Novel BODIPY-Based Photobase Generators for Photoinduced Polymerization.

Photobase generators (PBGs) are compounds that utilize light-sensitive chemical-protecting groups to offer spatiotemporal control of releasing organic bases upon targeted light irradiation. PBGs can be implemented as an external control to initiate anionic polymerizations such as thiol-ene Michael addition reactions. However, there are limitations for common PBGs, including a short absorption wavelength and weak base release that lead to poor efficiency in photopolymerization. Therefore, there is a great need for visible-light-triggered PBGs that are capable of releasing strong bases efficiently. Here, we report two novel BODIPY-based visible-light-sensitive PBGs for light-induced activation of the thiol-ene Michael "click" reaction and polymerization. These PBGs were designed by connecting the BODIPY-based light-sensitive protecting group with tetramethylguanidine (TMG), a strong base. Moreover, we exploited the heavy atom effect to increase the efficiency of releasing TMG and the polymerization rate. These BODIPY-based PBGs exhibit extraordinary activity toward thiol-ene Michael addition-based polymerization, and they can be used in surface coating and polymer network formation of different thiol and vinyl monomers.

Elastomeric platform with surface wrinkling patterns to control cardiac cell alignment.

There is a growing interest in creating 2D cardiac tissue models that display native extracellular matrix (ECM) cues of the heart tissue. Cellular alignment alone is known to be a crucial cue for cardiac tissue development by regulating cell-cell and cell-ECM interactions. In this study, we report a simple and robust approach to create lamellar surface wrinkling patterns enabling spatial control of pattern dimensions with a wide range of pattern amplitude (A ≈ 2-55 µm) and wavelength (λ ≈ 35-100 µm). For human cardiomyocytes (hCMs) and human cardiac fibroblasts (hCFs), our results indicate that the degree of cellular alignment and pattern recognition are correlated with pattern A and λ. We also demonstrate fabrication of devices composed of micro-well arrays with user-defined lamellar patterns on the bottom surface of each well for high-throughput screening studies. Results from a screening study indicate that cellular alignment is strongly diminished with increasing seeding density. In another study, we show our ability to vary hCM/hCF seeding ratio for each well to create co-culture systems where seeding ratio is independent of cellular alignment.

Dose Titration of Solid Dosage Forms via FDM 3D-Printed Mini-Tablets.

The robustness of 3D-printed mini-tablets as a platform to administer milligram dosages, intended for age-specific therapy, without the need of tablet splitting while maintaining similar release profiles, was investigated. Griseofulvin, as a model poorly water-soluble drug, and hydroxypropyl cellulose along with Kollicoat Protect as polymers were used to prepare filaments at 1-20% drug concentrations via hot-melt extrusion (HME). Higher drug concentrations served for testing the feasibility of a reduced number of mini-tablets to be administered. A reliable dose titration in the range 0.19-3.91 mg at a high accuracy (R2 of 0.999) was achieved through composite unit (multi-unit) mini-tablets. All mini-tablets produced had excellent content uniformity and their label claim values were within the acceptable range, proving that HME processing followed by 3D printing promotes content uniformity even for mini-tablets containing low drug doses (0.19 mg). Remarkably, the proposed approach allowed achieving similar drug release profiles via composite unit mini-tablets as well as single mini-tablets at high drug concentrations. In contrast, split tablets demonstrated different release behaviors, attributed to their size and shape differences. Overall, the distinct advantages of mini-tablets to provide dose flexibility while maintaining similar release profiles was demonstrated.

Cell-Laden Composite Hydrogel Bioinks with Human Bone Allograft Particles to Enhance Stem Cell Osteogenesis.

There is a growing demand for bone graft substitutes that mimic the extracellular matrix properties of the native bone tissue to enhance stem cell osteogenesis. Composite hydrogels containing human bone allograft particles are particularly interesting due to inherent bioactivity of the allograft tissue. Here, we report a novel photocurable composite hydrogel bioink for bone tissue engineering. Our composite bioink is formulated by incorporating human allograft bone particles in a methacrylated alginate formulation to enhance adult human mesenchymal stem cell (hMSC) osteogenesis. Detailed rheology and printability studies confirm suitability of our composite bioinks for extrusion-based 3D bioprinting technology. In vitro studies reveal high cell viability (~90%) for hMSCs up to 28 days of culture within 3D bioprinted composite scaffolds. When cultured within bioprinted composite scaffolds, hMSCs show significantly enhanced osteogenic differentiation as compared to neat scaffolds based on alkaline phosphatase activity, calcium deposition, and osteocalcin expression.

