A systematic approach to improve printability and cell viability of methylcellulose-based bioinks.

Printability in 3D extrusion bioprinting encompasses extrudability, filament formation, and shape fidelity. Rheological properties can predict the shape fidelity of printed hydrogels. In particular, tan(δ), the ratio between loss (G'') and storage (G') modulus (G''/G'), is a powerful indicator of printability. This study explores the effect of different salt, sucrose, and MC concentrations on tan(δ), and therefore the printability of methylcellulose (MC) hydrogels. Salt and sucrose increased G', lowering tan(δ) and enabling printing of scaffolds with high shape fidelity. Conversely, MC concentration increased G'' and G', having a lesser effect on tan(δ). Shape fidelity of three formulations with similar G' but varying tan(δ) values were compared. Higher tan(δ) led to reduced height, while lower tan(δ) improved shape fidelity. Cell viability increased when reducing MC content, extrusion rate, and nozzle gauge. Higher MC concentration (G' > 1.5 kPa) increased the influence of needle size and extrusion rate on cell viability. Hydrogels with G' < 1 kPa could be extruded at high rates with small nozzles, minimally affecting cell viability. This work shows a direct relationship between tan(δ) and printability of MC-based hydrogels. Lowering the complex modulus of hydrogels, mitigates extrusion stress, thus improving cell survival.

Advanced Binary Guanosine and Guanosine 5'-Monophosphate Cell-Laden Hydrogels for Soft Tissue Reconstruction by 3D Bioprinting.

Soft tissue defects or pathologies frequently necessitate the use of biomaterials that provide the volume required for subsequent vascularization and tissue formation as autrografts are not always a feasible alternative. Supramolecular hydrogels represent promising candidates because of their 3D structure, which resembles the native extracellular matrix, and their capacity to entrap and sustain living cells. Guanosine-based hydrogels have emerged as prime candidates in recent years since the nucleoside self-assembles into well-ordered structures like G-quadruplexes by coordinating K+ ions and π-π stacking, ultimately forming an extensive nanofibrillar network. However, such compositions were frequently inappropriate for 3D printing due to material spreading and low shape stability over time. Thus, the present work aimed to develop a binary cell-laden hydrogel capable of ensuring cell survival while providing enough stability to ensure scaffold biointegration during soft tissue reconstruction. For that purpose, a binary hydrogel made of guanosine and guanosine 5'-monophosphate was optimized, rat mesenchymal stem cells were entrapped, and the composition was bioprinted. To further increase stability, the printed structure was coated with hyperbranched polyethylenimine. Scanning electron microscopic studies demonstrated an extensive nanofibrillar network, indicating excellent G-quadruplex formation, and rheological analysis confirmed good printing and thixotropic qualities. Additionally, diffusion tests using fluorescein isothiocyanate labeled-dextran (70, 500, and 2000 kDa) showed that nutrients of various molecular weights may diffuse through the hydrogel scaffold. Finally, cells were evenly distributed throughout the printed scaffold, cell survival was 85% after 21 days, and lipid droplet formation was observed after 7 days under adipogenic conditions, indicating successful differentiation and proper cell functioning. To conclude, such hydrogels may enable the 3D bioprinting of customized scaffolds perfectly matching the respective soft tissue defect, thereby potentially improving the outcome of the tissue reconstruction intervention.

Optimization of guanosine-based hydrogels with boric acid derivatives for enhanced long-term stability and cell survival.

Tissue defects can lead to serious health problems and often require grafts or transplants to repair damaged soft tissues. However, these procedures can be complex and may not always be feasible due to a lack of available tissue. Hydrogels have shown potential as a replacement for tissue grafts due to their ability to support cell survival and encapsulate biomolecules such as growth factors. In particular, guanosine-based hydrogels have been explored as a potential solution, but they often exhibit limited stability which hampers their use in the biofabrication of complex grafts. To address this issue, we explored the use of borate ester chemistry and more complex boric acid derivatives to improve the stability and properties of guanosine-based hydrogels. We hypothesized that the aromatic rings in these derivatives would enhance the stability and printability of the hydrogels through added π-π stack interactions. After optimization, 13 compositions containing either 2-naphthylboronic acid or boric acid were selected. Morphology studies shows a well-defined nanofibrilar structure with good printable properties (thixotropic behaviour, print fidelity and printability). Moreover, the pH of all tested hydrogels was within the range suitable for cell viability (7.4-8.3). Nevertheless, only the boric acid-based formulations were stable for at least 7 days. Thus, our results clearly demonstrated that the presence of additional aromatic rings did actually impair the hydrogel properties. We speculate that this is due to steric hindrance caused by adjacent groups, which disrupt the correct orientation of the aromatic groups required for effective π-π stack interactions of the guanosine building block. Despite this drawback, the developed guanosine-boric acid hydrogel exhibited good thixotropic properties and was able to support cell survival, proliferation, and migration. For instance, SaOS-2 cells planted on these printed structures readily migrated into the hydrogel and showed nearly 100% cell viability after 7 days. In conclusion, our findings highlight the potential of guanosine-boric acid hydrogels as tissue engineering scaffolds that can be readily enhanced with living cells and bioactive molecules. Thus, our work represents a significant advancement towards the development of functionalized guanosine-based hydrogels.

