Miguel A. Mateos-Timoneda

Biofabrication of tissue constructs by 3D bioprinting of cell-laden microcarriers

Bioprinting allows the fabrication of living constructs with custom-made architectures by spatially controlled deposition of multiple bioinks. This is important for the generation of tissue, such as osteochondral tissue, which displays a zonal composition in the cartilage domain supported by the underlying subchondral bone. Challenges in fabricating functional grafts of clinically relevant size include the incorporation of cues to guide specific cell differentiation and the generation of sufficient cells, which is hard to obtain with conventional cell culture techniques. A novel strategy to address these demands is to combine bioprinting with microcarrier technology. This technology allows for the extensive expansion of cells, while they form multi-cellular aggregates, and their phenotype can be controlled. In this work, living constructs were fabricated via bioprinting of cell-laden microcarriers. Mesenchymal stromal cell (MSC)-laden polylactic acid microcarriers, obtained via static culture or spinner flask expansion, were encapsulated in gelatin methacrylamide-gellan gum bioinks, and the printability of the composite material was studied. This bioprinting approach allowed for the fabrication of constructs with high cell concentration and viability. Microcarrier encapsulation improved the compressive modulus of the hydrogel constructs, facilitated cell adhesion, and supported osteogenic differentiation and bone matrix deposition by MSCs. Bilayered osteochondral models were fabricated using microcarrier-laden bioink for the bone compartment. These findings underscore the potential of this new microcarrier-based biofabrication approach for bone and osteochondral constructs.

3D printed PLA-based scaffolds: A versatile tool in regenerative medicine

Rapid prototyping (RP), also known as additive manufacturing (AM), has been well received and adopted in the biomedical field. The capacity of this family of techniques to fabricate customized 3D structures with complex geometries and excellent reproducibility has revolutionized implantology and regenerative medicine. In particular, nozzle-based systems allow the fabrication of high-resolution polylactic acid (PLA) structures that are of interest in regenerative medicine. These 3D structures find interesting applications in the regenerative medicine field where promising applications including biodegradable templates for tissue regeneration purposes, 3D in vitro platforms for studying cell response to different scaffolds conditions and for drug screening are considered among others. Scaffolds functionality depends not only on the fabrication technique, but also on the material used to build the 3D structure, the geometry and inner architecture of the structure, and the final surface properties. All being crucial parameters affecting scaffolds success. This Commentary emphasizes the importance of these parameters in scaffolds’ fabrication and also draws the attention toward the versatility of these PLA scaffolds as a potential tool in regenerative medicine and other medical fields.

Disassembling bacterial extracellular matrix with DNase-coated nanoparticles to enhance antibiotic delivery in biofilm infections

Infections caused by biofilm-forming bacteria are a major threat to hospitalized patients and the main cause of chronic obstructive pulmonary disease and cystic fibrosis. There is an urgent necessity for novel therapeutic approaches, since current antibiotic delivery fails to eliminate biofilm-protected bacteria. In this study, ciprofloxacin-loaded poly(lactic-co-glycolic acid) nanoparticles, which were functionalized with DNase I, were fabricated using a green-solvent based method and their antibiofilm activity was assessed against Pseudomonas aeruginosa biofilms. Such nanoparticles constitute a paradigm shift in biofilm treatment, since, besides releasing ciprofloxacin in a controlled fashion, they are able to target and disassemble the biofilm by degrading the extracellular DNA that stabilize the biofilm matrix. These carriers were compared with free-soluble ciprofloxacin, and ciprofloxacin encapsulated in untreated and poly(lysine)-coated nanoparticles. DNase I-activated nanoparticles were not only able to prevent biofilm formation from planktonic bacteria, but they also successfully reduced established biofilm mass, size and living cell density, as observed in a dynamic environment in a flow cell biofilm assay. Moreover, repeated administration over three days of DNase I-coated nanoparticles encapsulating ciprofloxacin was able to reduce by 95% and then eradicate more than 99.8% of established biofilm, outperforming all the other nanoparticle formulations and the free-drug tested in this study. These promising results, together with minimal cytotoxicity as tested on J774 macrophages, allow obtaining novel antimicrobial nanoparticles, as well as provide clues to design the next generation of drug delivery devices to treat persistent bacterial infections.

