Bioengineering vascularized liver tissue for biomedical research and application.

The liver performs a wide range of biological functions that are essential to body homeostasis. Damage to liver tissue can result in reduced organ function, and if chronic in nature can lead to organ scarring and progressive disease. Currently, donor liver transplantation is the only longterm treatment for end-stage liver disease. However, orthotopic organ transplantation suffers from several drawbacks that include organ scarcity and lifelong immunosuppression. Therefore, new therapeutic strategies are required. One promising strategy is the engineering of implantable and vascularized liver tissue. This resource could also be used to build the next generation of liver tissue models to better understand human health, disease and aging in vitro. This article reviews recent progress in the field of liver tissue bioengineering, including microfluidic-based systems, bio-printed vascularized tissue, liver spheroids and organoid models, and the induction of angiogenesis in vivo.

The use of fluid-phase 3D printing to pattern alginate-gelatin hydrogel properties to guide cell growth and behaviourin vitro.

Three-dimensional (3D) (bio)printing technology has boosted the advancement of the biomedical field. However, tissue engineering is an evolving field and (bio)printing biomimetic constructions for tissue formation is still a challenge. As a new methodology to facilitate the construction of more complex structures, we suggest the use of the fluid-phase 3D printing to pattern the scaffold's properties. The methodology consists of an exchangeable fluid-phase printing medium in which the constructions are fabricated and patterned during the printing process. Using the fluid-phase methodology, the biological and mechanical properties can be tailored promoting cell behaviour guidance and compartmentalization. In this study, we first assessed different formulations of alginate/gelatin to create a stable substrate capable to promote massive cell colonizationin vitroover time. Overall, formulations with lower gelatin content and 2-(N-morpholino)ethanesulfonic acid (MES) buffer as a solvent showed better stability under cell culture conditions and enhanced U2OS cell growth. Next, the fluid-phase showed better printing fidelity and resolution in comparison to air printing as it diminished the collapsing and the spread of the hydrogel strand. In sequence, the fluid-phase methodology was used to create functionalized alginate-gelatin-arginylglycylaspartic acid peptide (RGD) hydrogels via carbodiimides chemistry. The alginate-gelatin-RGD hydrogels showed an increase of 2.97-fold in cell growth and more spread substrate colonization in comparison to alginate-gelatin hydrogel. Moreover, the fluid-phase methodology was used to add RGD molecules to pre-determined parts of the alginate-gelatin substrate during the printing process promoting U2OS cell compartmentalization. In addition, different substrate stiffnesses were also created via fluid-phase by crosslinking the hydrogel with different concentrations of CaCl2during the printing process. As a result, the U2OS cells were also compartmentalized on the stiffer parts of the printings. Finally, our results showed that by combining stiffer hydrogel with RGD increasing concentrations we can create a synergetic effect and boost cell metabolism by up to 3.17-fold. This work presents an idea of a new printing process for tailoring multiple parameters in hydrogel substrates by using fluid-phase to generate more faithful replication of thein vivoenvironment.

Additive Manufacturing Applications in Biosensors Technologies.

Three-dimensional (3D) printing technology, also known as additive manufacturing (AM), has emerged as an attractive state-of-the-art tool for precisely fabricating functional materials with complex geometries, championing several advancements in tissue engineering, regenerative medicine, and therapeutics. However, this technology has an untapped potential for biotechnological applications, such as sensor and biosensor development. By exploring these avenues, the scope of 3D printing technology can be expanded and pave the way for groundbreaking innovations in the biotechnology field. Indeed, new printing materials and printers would offer new possibilities for seamlessly incorporating biological functionalities within the growing 3D scaffolds. Herein, we review the additive manufacturing applications in biosensor technologies with a particular emphasis on extrusion-based 3D printing modalities. We highlight the application of natural, synthetic, and composite biomaterials as 3D-printed soft hydrogels. Emphasis is placed on the approach by which the sensing molecules are introduced during the fabrication process. Finally, future perspectives are provided.

