Injectable biodegradable carriers for the delivery of therapeutic agents and tissue engineering

The design of smart biomaterial devices plays a key role to improve the way conventional therapies are being delivered, and to promote the development of new approaches for advanced therapies, such as regenerative medicine and targeted drug release. Injectable biodegradable materials, such as those consisting of suspensions of polymeric particles, are highly versatile devices that can be delivered through minimally-invasive injections. The physic-chemical properties of the particles can be engineered to obtain smart scaffolds for tissue engineering, carriers for drug release and cell therapy. The aim of this Thesis is to develop a novel class of biodegradable and injectable particulate carriers based on polylactic acid (PLA), that are capable to trigger and guide specific responses from the cells and the biological milieu. First, a novel route to fabricate PLA-based microcarriers (MCs) was set and characterized. The production method involved green, non-harmful chemicals and it is easy to scale-up. Such technique allowed tuning MC size and size distribution in the range suitable for drug and cell delivery applications. The favorable regulatory status of the materials and reagents may also be beneficial for the translation of the MCs from bench to bedside. The principles guiding the fabrication procedure can inspire techniques to generate nanocarriers for controlled drug delivery. Recent studies point out the importance of drug-loaded and submicron-sized materials in the treatment of severe clinical conditions, such as persistent biofilm infections. These nanoparticles (NPs) can be endowed with smart functionalities to enhance drug delivery within the biofilm matrix. In this way, NPs encapsulating the antibiotic ciprofloxacin were produced and functionalized with DNase I. The NPs improved the antimicrobial activity of the encapsulated drug and promoted biofilm eradication, targeting and degrading directly the biofilm matrix. On the other hand, larger particles such as MC, display a high surface area for cell expansion. MCs can also deliver cells with therapeutic potential as ¿living drugs¿, ideally in a spatio-temporal controlled fashion. This is especially important, as, in standard cell therapies, direct injection of cells is accompanied by massive cell mortality that renders the treatment ineffective. PLA MCs suitable for Mesenchymal Stromal Cells (MSCs) homing have been produced and modified with different functionalization approaches. The physic-chemical properties of the MCs and bioactive coatings modulated cell adhesion, proliferation, and migratory potential in response to chemokines that regulate MSC tissue localization, like SDF-1a. The results highlight the importance of carriers design to control cell delivery, and provide important guidelines to instruct a new generation of efficient biomaterial carriers. Another exciting application of injectable, cell-laden MCs is to use them as building blocks to fabricate living tissues in vitro. Combining MC technology and bioprinting is an appealing strategy to generate tissues grafts with controlled architectures. The suspension of injectable PLA cell-laden MCs within gelatin-based hydrogels formed an extrudable, composite bioink. MCs acted as mechanical reinforcement for soft gels and as means for cell expansion to encapsulate high cell payload. MSCs were shown to form MC-MSCs aggregates, with enhanced cell-to-cell contact on surface functionalized PLA MCs, and differentiated towards the osteogenic lineage. This result suggests potential applications of MC-MSCs laden bioinks for bone tissue engineering, and the composite bioink is proposed as component to build multimaterial, 3D-printed osteochondral graft models. Taken together, the injectable devices developed in the Thesis constitute promising and highly versatile biomaterial platforms for biomedical applications, and can be employed in a wide array of tissue engineering, and cell and drug delivery strategies.El diseño de dispositivos basados en biomateriales inteligentes, juega un papel fundamental a la hora de mejorar las terapias convencionales, así como en el desarrollo de nuevas estrategias para la medicina regenerativa y la liberación controlada de fármacos. Materiales inyectables biodegradables, tales como las suspensiones de partículas poliméricas, constituyen dispositivos versátiles, que se pueden suministrar por medio de inyecciones