a layer of calcium phosphate (CaP) loaded with chlorhexidine digluconate (CHX). Scaffold characterization was assessed by scanning electron microscopy, Energy dispersive X-ray spectroscopy, X-ray diffraction, micro-Raman microscopy and compressive strength tests. Results determined that the surface of scaffolds was covered by plate-like and whisker-like calcium phosphate crystals, which main phases were octacalcium phosphate and brushite. Biological tests showed that the as-coated scaffolds reduced bacteria adhesion (73 +/- 3% for Staphylococcus aureus and 70 +/- 2% for Escherichia coli). In vitro cell studies and confocal analysis revealed the adhesion and spreading of osteoblast-like SaOS-2 on coated surfaces. Therefore, the proposed strategy can be a potential candidate in bone replacing surgeries.
Keywords: Antibacterial, Bacterial, Behavior, Biocompatibility, Calcium phosphate coating, Chlorhexidine, Chlorhexidine digluconate, Deposition, Electrodeposition, Hydroxyapatite coatings, Implants, One-step pulse electrodeposition, Plasma-spray, Release, Surface, Titanium scaffolds
Ben Hamouda S, Vargas A, Boivin R, Miglino MA, da Palma RK, Lavoie JP, (2021). Recellularization of Bronchial Extracellular Matrix With Primary Bronchial Smooth Muscle Cells Journal Of Equine Veterinary Science 96, 103313
© 2020 Elsevier Inc. Severe asthma is associated with an increased airway smooth muscle (ASM) mass and altered composition of the extracellular matrix (ECM). Studies have indicated that ECM-ASM cell interactions contribute to this remodeling and its limited reversibility with current therapy. Three-dimensional matrices allow the study of complex cellular responses to different stimuli in an almost natural environment. Our goal was to obtain acellular bronchial matrices and then develop a recellularization protocol with ASM cells. We studied equine bronchi as horses spontaneously develop a human asthma-like disease. The bronchi were decellularized using Triton/Sodium Deoxycholate. The obtained scaffolds retained their anatomical and histological properties. Using immunohistochemistry and a semi-quantitative score to compare native bronchi to scaffolds revealed no significant variation for matrixial proteins. DNA quantification and electrophoresis revealed that most DNA was 29.6 ng/mg of tissue ± 5.6, with remaining fragments of less than 100 bp. Primary ASM cells were seeded on the scaffolds. Histological analysis of the recellularizations showed that ASM cells migrated and proliferated primarily in the decellularized smooth muscle matrix, suggesting a chemotactic effect of the scaffolds. This is the first report of primary ASM cells preferentially repopulating the smooth muscle matrix layer in bronchial matrices. This protocol is now being used to study the molecular interactions occurring between the asthmatic ECMs and ASM to identify effectors of asthmatic bronchial remodeling.
Keywords: 2d, airway smooth muscle cells, asthma, decellularization, disease, elastin, extracellular matrix, lung scaffolds, migration, peptide, recellularization, tissues, Airway smooth muscle cells, Asthma, Culture-systems, Decellularization, Extracellular matrix, Recellularization
Soriente A, Amodio SP, Fasolino I, Raucci MG, Demitri C, Engel E, Ambrosio L, (2021). Chitosan/PEGDA based scaffolds as bioinspired materials to control in vitro angiogenesis Materials Science & Engineering C-Materials for Biological Applications 118
© 2020 Elsevier B.V. In the current work, our purpose was based on the assessment of bioactive chitosan (CS)/Poly(ethylene glycol) diacrylate (PEGDA) based scaffolds ability to stimulate in vitro angiogenesis process. The bioactivation of the scaffolds was accomplished by using organic (BMP-2 peptide) and inorganic (hydroxyapatite nanoparticles) cues. In particular, the properties of the materials in terms of biological response promotion on human umbilical vein endothelial cells (HUVECs) were studied by using in vitro angiogenesis tests based on cell growth and proliferation. Furthermore, our interest was to examine the scaffolds capability to modulate two important steps involved in angiogenesis process: migration and tube formation of cells. Our data underlined that bioactive signals on CS/PEGDA scaffolds surface induce a desirable effect on angiogenic response concerning angiogenic marker expression (CD-31) and endothelial tissue formation (tube formation). Taken together, the results emphasized the concept that bioactive CS/PEGDA scaffolds may be novel implants for stimulating neovascularization of tissue-engineered constructs in regenerative medicine field.
Keywords: angiogenesis, bmp-2 peptide, chitosan/pegda based scaffolds, human umbilical vein endothelial cells huvecs, Angiogenesis, Bmp-2 peptide, Chitosan/pegda based scaffolds, Human umbilical vein endothelial cells huvecs, Osteogenesis
Blanco-Fernandez, B, Castano, O, Mateos-Timoneda, MA, Engel, E, Perez-Amodio, S, (2021). Nanotechnology Approaches in Chronic Wound Healing Advances in Wound Care 10, 234-256
Significance: The incidence of chronic wounds is increasing due to our aging population and the augment of people afflicted with diabetes. With the extended knowledge on the biological mechanisms underlying these diseases, there is a novel influx of medical technologies into the conventional wound care market. Recent Advances: Several nanotechnologies have been developed demonstrating unique characteristics that address specific problems related to wound repair mechanisms. In this review, we focus on the most recently developed nanotechnology-based therapeutic agents and evaluate the efficacy of each treatment in in vivo diabetic models of chronic wound healing. Critical Issues: Despite the development of potential biomaterials and nanotechnology-based applications for wound healing, this scientific knowledge is not translated into an increase of commercially available wound healing products containing nanomaterials. Future Directions: Further studies are critical to provide insights into how scientific evidences from nanotechnology-based therapies can be applied in the clinical setting.
Keywords: chronic, diabetes, liposomes, nanofibers, nanoparticles, Chronic, Chronic wound, Diabetes, Diabetic wound, Diabetic-rats, Dressings, Drug mechanism, Extracellular-matrix, Growth-factor, Human, In-vitro, Liposome, Liposomes, Mesenchymal stem-cells, Metal nanoparticle, Nanofiber, Nanofibers, Nanofibrous scaffolds, Nanoparticles, Nanotechnology, Nonhuman, Polyester, Polymer, Polysaccharide, Priority journal, Protein, Review, Self assembled protein nanoparticle, Silk fibroin, Skin wounds, Wound healing, Wound healing promoting agent
Prat-Vidal, C., Rodríguez-Gómez, L., Aylagas, M., Nieto-Nicolau, N., Gastelurrutia, P., Agustí, E., Gálvez-Montón, C., Jorba, I., Teis, A., Monguió-Tortajada, M., Roura, S., Vives, J., Torrents-Zapata, S., Coca, M. I., Reales, L., Cámara-Rosell, M. L., Cediel, G., Coll, R., Farré, R., Navajas, D., Vilarrodona, A., García-López, J., Muñoz-Guijosa, C., Querol, S., Bayes-Genis, A., (2020). First-in-human PeriCord cardiac bioimplant: Scalability and GMP manufacturing of an allogeneic engineered tissue graft EBioMedicine 54, 102729
Small cardiac tissue engineering constructs show promise for limiting post-infarct sequelae in animal models. This study sought to scale-up a 2-cm2 preclinical construct into a human-size advanced therapy medicinal product (ATMP; PeriCord), and to test it in a first-in-human implantation.
The PeriCord is a clinical-size (12–16 cm2) decellularised pericardial matrix colonised with human viable Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs). WJ-MSCs expanded following good manufacturing practices (GMP) met safety and quality standards regarding the number of cumulative population doublings, genomic stability, and sterility. Human decellularised pericardial scaffolds were tested for DNA content, matrix stiffness, pore size, and absence of microbiological growth.