Nucleoside-Based Supramolecular Hydrogels: From Synthesis and Structural Properties to Biomedical and Tissue Engineering Applications.

Supramolecular hydrogels are of great interest in tissue scaffolding, diagnostics, and drug delivery due to their biocompatibility and stimuli-responsive properties. In particular, nucleosides are promising candidates as building blocks due to their manifold noncovalent interactions and ease of chemical modification. Significant progress in the field has been made over recent years to allow the use of nucleoside-based supramolecular hydrogels in the biomedical field, namely drug delivery and 3D bioprinting. For example, their long-term stability, printability, functionality, and bioactivity have been greatly improved by employing more than one gelator, incorporating different cations, including silver for antibacterial activity, or using additives such as boric acid or even biomolecules. This now permits their use as bioinks for 3D printing to produce cell-laden scaffolds with specified geometries and pore sizes as well as a homogeneous distribution of living cells and bioactive molecules. We have summarized the latest advances in nucleoside-based supramolecular hydrogels. Additionally, we discuss their synthesis, structural properties, and potential applications in tissue engineering and provide an outlook and future perspective on ongoing developments in the field.

Development of Cell-Derived Matrices for Three-Dimensional In Vitro Cancer Cell Models.

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.

Development and Angiogenic Potential of Cell-Derived Microtissues Using Microcarrier-Template.

Tissue engineering and regenerative medicine approaches use biomaterials in combination with cells to regenerate lost functions of tissues and organs to prevent organ transplantation. However, most of the current strategies fail in mimicking the tissue's extracellular matrix properties. In order to mimic native tissue conditions, we developed cell-derived matrix (CDM) microtissues (MT). Our methodology uses poly-lactic acid (PLA) and Cultispher® S microcarriers' (MCs') as scaffold templates, which are seeded with rat bone marrow mesenchymal stem cells (rBM-MSCs). The scaffold template allows cells to generate an extracellular matrix, which is then extracted for downstream use. The newly formed CDM provides cells with a complex physical (MT architecture) and biochemical (deposited ECM proteins) environment, also showing spontaneous angiogenic potential. Our results suggest that MTs generated from the combination of these two MCs (mixed MTs) are excellent candidates for tissue vascularization. Overall, this study provides a methodology for in-house fabrication of microtissues with angiogenic potential for downstream use in various tissue regenerative strategies.

Engineered microtissues for the bystander therapy against cancer.

Thymidine kinase expressing human adipose mesenchymal stem cells (TK-hAMSCs) in combination with ganciclovir (GCV) are an effective platform for antitumor bystander therapy in mice models. However, this strategy requires multiple TK-hAMSCs administrations and a substantial number of cells. Therefore, for clinical translation, it is necessary to find a biocompatible scaffold providing TK-hAMSCs retention in the implantation site against their rapid wash-out. We have developed a microtissue (MT) composed by TKhAMSCs and a scaffold made of polylactic acid microparticles and cell-derived extracellular matrix deposited by hAMSCs. The efficacy of these MTs as vehicles for TK-hAMSCs/GCV bystander therapy was evaluated in a rodent model of human prostate cancer. Subcutaneously implanted MTs were integrated in the surrounding tissue, allowing neovascularization and maintenance of TK-hAMSCs viability. Furthermore, MTs implanted beside tumors allowed TK-hAMSCs migration towards tumor cells and, after GCV administration, inhibited tumor growth. These results indicate that TK-hAMSCs-MTs are promising cell reservoirs for clinical use of therapeutic MSCs in bystander therapies.

