Methacrylate-Modified Gold Nanoparticles Enable Non-Invasive Monitoring of Photocrosslinked Hydrogel Scaffolds.

Photocrosslinked hydrogels, such as methacrylate-modified gelatin (gelMA) and hyaluronic acid (HAMA), are widely utilized as tissue engineering scaffolds and/or drug delivery vehicles, but lack a suitable means for non-invasive, longitudinal monitoring of surgical placement, biodegradation, and drug release. Therefore, we developed a novel photopolymerizable X-ray contrast agent, methacrylate-modified gold nanoparticles (AuMA NPs), to enable covalent-linking to methacrylate-modified hydrogels (gelMA and HAMA) in one-step during photocrosslinking and non-invasive monitoring by X-ray micro-computed tomography (micro-CT). Hydrogels exhibited a linear increase in X-ray attenuation with increased Au NP concentration to enable quantitative imaging by contrast-enhanced micro-CT. The enzymatic and hydrolytic degradation kinetics of gelMA-Au NP hydrogels were longitudinally monitored by micro-CT for up to one month in vitro, yielding results that were consistent with concurrent measurements by optical spectroscopy and gravimetric analysis. Importantly, AuMA NPs did not disrupt the hydrogel network, rheology, mechanical properties, and hydrolytic stability compared with gelMA alone. GelMA-Au NP hydrogels were thus able to be bioprinted into well-defined three-dimensional architectures supporting endothelial cell viability and growth. Overall, AuMA NPs enabled the preparation of both conventional photopolymerized hydrogels and bioprinted scaffolds with tunable X-ray contrast for noninvasive, longitudinal monitoring of placement, degradation, and NP release by micro-CT.

3D bioprinting of nanoparticle-laden hydrogel scaffolds with enhanced antibacterial and imaging properties.

Biomaterial-associated microbial contaminations in biologically conducive three-dimensional (3D) tissue-engineered constructs have significantly limited the clinical applications of scaffold systems. To prevent such infections, antimicrobial biomaterials are rapidly evolving. Yet, the use of such materials in bioprinting-based approaches of scaffold fabrication has not been examined. This study introduces a new generation of bacteriostatic gelatin methacryloyl (GelMA)-based bioinks, incorporated with varying doses of antibacterial superparamagnetic iron oxide nanoparticles (SPIONs). The SPION-laden GelMA scaffolds showed significant resistance against the Staphylococcus aureus growth, while providing a contrast in magnetic resonance imaging. We simulated the bacterial contamination of cellular 3D GelMA scaffolds in vitro and demonstrated the significant effect of functionalized scaffolds in inhibiting bacterial growth, while maintaining cell viability and growth. Together, these results present a new promising class of functionalized bioinks to 3D bioprint tissue-engineered scaffold with markedly enhanced properties for the use in a variety of in vitro and clinical applications.

Tissue engineered drug delivery vehicles: Methods to monitor and regulate the release behavior.

Tissue engineering is a rapidly evolving, multidisciplinary field that aims at generating or regenerating 3D functional tissues for in vitro disease modeling and drug screening applications or for in vivo therapies. A variety of advanced biological and engineering methods are increasingly being used to further enhance and customize the functionality of tissue engineered scaffolds. To this end, tunable drug delivery and release mechanisms are incorporated into tissue engineering modalities to promote different therapeutic processes, thus, addressing challenges faced in the clinical applications. In this review, we elaborate the mechanisms and recent developments in different drug delivery vehicles, including the quantum dots, nano/micro particles, and molecular agents. Different loading strategies to incorporate the therapeutic reagents into the scaffolding structures are explored. Further, we discuss the main mechanisms to tune and monitor/quantify the release kinetics of embedded drugs from engineered scaffolds. We also survey the current trend of drug delivery using stimuli driven biopolymer scaffolds to enable precise spatiotemporal control of the release behavior. Recent advancements, challenges facing current scaffold-based drug delivery approaches, and areas of future research are discussed.

