Uncovering hidden treasures: Mapping morphological changes in the differentiation of human mesenchymal stem cells to osteoblasts using deep learning.

Deep Learning (DL) is becoming an increasingly popular technology being employed in life sciences research due to its ability to perform complex and time-consuming tasks with significantly greater speed, accuracy, and reproducibility than human researchers - allowing them to dedicate their time to more complex tasks. One potential application of DL is to analyze cell images taken by microscopes. Quantitative analysis of cell microscopy images remain a challenge - with manual cell characterization requiring excessive amounts of time and effort. DL can address these issues, by quickly extracting such data and enabling rigorous, empirical analysis of images. Here, DL is used to quantitively analyze images of Mesenchymal Stem Cells (MSCs) differentiating into Osteoblasts (OBs), tracking morphological changes throughout this transition. The changes in morphology throughout the differentiation protocol provide evidence for a distinct path of morphological transformations that the cells undergo in their transition, with changes in perimeter being observable before changes in eceentricity. Subsequent differentiation experiments can be quantitatively compared with our dataset to concretely evaluate how different conditions affect differentiation and this paper can also be used as a guide for researchers on how to utilize DL workflows in their own labs.

A strategy for enhancing bioactivity and osseointegration with antibacterial effect by incorporating magnesium in polylactic acid based biodegradable orthopedic implant.

Biodegradable orthopedic implants are essential for restoring the physiological structure and function of bone tissue while ensuring complete degradation after recovery. Polylactic acid (PLA), a biodegradable polymer, is considered a promising material due to its considerable mechanical properties and biocompatibility. However, further improvements are necessary to enhance the mechanical strength and bioactivity of PLA for reliable load-bearing orthopedic applications. In this study, a multifunctional PLA-based composite was fabricated by incorporating tricalcium phosphate (TCP) microspheres and magnesium (Mg) particles homogenously at a volume fraction of 40 %. This approach aims to enhance mechanical strength, accelerate pore generation, and improve biological and antibacterial performance. Mg content was incorporated into the composite at varying values of 1, 3, and 5 vol% (referred to as PLA/TCP-1 Mg, PLA/TCP-3 Mg, and PLA/TCP-5 Mg, respectively). The compressive strength and stiffness were significantly enhanced in all composites, reaching 87.7, 85.9, and 84.1 MPa, and 2.7, 3.0, and 3.1 GPa, respectively. The degradation test indicated faster elimination of the reinforcers as the Mg content increased, resulting in accelerated pore generation to induce enhanced osseointegration. Because PLA/TCP-3 Mg and PLA/TCP-5 Mg exhibited cracks in the PLA matrix due to rapid corrosion of Mg forming corrosion byproducts, to optimize the Mg particle content, PLA/TCP-1 Mg was selected for further evaluation. As determined by in vitro biological and antibacterial testing, PLA/TCP-1 Mg showed enhanced bioactivity with pre-osteoblast cells and exhibited antibacterial properties by suppressing bacterial colonization. Overall, the multifunctional PLA/TCP-Mg composite showed improved mechanobiological performance, making it a promising material for biodegradable orthopedic implants.

Nanoengineered injectable hydrogels derived from layered double hydroxides and alginate for sustained release of protein therapeutics in tissue engineering applications.

Chronic Kidney Disease (CKD) which involves gradual loss of kidney function is characterized by low levels of a glycoprotein called Erythropoietin (EPO) that leads to red blood cell  deficiency and anemia. Recombinant human EPO (rhEPO) injections that are administered intravenously or subcutaneously is the current gold standard for treating CKD. The rhEPO injections have very short half-lives and thus demands frequent administration with a risk of high endogenous EPO levels leading to severe side effects that could prove fatal. To this effect, this work provides a novel approach of using lamellar inorganic solids with a brucite-like structure for controlling the release of protein therapeutics such as rhEPO in injectable hydrogels. The nanoengineered injectable system was formulated by incorporating two-dimensional layered double hydroxide (LDH) clay materials with a high surface area into alginate hydrogels for sustained delivery. The inclusion of LDH in the hydrogel network not only improved the mechanical properties of the hydrogels (5-30 times that of alginate hydrogel) but also exhibited a high binding affinity to proteins without altering their bioactivity and conformation. Furthermore, the nanoengineered injectable hydrogels (INHs) demonstrated quick gelation, injectability, and excellent adhesion properties on human skin. The in vitro release test of EPO from conventional alginate hydrogels (Alg-Gel) showed 86% EPO release within 108 h while INHs showed greater control over the initial burst and released only 24% of EPO in the same incubation time. INH-based ink was successfully used for 3D printing, resulting in scaffolds with good shape fidelity and stability in cell culture media. Controlled release of EPO from INHs facilitated superior angiogenic potential in ovo (chick chorioallantoic membrane) compared to Alg-Gel. When subcutaneously implanted in albino mice, the INHs formed a stable gel in vivo without inducing any adverse effects. The results suggest that the proposed INHs in this study can be utilized as a minimally invasive injectable platform or as 3D printed patches for the delivery of protein therapeutics to facilitate tissue regeneration.