Neurogenesis and vascularization of the damaged brain using a lactate-releasing biomimetic scaffold

Regenerative medicine strategies to promote recovery following traumatic brain injuries are currently focused on the use of biomaterials as delivery systems for cells or bioactive molecules. This study shows that cell-free biomimetic scaffolds consisting of radially aligned electrospun poly-l/dl lactic acid (PLA70/30) nanofibers release L-lactate and reproduce the 3D organization and supportive function of radial glia embryonic neural stem cells. The topology of PLA nanofibers supports neuronal migration while L-lactate released during PLA degradation acts as an alternative fuel for neurons and is required for progenitor maintenance. Radial scaffolds implanted into cavities made in the postnatal mouse brain fostered complete implant vascularization, sustained neurogenesis, and allowed the long-term survival and integration of the newly generated neurons. Our results suggest that the endogenous central nervous system is capable of regeneration through the in vivo dedifferentiation induced by biophysical and metabolic cues, with no need for exogenous cells, growth factors, or genetic manipulation.

3D printed PLA-based scaffolds

Rapid prototyping (RP), also known as additive manufacturing (AM), has been well received and adopted in the biomedical field. The capacity of this family of techniques to fabricate customized 3D structures with complex geometries and excellent reproducibility has revolutionized implantology and regenerative medicine. In particular, nozzle-based systems allow the fabrication of high-resolution polylactic acid (PLA) structures that are of interest in regenerative medicine. These 3D structures find interesting applications in the regenerative medicine field where promising applications including biodegradable templates for tissue regeneration purposes, 3D in vitro platforms for studying cell response to different scaffolds conditions and for drug screening are considered among others. Scaffolds functionality depends not only on the fabrication technique, but also on the material used to build the 3D structure, the geometry and inner architecture of the structure, and the final surface properties. All being crucial parameters affecting scaffolds success. This Commentary emphasizes the importance of these parameters in scaffolds’ fabrication and also draws the attention toward the versatility of these PLA scaffolds as a potential tool in regenerative medicine and other medical fields.

Enhanced cell-material interactions through the biofunctionalization of polymeric surfaces with engineered peptides

Research on surface modification of polymeric materials to guide the cellular activity in biomaterials designed for tissue engineering applications has mostly focused on the use of natural extracellular matrix (ECM) proteins and short peptides, such as RGD. However, the use of engineered proteins can gather the advantages of these strategies and avoid the main drawbacks. In this study, recombinant engineered proteins called elastin-like recombinamers (ELRs) have been used to functionalize poly(lactic) acid (PLA) model surfaces. The structure of the ELRs has been designed to include the integrin ligand RGDS and the cross-linking module VPGKG. Surface functionalization has been characterized and optimized by means of ELISA and atomic force microscopy (AFM). The results suggest that ELR functionalization creates a nonfouling canvas able to restrict unspecific adsorption of proteins. Moreover, AFM analysis reveals the conformation and disposition of ELRs on the surface. Biological performance of PLA surfaces functionalized with ELRs has been studied and compared with the use of short peptides. Cell response has been assessed for different functionalization conditions in the presence and absence of the bovine serum albumin (BSA) protein, which could interfere with the surface-cell interaction by adsorbing on the interface. Studies have shown that ELRs are able to elicit higher rates of cell attachment, stronger cell anchorages and faster levels of proliferation than peptides. This work has demonstrated that the use of engineered proteins is a more efficient strategy to guide the cellular activity than the use of short peptides, because they not only allow for better cell attachment and proliferation, but also can provide more complex properties such as the creation of nonfouling surfaces.