Sequential Fabrication of a Three-Layer Retina-like Structure.

Tissue engineering is considered a promising approach to treating advanced degenerative maculopathies such as nonexudative age-related macular degeneration (AMD), the leading cause of blindness worldwide. The retina consists of several hierarchical tissue layers, each of which is supported by a layer underneath. Each of these layers has a different morphology and requires distinct conditions for proper assembly. In fact, a prerequisite step for the assembly of each of these layers is the organization of the layer underneath. Advanced retinal degeneration includes degeneration of the other retina layers, including the choroid, the retinal pigmented epithelium (RPE), and the photoreceptors. Here, we report a step-by-step fabrication process of a three-layer retina-like structure. The process included the 3D printing of a choroid-like structure in an extracellular matrix (ECM) hydrogel, followed by deposition of the RPE monolayer. After the formation of the blood vessel-RPE interface, the photoreceptor cells were deposited to interact with the RPE layer. At the end of the fabrication process, each layer was characterized for its morphology and expression of specific markers, and the integration of the three-layer retina was evaluated. We envision that such a retina-like structure may be able to attenuate the deterioration of a degenerated retina and improve engraftment and regeneration. This retinal implant may potentially be suitable for a spectrum of macular degenerative diseases for which there are currently no cures and may save millions from complete blindness.

Biofabrication of Composite Bioink-Nanofiber Constructs: Effect of Rheological Properties of Bioinks on 3D (Bio)Printing and Cells Interaction with Aligned Touch Spun Nanofibers.

This paper reports on a novel approach for the fabrication of composite multilayered bioink-nanofibers construct. This work achieves this by using a hands-free 3D (bio)printing integrated touch-spinning approach. Additionally, this work investigates the interaction of fibroblasts in different bioinks with the highly aligned touch-spun nanofibers. This work conducts a comprehensive characterization of the rheological properties of the inks, starting with low-strain oscillatory rheology to analyze the viscoelastic behavior, when the material structure remains intact. Moreover, this work performs amplitude sweeps to investigate the stability of the inks under large deformations, rotational rheology to examine the shear thinning profile, and a three-step creep experiment to study time-dependent rheological behavior. The obtained rheological results are correlated to visual observation of the flow behavior of inks. These behaviors span from an ink with zero-shear viscosity, very weak shear thinning, and no thixotropic behavior to inks exhibiting flow stress, pronounced shear thinning, and thixotropy. It is demonstrated that inks have an essential effect on cell behavior. While all bioinks allow a preferred directionality of the fibroblasts along the fiber direction, cells tend to form aggregates in bioinks with higher viscosity, and a considerable number of agglomerates are observed in the presence of laponite-RD.

(Bio)printing in Personalized Medicine-Opportunities and Potential Benefits.

The global development of technologies now enters areas related to human health, with a transition from conventional to personalized medicine that is based to a significant extent on (bio)printing. The goal of this article is to review some of the published scientific literature and to highlight the importance and potential benefits of using 3D (bio)printing techniques in contemporary personalized medicine and also to offer future perspectives in this research field. The article is prepared according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Web of Science, PubMed, Scopus, Google Scholar, and ScienceDirect databases were used in the literature search. Six authors independently performed the search, study selection, and data extraction. This review focuses on 3D bio(printing) in personalized medicine and provides a classification of 3D bio(printing) benefits in several categories: overcoming the shortage of organs for transplantation, elimination of problems due to the difference between sexes in organ transplantation, reducing the cases of rejection of transplanted organs, enhancing the survival of patients with transplantation, drug research and development, elimination of genetic/congenital defects in tissues and organs, and surgery planning and medical training for young doctors. In particular, we highlight the benefits of each 3D bio(printing) applications included along with the associated scientific reports from recent literature. In addition, we present an overview of some of the challenges that need to be overcome in the applications of 3D bioprinting in personalized medicine. The reviewed articles lead to the conclusion that bioprinting may be adopted as a revolution in the development of personalized, medicine and it has a huge potential in the near future to become a gold standard in future healthcare in the world.