Bioprinting the future using light: A review on photocrosslinking reactions, photoreactive groups, and photoinitiators.

Light-based bioprinting is a type of additive manufacturing technologies that uses light to control the formation of biomaterials, tissues, and organs. It has the potential to revolutionize the adopted approach in tissue engineering and regenerative medicine by allowing the creation of functional tissues and organs with high precision and control. The main chemical components of light-based bioprinting are activated polymers and photoinitiators. The general photocrosslinking mechanisms of biomaterials are described, along with the selection of polymers, functional group modifications, and photoinitiators. For activated polymers, acrylate polymers are ubiquitous but are made of cytotoxic reagents. A milder option that exists is based on norbornyl groups which are biocompatible and can be used in self-polymerization or with thiol reagents for more precision. Polyethylene-glycol and gelatin activated with both methods can have high cell viability rates. Photoinitiators can be divided into types I and II. The best performances for type I photoinitiators are produced under ultraviolet light. Most alternatives for visible-light-driven photoinitiators were of type II, and changing the co-initiator along the main reagent can fine-tune the process. This field is still underexplored and a vast room for improvements still exist, which can open the way for cheaper complexes to be developed. The progress, advantages, and shortcomings of light-based bioprinting are highlighted in this review, with special emphasis on developments and future trends of activated polymers and photoinitiators.

Bioprinting of Human Neural Tissues Using a Sustainable Marine Tunicate-Derived Bioink for Translational Medicine Applications.

Bioprinting of nervous tissue is a major challenge in the bioprinting field due to its soft consistency and complex architecture. The first step in efficient neural bioprinting is the design and optimization of printable bioinks which favor the growth and differentiation of neural tissues by providing the mechanophysiological properties of the native tissue microenvironment. However, till date, limited studies have been conducted to make tissue specific bioinks. Here, we report a novel bioink formulation specifically designed for bioprinting and differentiation of neural stem cells (NSCs) to peripheral neurons, using a marine tunicate-derived hydrogel and Matrigel. The formulation resulted in seamless bioprinting of NSCs with minimal processing time from bioink preparation to in vitro culture. The tissues exhibited excellent post-printing viability and cell proliferation along with a precise peripheral nerve morphology on in vitro differentiation. The cultured tissues showed significant cell recovery after subjecting to a freeze-thaw cycle of -80 to 37°C, indicating the suitability of the method for developing tissues compatible for long-term storage and transportation for clinical use. The study provides a robust method to use a sustainable bioink for three-dimensional bioprinting of neural tissues for translational medicine applications.

Rapid and inexpensive process to fabricate paper based microfluidic devices using a cut and heat plastic lamination process.

Microfluidic paper-based analytical devices (microPADs) are emerging as simple-to-use, low-cost point-of-care testing platforms. Such devices are mostly fabricated at present by creating hydrophobic barriers using wax or photoresist patterning on porous paper sheets. Even though devices fabricated using these methods are used and tested with a wide variety of analytes, still they pose many serious practical limitations for low-cost automated mass fabrication for their widespread applicability. We present an affordable and simple two-step process - cut and heat (CH-microPADs) - for the selective fabrication of hydrophilic channels and reservoirs on a wide variety of porous media such as tissue/printing/filter paper and cloth types, such as cotton and polyester, by a lamination process. The technique presents many advantages as compared to existing commonly used methods. The devices possess excellent mechanical strength against bending, folding and twisting, making them virtually unbreakable. They are structurally flexible and show good chemical resistance to various solvents, acids and bases, presenting widespread applicability in areas such as clinical diagnostics, biological sensing applications, food processing, and the chemical industry. Fabricated paper media 96 well-plate CH-microPAD configurations were tested for cell culture applications using mice embryonic fibroblasts and detection of proteins and enzymes using ELISA. With a simple two-step process and minimal human intervention, the technique presents a promising step towards mass fabrication of inexpensive disposable diagnostic devices for both resource-limited and developed regions.