mínimamente invasivas. Las propiedades físico-químicas de las partículas pueden ser modificadas para obtener andamios inteligentes para la ingeniería de tejidos, transportadores para liberación de fármacos y cultivo y terapia celular. El objetivo de esta Tesis es el desarrollo de una nueva clase de partículas transportadoras inyectables y biodegradables, basadas en ácido poliláctico (PLA), que sean capaces de desencadenar y guiar respuestas específicas por parte de las células y del entorno biológico. Primero, se ha creado y caracterizado una nueva ruta para fabricar microstransportadores (MCs) basados en PLA. Este método de producción utiliza reactivos verdes y no-tóxicos, y es sencillo de adaptar para la fabricación a gran escala. Esta técnica permite controlar parámetros fundamentales en las MCs, tales como su tamaño y dispersión, que pueden ser controlados dentro de los rangos adecuados para aplicaciones de liberación de fármacos y células. El hecho que los materiales y reactivos utilizados están bien aceptados por las agencias reguladoras, puede favorecer el traslado de las partículas fabricadas desde la investigación hasta la práctica clínica. Los principios de este método pueden adaptarse a otras técnicas de fabricación para generar nanotransportadores (nanopartículas, NPs) de fármacos. Estudio recientes subrayan la importancia de biomateriales submicrométricos cargados con compuestos bioactivos en el tratamiento de enfermedades, tal como las infecciones provocadas por biofilms. Estas NPs pueden ser modificadas con funcionalidades inteligentes, para mejorar la distribución del fármaco en la matriz del biofilm. De esta manera, se han producido NPs que encapsulan el antibiótico ciprofloxacino, modificadas superficialmente con DNasa I. Estos transportadores tienen como diana la matriz que compone el biofilm y pueden degradarla, incrementando la actividad antibacteriana del ciprofloxacino y promoviendo la erradicación de los biofilms. Por otra banda, las partículas más grandes, como las MCs, poseen una superficie adecuada para la expansión celular. Las MCs se pueden usar para transportar “drogas vivas”, es decir células con potencial terapéutico, posiblemente controlando su distribución espacial y su cinética de liberación. Esto es de particular importancia, porque la ineficiencia de muchas terapias celulares actuales se debe a la gran cantidad de células que no sobreviven una vez inyectadas in vivo. Se han producido MCs de PLA modificadas por diferentes estrategias de funcionalización y aptas para suportar en su superficie células madres mesenquimales (MSCs). La biofuncionalización y las propiedades físico-químicas de las MCs juegan un papel fundamental en la adhesión y proliferación célular, así como la capacidad de las MSCs de migrar en respuesta a estímulos quimiotácticos, que regulan su localización en los tejidos, tal como el SDF-1α. Los resultados subrayan la importancia del diseño de las MCs para controlar la liberación de las células, y a la vez aportan información para desarrollar una nueva y más eficiente generación de transportadores de células. Otra aplicación prometedora de las MCs inyectables es su uso como bloques de construcción para fabricar tejidos vivos in vitro. La combinación de la tecnología de las MCs con la bioimpresión 3D constituye una estrategia atractiva para obtener injertos de tejidos multimateriales con arquitectura controlada. Se han obtenido biotintas compuestas y capaces de ser extruidas mezclando materiales basados en hidrogeles de gelatina con las MCs de PLA cargadas con células. Las MCs actúan de refuerzo mecánico para el hidrogel y como vehículo para la expansión celular (por ejemplo, en un bioreactor “spinner flask”) para encapsular elevadas cantidades de células. Las MSCs forman agregados células-particulas, una vez sembradas en las superficies de las MCs, y estos complejos, ricos en contactos célula-célula, se demostraron capaces de suportar la diferenciación osteogénica de las MSCs. Este resultado sugiere potenciales aplicaciones de las biotintas cargadas de agregados de MCs y MSCs para la ingeniería del tejido óseo. Esta biotinta ha sido también utilizada como componiente para generar un modelo de injerto osteocondral, por medio de una técnica de impresión 3D. El conjunto de dispositivos inyectables desarrollados en esta Tesis constituyen una plataforma muy versátil y prometedora para aplicaciones biomédicas, en particular en estrategias de ingeniería de tejidos, y liberación de células y fármaco