Recent Developments in 3D Bio-Printing and Its Biomedical Applications.

The fast-developing field of 3D bio-printing has been extensively used to improve the usability and performance of scaffolds filled with cells. Over the last few decades, a variety of tissues and organs including skin, blood vessels, and hearts, etc., have all been produced in large quantities via 3D bio-printing. These tissues and organs are not only able to serve as building blocks for the ultimate goal of repair and regeneration, but they can also be utilized as in vitro models for pharmacokinetics, drug screening, and other purposes. To further 3D-printing uses in tissue engineering, research on novel, suitable biomaterials with quick cross-linking capabilities is a prerequisite. A wider variety of acceptable 3D-printed materials are still needed, as well as better printing resolution (particularly at the nanoscale range), speed, and biomaterial compatibility. The aim of this study is to provide expertise in the most prevalent and new biomaterials used in 3D bio-printing as well as an introduction to the associated approaches that are frequently considered by researchers. Furthermore, an effort has been made to convey the most pertinent implementations of 3D bio-printing processes, such as tissue regeneration, etc., by providing the most significant research together with a comprehensive list of material selection guidelines, constraints, and future prospects.

Towards the personalization of gelatin-based 3D patches: a tunable porous carrier for topical applications.

Cell-free based therapies, for example, the use of the cell secretome, have emerged as a promising alternative to conventional skin therapies using bioactive and, when combined with 3D printing technologies, allow the development of personalized dosage forms. This research work aimed to develop gelatin-based patches with controlled network topology via extrusion 3D printing, loaded with cell culture medium as a model of the secretome, and applicable as vehicles for topical delivery. Inks were optimized through rheological and printing assays, and the incorporation of medium had minor effects in printability. Regarding network topology, grid infills rendered more defined structures than the triangular layout, depicting clearer pores and pore area consistency. Release studies showed that filament spacing and infill pattern influenced the release of rhodamine B (model bioactive) and bovine serum albumin (model protein). Moreover, the grid patches (G-0.7/1/0.7), despite having around a seven-fold higher mean pore area than 0.7-mm triangular ones (T-0.7), showed a similar release profile, which can be linked to the network topology of the printed structures This work provided insight on employing (bio)printing in the production of carriers with reproducible and controlled pore area, able to incorporate cell-derived secretome and to be quickly tailored to the patient's lesions.

3D bio-printed endometrial construct restores the full-thickness morphology and fertility of injured uterine endometrium.

Severe damage to the uterine endometrium, which results in scar formation and endometrial dysfunction, eventually leads to infertility or pregnancy-related complications. No effective therapeutic treatment is currently available for such injuries owing to the structural complexity, internal environment, and function of the uterus. Three-dimensional (3D) bio-printing to engineer biomimetic structural constructs provides a unique opportunity for tissue regeneration. Herein, using 3D extrusion-based bioprinting (EBB), we constructed a bilayer endometrial construct (EC) based on a sodium alginate-hyaluronic acid (Alg-HA) hydrogel for functional regeneration of the endometrium. The upper layer of the 3D bio-printed EC is a monolayer of endometrial epithelial cells (EECs), while the lower layer has a grid-like microstructure loaded with endometrial stromal cells (ESCs). In a partial full-thickness uterine excision rat model, our bilayer EC not only restored the morphology and structure of the endometrial wall (including organized luminal/ glandular epithelium, stroma, vasculature and the smooth muscle layer), but also significantly improved the reproductive outcome in the surgical area after implantation (75%, 12/16, p < 0.01). Therefore, repair of the uterine endometrium using the developed 3D bio-printed bilayer EC may represent an effective regenerative treatment for severe endometrial injury. STATEMENT OF SIGNIFICANCE: Achieving structural and functional recovery of the endometrium following severe injury is still a challenge. Here, we designed a 3D bio-printed endometrial construct (EC) to mimic the native bilayer structure and cellular components of the endometrium. The bio-printed EC consists of a dense upper layer with endometrial epithelial cells and a lower layer with endometrial stromal cells. In particular, the 3D bio-printed EC significantly improved the reproductive outcome in the surgical area (75%, 12/16) compared to that of the cell-loaded non-printed group (12.5%, 2/16). This study demonstrates that a biomimetic bilayer construct can facilitate endometrial repair and regeneration. Therefore, an endometrial cells-loaded 3D-bioprinted EC is a promising therapeutic option for patients suffering from severe endometrial damage.