Development of a novel automatable fabrication method based on electrospinning co electrospraying for rotator cuff augmentation patches.

Rotator cuff tear is one of the most common shoulder diseases. Rotator cuff augmentation (RCA) is trying to solve the high retear failure percentage after the surgery procedures (20-90%). The ideal augmentation patch must provide a temporal mechanical support during the healing process. In this work, we proposed a simple method for the fabrication of synthetic RCA patches. This method combines the use of electrospraying to produce poly-L-lactic-co-ε-caprolactone (PLC) films in an organogel form and electrospinning to produce poly(lactic) acid (PLA) nanofibers. The device consists in a combination of layers, creating a multilayered construct, enabling the possibility of tuning its mechanical properties and thickness. Besides, both techniques are simple to escalate for industrial production. A complete characterization has been performed to optimize the involved number of layers and production time of PLC films and PLA nanofibers fabrication, obtaining a final optimal configuration for RCA devices. Structural, mechanical and suture properties were evaluated. Also, the possibility of surface functionalization to improve the bioactivity of the scaffold was studied, adding aligned electrospun PLA nanofibers on the surface of the device to mimic the natural tendon topography. Surface modification was characterized by culturing adult normal human dermal fibroblasts. Lack of toxicity was detected for material presented, and cell alignment shape orientation guided by aligned fibers, mimicking tendon structure, was obtained. Cell proliferation and protein production were also evaluated.

Poly-l/dl-lactic acid films functionalized with collagen IV as carrier substrata for corneal epithelial stem cells.

Limbal epithelial stem cells (LESCs) are responsible for the renewal of corneal epithelium. Cultivated limbal epithelial transplantation is the current treatment of choice for restoring the loss or dysfunction of LESCs. To perform this procedure, a substratum is necessary for in vitro culturing of limbal epithelial cells and their subsequent transplantation onto the ocular surface. In this work, we evaluated poly-L/DL-lactic acid 70:30 (PLA) films functionalized with type IV collagen (col IV) as potential in vitro carrier substrata for LESCs. We first demonstrated that PLA-col IV films were biocompatible and suitable for the proliferation of human corneal epithelial cells. Subsequently, limbal epithelial cell suspensions, isolated from human limbal rings, were cultivated using culture medium that did not contain animal components. The cells adhered significantly faster to PLA-col IV films than to tissue culture plastic (TCP). The mRNA expression levels for the LESC specific markers, K15, P63α and ABCG2 were similar or greater (significantly in the case of K15) in limbal epithelial cells cultured on PLA-col IV films than limbal epithelial cells cultured on TCP. The percentage of cells expressing the corneal (K3, K12) and the LESC (P63α, ABCG2) specific markers was similar for both substrata. These results suggest that the PLA-col IV films promoted LESC attachment and helped to maintain their undifferentiated stem cell phenotype. Consequently, these substrata offer an alternative for the transplantation of limbal cells onto the ocular surface.

Differential neuronal and glial behavior on flat and micro patterned chitosan films.

Chitosan is a biodegradable natural polysaccharide that has been widely studied for regenerative purposes in the central nervous system. In this study we assessed the in vitro glial and neuronal cells response to chitosan either flat or patterned with grooves in the micrometric range. Chitosan demonstrated to be a good substrate for the attachment and growth of both neurons and glial cells. Chitosan micropatterns promoted glial cell maturation, suggesting astroglial activation. Nevertheless, those mature/reactive glial cells were permissive for axonal growth. Axons aligned and organized along the patterned grooves and the size of the linear topographic patterns is also affecting neurite and cell response. Patterns with 10µm width induced fasciculation of axons, which can be useful for CNS tissue engineering substrates when precise orientation of the axonal outgrowth is desired.

Development of tailored and self-mineralizing citric acid-crosslinked hydrogels for in situ bone regeneration.