A 3D Bioprinted In Vitro Model of Pulmonary Artery Atresia to Evaluate Endothelial Cell Response to Microenvironment.

Vascular atresia are often treated via transcatheter recanalization or surgical vascular anastomosis due to congenital malformations or coronary occlusions. The cellular response to vascular anastomosis or recanalization is, however, largely unknown and current techniques rely on restoration rather than optimization of flow into the atretic arteries. An improved understanding of cellular response post anastomosis may result in reduced restenosis. Here, an in vitro platform is used to model anastomosis in pulmonary arteries (PAs) and for procedural planning to reduce vascular restenosis. Bifurcated PAs are bioprinted within 3D hydrogel constructs to simulate a reestablished intervascular connection. The PA models are seeded with human endothelial cells and perfused at physiological flow rate to form endothelium. Particle image velocimetry and computational fluid dynamics modeling show close agreement in quantifying flow velocity and wall shear stress within the bioprinted arteries. These data are used to identify regions with greatest levels of shear stress alterations, prone to stenosis. Vascular geometry and flow hemodynamics significantly affect endothelial cell viability, proliferation, alignment, microcapillary formation, and metabolic bioprofiles. These integrated in vitro-in silico methods establish a unique platform to study complex cardiovascular diseases and can lead to direct clinical improvements in surgical planning for diseases of disturbed flow.

Nanomaterials for bioprinting: functionalization of tissue-specific bioinks.

Three-dimensional (3D) bioprinting is rapidly evolving, offering great potential for manufacturing functional tissue analogs for use in diverse biomedical applications, including regenerative medicine, drug delivery, and disease modeling. Biomaterials used as bioinks in printing processes must meet strict physiochemical and biomechanical requirements to ensure adequate printing fidelity, while closely mimicking the characteristics of the native tissue. To achieve this goal, nanomaterials are increasingly being investigated as a robust tool to functionalize bioink materials. In this review, we discuss the growing role of different nano-biomaterials in engineering functional bioinks for a variety of tissue engineering applications. The development and commercialization of these nanomaterial solutions for 3D bioprinting would be a significant step towards clinical translation of biofabrication.

Restoring Endogenous Repair Mechanisms to Heal Chronic Wounds with a Multifunctional Wound Dressing.

Current treatment of chronic wounds has been critically limited by various factors, including bacterial infection, biofilm formation, impaired angiogenesis, and prolonged inflammation. Addressing these challenges, we developed a multifunctional wound dressing-based three-pronged approach for accelerating wound healing. The multifunctional wound dressing, composed of nanofibers, functional nanoparticles, natural biopolymers, and selected protein and peptide, can target multiple endogenous repair mechanisms and represents a promising alternative to current wound healing products.

3D Bioprinted Bacteriostatic Hyperelastic Bone Scaffold for Damage-Specific Bone Regeneration.

Current strategies for regeneration of large bone fractures yield limited clinical success mainly due to poor integration and healing. Multidisciplinary approaches in design and development of functional tissue engineered scaffolds are required to overcome these translational challenges. Here, a new generation of hyperelastic bone (HB) implants, loaded with superparamagnetic iron oxide nanoparticles (SPIONs), are 3D bioprinted and their regenerative effect on large non-healing bone fractures is studied. Scaffolds are bioprinted with the geometry that closely correspond to that of the bone defect, using an osteoconductive, highly elastic, surgically friendly bioink mainly composed of hydroxyapatite. Incorporation of SPIONs into HB bioink results in enhanced bacteriostatic properties of bone grafts while exhibiting no cytotoxicity. In vitro culture of mouse embryonic cells and human osteoblast-like cells remain viable and functional up to 14 days on printed HB scaffolds. Implantation of damage-specific bioprinted constructs into a rat model of femoral bone defect demonstrates significant regenerative effect over the 2-week time course. While no infection, immune rejection, or fibrotic encapsulation is observed, HB grafts show rapid integration with host tissue, ossification, and growth of new bone. These results suggest a great translational potential for 3D bioprinted HB scaffolds, laden with functional nanoparticles, for hard tissue engineering applications.