Computational Fluid Dynamics Assessment of the Effect of Bioprinting Parameters in Extrusion Bioprinting.

Wall shear stress is the most critical factor in determining the viability of cells during the bioprinting process, and controlling wall shear stress remains a challenge in extrusion bioprinting. We investigated the effect of various bioprinting parameters using computational simulations on maximum wall shear stress (MWSS) in the nozzle to optimize the bioprinting process. Steady-state simulations were done for three nozzle geometries (conical, tapered conical, and cylindrical) with varying nozzle diameters (0.1 mm-0.5 mm) at different inlet pressure (0.025 MPa-0.25 MPa) as inlet conditions. Non-Newtonian power law was used to model the bioink rheology and four different bioinks with power-law constants ranging from 0.0863 to 0.5050 were examined. To capture the dynamic behavior of the bioink and the thread profile of the extruded bioink, transient simulations were carried out. Our results indicate that although the MWSS is lowest in the cylindrical nozzle, this stress condition lasts for a longer portion of the nozzle and for the same inlet pressure and nozzle diameter, the mass flow rate is lower compared to the tapered conical and conical nozzle, contributing to lower cell viability.

Novel vision restoration techniques: 3D bioprinting, gene and stem cell therapy, optogenetics, and the bionic eye.

BACKGROUND: Vision restoration has been one of the most sought-after goals of ophthalmology because of its inception. Despite these problems being tackled from numerous different perspectives, a concrete solution has not yet been achieved. An optimal solution will have significant implications on the patient's quality of life, socioeconomic status, and mental health. METHODS: This article will explore new and innovative approaches with one common aim-to restore functional vision for the visually impaired. These novel techniques include 3D bioprinting, stem cell therapy, gene therapy, implantable devices, and optogenetics. RESULTS: While the techniques mentioned above show significant promise, they are currently in various stages of development ranging from clinical trials to commercial availability. Restoration of minimal vision in specific cases has already been achieved by the different methods but optimization of different parameters like biocompatibility, spatiotemporal resolution, and minimizing the costs are essential for widespread use. CONCLUSION: The developments over the past decade have resulted in multiple milestones in each of the techniques with many solutions getting approved by the FDA. This article will compare these novel techniques and highlight the major advantages and drawbacks of each of them.

A 'Relay'-Type Drug-Eluting Nerve Guide Conduit: Computational Fluid Dynamics Modeling of the Drug Eluting Efficiency of Various Drug Release Systems.

Nerve guidance conduits (NGCs) are tubular scaffolds that act as a bridge between the proximal and distal ends of the native nerve to facilitate the nerve regeneration. The application of NGCs is mostly limited to nerve defects less than 3 mm due to the lack of sufficient cells in the lumen. The development of drug-release-system-embedded NGCs has the potential to improve the nerve regeneration performance by providing long-term release of growth factors. However, most of the past works only focused on one type of drug release system, limiting the variation in drug release system types and features. Therefore, in this study, computer-aided design (CAD) models were constructed and Computational Fluid Dynamics (CFD) simulations were carried out to investigate the effect of growth factor transporting efficiency on different drug release systems. To overcome the challenges posed by the current NGCs in treating long nerve gap injuries (>4 cm), a novel 'relay' NGC design is first proposed in this paper and has the potential to improve the nerve regeneration performance to next level. The intermediate cavities introduced along the length of the multi-channel NGCs act as a relay to further enhance the cell concentrations or growth factor delivery as well as the regeneration performance. Four different drug release systems, namely, a single-layer microsphere system, a double-layer microsphere system, bulk hydrogel, and hydrogel film, were chosen for the simulation. The results show that the double-layer microsphere system achieves the highest growth factor volume fraction among all the drug release systems. For the single-layer microsphere system, growth factor concentration can be significantly improved by increasing the microsphere quantities and decreasing the diameter and adjacent distance of microspheres. Bulk hydrogel systems hold the lowest growth factor release performance, and the growth factor concentration monotonically increased with the increase of film thickness in the hydrogel film system. Owing to the easy fabrication of hydrogel film and the even distribution of growth factors, the hydrogel film system can be regarded as a strong candidate in drug-eluting NGCs. The use of computational simulations can be regarded as a guideline for the design and application of drug release systems, as well as a promising tool for further nerve tissue engineering study.

