Layer-by-layer bioassembly of cellularized polylactic acid porous membranes for bone tissue engineering.

The conventional tissue engineering is based on seeding of macroporous scaffold on its surface ("top-down" approach). The main limitation is poor cell viability in the middle of the scaffold due to poor diffusion of oxygen and nutrients and insufficient vascularization. Layer-by-Layer (LBL) bioassembly is based on "bottom-up" approach, which considers assembly of small cellularized blocks. The aim of this work was to evaluate proliferation and differentiation of human bone marrow stromal cells (HBMSCs) and endothelial progenitor cells (EPCs) in two and three dimensions (2D, 3D) using a LBL assembly of polylactic acid (PLA) scaffolds fabricated by 3D printing. 2D experiments have shown maintain of cell viability on PLA, especially when a co-cuture system was used, as well as adequate morphology of seeded cells. Early osteoblastic and endothelial differentiations were observed and cell proliferation was increased after 7 days of culture. In 3D, cell migration was observed between layers of LBL constructs, as well as an osteoblastic differentiation. These results indicate that LBL assembly of PLA layers could be suitable for BTE, in order to promote homogenous cell distribution inside the scaffold and gene expression specific to the cells implanted in the case of co-culture system.

Harnessing light in biofabrication.

The integration of light-driven technologies into biofabrication has revolutionized the field of tissue engineering and regenerative medicine, with numerous breakthroughs in the last few years. Light-based bioprinting approaches (lithography, multiphoton and volumetric bioprinting) have shown the potential to fabricate large scale tissue engineering constructs of high resolution, with great flexibility and control over the cellular organization. Given the unprecedented degree of freedom in fabricating convoluted structures, key challenges in regenerative medicine, such as introducing complex channels and pre-vascular networks in 3D constructs have also been addressed. Light has also been proven as a powerful tool, leading to novel photo-chemistry in designing bioinks, but also able to impart spatial-temporal control over cellular functions through photo-responsive chemistry. For instance, smart constructs able to undergo remotely controlled shape changes, stiffening, softening and degradation can be produced. The non-invasive nature of light stimulation also enables to trigger such responses post-fabrication, during the maturation phase of a construct. Such unique ability can be used to mimic the dynamic processes occurring in tissue regeneration, as well as in disease progression and degenerative processes in vivo. Bringing together these novel multidisciplinary expertise, the present Special Issue aims to discuss the most recent trends, strategies and novel light-based technologies in the field of biofabrication. These include: 1) using light-based bioprinting to develop in vitro models for drug screening, developmental biology models, disease models, and also functional tissues for implantation; 2) novel light-based biofabrication technologies; 3) development of new photo-responsive bioinks or biomaterial inks.

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.

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 angiogenesis.

Volumetric Printing Across Melt Electrowritten Scaffolds Fabricates Multi-Material Living Constructs with Tunable Architecture and Mechanics.

Major challenges in biofabrication revolve around capturing the complex, hierarchical composition of native tissues. However, individual 3D printing techniques have limited capacity to produce composite biomaterials with multi-scale resolution. Volumetric bioprinting recently emerged as a paradigm-shift in biofabrication. This ultrafast, light-based technique sculpts cell-laden hydrogel bioresins into 3D structures in a layerless fashion, providing enhanced design freedom over conventional bioprinting. However, it yields prints with low mechanical stability, since soft, cell-friendly hydrogels are used. Herein, the possibility to converge volumetric bioprinting with melt electrowriting, which excels at patterning microfibers, is shown for the fabrication of tubular hydrogel-based composites with enhanced mechanical behavior. Despite including non-transparent melt electrowritten scaffolds in the volumetric printing process, high-resolution bioprinted structures are successfully achieved. Tensile, burst, and bending mechanical properties of printed tubes are tuned altering the electrowritten mesh design, resulting in complex, multi-material tubular constructs with customizable, anisotropic geometries that better mimic intricate biological tubular structures. As a proof-of-concept, engineered tubular structures are obtained by building trilayered cell-laden vessels, and features (valves, branches, fenestrations) that can be rapidly printed using this hybrid approach. This multi-technology convergence offers a new toolbox for manufacturing hierarchical and mechanically tunable multi-material living structures.

Shaping Synthetic Multicellular and Complex Multimaterial Tissues via Embedded Extrusion-Volumetric Printing of Microgels.