Patient-Specific 3D Bioprinted Models of Developing Human Heart.

The heart is the first organ to develop in the human embryo through a series of complex chronological processes, many of which critically rely on the interplay between cells and the dynamic microenvironment. Tight spatiotemporal regulation of these interactions is key in heart development and diseases. Due to suboptimal experimental models, however, little is known about the role of microenvironmental cues in the heart development. This study investigates the use of 3D bioprinting and perfusion bioreactor technologies to create bioartificial constructs that can serve as high-fidelity models of the developing human heart. Bioprinted hydrogel-based, anatomically accurate models of the human embryonic heart tube (e-HT, day 22) and fetal left ventricle (f-LV, week 33) are perfused and analyzed both computationally and experimentally using ultrasound and magnetic resonance imaging. Results demonstrate comparable flow hemodynamic patterns within the 3D space. We demonstrate endothelial cell growth and function within the bioprinted e-HT and f-LV constructs, which varied significantly in varying cardiac geometries and flow. This study introduces the first generation of anatomically accurate, 3D functional models of developing human heart. This platform enables precise tuning of microenvironmental factors, such as flow and geometry, thus allowing the study of normal developmental processes and underlying diseases.

Bioprintability: Physiomechanical and Biological Requirements of Materials for 3D Bioprinting Processes.

Three-dimensional (3D) bioprinting is an additive manufacturing process that utilizes various biomaterials that either contain or interact with living cells and biological systems with the goal of fabricating functional tissue or organ mimics, which will be referred to as bioinks. These bioinks are typically hydrogel-based hybrid systems with many specific features and requirements. The characterizing and fine tuning of bioink properties before, during, and after printing are therefore essential in developing reproducible and stable bioprinted constructs. To date, myriad computational methods, mechanical testing, and rheological evaluations have been used to predict, measure, and optimize bioinks properties and their printability, but none are properly standardized. There is a lack of robust universal guidelines in the field for the evaluation and quantification of bioprintability. In this review, we introduced the concept of bioprintability and discussed the significant roles of various physiomechanical and biological processes in bioprinting fidelity. Furthermore, different quantitative and qualitative methodologies used to assess bioprintability will be reviewed, with a focus on the processes related to pre, during, and post printing. Establishing fully characterized, functional bioink solutions would be a big step towards the effective clinical applications of bioprinted products.

Embedded 3D Bioprinting of Gelatin Methacryloyl-Based Constructs with Highly Tunable Structural Fidelity.

Three-dimensional (3D) bioprinting of hydrogel-based constructs at adequate consistency and reproducibility can be obtained through a compromise between the hydrogel's inherent instability and printing fidelity. There is an increasing demand to develop bioprinting modalities that enable high-fidelity fabrication of 3D hydrogel structures that closely correspond to the envisioned design. In this work, we performed a systematic, in-depth characterization and optimization of embedded 3D bioprinting to create 3D gelatin-methacryloyl (gelMA) structures with highly controlled fidelity using Carbopol as suspension bath. The role of various embedded printing process parameters in bioprinting fidelity was investigated using a combination of experimental and theoretical approaches. We examined the effect of rheological properties of gelMA and Carbopol at varying concentrations, as well as printing conditions on the volumetric flow rate of gelMA bioink. Printing speed was examined and optimized to successfully print gelMA into the support bath at varying Carbopol concentrations. Printing fidelity was characterized in terms of printed strand diameter, uniformity, angle, and area. The optimal Carbopol solution that retained filament shape at highest fidelity was determined. The efficacy of developed bioprinting approach was then demonstrated by fabricating 3D hydrogel constructs with varying geometries and visualized using an advanced synchrotron-based imaging technique. We also investigated the influence of the Carbopol medium on cross-linking and the resulting stiffness of gelMA constructs. Finally, in vitro cytotoxicity of the developed bioprinting approach was assessed by printing human umbilical vein endothelial cells encapsulated in the gelMA bioink. These results demonstrate the significance of the close interplay between bioink-support bath rheology and printing parameters and help to establish an optimized workflow for creating 3D hydrogel structures with high fidelity and cytocompatibility via embedded bioprinting techniques. This robust platform could further expand the application of bioprinted soft tissue constructs in a wide variety of biomedical applications.