Biofabrication in Congenital Cardiac Surgery: A Plea from the Operating Theatre, Promise from Science.

Despite significant advances in numerous fields of biofabrication, clinical application of biomaterials combined with bioactive molecules and/or cells largely remains a promise in an individualized patient settings. Three-dimensional (3D) printing and bioprinting evolved as promising techniques used for tissue-engineering, so that several kinds of tissue can now be printed in layers or as defined structures for replacement and/or reconstruction in regenerative medicine and surgery. Besides technological, practical, ethical and legal challenges to solve, there is also a gap between the research labs and the patients' bedside. Congenital and pediatric cardiac surgery mostly deal with reconstructive patient-scenarios when defects are closed, various segments of the heart are connected, valves are implanted. Currently available biomaterials lack the potential of growth and conduits, valves derange over time surrendering patients to reoperations. Availability of viable, growing biomaterials could cancel reoperations that could entail significant public health benefit and improved quality-of-life. Congenital cardiac surgery is uniquely suited for closing the gap in translational research, rapid application of new techniques, and collaboration between interdisciplinary teams. This article provides a succinct review of the state-of-the art clinical practice and biofabrication strategies used in congenital and pediatric cardiac surgery, and highlights the need and avenues for translational research and collaboration.

Progress in cardiovascular bioprinting.

Cardiovascular disease has been the leading cause of death globally for the past 15 years. Following a major cardiac disease episode, the ideal treatment would be the replacement of the damaged tissue, due to the limited regenerative capacity of cardiac tissues. However, we suffer from a chronic organ donor shortage which causes approximately 20 people to die each day waiting to receive an organ. Bioprinting of tissues and organs can potentially alleviate this burden by fabricating low cost tissue and organ replacements for cardiac patients. Clinical adoption of bioprinting in cardiovascular medicine is currently limited by the lack of systematic demonstration of its effectiveness, high costs, and the complexity of the workflow. Here, we give a concise review of progress in cardiovascular bioprinting and its components. We further discuss the challenges and future prospects of cardiovascular bioprinting in clinical applications.

Applications of 3D Bioprinted-Induced Pluripotent Stem Cells in Healthcare.

Induced pluripotent stem cell (iPSC) technology and advancements in three-dimensional (3D) bioprinting technology enable scientists to reprogram somatic cells to iPSCs and 3D print iPSC-derived organ constructs with native tissue architecture and function. iPSCs and iPSC-derived cells suspended in hydrogels (bioinks) allow to print tissues and organs for downstream medical applications. The bioprinted human tissues and organs are extremely valuable in regenerative medicine as bioprinting of autologous iPSC-derived organs eliminates the risk of immune rejection with organ transplants. Disease modeling and drug screening in bioprinted human tissues will give more precise information on disease mechanisms, drug efficacy, and drug toxicity than experimenting on animal models. Bioprinted iPSC-derived cancer tissues will aid in the study of early cancer development and precision oncology to discover patient-specific drugs. In this review, we present a brief summary of the combined use of two powerful technologies, iPSC technology, and 3D bioprinting in health-care applications.

Perspectives on 3D Bioprinting of Peripheral Nerve Conduits.