PeriCord implantation was surgically performed on a large non-revascularisable scar in the inferior wall of a 63-year-old male patient. Coronary artery bypass grafting was concomitantly performed in the non-infarcted area. At implantation, the 16-cm2 pericardial scaffold contained 12·5 × 106 viable WJ-MSCs (85·4% cell viability; <0·51 endotoxin units (EU)/mL). Intraoperative PeriCord delivery was expeditious, and secured with surgical glue. The post-operative course showed non-adverse reaction to the PeriCord, without requiring host immunosuppression. The three-month clinical follow-up was uneventful, and three-month cardiac magnetic resonance imaging showed ~9% reduction in scar mass in the treated area.
This preliminary report describes the development of a scalable clinical-size allogeneic PeriCord cardiac bioimplant, and its first-in-human implantation.
La Marató de TV3 Foundation, Government of Catalonia, Catalan Society of Cardiology, “La Caixa” Banking Foundation, Spanish Ministry of Science, Innovation and Universities, Institute of Health Carlos III, and the European Regional Development Fund.
Keywords: Advanced therapy medicinal product (ATMP), Biofabrication, Cardiac tissue engineering, Myocardial infarction, Scaffold, Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs)
Altay, Gizem, Tosi, Sébastien, García-Díaz, María, Martínez, Elena, (2020). Imaging the cell morphological response to 3D topography and curvature in engineered intestinal tissues Frontiers in Bioengineering and Biotechnology 8, 294
While conventional cell culture methodologies have relied on flat, two-dimensional cell monolayers, three-dimensional engineered tissues are becoming increasingly popular. Often, engineered tissues can mimic the complex architecture of native tissues, leading to advancements in reproducing physiological functional properties. In particular, engineered intestinal tissues often use hydrogels to mimic villi structures. These finger-like protrusions of a few hundred microns in height have a well-defined topography and curvature. Here, we examined the cell morphological response to these villus-like microstructures at single-cell resolution using a novel embedding method that allows for the histological processing of these delicate hydrogel structures. We demonstrated that by using photopolymerisable poly(ethylene) glycol as an embedding medium, the villus-like microstructures were successfully preserved after sectioning with vibratome or cryotome. Moreover, high-resolution imaging of these sections revealed that cell morphology, nuclei orientation, and the expression of epithelial polarization markers were spatially encoded along the vertical axis of the villus-like microstructures and that this cell morphological response was dramatically affected by the substrate curvature. These findings, which are in good agreement with the data reported for in vivo experiments on the native tissue, are likely to be the origin of more physiologically relevant barrier properties of engineered intestinal tissues when compared with standard monolayer cultures. By showcasing this example, we anticipate that the novel histological embedding procedure will have a positive impact on the study of epithelial cell behavior on three-dimensional substrates in both physiological and pathological situations.
Keywords: Hydrogel scaffold, Confocal microscopy, Substrate curvature, Cell morphology, Cell orientation, Histological section, Small intestine, Villus
Vidal, E., Torres, D., Guillem-Marti, J., Scionti, G., Manero, J. M., Ginebra, M. P., Rodríguez, D., Rupérez, E., (2020). Titanium scaffolds by direct ink writing: Fabrication and functionalization to guide osteoblast behavior Metals 10, (9), 1156
Titanium (Ti) and Ti alloys have been used for decades for bone prostheses due to its mechanical reliability and good biocompatibility. However, the high stiffness of Ti implants and the lack of bioactivity are pending issues that should be improved to minimize implant failure. The stress shielding effect, a result of the stiffness mismatch between titanium and bone, can be reduced by introducing a tailored structural porosity in the implant. In this work, porous titanium structures were produced by direct ink writing (DIW), using a new Ti ink formulation containing a thermosensitive hydrogel. A thermal treatment was optimized to ensure the complete elimination of the binder before the sintering process, in order to avoid contamination of the titanium structures. The samples were sintered in argon atmosphere at 1200 °C, 1300 °C or 1400 °C, resulting in total porosities ranging between 72.3% and 77.7%. A correlation was found between the total porosity and the elastic modulus of the scaffolds. The stiffness and yield strength were similar to those of cancellous bone. The functionalization of the scaffold surface with a cell adhesion fibronectin recombinant fragment resulted in enhanced adhesion and spreading of osteoblastic-like cells, together with increased alkaline phosphatase expression and mineralization.
Keywords: Direct ink writing, Osseointegration, Recombinant protein, Thermoresponsive binder, Titanium, Titanium scaffold
Casanellas, Ignasi, García-Lizarribar, Andrea, Lagunas, Anna, Samitier, Josep, (2018). Producing 3D biomimetic nanomaterials for musculoskeletal system regeneration Frontiers in Bioengineering and Biotechnology 6, Article 128
The human musculoskeletal system is comprised mainly of connective tissues such as cartilage, tendon, ligaments, skeletal muscle and skeletal bone. These tissues support the structure of the body, hold and protect the organs, and are responsible of movement. Since it is subjected to continuous strain, the musculoskeletal system is prone to injury by excessive loading forces or aging, whereas currently available treatments are usually invasive and not always effective. Most of the musculoskeletal injuries require surgical intervention facing a limited post-surgery tissue regeneration, especially for widespread lesions. Therefore, many tissue engineering approaches have been developed tackling musculoskeletal tissue regeneration. Materials are designed to meet the chemical and mechanical requirements of the native tissue three-dimensional (3D) environment, thus facilitating implant integration while providing a good reabsorption rate. With biological systems operating at the nanoscale, nanoengineered materials have been developed to support and promote regeneration at the interprotein communication level. Such materials call for a great precision and architectural control in the production process fostering the development of new fabrication techniques. In this mini review, we would like to summarize the most recent advances in 3D nanoengineered biomaterials for musculoskeletal tissue regeneration, with especial emphasis on the different techniques used to produce them.
Keywords: Nanofiber, 3D printing, Musculoskeletal, Regeneration, Scaffold, Tissue Engineering, Stimuli-responsive
Barbeck, Mike, Serra, Tiziano, Booms, Patrick, Stojanovic, Sanja, Najman, Stevo, Engel, Elisabeth, Sader, Robert, Kirkpatrick, Charles James, Navarro, Melba, Ghanaati, Shahram, (2017). Analysis of the in vitro degradation and the in vivo tissue response to bi-layered 3D-printed scaffolds combining PLA and biphasic PLA/bioglass components – Guidance of the inflammatory response as basis for osteochondral regeneration Bioactive Materials , 2, (4), 208-223
Abstract The aim of the present study was the inÂ vitro and inÂ vivo analysis of a bi-layered 3D-printed scaffold combining a PLA layer and a biphasic PLA/bioglass G5 layer for regeneration of osteochondral defects inÂ vivo Focus of the inÂ vitro analysis was on the (molecular) weight loss and the morphological and mechanical variations after immersion in SBF. The inÂ vivo study focused on analysis of the tissue reactions and differences in the implant bed vascularization using an established subcutaneous implantation model in CD-1 mice and established histological and histomorphometrical methods. Both scaffold parts kept their structural integrity, while changes in morphology were observed, especially for the PLA/G5 scaffold. Mechanical properties decreased with progressive degradation, while the PLA/G5 scaffolds presented higher compressive modulus than PLA scaffolds. The tissue reaction to PLA included low numbers of BMGCs and minimal vascularization of its implant beds, while the addition of G5 lead to higher numbers of BMGCs and a higher implant bed vascularization. Analysis revealed that the use of a bi-layered scaffold shows the ability to observe distinct inÂ vivo response despite the physical proximity of PLA and PLA/G5 layers. Altogether, the results showed that the addition of G5 enables to reduce scaffold weight loss and to increase mechanical strength. Furthermore, the addition of G5 lead to a higher vascularization of the implant bed required as basis for bone tissue regeneration mediated by higher numbers of BMGCs, while within the PLA parts a significantly lower vascularization was found optimally for chondral regeneration. Thus, this data show that the analyzed bi-layered scaffold may serve as an ideal basis for the regeneration of osteochondral tissue defects. Additionally, the results show that it might be able to reduce the number of experimental animals required as it may be possible to analyze the tissue response to more than one implant in one experimental animal.