In living tissues, cells express their functions following complex signals from their surrounding microenvironment. Capturing both hierarchical architectures at the micro- and macroscale, and anisotropic cell patterning remains a major challenge in bioprinting, and a bottleneck toward creating physiologically-relevant models. Addressing this limitation, a novel technique is introduced, termed Embedded Extrusion-Volumetric Printing (EmVP), converging extrusion-bioprinting and layer-less, ultra-fast volumetric bioprinting, allowing spatially pattern multiple inks/cell types. Light-responsive microgels are developed for the first time as bioresins (µResins) for light-based volumetric bioprinting, providing a microporous environment permissive for cell homing and self-organization. Tuning the mechanical and optical properties of gelatin-based microparticles enables their use as support bath for suspended extrusion printing, in which features containing high cell densities can be easily introduced. µResins can be sculpted within seconds with tomographic light projections into centimeter-scale, granular hydrogel-based, convoluted constructs. Interstitial microvoids enhanced differentiation of multiple stem/progenitor cells (vascular, mesenchymal, neural), otherwise not possible with conventional bulk hydrogels. As proof-of-concept, EmVP is applied to create complex synthetic biology-inspired intercellular communication models, where adipocyte differentiation is regulated by optogenetic-engineered pancreatic cells. Overall, EmVP offers new avenues for producing regenerative grafts with biological functionality, and for developing engineered living systems and (metabolic) disease models.

From Shape to Function: The Next Step in Bioprinting.

In 2013, the "biofabrication window" was introduced to reflect the processing challenge for the fields of biofabrication and bioprinting. At that time, the lack of printable materials that could serve as cell-laden bioinks, as well as the limitations of printing and assembly methods, presented a major constraint. However, recent developments have now resulted in the availability of a plethora of bioinks, new printing approaches, and the technological advancement of established techniques. Nevertheless, it remains largely unknown which materials and technical parameters are essential for the fabrication of intrinsically hierarchical cell-material constructs that truly mimic biologically functional tissue. In order to achieve this, it is urged that the field now shift its focus from materials and technologies toward the biological development of the resulting constructs. Therefore, herein, the recent material and technological advances since the introduction of the biofabrication window are briefly summarized, i.e., approaches how to generate shape, to then focus the discussion on how to acquire the biological function within this context. In particular, a vision of how biological function can evolve from the possibility to determine shape is outlined.

Combining multi-scale 3D printing technologies to engineer reinforced hydrogel-ceramic interfaces.

Multi-material 3D printing technologies that resolve features at different lengths down to the microscale open new avenues for regenerative medicine, particularly in the engineering of tissue interfaces. Herein, extrusion printing of a bone-biomimetic ceramic ink and melt electrowriting (MEW) of spatially organized polymeric microfibres are integrated for the biofabrication of an osteochondral plug, with a mechanically reinforced bone-to-cartilage interface. A printable physiological temperature-setting bioceramic, based on α-tricalcium phosphate, nanohydroxyapatite and a custom-synthesized biodegradable and crosslinkable poloxamer, was developed as bone support. The mild setting reaction of the bone ink enabled us to print directly within melt electrowritten polycaprolactone meshes, preserving their micro-architecture. Ceramic-integrated MEW meshes protruded into the cartilage region of the composite plug, and were embedded with mechanically soft gelatin-based hydrogels, laden with articular cartilage chondroprogenitor cells. Such interlocking design enhanced the hydrogel-to-ceramic adhesion strength >6.5-fold, compared with non-interlocking fibre architectures, enabling structural stability during handling and surgical implantation in osteochondral defects ex vivo. Furthermore, the MEW meshes endowed the chondral compartment with compressive properties approaching those of native cartilage (20-fold reinforcement versus pristine hydrogel). The osteal and chondral compartment supported osteogenesis and cartilage matrix deposition in vitro, and the neo-synthesized cartilage matrix further contributed to the mechanical reinforcement at the ceramic-hydrogel interface. This multi-material, multi-scale 3D printing approach provides a promising strategy for engineering advanced composite constructs for the regeneration of musculoskeletal and connective tissue interfaces.

Bio-resin for high resolution lithography-based biofabrication of complex cell-laden constructs.

Lithography-based three-dimensional (3D) printing technologies allow high spatial resolution that exceeds that of typical extrusion-based bioprinting approaches, allowing to better mimic the complex architecture of biological tissues. Additionally, lithographic printing via digital light processing (DLP) enables fabrication of free-form lattice and patterned structures which cannot be easily produced with other 3D printing approaches. While significant progress has been dedicated to the development of cell-laden bioinks for extrusion-based bioprinting, less attention has been directed towards the development of cyto-compatible bio-resins and their application in lithography-based biofabrication, limiting the advancement of this promising technology. In this study, we developed a new bio-resin based on methacrylated poly(vinyl alcohol) (PVA-MA), gelatin-methacryloyl (Gel-MA) and a transition metal-based visible light photoinitiator. The utilization of a visible light photo-initiating system displaying high molar absorptivity allowed the bioprinting of constructs with high resolution features, in the range of 25-50 µm. Biofunctionalization of the resin with 1 wt% Gel-MA allowed long term survival (>90%) of encapsulated cells up to 21 d, and enabled attachment and spreading of endothelial cells seeded on the printed hydrogels. Cell-laden hydrogel constructs of high resolution with complex and ordered architecture were successfully bioprinted, where the encapsulated cells remained viable, homogenously distributed and functional. Bone and cartilage tissue synthesis was confirmed by encapsulated stem cells, underlining the potential of these DLP-bioprinted hydrogels for tissue engineering and biofabrication. Overall, the PVA-MA/Gel-MA bio-resin is a promising material for biofabrication and provides important cues for the further development of lithography-based bioprinting of complex, free-form living tissue analogues.