The peripheral nervous system controls the functions of sensation, movement and motor coordination of the body. Peripheral nerves can get damaged easily by trauma or neurodegenerative diseases. The injury can cause a devastating effect on the affected individual and his aides. Treatment modalities include anti-inflammatory medications, physiotherapy, surgery, nerve grafting and rehabilitation. 3D bioprinted peripheral nerve conduits serve as nerve grafts to fill the gaps of severed nerve bodies. The application of induced pluripotent stem cells, its derivatives and bioprinting are important techniques that come in handy while making living peripheral nerve conduits. The design of nerve conduits and bioprinting require comprehensive information on neural architecture, type of injury, neural supporting cells, scaffold materials to use, neural growth factors to add and to streamline the mechanical properties of the conduit. This paper gives a perspective on the factors to consider while bioprinting the peripheral nerve conduits.

Nerve guide conduits for peripheral nerve injury repair: A review on design, materials and fabrication methods.

Peripheral nerves can sustain injuries due to loss of structure and/or function of peripheral nerves because of accident, trauma and other causes, which leads to partial or complete loss of sensory, motor, and autonomic functions and neuropathic pain. Even with the extensive knowledge on the pathophysiology and regeneration mechanisms of peripheral nerve injuries (PNI), reliable treatment methods that ensure full functional recovery are scant. Nerve autografting is the current gold standard for treatment of PNI. Given the limitations of autografts including donor site morbidity and limited supply, alternate treatment methods are being pursued by the researchers. Neural guide conduits (NGCs) are increasingly being considered as a potential alternative to nerve autografts. The anatomy of peripheral nerves, classification of PNI, and current treatment methods are briefly yet succinctly reviewed. A detailed review on the various designs of NGCs, the different materials used for making the NGCs, and the fabrication methods adopted is presented in this work. Much progress had been made in all the aspects of making an NGC, including the design, materials and fabrication techniques. The advent of advanced technologies such as additive manufacturing and 3D bioprinting could be beneficial in easing the production of patient-specific NGCs. NGCs with supporting cells or stem cells, NGCs loaded with neurotropic factors and drugs, and 4D printed NGCs are some of the futuristic areas of interest. STATEMENT OF SIGNIFICANCE: Neural guide conduits (NGCs) are increasingly being considered as a potential alternative to nerve autografts in the treatment of peripheral nerve injuries. A detailed review on the various designs of NGCs, the different materials used for making the NGCs, and the fabrication methods (including Additive Manufacturing) adopted is presented in this work.

3D-Printed PCL/PPy Conductive Scaffolds as Three-Dimensional Porous Nerve Guide Conduits (NGCs) for Peripheral Nerve Injury Repair.

Conductivity is a desirable property of an ideal nerve guide conduit (NGC) that is being considered for peripheral nerve regeneration. Most of the conductive polymers reported in use for fabrication of tissue engineering scaffolds such as polypyrrole (PPy), polyaniline, polythiophene, and poly(3,4-ethylenedioxythiophene) are non-biodegradable and possess weak mechanical properties to be fabricated into 3D structures. In this study, a biodegradable and conductive block copolymer of PPy and Polycaprolactone (PPy-b-PCL) was used to fabricate 3D porous NGCs using a novel electrohydrodynamic jet 3D printing process which offers superior control over fiber diameter, pore size, porosity, and fiber alignment. PCL/PPy scaffolds with three different concentrations of PPy-b-PCL (0.5, 1, and 2% v/v) were fabricated as a mesh (pore size 125 ± 15 µm) and the effect of incorporation of PPy-b-PCL on mechanical properties, biodegradability, and conductivity of the NGCs were studied. The mechanical properties of the scaffolds decreased with the addition of PPy-b-PCL which aided the ability to fabricate softer scaffolds that are closer to the properties of the native human peripheral nerve. With increasing concentrations of PPy-b-PCL, the scaffolds displayed a marked increase in conductivity (ranging from 0.28 to 1.15 mS/cm depending on concentration of PPy). Human embryonic stem cell-derived neural crest stem cells (hESC-NCSCs) were used to investigate the impact of PPy-b-PCL based conductive scaffolds on the growth and differentiation to peripheral neuronal cells. The hESC-NCSCs were able to attach and differentiate to peripheral neurons on PCL and PCL/PPy scaffolds, in particular the PCL/PPy (1% v/v) scaffolds supported higher growth of neural cells and a stronger maturation of hESC-NCSCs to peripheral neuronal cells. Overall, these results suggest that PPy-based conductive scaffolds have potential clinical value as cell-free or cell-laden NGCs for peripheral neuronal regeneration.