Keywords: Bioactive glass, Polylactic acid (PLA), Bi-layer scaffold, Multinucleated giant cells, Bone substitute, Vascularization, Calcium phosphate glass
Punet, X., Levato, R., Bataille, I., Letourneur, D., Engel, E., Mateos-Timoneda, M. A., (2017). Polylactic acid organogel as versatile scaffolding technique Polymer 113, 81-91
Tissue engineering requires scaffolding techniques based on non-toxic processes that permits the fabrication of constructs with tailored properties. Here, a two-step methodology based on the gelation and precipitation of the poly(lactic) acid/ethyl lactate organogel system is presented. With this technique nanofibrous matrices that resemble natural extracellular matrix can be easily obtained, while allowing control over the mechanical properties of the device. Gelation temperature and the dynamics of the gelation of the organogel system are characterized, and the final mechanical and viscoelastic properties, as well as porosity, as function of the initial polymer concentration are described. We show that gelation temperature of the system is concentration independent and below 44.5 °C, which permits gelation at room temperature. Furthermore, mechanical properties are found in the range of the soft organic tissues, and the obtained micro-network architecture gives place to a flexible structure. Such structure presents tuneable elastic modulus and viscoelastic properties as function of nanofibers density. Moreover, centimetre-long tubular scaffolds with the diameter of medium-caliber blood vessels were produced. The fibrous nano-architecture mimics the native extracellular matrix fibres diameter and morphology was proven to be suitable to support endothelialization of the lumen of the tube. Thus, this strategy, based on biocompatible green compound might be promising for the fabrication of large 3D scaffolds for tissue engineering applications.
Keywords: Gel, Gelation, Nanofibrous, Organogel, PLA, Poly(lactic) acid, Scaffold
Planell, J. A., Navarro, M., Engel, E., (2017). Developing targeted biocomposites in tissue engineering and regenerative medicine Biomedical Composites (ed. Ambrosio, L.), Woodhead Publishing (Duxfor, UK) Biomaterials, 569-587
Regenerative medicine is a relatively new field with new requirements for smart materials, where composites will have a strong role to play. The new paradigm of regenerative medicine and tissue engineering requires biomaterials with high specificity, where physical and chemical properties are duly tailored and combined with appropriate mechanical and degradation features in order to trigger specific cell events and functions involved in the regenerative process. In this chapter, the chemical, physical, and biological elements that have to be targeted by biocomposites in regenerative medicine are described.
Keywords: Biocomposite, Regenerative medicine, Tissue engineering, Scaffolds, Cell/material interactions
Vila, M., García, A., Girotti, A., Alonso, M., Rodríguez-Cabello, J. C., González-Vázquez, A., Planell, J. A., Engel, E., Buján, J., Garcíaa-Honduvilla, N., Vallet-Regí, M., (2016). 3D silicon doped hydroxyapatite scaffolds decorated with Elastin-like Recombinamers for bone regenerative medicine Acta Biomaterialia 45, 349-356
The current study reports on the manufacturing by rapid prototyping technique of three-dimensional (3D) scaffolds based on silicon substituted hydroxyapatite with Elastin-like Recombinamers (ELRs) functionalized surfaces. Silicon doped hydroxyapatite (Si-HA), with Ca10(PO4)5.7(SiO4)0.3(OH)1.7h0.3 nominal formula, was surface functionalized with two different types of polymers designed by genetic engineering: ELR-RGD that contain cell attachment specific sequences and ELR-SNA15/RGD with both hydroxyapatite and cells domains that interact with the inorganic phase and with the cells, respectively. These hybrid materials were subjected to in vitro assays in order to clarify if the ELRs coating improved the well-known biocompatible and bone regeneration properties of calcium phosphates materials. The in vitro tests showed that there was a total and homogeneous colonization of the 3D scaffolds by Bone marrow Mesenchymal Stromal Cells (BMSCs). In addition, the BMSCs were viable and able to proliferate and differentiate into osteoblasts. Statement of Significance Bone tissue engineering is an area of increasing interest because its main applications are directly related to the rising life expectancy of the population, which promotes higher rates of several bone pathologies, so innovative strategies are needed for bone tissue regeneration therapies. Here we use the rapid prototyping technology to allow moulding ceramic 3D scaffolds and we use different bio-polymers for the functionalization of their surfaces in order to enhance the biological response. Combining the ceramic material (silicon doped hydroxyapatite, Si-HA) and the Elastin like Recombinamers (ELRs) polymers with the presence of the integrin-mediate adhesion domain alone or in combination with SNA15 peptide that possess high affinity for hydroxyapatite, provided an improved Bone marrow Mesenchymal Stromal Cells (BMSCs) differentiation into osteoblastic linkage.
Keywords: Bone marrow Mesenchymal Stromal Cells (BMSCs), Bone repair, Elastin-like Recombinamers (ELRs), Rapid prototyped 3D scaffolds, Silicon doped hydroxyapatite (Si-HA), Tissue engineering
da Palma, R. K., Nonaka, P. N., Campillo, N., Uriarte, J. J., Urbano, J. J., Navajas, D., Farré, R., Oliveira, L. V. F., (2016). Behavior of vascular resistance undergoing various pressure insufflation and perfusion on decellularized lungs Journal of Biomechanics 49, (7), 1230-1232
Bioengineering of functional lung tissue by using whole lung scaffolds has been proposed as a potential alternative for patients awaiting lung transplant. Previous studies have demonstrated that vascular resistance (Rv) could be altered to optimize the process of obtaining suitable lung scaffolds. Therefore, this work was aimed at determining how lung inflation (tracheal pressure) and perfusion (pulmonary arterial pressure) affect vascular resistance. This study was carried out using the lungs excised from 5 healthy male Sprague-Dawley rats. The trachea was cannulated and connected to a continuous positive airway pressure (CPAP) device to provide a tracheal pressure ranging from 0 to 15cmH2O. The pulmonary artery was cannulated and connected to a controlled perfusion system with continuous pressure (gravimetric level) ranging from 5 to 30cmH2O. Effective Rv was calculated by ratio of pulmonary artery pressure (P PA) by pulmonary artery flow (V'PA). Rv in the decellularized lungs scaffolds decreased at increasing V' PA, stabilizing at a pulmonary arterial pressure greater than 20cmH2O. On the other hand, CPAP had no influence on vascular resistance in the lung scaffolds after being subjected to pulmonary artery pressure of 5cmH2O. In conclusion, compared to positive airway pressure, arterial lung pressure markedly influences the mechanics of vascular resistance in decellularized lungs.