A multiangular approach towards biofabrication of an auricular cartilage implant

Cartilage tissue engineering opens new avenues for reconstruction of auricular deformities. Nevertheless, a number of challenges hinder the development of an auricular cartilage implant, including an appropriate cell source, nutrient limitation in large non-vascularized constructs, and maintenance of the complex auricular shape. This work uses a multiangular approach including biofabrication strategies to address these challenges. Firstly, we investigated the regenerative potential of novel auricular cartilage progenitor cells in 3D printable hydrogels. Furthermore, we proposed a modular construct to decrease the diffusion distance throughout the implant. In addition, the mechanical integrity of the developing construct is warranted by a polymer fiber-reinforced network integrated into a cell-laden hydrogel. Equine auricular cartilage progenitor cells (AuCPC) were encapsulated in 10% gelatin methacrylate (gelMA) hydrogel cylinders and chondrogenically differentiated up to 56 days in vitro. The neocartilage produced by AuCPC displayed GAG/DNA composition and mechanical integrity comparable to auricular chondrocytes (AuCH), and the production of cartilage-like extracellular matrix was confirmed by histology. Polycaprolactone (PCL) scaffolds for custom-designed modular parts of the auricle were fabricated using a Bioscaffolder and combined with gelMA to form hybrid constructs. Light microscopy confirmed homogenous distribution of the hydrogel through the reinforcing network, and the assembled modules displayed a convincing aesthetical appearance under a rubber skin. Bioprinted cell-laden constructs demonstrated homogenous cell distribution and good cell viability after printing up to 7 days of in vitro culture. These results indicate that a multi-faceted approach in creating large tissue constructs is a promising method that warrants further investigation

Application of different cell populations in hydrogel bioinks for zonal Cartilage biofabrication

Functional regeneration of articular cartilage is still a major challenge in human. Bioprinting permits to mimic the complex architecture of articular cartilage, by coordinating the deposition of multiple cell types and materials, termed bioinks. For this purpose, cells with high potential for zonal differentiation need to be encapsulated in bioinks that provide an instructive niche for extracellular matrix (ECM) synthesis. The recent identification of multipotent articular cartilage chondroprogenitor cells (ACPCs) represents a new opportunity to generate bioinks with defined zonal affinity. The aim of this work was to print zonal constructs using hydrogel bioinks encapsulating ACPCs, alone or in combination with other cell types, obtained from equine donors. Gelatin methacryloyl (gelMA)-based inks were used to culture ACPCs, bone marrow mesenchymal stromal cells (MSCs) and chondrocytes (CHs) in casted gels. The expression of zonal markers and ECM molecules by each cell type was studied. Constructs composed of two adjacent regions, each containing a single cell type were also fabricated, as models for zonal co-culture of the possible MSCs, CHs, and ACPCs pairings. Finally, zonal constructs were printed using ACPC-laden gelMA as superficial zone-competent bioink, and a MSC-laden ink for the deeper zones, via bioink extrusion in a sacrificial poloxamer frame. The effect of printing on long-term cell performance was evaluated during 56 days of culture. GAG/DNA quantification, histological and qPCR analysis revealed that all cell types underwent chondrogenic differentiation in gelMA bioinks. Additionally, a differential expression of zonal markers was detected between MSCs and ACPCs, the latter significantly upregulating the superficial zone marker PRG4. Conversely, MSCs had higher expression of collagen type X, a marker for the calcified zone. Differential distribution of ECM molecules was preserved also in zonal co-cultures. These results pave the way to the biofabrication of multicellular, functional constructs with zone-mimicking composition to be used for cartilage regeneration or as in vitro tissue models

Convergence of printing technologies to engineer an interface between bone and cartilage

The combination of multiple three dimensional printing technologies can aid the generation of osteochondral grafts that display a strong interface between the cartilage and the bone compartment. In this study, the integration between bone biomimetic a three-dimensional (3D) printed calcium phosphate paste (PCP) and a gelatin methacryloyl (gelMA) hydrogel substrate for cartilage, was reinforced with a PCL mesh produced by melt electrospinning writing (MEW). Please download the file below for full content