A new mechanism of fibronectin fibril assembly revealed by live imaging and super-resolution microscopy.

Fibronectin (Fn1) fibrils have long been viewed as continuous fibers composed of extended, periodically aligned Fn1 molecules. However, our live-imaging and single-molecule localization microscopy data are inconsistent with this traditional view and show that Fn1 fibrils are composed of roughly spherical nanodomains containing six to eleven Fn1 dimers. As they move toward the cell center, Fn1 nanodomains become organized into linear arrays, in which nanodomains are spaced with an average periodicity of 105±17 nm. Periodical Fn1 nanodomain arrays can be visualized between cells in culture and within tissues; they are resistant to deoxycholate treatment and retain nanodomain periodicity in the absence of cells. The nanodomain periodicity in fibrils remained constant when probed with antibodies recognizing distinct Fn1 epitopes or combinations of antibodies recognizing epitopes spanning the length of Fn1. Treatment with FUD, a peptide that binds the Fn1 N-terminus and disrupts Fn1 fibrillogenesis, blocked the organization of Fn1 nanodomains into periodical arrays. These studies establish a new paradigm of Fn1 fibrillogenesis. This article has an associated First Person interview with the first author of the paper.

Interlayer bonding strength of 3D printed PEEK specimens.

Recent advances in extrusion-based filament 3D printing technology enable the processability of high-performance polymers. Poly(ether ether ketone) (PEEK) is an important group of high-performance polymer that has been widely used in aerospace, automotive, and biomedical applications. The interlayer bonding strength of 3D printed PEEK is crucial for load-bearing applications, yet studies on 3D printed PEEK are sparse due to processing challenges. In this study, the three-point flexural test is used to study the interlayer bonding strength of 3D-printed PEEK specimens with respect to the printing process parameters, including nozzle temperature, print speed, layer height, and wait-time. A design of experiment (DOE) approach is developed to study correlations between printing parameters and the end-use properties, including flexural stress (σf) and strain at break (εf), flexural modulus (Ef), and crystallinity (χ). Our results show that the nozzle temperature, layer height, and wait-time significantly affect the interlayer bonding strength, with nozzle temperature being the most influential parameter to enhance interlayer bonding strength indicated by a significant increase in σf, εf, and χ. Thermal annealing post-printing is shown to increase the degree of χ and Ef, yet its effect on interlayer bonding strength is minimal, indicating that the interlayer bonding strength is primarily determined during the printing process. This study demonstrates the use of a three-point flexural test integrated with a versatile and robust DOE approach to study the interlayer bonding strength of PEEK to reduce product development time while improving mechanical properties.

Complex 3D bioprinting methods.

3D bioprinting technology is evolving in complexity to enable human-scale, high-resolution, and multi-cellular constructs to better mimic the native tissue microenvironment. The ultimate goal is to achieve necessary complexity in the bioprinting process to biomanufacture fully-functional tissues and organs to address organ shortage and lack of patient-specific disease models. In this Review, we presented an in-depth overview of complex 3D bioprinting approaches including evolution of complex bioprinting, from simple gel-casting approach to multi-material bioprinting to omnidirectional bioprinting approaches, and emerging bioprinting approaches, including 4D bioprinting and in situ bioprinting technologies.

Designing Biomaterial Platforms for Cardiac Tissue and Disease Modeling.