Keywords: Decellularized lung, Scaffolds, Vascular resistance
Perea-Gil, I., Uriarte, J. J., Prat-Vidal, C., Gálvez-Montón, C., Roura, S., Llucià-Valldeperas, A., Soler-Botija, C., Farré, R., Navajas, D., Bayes-Genis, A., (2015). In vitro comparative study of two decellularization protocols in search of an optimal myocardial scaffold for recellularization American Journal of Translational Research , 7, (3), 558-573
Introduction. Selection of a biomaterial-based scaffold that mimics native myocardial extracellular matrix (ECM) architecture can facilitate functional cell attachment and differentiation. Although decellularized myocardial ECM accomplishes these premises, decellularization processes may variably distort or degrade ECM structure. Materials and methods. Two decellularization protocols (DP) were tested on porcine heart samples (epicardium, mid myocardium and endocardium). One protocol, DP1, was detergent-based (SDS and Triton X-100), followed by DNase I treatment. The other protocol, DP2, was focused in trypsin and acid with Triton X-100 treatments. Decellularized myocardial scaffolds were reseeded by embedding them in RAD16-I peptidic hydrogel with adipose tissue-derived progenitor cells (ATDPCs). Results. Both protocols yielded acellular myocardial scaffolds (~82% and ~94% DNA reduction for DP1 and DP2, respectively). Ultramicroscopic assessment of scaffolds was similar for both protocols and showed filamentous ECM with preserved fiber disposition and structure. DP1 resulted in more biodegradable scaffolds (P = 0.04). Atomic force microscopy revealed no substantial ECM stiffness changes post-decellularization compared to native tissue. The Youngâ€™s modulus did not differ between heart layers (P = 0.69) or decellularization protocols (P = 0.15). After one week, recellularized DP1 scaffolds contained higher cell density (236 Â± 106 and 98 Â± 56 cells/mm2 for recellularized DP1 and DP2 scaffolds, respectively; P = 0.04). ATDPCs in both DP1 and DP2 scaffolds expressed the endothelial marker isolectin B4, but only in the DP1 scaffold ATDPCs expressed the cardiac markers GATA4, connexin43 and cardiac troponin T. Conclusions. In our hands, DP1 produced myocardial scaffolds with higher cell repopulation and promotes ATDPCs expression of endothelial and cardiomyogenic markers.
Keywords: Acellular myocardial scaffold, Adipose tissue-derived progenitor cells, Decellularization protocols, Extracellular matrix, Myocardial infarction, Recellularization
da Palma, R. K., Campillo, N., Uriarte, J. J., Oliveira, L. V. F., Navajas, D., Farré, R., (2015). Pressure- and flow-controlled media perfusion differently modify vascular mechanics in lung decellularization Journal of the Mechanical Behavior of Biomedical Materials , 49, 69-79
Organ biofabrication is a potential future alternative for obtaining viable organs for transplantation. Achieving intact scaffolds to be recellularized is a key step in lung bioengineering. Perfusion of decellularizing media through the pulmonary artery has shown to be effective. How vascular perfusion pressure and flow vary throughout lung decellularization, which is not well known, is important for optimizing the process (minimizing time) while ensuring scaffold integrity (no barotrauma). This work was aimed at characterizing the pressure/flow relationship at the pulmonary vasculature and at how effective vascular resistance depends on pressure- and flow-controlled variables when applying different methods of media perfusion for lung decellularization. Lungs from 43 healthy mice (C57BL/6; 7-8 weeks old) were investigated. After excision and tracheal cannulation, lungs were inflated at 10cmH
Keywords: Acellular lung, Fluid mechanics, Lung bioengineering, Lung scaffold, Organ biofabrication, Tissue engineering, Vascular resistance
Won, J. E., Mateos-Timoneda, M. A., Castaño, O., Planell, J. A., Seo, S. J., Lee, E. J., Han, C. M., Kim, H. W., (2015). Fibronectin immobilization on to robotic-dispensed nanobioactive glass/polycaprolactone scaffolds for bone tissue engineering Biotechnology Letters , 37, (4), 935-342
Bioactive nanocomposite scaffolds with cell-adhesive surface have excellent bone regeneration capacities. Fibronectin (FN)-immobilized nanobioactive glass (nBG)/polycaprolactone (PCL) (FN-nBG/PCL) scaffolds with an open pore architecture were generated by a robotic-dispensing technique. The surface immobilization level of FN was significantly higher on the nBG/PCL scaffolds than on the PCL scaffolds, mainly due to the incorporated nBG that provided hydrophilic chemical-linking sites. FN-nBG/PCL scaffolds significantly improved cell responses, including initial anchorage and subsequent cell proliferation. Although further in-depth studies on cell differentiation and the in vivo animal responses are required, bioactive nanocomposite scaffolds with cell-favoring surface are considered to provide promising three-dimensional substrate for bone regeneration.
Keywords: Bone scaffolds, Cell response, Fibronectin, Nanobioactive glass, Nanocomposites, Polycaprolactone, Bone, Cell proliferation, Cells, Cytology, Glass, Nanocomposites, Polycaprolactone, Robotics, Bone scaffolds, Bone tissue engineering, Cell response, Fibronectin, Fibronectin immobilizations, Nano bioactive glass, Nanocomposite scaffolds, Three-dimensional substrates, Scaffolds (biology)
Pérez-Madrigal, M. M., Giannotti, M. I., Del Valle, L. J., Franco, L., Armelin, E., Puiggalí, J., Sanz, F., Alemán, C., (2014). Thermoplastic polyurethane:polythiophene nanomembranes for biomedical and biotechnological applications ACS Applied Materials & Interfaces 6, (12), 9719-9732
Nanomembranes have been prepared by spin-coating mixtures of a polythiophene (P3TMA) derivative and thermoplastic polyurethane (TPU) using 20:80, 40:60, and 60:40 TPU:P3TMA weight ratios. After structural, topographical, electrochemical, and thermal characterization, properties typically related with biomedical applications have been investigated: swelling, resistance to both hydrolytic and enzymatic degradation, biocompatibility, and adsorption of type I collagen, which is an extra cellular matrix protein that binds fibronectin favoring cell adhesion processes. The swelling ability and the hydrolytic and enzymatic degradability of TPU:P3TMA membranes increases with the concentration of P3TMA. Moreover, the degradation of the blends is considerably promoted by the presence of enzymes in the hydrolytic medium, TPU:P3TMA blends behaving as biodegradable materials. On the other hand, TPU:P3TMA nanomembranes behave as bioactive platforms stimulating cell adhesion and, especially, cell viability. Type I collagen adsorption largely depends on the substrate employed to support the nanomembrane, whereas it is practically independent of the chemical nature of the polymeric material used to fabricate the nanomembrane. However, detailed microscopy study of the morphology and topography of adsorbed collagen evidence the formation of different organizations, which range from fibrils to pseudoregular honeycomb networks depending on the composition of the nanomembrane that is in contact with the protein. Scaffolds made of electroactive TPU:P3TMA nanomembranes are potential candidates for tissue engineering biomedical applications.
Keywords: Bioactive platform, Biodegradable blend, Collaged adsorption, Scaffolds, Tissue engineering, Ultrathin films
Andreu, I., Luque, T., Sancho, A., Pelacho, B., Iglesias-García, O., Melo, E., Farré, R., Prósper, F., Elizalde, M. R., Navajas, D., (2014). Heterogeneous micromechanical properties of the extracellular matrix in healthy and infarcted hearts Acta Biomaterialia 10, (7), 3235-3242
Infarcted hearts are macroscopically stiffer than healthy organs. Nevertheless, although cell behavior is mediated by the physical features of the cell niche, the intrinsic micromechanical properties of healthy and infarcted heart extracellular matrix (ECM) remain poorly characterized. Using atomic force microscopy, we studied ECM micromechanics of different histological regions of the left ventricle wall of healthy and infarcted mice. Hearts excised from healthy (n = 8) and infarcted mice (n = 8) were decellularized with sodium dodecyl sulfate and cut into 12 Î¼m thick slices. Healthy ventricular ECM revealed marked mechanical heterogeneity across histological regions of the ventricular wall with the effective Young's modulus ranging from 30.2 Â± 2.8 to 74.5 Â± 8.7 kPa in collagen- and elastin-rich regions of the myocardium, respectively. Infarcted ECM showed a predominant collagen composition and was 3-fold stiffer than collagen-rich regions of the healthy myocardium. ECM of both healthy and infarcted hearts exhibited a solid-like viscoelastic behavior that conforms to two power-law rheology. Knowledge of intrinsic micromechanical properties of the ECM at the length scale at which cells sense their environment will provide further insight into the cell-scaffold interplay in healthy and infarcted hearts.