Evaluation of bioink printability with quantitative methods to aid material development

During extrusion-based bioprinting, the deposited bioink filaments are subjected to deformations, such as collapse of overhanging filaments and fusion between adjacent filaments, which compromise shape fidelity of printed constructs. The degree of deformation of printed filaments could be used to quantitatively assess the printability of newly developed bioinks. This approach would be an alternative to current assessment through qualitative visual inspection after printing, which have been hampering any comparison between different bioinks. For this reason, we propose two quantitative printability tests based on the mentioned filament deformations: filament collapse of overhanging structures (Fig 1a) and filament fusion on parallel filaments (Fig 1b). Both printability tests were applied on two printable hydrogel platforms: poloxamer 407 and poly(ethylene glycol) blends (poloxamer/PEG), displaying a range of yield stress values. We also propose theoretical models for each test to predict printability from bioink yield stress. The results on poloxamer/PEG hydrogels show that as the yield stress decreases, the filament collapse is greater, decreasing the ability to maintain the shape of suspended filaments. Similarly, filament fusion occurs at bigger filament distances, decreasing resolution on the x-y plane. These results confirm that printability is largely dependent on yield stress. Our bioink printability testing is straightforward, assessible with any extrusion-based bioprinting system. The proposed method provides a quantitative evaluation based on physical deformation of printed filaments, potentially reducing long experimental trial-and-error printing with newly developed bioinks and allowing reproducible comparisons between different inks. Please click Additional Files below to see the full abstract

Bioprinting Neural Systems to Model Central Nervous System Diseases

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A Multifunctional Nanocomposite Hydrogel for Endoscopic Tracking and Manipulation

Herein, the fabrication of multi‐responsive and hierarchically organized nanomaterial using core‐shell SrF2 upconverting nanoparticles, doped with Yb3+, Tm3+, Nd3+ incorporated into gelatin methacryloyl matrix, is reported. Upon 800 nm excitation, deep monitoring of 3D‐printed constructs is demonstrated. Addition of magnetic self‐assembly of iron oxide nanoparticles within the hydrogel provides anisotropic structuration from the nano‐ to the macro‐scale and magnetic responsiveness permitting remote manipulation. The present study provides a new strategy for the fabrication of a novel highly organized multi‐responsive material using additive manufacturing, which can have important implications in biomedicine

Biofabricating the vascular tree in engineered bone tissue

The development of tissue engineering strategies for treatment of large bone defects has become increasingly relevant, given the growing demand for bone substitutes. Native bone is composed of a dense vascular network necessary for the regulation of bone development, regeneration and homeostasis. A major obstacle in fabricating living, clinically relevant-sized bone mimics (1-10 cm3) is the limited supply of nutrients, including oxygen to the core of the construct. Therefore, strategies to support vascularization are pivotal for the development of tissue engineered bone constructs. Creating a functional bone construct integrated with a vascular network, capable of delivering the necessary nutrients for optimal tissue development is imperative for translation into the clinics. The vascular system is composed of a complex network that runs throughout the body in a tree-like hierarchical branching fashion. A significant challenge for tissue engineering approaches lies in mimicking the intricate, multi-scale structures consisting of larger vessels (macro-vessels) which interconnect with multiple sprouting vessels (microvessels) in a closed network. The advent of biofabrication has enabled complex, out of plane channels to be generated and has laid the groundwork for the creation of multi-scale vasculature in recent years. This review highlights the key state-of-the-art achievements for the development of vascular networks of varying scales in the field of biofabrication with a particular focus for its application in developing a functional tissue engineered bone construct. STATEMENT OF SIGNIFICANCE: There is a growing need for bone substitutes to overcome the limited supply of patient-derived bone. Bone tissue engineering aims to overcome this by combining stem cells with scaffolds to restore missing bone. The current bottleneck in upscaling is the lack of an integrated vascular network, required for the delivery of nutrients to cells. 3D bioprinting techniques has enabled the creation of complex hollow structures of varying dimensions that resemble native blood vessels. The convergence of multiple materials, cell types and fabrication approaches, opens the possibility of developing clinically-relevant sized vascularized bone constructs. This review provides an up-to-date insight of the technologies currently available for the generation of complex vascular networks, with a focus on their application in bone tissue engineering