Heart disease is one of the leading causes of death in the world. There is a growing demand for in vitro cardiac models that can recapitulate the complex physiology of the cardiac tissue. These cardiac models can provide a platform to better understand the underlying mechanisms of cardiac development and disease and aid in developing novel treatment alternatives and platforms towards personalized medicine. In this review, a summary of engineered cardiac platforms is presented. Basic design considerations for replicating the heart's microenvironment are discussed considering the anatomy of the heart. This is followed by a detailed summary of the currently available biomaterial platforms for modeling the heart tissue in vitro. These in vitro models include 2D surface modified structures, 3D molded structures, porous scaffolds, electrospun scaffolds, bioprinted structures, and heart-on-a-chip devices. The challenges faced by current models and the future directions of in vitro cardiac models are also discussed. Engineered in vitro tissue models utilizing patients' own cells could potentially revolutionize the way we develop treatment and diagnostic alternatives.

A 3D Bioprinted Material That Recapitulates the Perivascular Bone Marrow Structure for Sustained Hematopoietic and Cancer Models.

Translational medicine requires facile experimental systems to replicate the dynamic biological systems of diseases. Drug approval continues to lag, partly due to incongruencies in the research pipeline that traditionally involve 2D models, which could be improved with 3D models. The bone marrow (BM) poses challenges to harvest as an intact organ, making it difficult to study disease processes such as breast cancer (BC) survival in BM, and to effective evaluation of drug response in BM. Furthermore, it is a challenge to develop 3D BM structures due to its weak physical properties, and complex hierarchical structure and cellular landscape. To address this, we leveraged 3D bioprinting to create a BM structure with varied methylcellulose (M): alginate (A) ratios. We selected hydrogels containing 4% (w/v) M and 2% (w/v) A, which recapitulates rheological and ultrastructural features of the BM while maintaining stability in culture. This hydrogel sustained the culture of two key primary BM microenvironmental cells found at the perivascular region, mesenchymal stem cells and endothelial cells. More importantly, the scaffold showed evidence of cell autonomous dedifferentiation of BC cells to cancer stem cell properties. This scaffold could be the platform to create BM models for various diseases and also for drug screening.

Additive Manufacturing of Oral Tablets: Technologies, Materials and Printed Tablets.

Additive manufacturing (AM), also known as three-dimensional (3D) printing, enables fabrication of custom-designed and personalized 3D constructs with high complexity in shape and composition. AM has a strong potential to fabricate oral tablets with enhanced customization and complexity as compared to tablets manufactured using conventional approaches. Despite these advantages, AM has not yet become the mainstream manufacturing approach for fabrication of oral solid dosage forms mainly due to limitations of AM technologies and lack of diverse printable drug formulations. In this review, AM of oral tablets are summarized with respect to AM technology. A detailed review of AM methods and materials used for the AM of oral tablets is presented. This article also reviews the challenges in AM of pharmaceutical formulations and potential strategies to overcome these challenges.

Designing Decellularized Extracellular Matrix-Based Bioinks for 3D Bioprinting.

3D bioprinting is an emerging technology to fabricate tissues and organs by precisely positioning cells into 3D structures using printable cell-laden formulations known as bioinks. Various bioinks are utilized in 3D bioprinting applications; however, developing the perfect bioink to fabricate constructs with biomimetic microenvironment and mechanical properties that are similar to native tissues is a challenging task. In recent years, decellularized extracellular matrix (dECM)-based bioinks have received an increasing attention in 3D bioprinting applications, since they are derived from native tissues and possess unique, complex tissue-specific biochemical properties. This review focuses on designing dECM-based bioinks for tissue and organ bioprinting, including commonly used decellularization and decellularized tissue characterization methods, bioink formulation and characterization, applications of dECM-based bioinks, and most recent advancements in dECM-based bioink design.

Editorial for the Special Issue on 3D Printing for Tissue Engineering and Regenerative Medicine.

Three-dimensional (3D) bioprinting uses additive manufacturing techniques to fabricate 3Dstructures consisting of heterogenous selections of living cells, biomaterials, and active biomolecules[1,2] [...].

3D Printed Wavy Scaffolds Enhance Mesenchymal Stem Cell Osteogenesis.