Biomechanical factors in three-dimensional tissue bioprinting.

3D bioprinting techniques have shown great promise in various fields of tissue engineering and regenerative medicine. Yet, creating a tissue construct that faithfully represents the tightly regulated composition, microenvironment, and function of native tissues is still challenging. Among various factors, biomechanics of bioprinting processes play fundamental roles in determining the ultimate outcome of manufactured constructs. This review provides a comprehensive and detailed overview on various biomechanical factors involved in tissue bioprinting, including those involved in pre, during, and post printing procedures. In preprinting processes, factors including viscosity, osmotic pressure, and injectability are reviewed and their influence on cell behavior during the bioink preparation is discussed, providing a basic guidance for the selection and optimization of bioinks. In during bioprinting processes, we review the key characteristics that determine the success of tissue manufacturing, including the rheological properties and surface tension of the bioink, printing flow rate control, process-induced mechanical forces, and the in situ cross-linking mechanisms. Advanced bioprinting techniques, including embedded and multi-material printing, are explored. For post printing steps, general techniques and equipment that are used for characterizing the biomechanical properties of printed tissue constructs are reviewed. Furthermore, the biomechanical interactions between printed constructs and various tissue/cell types are elaborated for both in vitro and in vivo applications. The review is concluded with an outlook regarding the significance of biomechanical processes in tissue bioprinting, presenting future directions to address some of the key challenges faced by the bioprinting community.

Engineering Functional Cardiac Tissues for Regenerative Medicine Applications.

PURPOSE OF REVIEW: Tissue engineering has expanded into a highly versatile manufacturing landscape that holds great promise for advancing cardiovascular regenerative medicine. In this review, we provide a summary of the current state-of-the-art bioengineering technologies used to create functional cardiac tissues for a variety of applications in vitro and in vivo. RECENT FINDINGS: Studies over the past few years have made a strong case that tissue engineering is one of the major driving forces behind the accelerating fields of patient-specific regenerative medicine, precision medicine, compound screening, and disease modeling. To date, a variety of approaches have been used to bioengineer functional cardiac constructs, including biomaterial-based, cell-based, and hybrid (using cells and biomaterials) approaches. While some major progress has been made using cellular approaches, with multiple ongoing clinical trials, cell-free cardiac tissue engineering approaches have also accomplished multiple breakthroughs, although drawbacks remain. This review summarizes the most promising methods that have been employed to generate cardiovascular tissue constructs for basic science or clinical applications. Further, we outline the strengths and challenges that are inherent to this field as a whole and for each highlighted technology.

In Vivo Tracking of Tissue Engineered Constructs.

To date, the fields of biomaterials science and tissue engineering have shown great promise in creating bioartificial tissues and organs for use in a variety of regenerative medicine applications. With the emergence of new technologies such as additive biomanufacturing and 3D bioprinting, increasingly complex tissue constructs are being fabricated to fulfill the desired patient-specific requirements. Fundamental to the further advancement of this field is the design and development of imaging modalities that can enable visualization of the bioengineered constructs following implantation, at adequate spatial and temporal resolution and high penetration depths. These in vivo tracking techniques should introduce minimum toxicity, disruption, and destruction to treated tissues, while generating clinically relevant signal-to-noise ratios. This article reviews the imaging techniques that are currently being adopted in both research and clinical studies to track tissue engineering scaffolds in vivo, with special attention to 3D bioprinted tissue constructs.