Platelet-Rich Plasma Does Not Inhibit Inflammation or Promote Regeneration in Human Osteoarthritic ChondrocytesIn VitroDespite Increased Proliferation

Objective The aims of the study were to assess the anti-inflammatory properties of platelet-rich plasma (PRP) and investigate its regenerative potential in osteoarthritic (OA) human chondrocytes. We hypothesized that PRP can modulate the inflammatory response and stimulate cartilage regeneration. Design Primary human chondrocytes from OA knees were treated with manually prepared PRP, after which cell migration and proliferation were assessed. Next, tumor necrosis factor-alpha-stimulated chondrocytes were treated with a range of concentrations of PRP. Expression of genes involved in inflammation and chondrogenesis was determined by real-time polymerase chain reaction. In addition, chondrocytes were cultured in PRP gels and fibrin gels consisting of increasing concentrations of PRP. The production of cartilage extracellular matrix (ECM) was assessed. Deposition and release of glycosaminoglycans (GAG) and collagen was quantitatively determined and visualized by (immuno)histochemistry. Proliferation was assessed by quantitative measurement of DNA. Results Both migration and the inflammatory response were altered by PRP, while proliferation was stimulated. Expression of chondrogenic markers COL2A1 and ACAN was downregulated by PRP, independent of PRP concentration. Chondrocytes cultured in PRP gel for 28 days proliferated significantly more when compared with chondrocytes cultured in fibrin gels. This effect was dose dependent. Significantly less GAGs and collagen were produced by chondrocytes cultured in PRP gels when compared with fibrin gels. This was qualitatively confirmed by histology. Conclusions PRP stimulated chondrocyte proliferation, but not migration. Also, production of cartilage ECM was strongly downregulated by PRP. Furthermore, PRP did not act anti-inflammatory on chondrocytes in anin vitroinflammation model

Biofunctionalization of 3D printed collagen with bevacizumab-loaded microparticles targeting pathological angiogenesis

Pathological angiogenesis is a crucial attribute of several chronic diseases such as cancer, age-related macular degeneration, and osteoarthritis (OA). In the case of OA, pathological angiogenesis mediated by the vascular endothelial growth factor (VEGF), among other factors, contributes to cartilage degeneration and to implants rejection. In line with this, the use of the anti-VEGF bevacizumab (BVZ) has been shown to prevent OA progression and support cartilage regeneration. The aim of this work was to functionalize a medical grade collagen with poly (lactic-co-glycolic acid) (PLGA) microparticles containing BVZ via three-dimensional (3D) printing to target pathological angiogenesis. First, the effect of several formulation parameters on the encapsulation and release of BVZ from PLGA microparticles was studied. Then, the anti-angiogenic activity of released BVZ was tested in a 3D cell model. The 3D printability of the microparticle-loaded collagen ink was tested by evaluating the shape fidelity of 3D printed structures. Results showed that the release and the encapsulation efficiency of BVZ could be tuned as a function of several formulation parameters. In addition, the released BVZ was observed to reduce vascularization by human umbilical vein endothelial cells. Finally, the collagen ink with embedded BVZ microparticles was successfully printed, leading to shape-stable meniscus-, nose- and auricle-like structures. Taken altogether, we defined the conditions for the successful combination of BVZ-loaded microparticles with the 3D printing of a medical grade collagen to target pathological angiogenesisThis project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 814444 (MEFISTO). The authors thank mAbxience-GH Genhelix for the kind donation of Bevacizumab (Avastin®) and Geistlich Pharma AG for providing the medical grade collagen. AA acknowledges funding from “la Caixa” Foundation (ID 100010434) with a fellowship code LCF/BQ/PR22/11920003. RL acknowledges funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No. 949806, VOLUME-BIO). RL and JM acknowledge funding from the Dutch Artritis Foundation (LLP-12 and LLP-22)S