There is a growing interest in developing 3D porous scaffolds with tunable architectures for bone tissue engineering. Surface topography has been shown to control stem cell behavior including differentiation. In this study, we printed 3D porous scaffolds with wavy or linear patterns to investigate the effect of wavy scaffold architecture on human mesenchymal stem cell (hMSC) osteogenesis. Five distinct wavy scaffolds were designed using sinusoidal waveforms with varying wavelengths and amplitudes, and orthogonal scaffolds were designed using linear patterns. We found that hMSCs attached to wavy patterns, spread by taking the shape of the curvatures presented by the wavy patterns, exhibited an elongated shape and mature focal adhesion points, and differentiated into the osteogenic lineage. When compared to orthogonal scaffolds, hMSCs on wavy scaffolds showed significantly enhanced osteogenesis, indicated by higher calcium deposition, alkaline phosphatase activity, and osteocalcin staining. This study aids in the development of 3D scaffolds with novel architectures to direct stem osteogenesis for bone tissue engineering.

3D bioprinting of complex channels within cell-laden hydrogels.

3D bioprinting is an emerging manufacturing approach to fabricate (cell-laden) hydrogel constructs with embedded microchannels, which are potentially useful for fundamental studies to understand vascularization and angiogenesis, and for developing organ-on-a-chip devices for disease modeling. Although numerous printing approaches have been developed, novel approaches are still needed that enable printing of channels with user-defined and tunable size, morphology, and complexity. Here, we report a novel bioprinting approach enabling printing of a sacrificial ink within commonly used photocurable hydrogels using a sequential printing approach. To achieve this, photocurable hydrogel is printed layer-by-layer as usual, but each layer is exposed to light briefly (seconds) to create partially crosslinked, self-supporting layers. At a desired thickness, immediately after the layer is printed (prior to partial crosslinking step), sacrificial hydrogel is directly printed within this viscous uncrosslinked layer. The layer was then exposed to light to confine and support the sacrificial hydrogel. After fully crosslinking the system, the sacrificial hydrogel is washed away, forming a channel. This approach allows bioprinting of cells with the matrix material and seeding of cells into channels after the sacrificial ink is removed. This approach can potentially provide a robust platform for fabricating vascularized tissues and studying cell behaviors on diverse channel surfaces. STATEMENT OF SIGNIFICANCE: 3D bioprinting is an emerging platform for the fabrication of hydrogel-based constructs for in vitro tissue/disease modelling or tissue and organ printing. Although several approaches have been developed to print channels within these constructs, it is still challenging to incorporate microchannels (for vascularization) within 3D bioprinted constructs. This study presents a novel bioprinting approach to create user-defined and tunable channels embedded within cell-laden hydrogel constructs. We report an important advance as our approach does not require complex device modifications for bioprinters or complex synthesis and processing hurdles for the inks. Since our approach does not require special chemistries, there are potentially a greater number of commercially available options for ink materials.

Polyester-based ink platform with tunable bioactivity for 3D printing of tissue engineering scaffolds.

In this work, we synthesized a novel polymeric biomaterial platform with tunable functionalizability for extrusion-based 3D printing. Biodegradable polymers were synthesized using 4-hydroxyphenethyl 2-(4-hydroxyphenyl)acetate (HTy), which is derived from Tyrosol and 2-(4-hydroxyphenyl)acetic acid. p-Phenylenediacetic acid (PDA) was introduced to enhance crystallinity. To enable functionalizability without deteriorating printability, glutamic acid derivatives were introduced into the polymer design, forming copolymers including poly(HTy-co-45%PDA-co-5%Gluhexenamide ester) (HP5GH), poly(HTy-co-45%PDA-co-5%Glupentynamide ester) (HP5GP), and poly(HTy-co-45%PDA-co-5%BocGlu ester) (HP5BG). The resulting polymers have: two melting temperatures (125-131 °C and 141-147 °C), Young's moduli of 1.9-2.4 GPa, and print temperatures of 170-190 °C. The molecular weight (Mw) loss due to hydrolytic degradation was gradual with ∼30% Mw retained after 25 weeks for HP5BG, whereas it was much faster for HP5GP and HP5GH with only 18% Mw retained after 8 weeks. HP5GH and HP5GP were successfully functionalized in solution (bulk) or on the surface using click-based chemistry. Finally, the utilization of this novel platform was demonstrated by studying osteogenic differentiation of human mesenchymal stem cells (hMSCs) using 3D printed scaffolds from HP5GP. Scaffolds were functionalized with azide-Heparin (az-Heparin) to bind and deliver bone morphogenetic protein 2 (BMP-2). This sample group significantly enhanced osteogenic differentiation of hMSCs as compared to unfunctionalized scaffolds incubated directly with az-Heparin or BMP-2 prior to cell culture. This novel polymer platform with tunable functionalizability could be utilized for additive manufacturing of biodegradable devices and scaffolds with tailored mechanical and bioactive properties for a wide range of medical applications including bone fixation devices and scaffolds for bone regeneration.