Keywords: Atomic force microscopy, Extracellular matrix, Heart scaffold, Nanoindentation, Viscoelasticity
Uriarte, J. J., Nonaka, P. N., Campillo, N., Palma, R. K., Melo, E., de Oliveira, L. V. F., Navajas, D., Farré, R., (2014). Mechanical properties of acellular mouse lungs after sterilization by gamma irradiation Journal of the Mechanical Behavior of Biomedical Materials , 40, 168-177
Lung bioengineering using decellularized organ scaffolds is a potential alternative for lung transplantation. Clinical application will require donor scaffold sterilization. As gamma-irradiation is a conventional method for sterilizing tissue preparations for clinical application, the aim of this study was to evaluate the effects of lung scaffold sterilization by gamma irradiation on the mechanical properties of the acellular lung when subjected to the artificial ventilation maneuvers typical within bioreactors. Twenty-six mouse lungs were decellularized by a sodium dodecyl sulfate detergent protocol. Eight lungs were used as controls and 18 of them were submitted to a 31kGy gamma irradiation sterilization process (9 kept frozen in dry ice and 9 at room temperature). Mechanical properties of acellular lungs were measured before and after irradiation. Lung resistance (RL) and elastance (EL) were computed by linear regression fitting of recorded signals during mechanical ventilation (tracheal pressure, flow and volume). Static (Est) and dynamic (Edyn) elastances were obtained by the end-inspiratory occlusion method. After irradiation lungs presented higher values of resistance and elastance than before irradiation: RL increased by 41.1% (room temperature irradiation) and 32.8% (frozen irradiation) and EL increased by 41.8% (room temperature irradiation) and 31.8% (frozen irradiation). Similar increases were induced by irradiation in Est and Edyn. Scanning electron microscopy showed slight structural changes after irradiation, particularly those kept frozen. Sterilization by gamma irradiation at a conventional dose to ensure sterilization modifies acellular lung mechanics, with potential implications for lung bioengineering.
Keywords: Gamma irradiation, Lung bioengineering, Lung decellularization, Organ scaffold, Pulmonary mechanics, Decellularization, Gamma irradiation, Mouse lung, Pulmonary mechanics, dodecyl sulfate sodium, animal tissue, Article, artificial ventilation, bioengineering, bioreactor, compliance (physical), controlled study, freezing, gamma irradiation, lung, lung mechanics, lung resistance, male, mouse, nonhuman, room temperature, scanning electron microscopy, tissue scaffold, trachea pressure
Nonaka, P. N., Campillo, N., Uriarte, J. J., Garreta, E., Melo, E., de Oliveira, L. V. F., Navajas, D., Farré, R., (2014). Effects of freezing/thawing on the mechanical properties of decellularized lungs Journal of Biomedical Materials Research - Part A , 102, (2), 413-419
Lung bioengineering based on decellularized organ scaffolds is a potential alternative for transplantation. Freezing/thawing, a usual procedure in organ decellularization and storage could modify the mechanical properties of the lung scaffold and reduce the performance of the bioengineered lung when subjected to the physiological inflation-deflation breathing cycles. The aim of this study was to determine the effects of repeated freezing/thawing on the mechanical properties of decellularized lungs in the physiological pressure-volume regime associated with normal ventilation. Fifteen mice lungs (C57BL/6) were decellularized using a conventional protocol not involving organ freezing and based on sodium dodecyl sulfate detergent. Subsequently, the mechanical properties of the acellular lungs were measured before and after subjecting them to three consecutive cycles of freezing/thawing. The resistance (RL) and elastance (EL) of the decellularized lungs were computed by linear regression fitting of the recorded signals (tracheal pressure, flow, and volume) during mechanical ventilation. RL was not significantly modified by freezing-thawing: from 0.88 Â± 0.37 to 0.90 Â± 0.38 cmH2OÂ·sÂ·mL-1 (mean Â± SE). EL slightly increased from 64.4 Â± 11.1 to 73.0 Â± 16.3 cmH2OÂ·mL-1 after the three freeze-thaw cycles (p = 0.0013). In conclusion, the freezing/thawing process that is commonly used for both organ decellularization and storage induces only minor changes in the ventilation mechanical properties of the organ scaffold.
Keywords: Elastance, Freezing/thawing, Lung bioengineering, Lung decellularization, Mechanical ventilation, Organ scaffold
Sanzana, E. S., Navarro, M., Ginebra, M. P., Planell, J. A., Ojeda, A. C., Montecinos, H. A., (2014). Role of porosity and pore architecture in the in vivo bone regeneration capacity of biodegradable glass scaffolds Journal of Biomedical Materials Research - Part A , 102, (6), 1767-1773
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 Î¼m 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.
Keywords: Bone substitutes, Calcium phosphate glasses, in vivo, Scaffolds, Tissue engineering
Rajzer, I., Menaszek, E., Kwiatkowski, R., Planell, J. A., Castaño, O., (2014). Electrospun gelatin/poly(ε-caprolactone) fibrous scaffold modified with calcium phosphate for bone tissue engineering Materials Science and Engineering: C 44, 183-190
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.
Keywords: Bilayer fibrous scaffold, Ceramic nanoparticles, Electrospinning, Gelatin, Polycaprolactone, Biomechanics, Bone, Calcium phosphate, Cell culture, Electrospinning, Fourier transform infrared spectroscopy, Mechanical properties, Mineralogy, Nanoparticles, Phosphatases, Polycaprolactone, Scanning electron microscopy, X ray diffraction, Polycaprolactone, Alkaline phosphatase activity, Bone tissue engineering, Calcium phosphate nanoparticles, Ceramic nanoparticles, Fibrous scaffolds, Gelatin, Simulated body fluids, Wide-angle x-ray diffraction, Electrospuns, Scaffolds (biology), Electrospinning
Serra, T., Ortiz-Hernandez, M., Engel, E., Planell, J. A., Navarro, M., (2014). Relevance of PEG in PLA-based blends for tissue engineering 3D-printed scaffolds Materials Science and Engineering: C 38, (1), 55-62
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.
Keywords: 3D-printing, Polylactic acid, Rapid prototyping, Scaffold, Surface characterization
Nonaka, P. N., Uriarte, J. J., Campillo, N., Melo, E., Navajas, D., Farré, R., Oliveira, L. V. F., (2014). Mechanical properties of mouse lungs along organ decellularization by sodium dodecyl sulfate Respiratory Physiology & Neurobiology , 200, 1-5
Lung decellularization is based on the use of physical, chemical, or enzymatic methods to break down the integrity of the cells followed by a treatment to extract the cellular material from the lung scaffold. The aim of this study was to characterize the mechanical changes throughout the different steps of lung decellularization process. Four lungs from mice (C57BL/6) were decellularized by using a conventional protocol based on sodium dodecyl sulfate. Lungs resistance (RL) and elastance (EL) were measured along decellularization steps and were computed by linear regression fitting of tracheal pressure, flow, and volume during mechanical ventilation. Transients differences found were more distinct in an intermediate step after the lungs were rinsed with deionized water and treated with 1% SDS, whereupon the percentage of variation reached approximately 80% for resistance values and 30% for elastance values. In conclusion, although a variation in extracellular matrix stiffness was observed during the decellularization process, this variation can be considered negligible overall because the resistance and elastance returned to basal values at the final decellularization step.