Bone tissue engineering demands alternatives overcoming the limitations of traditional approaches in the context of a constantly aging global population. In the present study, elastin-like recombinamers hydrogels were produced by means of carbodiimide-catalyzed crosslinking with citric acid, a molecule suggested to be essential for bone nanostructure. By systematically studying the effect of the relative abundance of reactive species on gelation and hydrogel properties such as functional groups content, degradation and structure, we were able to understand and to control the crosslinking reaction to achieve hydrogels mimicking the fibrillary nature of the extracellular matrix. By studying the effect of polymer concentration on scaffold mechanical properties, we were able to produce hydrogels with a stiffness value of 36.13 ± 10.72 kPa, previously suggested to be osteoinductive. Microstructured and mechanically-tailored hydrogels supported the growth of human mesenchymal stem cells and led to higher osteopontin expression in comparison to their non-tailored counterparts. Additionally, tailored hydrogels were able to rapidly self-mineralize in biomimetic conditions, evidencing that citric acid was successfully used both as a crosslinker and a bioactive molecule providing polymers with calcium phosphate nucleation capacity.

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.

Biomolecular functionalization for enhanced cell-material interactions of poly(methyl methacrylate) surfaces.

The integration of implants or medical devices into the body tissues requires of good cell-material interactions. However, most polymeric materials used for these applications lack on biological cues, which enhanced mid- and long-term implant failure due to weak integration with the surrounding tissue. Commonly used strategies for tissue-material integration focus on functionalization of the material surface by means of natural proteins or short peptides. However, the use of these biomolecules involves major drawbacks such as immunogenic problems and oversimplification of the constructs. Here, designed elastin-like recombinamers (ELRs) are used to enhance poly(methyl methacrylate) surface properties and compared against the use of short peptides. In this study, cell response has been analysed for different functionalization conditions in the presence and absence of a competing protein, which interferes on surface-cell interaction by unspecific adsorption on the interface. The study has shown that ELRs can induce higher rates of cell attachment and stronger cell anchorages than short peptides, being a better choice for surface functionalization.

Biofunctionalization of polymeric surfaces.

Most of the synthetic polymeric biomaterials used for biomedical applications lack of functional groups able to specifically instruct cells to unlock their potential for tissue regeneration. Surface modification strategies are able to overcome this limitation by introducing bioactive cues. In this study, several functionalization approaches are analyzed. Wet chemical methods such as controlled hydrolysis of polyesters followed by biomolecules grafting by carbodiimide chemistry are simple and versatile approaches, able to succesfully improve the bioactivity of devices with virtually any architecture. Grafting of short peptides, extracellular matrix proteins (ECM) or engineered protein-like recombinamers are promising techniques to improve cell adhesion to biomaterials, including polylactic acid (PLA) and its derivatives. ECM molecules and recombinamers can present more effectively bioactive signals, even in presence of competing, nonadhesive serum proteins. Besides adhesion, surface modifications intended to improve cell attachment, play a role on other cell responses, such as migratory potential. Collagen coating were shown to enhance the expression of the migratory receptor CXCR4 in mesenchymal stromal cells, when compared to short RGD peptides, while the modality of functionalization (covalent vs. physisorbed) tuned the rate of cell migration from PLA-based microcarriers. This multiple effects have to be taken into account when designing biomaterials for cell delivery and tissue engineering. Furthermore, as we aim to recapitulate in vitro the complexity of native tissues, alternative strategies based on the generation of decellularized polymer scaffold rich in cell-deposited ECM are proposed.

Fibronectin immobilization on to robotic-dispensed nanobioactive glass/polycaprolactone scaffolds for bone tissue engineering.

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.

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.

Effect of structure, topography and chemistry on fibroblast adhesion and morphology.

Surface biofunctionalisation of many biodegradable polymers is one of the used strategies to improve the biological activity of such materials. In this work, the introduction of collagen type I over the surface of a biodegradable polymer (poly lactic acid) processed in the forms of films and fibers leads to an enhancing of the cellular adhesion of human dermal fibroblast when compared to unmodified polymer and biomolecule-physisorbed polymer surface. The change of topography of the material does not affect the cellular adhesion but results in a higher proliferation of the fibroblast cultured over the fibers. Moreover, the difference of topography governs the cellular morphology, i.e. cells adopt a more stretched conformation where cultured over the films while a more elongated with lower area morphology are obtained for the cells grown over the fibers. This study is relevant for designing and modifying different biodegradable polymers for their use as scaffolds for different applications in the field of Tissue Engineering and Regenerative Medicine.

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.

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 Young's modulus sixfold higher than non-coated ones, while remaining fully flexible and easy to handle. Rat mesenchymal stem cells cultured on these fibres showed great cellular spreading and interactions with the material. This protocol can be transferred to other structures and glasses, allowing the fabrication of 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.

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.