Hierarchically engineered fibrous scaffolds for bone regeneration

Surface properties of biomaterials play a major role in the governing of cell functionalities. It is well known that mechanical, chemical and nanotopographic cues, for example, influence cell proliferation and differentiation. Here, we present a novel coating protocol to produce hierarchically engineered fibrous scaffolds with tailorable surface characteristics, which mimic bone extracellular matrix. Based on the sol–gel method and a succession of surface treatments, hollow electrospun polylactic acid fibres were coated with a silicon–calcium–phosphate bioactive organic–inorganic glass. Compared with pure polymeric fibres that showed a completely smooth surface, the coated fibres exhibited a nanostructured topography and greater roughness. They also showed improved hydrophilic properties and a Youngs modulus sixfold higher than non-coated ones, while remaining fully flexible and easy to handle. Rat mesenchymal stem cells cultured on these fibres showed great cellular spreading and interactions with the material. This protocol can be transferred to other structures and glasses, allowing the fabrication of various materials with well-defined features. This novel approach represents therefore a valuable improvement in the production of artificial matrices able to direct stem cell fate through physical and chemical interactions.

The effect of the composition of PLA films and lactate release on glial and neuronal maturation and the maintenance of the neuronal progenitor niche.

To develop tissue engineering strategies useful for repairing damage in the central nervous system (CNS) it is essential to design scaffolds that emulate the NSC niche and its tight control of neural cell genesis, growth, and differentiation. In this study we tested two types of poly L/DL lactic acid (PLA95/5 and PLA70/30), a biodegradable material permissive for neural cell adhesion and growth, as materials for nerve regeneration. Both PLA were slightly hydrophobic and negatively charged but differed in crystallinity, stiffness and degradation rate. PLA95/5 films were highly crystalline, stiff (GPa), and did not degrade significantly in the one-month period analyzed in culture. In contrast, PLA70/30 films were more amorphous, softer (MPa) and degraded faster, releasing significant amounts of lactate into the culture medium. PLA70/30 performs better than PLA95/5 for primary cortical neural cell adhesion, proliferation and differentiation, maintaining the pools of neuronal and glial progenitor cells in vitro. L-lactate in the medium recapitulated PLA70/30s maintenance of neuronal restricted progenitors but did not sustain bipotential or glial restricted progenitors in the cultures, as occurred when neural cells were grown on PLA70/30. Our results suggest that PLA70/30 may mimic some of the physical and biochemical characteristics of the NSC niche. Its mechanical and surface properties may act synergistically in the modulation of bipotential and glial restricted progenitor phenotypes, while it is L-lactate, either added to the medium or released by the film that drives the maintenance of neuronal restricted progenitor cell phenotypes.

Modular polylactic acid microparticle-based scaffolds prepared via microfluidic emulsion/solvent displacement process: Fabrication, characterization, and in vitro mesenchymal stem cells interaction study

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.

Role of ECM/peptide coatings on SDF-1α triggered mesenchymal stromal cell migration from microcarriers for cell therapy

Many cell therapies rely on the ability of mesenchymal stromal cells (MSCs) to diffuse and localize throughout the target tissue - such as tumoral and ischemic tissues-, in response to specific cytokine signals, rather than being concentrated at the site of implantation. Therefore, it is fundamental to engineer biomaterial carriers as reservoirs, from which cells can migrate, possibly in a controlled manner. In this work, microcarriers (μCs) made of polylactic acid are characterized as MSC delivery vehicles capable of modulating key chemotactic pathways. The effect of different functionalization strategies on MSC migratory behavior from the μCs is studied in vitro in relation to SDF-1α/CXCR4 axis, - a major actor in MSC recruitment, chemotaxis and homing. Collagen and arginine-glycine-aspartic acid (RGD) peptides were either covalently grafted or physisorbed on μC surface. While stable covalent modifications promoted better cell adhesion and higher proliferation compared to physisorption, the functionalization method of the μCs also affected the cells migratory behavior in response to SDF-1α (CXCL12) stimulation. Less stable coatings (physisorbed) showed sensibly higher number of migrating cells than covalent collagen/RGD coatings. The combination of physic-chemical cues provided by protein/peptide functionalization and stimuli induced by 3D culture on μCs improved MSC expression of CXCR4, and exerted a control over cell migration, a condition suitable to promote cell homing after transplantation in vivo. These are key findings to highlight the impact of surface modification approaches on chemokine-triggered cell release, and allow designing biomaterials for efficient and controlled cell delivery to damaged tissues.