Keywords: Lung bioengineering, Lung decellularization, Organ scaffold, dodecyl sulfate sodium, animal tissue, article, artificial ventilation, compliance (physical), controlled study, enzyme chemistry, extracellular matrix, female, flow, lung, lung decellularization, lung pressure, lung resistance, mouse, nonhuman, positive end expiratory pressure, priority journal, rigidity, tissue engineering, trachea pressure
Mendes, A. C., Smith, K. H., Tejeda-Montes, E., Engel, E., Reis, R. L., Azevedo, H. S., Mata, Alvaro, (2013). Co-assembled and microfabricated bioactive membranes Advanced Functional Materials 23, (4), 430-438
The fabrication of hierarchical and bioactive self-supporting membranes, which integrate physical and biomolecular elements, using a single-step process that combines molecular self-assembly with soft lithography is reported. A positively charged multidomain peptide (with or without the cell-adhesive sequence arginine-glycine-aspartic acid-serine (RGDS)) self-assembles with hyaluronic acid (HA), an anionic biopolymer. Optimization of the assembling conditions enables the realization of membranes with well-controlled and easily tunable features at multiple size scales including peptide sequence, building-block co-assembly, membrane thickness, bioactive epitope availability, and topographical pattern morphology. Membrane structure, morphology, and bioactivity are investigated according to temperature, assembly time, and variations in the experimental setup. Furthermore, to evaluate the physical and biomolecular signaling of the self-assembled microfabricated membranes, rat mesenchymal stem cells are cultured on membranes exhibiting various densities of RGDS and different topographical patterns. Cell adhesion, spreading, and morphology are significantly affected by the surface topographical patterns and the different concentrations of RGDS. The versatility of the combined bottom-up and top-down fabrication processes described may permit the development of hierarchical macrostructures with precise biomolecular and physical properties and the opportunity to fine tune them with spatiotemporal control.
Keywords: Membrane scaffolds, Mesenchymal stem cells, Microfabrication, Self-assembly, Topography
Serra, T., Planell, J. A., Navarro, M., (2013). High-resolution PLA-based composite scaffolds via 3-D printing technology Acta Biomaterialia 9, (3), 5521-5530
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.
Keywords: Rapid prototyping, Scaffold, Polylactic acid, Biodegradable, Composite
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
Salerno, A., Levato, R., Mateos-Timoneda, M. A., Engel, E., Netti, P. A., Planell, J. A., (2013). Modular polylactic acid microparticle-based scaffolds prepared via microfluidic emulsion/solvent displacement process: Fabrication, characterization, and in vitro mesenchymal stem cells interaction study Journal of Biomedical Materials Research - Part A , 101A, (3), 720-732
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 semicontinuous process involving (1) microfluidic emulsification of a PLA/ethyl lactate solution (5% w/v) in a span 80/paraffin oil solution (3% v/v) followed by (2) particles coagulation/assembly in an acetone/water solution for the development of a continuous 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 manufactured by means of flow rate modulation of of the continuous phase during emulsion. The biological response of the scaffolds was assessed in vitro by using bone marrow-derived rat mesenchymal stem cells (MSCs). The results demonstrated the ability of the scaffolds to support the extensive and uniform three-dimensional adhesion, colonization, and proliferation of MSCs within the entire construct.
Keywords: Green solvent, Microfluidic, Microstructure, Stem cells, Scaffold
Tejeda-Montes, E., Smith, K. H., Poch, M., López-Bosque, M. J., Martín, L., Alonso, M., Engel, E., Mata, Alvaro., (2012). Engineering membrane scaffolds with both physical and biomolecular signaling Acta Biomaterialia 8, (3), 998-1009
We report on the combination of a top-down and bottom-up approach to develop thin bioactive membrane scaffolds based on functional elastin-like polymers (ELPs). Our strategy combines ELP cross-linking and assembly, and a variety of standard and novel micro/nanofabrication techniques to create self-supporting membranes down to âˆ¼500 nm thick that incorporate both physical and biomolecular signals, which can be easily tailored for a specific application. In this study we used an ELP that included the cell-binding motif arginine-glycine-aspartic acid-serine (RGDS). Furthermore, fabrication processes were developed to create membranes that exhibited topographical patterns with features down to 200 nm in lateral dimensions and up to 10 Î¼m in height on either one or both sides, uniform and well-defined pores, or multiple ELP layers. A variety of processing parameters were tested in order to optimize membrane fabrication, including ELP and cross-linker concentration, temperature, reaction time and ambient humidity. Membrane micro/nanopatterning, swelling and stiffness were characterized by atomic force microscopy, nanoindentation tests and scanning electron microscopy. Upon immersion in phosphate-buffered saline and an increase in temperature from 25 to 40Â°C, membranes exhibited a significant increase in surface stiffness, with the reduced Young's modulus increasing with temperature. Finally, rat mesenchymal stem cells were cultured on thin RGDS-containing membranes, which allowed cell adhesion, qualitatively enhanced spreading compared to membranes without RGDS epitopes and permitted proliferation. Furthermore, cell morphology was drastically affected by topographical patterns on the surface of the membranes.
Keywords: Elastin-like polymers, Membranes, Nanotechnology, Scaffolds, Tissue engineering
Serra, T., Navarro, M., Planell, J. A., (2012). Fabrication and characterization of biodegradable composite scaffolds for tissue engineering Innovative Developments in Virtual and Physical Prototyping 5th International Conference on Advanced Research and Rapid Prototyping (ed. Margarida, T., Ferreira, D.), Taylor & Francis (Leiria, Portugal) VR@P, 67-72
In this study, polylactic acid (PLA) and polyethylene glycol (PEG) were combined with soluble CaP glass particles and processed by rapid prototyping to obtain fully biodegradable structures for Tissue Engineering applications. The obtained 3D biodegradable structures were characterized in terms of their architecture and mechanical properties. The scaffold morphology, internal micro-architecture and mechanical properties were evaluated using Scanning Electron Microscopy (SEM), micro-computed tomography (micro-CT) and mechanical testing, respectively. Well defined structures with pore size of 350-400Î¼m (in the axial view), struts width of approximately 70-80Î¼m, and a porosity ranging between 60-65% were obtained. The combination RP and PLA/PEG/CaP glass turned into promising fully degradable, mechanically stable, bioactive and biocompatible composite scaffolds for TE.
Keywords: Axial view, Biodegradable composites, Composite scaffolds, Glass particles, Mechanically stable, Micro architectures, Micro computed tomography (micro-CT), Poly lactic acid, Scaffold morphology, Tissue engineering applications, Well-defined structures, Bioactive glass, Mechanical properties, Mechanical testing, Polyethylene glycols, Polymer blends, Rapid prototyping, Scaffolds (biology), Scanning electron microscopy, Computerized tomography
Montufar, Edgar B., Traykova, Tania, Planell, Josep A., Ginebra, Maria-Pau, (2011). Comparison of a low molecular weight and a macromolecular surfactant as foaming agents for injectable self setting hydroxyapatite foams: Polysorbate 80 versus gelatine Materials Science and Engineering: C 31, (7), 1498-1504
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.
Keywords: Hydroxyapatite, Porosity, Calcium phosphate cement, Scaffolds, Foaming, Bone regeneration
Melchels, Ferry P. W., Tonnarelli, Beatrice, Olivares, Andy L., Martin, Ivan, Lacroix, Damien, Feijen, Jan, Wendt, David J., Grijpma, Dirk W., (2011). The influence of the scaffold design on the distribution of adhering cells after perfusion cell seeding Biomaterials 32, (11), 2878-2884
In natural tissues, the extracellular matrix composition, cell density and physiological properties are often non-homogeneous. Here we describe a model system, in which the distribution of cells throughout tissue engineering scaffolds after perfusion seeding can be influenced by the pore architecture of the scaffold. Two scaffold types, both with gyroid pore architectures, were designed and built by stereolithography: one with isotropic pore size (412 ± 13 [mu]m) and porosity (62 ± 1%), and another with a gradient in pore size (250-500 [mu]m) and porosity (35%-85%). Computational fluid flow modelling showed a uniform distribution of flow velocities and wall shear rates (15-24 s-1) for the isotropic architecture, and a gradient in the distribution of flow velocities and wall shear rates (12-38 s-1) for the other architecture. The distribution of cells throughout perfusion-seeded scaffolds was visualised by confocal microscopy. The highest densities of cells correlated with regions of the scaffolds where the pores were larger, and the fluid velocities and wall shear rates were the highest. Under the applied perfusion conditions, cell deposition is mainly determined by local wall shear stress, which, in turn, is strongly influenced by the architecture of the pore network of the scaffold.