The bio in the ink: cartilage regeneration with bioprintable hydrogels and articular cartilage-derived progenitor cells.

Cell-laden hydrogels are the primary building blocks for bioprinting, and, also termed bioinks, are the foundations for creating structures that can potentially recapitulate the architecture of articular cartilage. To be functional, hydrogel constructs need to unlock the regenerative capacity of encapsulated cells. The recent identification of multipotent articular cartilage-resident chondroprogenitor cells (ACPCs), which share important traits with adult stem cells, represents a new opportunity for cartilage regeneration. However, little is known about the suitability of ACPCs for tissue engineering, especially in combination with biomaterials. This study aimed to investigate the potential of ACPCs in hydrogels for cartilage regeneration and biofabrication, and to evaluate their ability for zone-specific matrix production. Gelatin methacryloyl (gelMA)-based hydrogels were used to culture ACPCs, bone marrow mesenchymal stromal cells (MSCs) and chondrocytes, and as bioinks for printing. Our data shows ACPCs outperformed chondrocytes in terms of neo-cartilage production and unlike MSCs, ACPCs had the lowest gene expression levels of hypertrophy marker collagen type X, and the highest expression of PRG4, a key factor in joint lubrication. Co-cultures of the cell types in multi-compartment hydrogels allowed generating constructs with a layered distribution of collagens and glycosaminoglycans. By combining ACPC- and MSC-laden bioinks, a bioprinted model of articular cartilage was generated, consisting of defined superficial and deep regions, each with distinct cellular and extracellular matrix composition. Taken together, these results provide important information for the use of ACPC-laden hydrogels in regenerative medicine, and pave the way to the biofabrication of 3D constructs with multiple cell types for cartilage regeneration or in vitro tissue models. STATEMENT OF SIGNIFICANCE: Despite its limited ability to repair, articular cartilage harbors an endogenous population of progenitor cells (ACPCs), that to date, received limited attention in biomaterials and tissue engineering applications. Harnessing the potential of these cells in 3D hydrogels can open new avenues for biomaterial-based regenerative therapies, especially with advanced biofabrication technologies (e.g. bioprinting). This study highlights the potential of ACPCs to generate neo-cartilage in a gelatin-based hydrogel and bioink. The ACPC-laden hydrogel is a suitable substrate for chondrogenesis and data shows it has a bias in directing cells towards a superficial zone phenotype. For the first time, ACPC-hydrogels are evaluated both as alternative for and in combination with chondrocytes and MSCs, using co-cultures and bioprinting for cartilage regeneration in vitro. This study provides important cues on ACPCs, indicating they represent a promising cell source for the next generation of cartilage constructs with increased biomimicry.

Double printing of hyaluronic acid/poly(glycidol) hybrid hydrogels with poly(ε-caprolactone) for MSC chondrogenesis.

This study investigates the use of allyl-functionalized poly(glycidol)s (P(AGE-co-G)) as a cytocompatible cross-linker for thiol-functionalized hyaluronic acid (HA-SH) and the optimization of this hybrid hydrogel as bioink for 3D bioprinting. The chemical cross-linking of gels with 10 wt.% overall polymer concentration was achieved by a UV-induced radical thiol-ene coupling between the thiol and allyl groups. The addition of unmodified high molecular weight HA (1.36 MDa) enabled the rheology to be tuned for extrusion-based bioprinting. The incorporation of additional HA resulted in hydrogels with a lower Young's modulus and a higher swelling ratio, especially in the first 24 h, but a comparable equilibrium swelling for all gels after 24 h. Embedding of human and equine mesenchymal stem cells (MSCs) in the gels and subsequent in vitro culture showed promising chondrogenic differentiation after 21 d for cells from both origins. Moreover, cells could be printed with these gels, and embedded hMSCs showed good cell survival for at least 21 d in culture. To achieve mechanically stable and robust constructs for the envisioned application in articular cartilage, the formulations were adjusted for double printing with thermoplastic poly(ε-caprolactone) (PCL).

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.

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.

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.

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.

Preparation of biodegradable polylactide microparticles via a biocompatible procedure.

PLA MPs are prepared via a novel and toxic-chemical-free fabrication route using ethyl lactate, a green solvent and FDA-approved aroma. MPs are obtained by a solution jet break-up and solvent displacement method. Adjusting flow parameters allows the tuning of MPs size between 60 and 180 µm, with reduced polydispersity. Morphological analysis shows microporous particles with Janus-like surface. A fluorophore is successfully loaded into the MPs during their formation step. This versatile green solvent-based procedure is proven to be suitable for drug encapsulation and delivery applications. The method may be extended to different droplet generation techniques.