Computational Design and Optimization of Nerve Guidance Conduits for Improved Mechanical Properties and Permeability.

Nerve guidance conduits (NGCs) are tubular tissue engineering scaffolds used for nerve regeneration. The poor mechanical properties and porosity have always compromised their performances for guiding and supporting axonal growth. Therefore, in order to improve the properties of NGCs, the computational design approach was adopted to investigate the effects of different NGC structural features on their various properties, and finally, design an ideal NGC with mechanical properties matching human nerves and high porosity and permeability. Three common NGC designs, namely hollow luminal, multichannel, and microgrooved, were chosen in this study. Simulations were conducted to study the mechanical properties and permeability. The results show that pore size is the most influential structural feature for NGC tensile modulus. Multichannel NGCs have higher mechanical strength but lower permeability compared to other designs. Square pores lead to higher permeability but lower mechanical strength than circular pores. The study finally selected an optimized hollow luminal NGC with a porosity of 71% and a tensile modulus of 8 MPa to achieve multiple design requirements. The use of computational design and optimization was shown to be promising in future NGC design and nerve tissue engineering research.

Conductive collagen/polypyrrole-b-polycaprolactone hydrogel for bioprinting of neural tissue constructs.

Bioprinting is increasingly being used for fabrication of engineered tissues for regenerative medicine, drug testing, and other biomedical applications. The success of this technology lies with the development of suitable bioinks and hydrogels that are specific to the intended tissue application. For applications such as neural tissue engineering, conductivity plays an important role in determining the neural differentiation and neural tissue regeneration. Although several conductive hydrogels based on metal nanoparticles (NPs) such as gold and silver, carbon-based materials such as graphene and carbon nanotubes and conducting polymers such as polypyrrole (PPy) and polyaniline were used, they possess several disadvantages. The long-term cytotoxicity of metal nanoparticles (NPs) and carbon-based materials restricts their use in regenerative medicine. The conductive polymers, on the other hand, are non-biodegradable and possess weak mechanical properties limiting their printability into three-dimensional constructs. The aim of this study is to develop a biodegradable, conductive, and printable hydrogel based on collagen and a block copolymer of PPy and polycaprolactone (PCL) (PPy-block-poly(caprolactone) [PPy-b-PCL]) for bioprinting of neural tissue constructs. The printability, including the influence of the printing speed and material flow rate on the printed fiber width; rheological properties; and cytotoxicity of these hydrogels were studied. The results prove that the collagen/PPy-b-PCL hydrogels possessed better printability and biocompatibility. Thus, the collagen/PPy-b-PCL hydrogels reported this study has the potential to be used in the bioprinting of neural tissue constructs for the repair of damaged neural tissues and drug testing or precision medicine applications.

A review on the use of computational methods to characterize, design, and optimize tissue engineering scaffolds, with a potential in 3D printing fabrication.

The design and fabrication of tissue engineering scaffolds is a highly complex process. In order to provide a proper architecture for cells to grow, proliferate, and differentiate to form tissues, scaffolds have to be made with suitable properties. However, the limited structural designs and conventional fabrication techniques severely cripple the improvement of scaffold properties. To overcome these limitations, many researchers have recently adopted computational methods combined with 3D printing techniques as a new approach for scaffold design and fabrication. This approach allows scaffolds to be designed and fabricated with highly complex microstructures and good control and accuracy. Previous works have also shown this approach to be a very useful tool to predict the scaffold properties and to optimize the scaffold designs with a great reduction of experimental iterations. As this approach combining computational methods and 3D printing techniques for scaffold design and fabrication has many advantages over the conventional trial-and-error based approach, it is imperative to provide a state-of-the-art review on the topic. To this end, this article reviews the various applications of computational methods in scaffold design and simulation; it also briefly reviews the application of 3D printing techniques to fabricate the computationally designed scaffolds. Finally, the limitations and future trends of this approach are discussed. Overall, this review will enable readers to understand the benefits of using computational methods coupled with 3D printing to design and fabricate scaffolds, and thus help researchers to improve and optimize the scaffold properties for future tissue engineering research. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1329-1351, 2019.