Keywords: Scaffolds, Microstructure, Cell adhesion, Confocal microscopy, Image analysis, Computational fluid dynamics
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
Montufar, E. B., Traykova, T., Gil, C., Harr, I., Almirall, A., Aguirre, A., Engel, E., Planell, J. A., Ginebra, M. P., (2010). Foamed surfactant solution as a template for self-setting injectable hydroxyapatite scaffolds for bone regeneration Acta Biomaterialia 6, (3), 876-885
The application of minimally invasive surgical techniques in the field of orthopaedic surgery has created a growing need for new injectable synthetic materials that can be used for bone grafting In this work a novel fully synthetic injectable calcium phosphate foam was developed by mixing alpha-tricalcium phosphate (alpha-TCP) powder with a foamed polysorbate 80 solution Polysorbate 80 is a non-ionic surfactant approved for parenteral applications The foam was able to retain the porous structure after injection provided that the foamed paste was injected shortly after mixing (typically 2 5 min), and set through the hydrolysis of alpha-TCP to a calcium-deficient hydroxyapatite, thus producing a hydroxyapatite solid foam in situ The effect of different processing parameters on the porosity. microstructure, injectability and mechanical properties of the hydroxyapatite foams was analysed, and the ability of the pre-set foam to support osteoblastic-like cell proliferation and differentiation was assessed. Interestingly, the concentration of surfactant needed to obtain the foams was lower than that considered safe in drug formulations for parenteral administration The possibility of combining bioactivity, injectability, macroporosity and self-setting ability in a single fully synthetic material represents a step forward in the design of new materials for bone regeneration compatible with minimally invasive surgical techniques.
Keywords: Calcium phosphate cement, Hydroxyapatite foam, Scaffold, Surfactant, Injectable material
Ginebra, M. P., Espanol, M., Montufar, E. B., Perez, R. A., Mestres, G., (2010). New processing approaches in calcium phosphate cements and their applications in regenerative medicine Acta Biomaterialia 6, (8), 2863-2873
The key feature of calcium phosphate cements (CPCs) lies in the setting reaction triggered by mixing one or more solid calcium phosphate salts with an aqueous solution. Upon mixture, the reaction takes place through a dissolution-precipitation process which is macroscopically observed by a gradual hardening of the cement paste. The precipitation of hydroxyapatite nanocrystals at body or room temperature, and the fact that those materials can be used as self-setting pastes, have for many years been the most attractive features of CPCs. However, the need to develop materials able to sustain bone tissue ingrowth and be capable of delivering drugs and bioactive molecules, together with the continuous requirement from surgeons to develop more easily handling cements, has pushed the development of new processing routes that can accommodate all these requirements, taking advantage of the possibility of manipulating the self-setting CPC paste. It is the goal of this paper to provide a brief overview of the new processing developments in the area of CPCs and to identify the most significant achievements.
Keywords: Bone regeneration, Calcium phosphate cements, Granules, Microcarriers, Scaffolds
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
Koch, M. A., Vrij, E. J., Engel, E., Planell, J. A., Lacroix, D., (2010). Perfusion cell seeding on large porous PLA/calcium phosphate composite scaffolds in a perfusion bioreactor system under varying perfusion parameters Journal of Biomedical Materials Research - Part A , 95A, (4), 1011-1018
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.
Keywords: Bioreactor, Bone tissue engineering, Scaffolds, In vitro
Rajzer, I., Castano, O., Engel, E., Planell, J. A., (2010). Injectable and fast resorbable calcium phosphate cement for body-setting bone grafts Journal of Materials Science-Materials in Medicine , 21, (7), 2049-2056
In this work a calcium phosphate (CPC)/polymer blend was developed with the advantage of being moldable and capable of in situ setting to form calcium deficient hydroxyapatite under physiological conditions in an aqueous environment at body temperature. The CPC paste consists in a mix of R cement, glycerol as a liquid phase carrier and a biodegradable hydrogel such as Polyvinyl alcohol, which acts as a binder. Microstructure and mechanical analysis shows that the CPC blend can be used as an injectable implant for low loaded applications and fast adsorption requirements. The storage for commercial distribution was also evaluated and the properties of the materials obtained do not significantly change during storage at -18A degrees C.
Keywords: Clinical-applications, Composites, Regeneration, Behavior, Scaffold, Repair
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
Jang, J. H., Castano, O., Kim, H. W., (2009). Electrospun materials as potential platforms for bone tissue engineering Advanced Drug Delivery Reviews 61, (12), 1065-1083
Nanofibrous materials produced by electrospinning processes have attracted considerable interest in tissue regeneration, including bone reconstruction. A range of novel materials and processing tools have been developed to mimic the native bone extracellular matrix for potential applications as tissue engineering scaffolds and ultimately to restore degenerated functions of the bone. Degradable polymers, bioactive inorganics and their nanocomposites/hybrids nanofibers with suitable mechanical properties and bone bioactivity for osteoblasts and progenitor/stem cells have been produced. The surface functionalization with apatite minerals and proteins/peptides as well as drug encapsulation within the nanofibers is a promising strategy for achieving therapeutic functions with nanofibrous materials. Recent attempts to endow a 3D scaffolding technique to the electrospinning regime have shown some promise for engineering 3D tissue constructs. With the improvement in knowledge and techniques of bone-targeted nanofibrous matrices, bone tissue engineering is expected to be realized in the near future.
Keywords: Electrospun nanofiber, Bone tissue engineering, Biomimetic matrix, Bone bioactivity, 3D scaffolding
Milan, J. L., Planell, J. A., Lacroix, D., (2009). Computational modelling of the mechanical environment of osteogenesis within a polylactic acid-calcium phosphate glass scaffold Biomaterials 30, (25), 4219-4226
A computational model based on finite element method (FEM) and computational fluid dynamics (CFD) is developed to analyse the mechanical stimuli in a composite scaffold made of polylactic acid (PLA) matrix with calcium phosphate glass (Glass) particles. Different bioreactor loading conditions were simulated within the scaffold. In vitro perfusion conditions were reproduced in the model. Dynamic compression was also reproduced in an uncoupled fluid-structure scheme: deformation level was studied analyzing the mechanical response of scaffold alone under static compression while strain rate was studied considering the fluid flow induced by compression through fixed scaffold. Results of the model show that during perfusion test an inlet velocity of 25mum/s generates on scaffold surface a fluid flow shear stress which may stimulate osteogenesis. Dynamic compression of 5% applied on the PLA-Glass scaffold with a strain rate of 0.005s(-1) has the benefit to generate mechanical stimuli based on both solid shear strain and fluid flow shear stress on large scaffold surface area. Values of perfusion inlet velocity or compression strain rate one order of magnitude lower may promote cell proliferation while values one order of magnitude higher may be detrimental for cells. FEM-CFD scaffold models may help to determine loading conditions promoting bone formation and to interpret experimental results from a mechanical point of view.