Engineering 3D Hydrogels for Personalized In Vitro Human Tissue Models.

There is a growing interest in engineering hydrogels for 3D tissue and disease models. The major motivation is to better mimic the physiological microenvironment of the disease and human condition. 3D tissue models derived from patients' own cells can potentially revolutionize the way treatment and diagnostic alternatives are developed. This requires development of tissue mimetic hydrogels with user defined and tunable properties. In this review article, a recent summary of 3D hydrogel platforms for in vitro tissue and disease modeling is given. Hydrogel design considerations and available hydrogel systems are summarized, followed by the types of currently available hydrogel models, such as bulk hydrogels, porous scaffolds, fibrous scaffolds, hydrogel microspheres, hydrogel sandwich systems, microwells, and 3D bioprinted constructs. Although hydrogels are utilized for a wide range of tissue models, this article focuses on liver and cancer models. This article also provides a detailed section on current challenges and future perspectives of hydrogel-based tissue models.

Functionally-Relevant Morphological Profiling: A Tool to Assess Cellular Heterogeneity.

Heterogeneity in cell function has presented a significant hurdle to the successful clinical translation of many cellular therapies. Current techniques for assessing cell quality and the effects of microenvironmental cues and manufacturing processes on cell behavior often inadequately address heterogeneity due to issues such as population versus single-cell measurements and the therapeutic relevance and throughput/robustness of the assay. Due to the well-established relationship between morphology and cellular function, morphological profiling has become increasingly utilized to better understand functional heterogeneity and its impact on therapeutic development. In this review, we introduce an emerging field we term functionally-relevant morphological profiling with great potential to improve our understanding of cellular heterogeneity through discovering novel quality attributes, optimizing manufacturing, and screening drugs/biomaterials.

Current and emerging applications of 3D printing in medicine.

Three-dimensional (3D) printing enables the production of anatomically matched and patient-specific devices and constructs with high tunability and complexity. It also allows on-demand fabrication with high productivity in a cost-effective manner. As a result, 3D printing has become a leading manufacturing technique in healthcare and medicine for a wide range of applications including dentistry, tissue engineering and regenerative medicine, engineered tissue models, medical devices, anatomical models and drug formulation. Today, 3D printing is widely adopted by the healthcare industry and academia. It provides commercially available medical products and a platform for emerging research areas including tissue and organ printing. In this review, our goal is to discuss the current and emerging applications of 3D printing in medicine. A brief summary on additive manufacturing technologies and available printable materials is also given. The technological and regulatory barriers that are slowing down the full implementation of 3D printing in the medical field are also discussed.

Recent Advances in Bioink Design for 3D Bioprinting of Tissues and Organs.

There is a growing demand for alternative fabrication approaches to develop tissues and organs as conventional techniques are not capable of fabricating constructs with required structural, mechanical, and biological complexity. 3D bioprinting offers great potential to fabricate highly complex constructs with precise control of structure, mechanics, and biological matter [i.e., cells and extracellular matrix (ECM) components]. 3D bioprinting is an additive manufacturing approach that utilizes a "bioink" to fabricate devices and scaffolds in a layer-by-layer manner. 3D bioprinting allows printing of a cell suspension into a tissue construct with or without a scaffold support. The most common bioinks are cell-laden hydrogels, decellulerized ECM-based solutions, and cell suspensions. In this mini review, a brief description and comparison of the bioprinting methods, including extrusion-based, droplet-based, and laser-based bioprinting, with particular focus on bioink design requirements are presented. We also present the current state of the art in bioink design including the challenges and future directions.