Keywords: Bone tissue engineering, Scaffold, Finite element analysis, Computational fluid dynamics, Mechanical stimuli
Olivares, A. L., Marshal, E., Planell, J. A., Lacroix, D., (2009). Finite element study of scaffold architecture design and culture conditions for tissue engineering Biomaterials 30, (30), 6142-6149
Tissue engineering scaffolds provide temporary mechanical support for tissue regeneration and transfer global mechanical load to mechanical stimuli to cells through its architecture. In this study the interactions between scaffold pore morphology, mechanical stimuli developed at the cell microscopic level, and culture conditions applied at the macroscopic scale are studied on two regular scaffold structures. Gyroid and hexagonal scaffolds of 55% and 70% porosity were modeled in a finite element analysis and were submitted to an inlet fluid flow or compressive strain. A mechanoregulation theory based on scaffold shear strain and fluid shear stress was applied for determining the influence of each structures on the mechanical stimuli on initial conditions. Results indicate that the distribution of shear stress induced by fluid perfusion is very dependent on pore distribution within the scaffold. Gyroid architectures provide a better accessibility of the fluid than hexagonal structures. Based on the mechanoregulation theory, the differentiation process in these structures was more sensitive to inlet fluid flow than axial strain of the scaffold. This study provides a computational approach to determine the mechanical stimuli at the cellular level when cells are cultured in a bioreactor and to relate mechanical stimuli with cell differentiation.
Keywords: Tissue engineering, Scaffold, Rapid prototyping, Computational fluid dynamics, Finite element
Engel, E., Michiardi, A., Navarro, M., Lacroix, D., Planell, J. A., (2008). Nanotechnology in regenerative medicine: the materials side Trends in Biotechnology , 26, (1), 39-47
Regenerative medicine is an emerging multidisciplinary field that aims to restore, maintain or enhance tissues and hence organ functions. Regeneration of tissues can be achieved by the combination of living cells, which will provide biological functionality, and materials, which act as scaffolds to support cell proliferation. Mammalian cells behave in vivo in response to the biological signals they receive from the surrounding environment, which is structured by nanometre-scaled components. Therefore, materials used in repairing the human body have to reproduce the correct signals that guide the cells towards a desirable behaviour. Nanotechnology is not only an excellent tool to produce material structures that mimic the biological ones but also holds the promise of providing efficient delivery systems. The application of nanotechnology to regenerative medicine is a wide issue and this short review will only focus on aspects of nanotechnology relevant to biomaterials science. Specifically, the fabrication of materials, such as nanoparticles and scaffolds for tissue engineering, and the nanopatterning of surfaces aimed at eliciting specific biological responses from the host tissue will be addressed.
Keywords: Animals, Biocompatible Materials/ metabolism, Humans, Nanoparticles, Nanotechnology/ methods, Regenerative Medicine/ methods, Tissue Scaffolds
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
Sandino, C., Planell, J. A., Lacroix, D., (2008). A finite element study of mechanical stimuli in scaffolds for bone tissue engineering Journal of Biomechanics 41, (5), 1005-1014
Mechanical stimuli are one of the factors that affect cell proliferation and differentiation in the process of bone tissue regeneration. Knowledge on the specific deformation sensed by cells at a microscopic level when mechanical loads are applied is still missing in the development of biomaterials for bone tissue engineering. The objective of this study was to analyze the behavior of the mechanical stimuli within some calcium phosphate-based scaffolds in terms of stress and strain distributions in the solid material phase and fluid velocity, fluid pressure and fluid shear stress distributions in the pores filled of fluid, by means of micro computed tomographed (CT)-based finite element (FE) models. Two samples of porous materials, one of calcium phosphate-based cement and another of biodegradable glass, were used. Compressive loads equivalent to 0.5% of compression applied to the solid material phase and interstitial fluid flows with inlet velocities of 1, 10 and 100 mu m/s applied to the interconnected pores were simulated, changing also the inlet side and the viscosity of the medium. Similar strain distributions for both materials were found, with compressive and tensile strain maximal values of 1.6% and 0.6%, respectively. Mean values were consistent with the applied deformation. When 10 mu m/s of inlet fluid velocity and 1.45 Pa s viscosity, maximal values of fluid velocity were 12.76 mm/s for CaP cement and 14.87 mm/s for glass. Mean values were consistent with the inlet ones applied, and mean values of shear stress were around 5 x 10(-5) Pa. Variations on inlet fluid velocity and fluid viscosity produce proportional and independent changes in fluid velocity, fluid shear stress and fluid pressure. This study has shown how mechanical loads and fluid flow applied on the scaffolds cause different levels of mechanical stimuli within the samples according to the morphology of the materials.
Keywords: Bone tissue engineering, Finite element analysis, Scaffolds, Mechanical stimuli
Montufar, E. B., Gil, C., Traykova, T., Ginebra, M. P., Planell, J., (2008). Foamed beta-tricalcium phosphate scaffolds Bioceramics: Key Engineering Materials 20th International Symposium on Ceramics in Medicine (ed. Daculsi, G., Layrolle, P.), Trans Tech Publications Ltd (Nantes, France) 20, 323-326
The design and processing of 3D macroporous bioactive scaffolds is one of the milestones for the progress of bone tissue engineering and bone regeneration. Calcium phosphate based ceramics are among the most suitable materials, due to their similarity to the bone mineral. Specifically, beta-tricalcium phosphate (beta-TCP) is known to be a resorbable and bioactive material, with well established applications as bone regeneration material. The aim of this work is to explore a new OF route to obtain beta-TCP macroporous scaffolds starting from calcium phosphate cements. To this end foamed calcium phosphate cement.. composed of alpha tricalcium phosphate as starting powder was used as initial material. The set foamed structures, made of calcium deficient hydroxyapatite (CDHA) were sintered to obtain the final beta-TCP macroporous architecture. The interconnected macroporosity was maintained.. whereas the porosity in the nanometric range was strongly reduced by the sintering process. The sintering produced also an increase in the mechanical properties of the scaffold.
Keywords: Calcium-phosphate ceramics, Cements, Scaffolds, Foams, Macroporous, Tissue engineering
Charles-Harris, M., del Valle, S., Hentges, E., Bleuet, P., Lacroix, D., Planell, J. A., (2007). Mechanical and structural characterisation of completely degradable polylactic acid/calcium phosphate glass scaffolds Biomaterials 28, (30), 4429-4438
This study involves the mechanical and structural characterisation of completely degradable scaffolds for tissue engineering applications. The scaffolds are a composite of polylactic acid (PLA) and a soluble calcium phosphate glass, and are thus completely degradable. A factorial experimental design was applied to optimise scaffold composition prior to simultaneous microtomography and micromechanical testing. Synchrotron X-ray microtomography combined with in situ micromechanical testing was performed to obtain three-dimensional 3D images of the scaffolds under compression. The 3D reconstruction was converted into a finite element mesh which was validated by simulating a compression test and comparing it with experimental results. The experimental design reveals that larger glass particle and pore sizes reduce the stiffness of the scaffolds, and that the porosity is largely unaffected by changes in pore sizes or glass weight content. The porosity ranges between 93% and 96.5%, and the stiffness ranges between 50 and 200 kPa. X-ray projections show a homogeneous distribution of the glass particles within the PLA matrix, and illustrate pore-wall breakage under strain. The 3D reconstructions are used qualitatively to visualise the distribution of the phases of the composite material, and to follow pore deformation under compression. Quantitatively, scaffold porosity, pore interconnectivity and surface/volume ratios have been calculated. Finite element analysis revealed the stress and strain distribution in the scaffold under compression, and could be used in the future to characterise the mechanical properties of the scaffolds.
Keywords: Synchrotron x-ray microtomography, Mechanical test, Biodegradable, Glass, Scaffold, Finite element analysis