Biomimetic Peridontium Patches for Functional Periodontal Regeneration.

The unique structure of the periodontium, including the alveolar bone, cementum, and periodontal ligament (PDL), presents difficulties for the regeneration of its intricate organization. Irreversible structural breakdown of the periodontium increases the risk of tooth loosening and loss. Although the current therapies can restore the periodontal hard tissues to a certain extent, the PDL with its high directionality of multiple groups with different orientations and functions cannot be reconstructed. Here, biomimetic peridontium patches (BPPs) for functional periodontal regeneration using a microscale continuous digital light projection bioprinting method is reported. Orthotopic transplantation in the mandibles shows effective periodontal reconstruction. The resulting bioengineered tissues closely resembles natural periodontium in terms of the "sandwich structures," especially the correctly oriented fibers, showing different and specific orientation in different regions of the tooth root, which has never been found in previous studies. Furthermore, after the assessment of clinically functional properties it is found that the regenerative periodontium can achieve stable tooth movement under orthodontic migration force with no adverse consequences. Overall, the BPPs promote reconstruction of the functional periodontium and the complex microstructure of the periodontal tissue, providing a proof of principle for the clinical functional treatment of periodontal defects.

The Progress in Bioprinting and Its Potential Impact on Health-Related Quality of Life.

The intensive development of technologies related to human health in recent years has caused a real revolution. The transition from conventional medicine to personalized medicine, largely driven by bioprinting, is expected to have a significant positive impact on a patient's quality of life. This article aims to conduct a systematic review of bioprinting's potential impact on health-related quality of life. A literature search was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A comprehensive literature search was undertaken using the PubMed, Scopus, Google Scholar, and ScienceDirect databases between 2019 and 2023. We have identified some of the most significant potential benefits of bioprinting to improve the patient's quality of life: personalized part production; saving millions of lives; reducing rejection risks after transplantation; accelerating the process of skin tissue regeneration; homocellular tissue model generation; precise fabrication process with accurate specifications; and eliminating the need for organs donor, and thus reducing patient waiting time. In addition, these advances in bioprinting have the potential to greatly benefit cancer treatment and other research, offering medical solutions tailored to each individual patient that could increase the patient's chance of survival and significantly improve their overall well-being. Although some of these advancements are still in the research stage, the encouraging results from scientific studies suggest that they are on the verge of being integrated into personalized patient treatment. The progress in bioprinting has the power to revolutionize medicine and healthcare, promising to have a profound impact on improving the quality of life and potentially transforming the field of medicine and healthcare.

Platelet-rich plasma-loaded bioactive chitosan@sodium alginate@gelatin shell-core fibrous hydrogels with enhanced sustained release of growth factors for diabetic foot ulcer healing.

The ability of autologous platelet-rich plasma (PRP) gel to promote rapid wound healing without immunological rejection has opened new avenues for the treatment of diabetic foot wounds. However, PRP gel still suffers from the quick release of growth factors (GFs) and requires frequent administration, thus resulting in decreased wound healing efficiency, higher cost as well as greater pain and suffering for the patients. In this study, the flow-assisted dynamic physical cross-linked coaxial microfluidic three-dimensional (3D) bio-printing technology, combined with the calcium ion chemical dual cross-linking method was developed to design PRP-loaded bioactive multi-layer shell-core fibrous hydrogels. The prepared hydrogels exhibited outstanding water absorption-retention capacity, good biocompatibility as well as a broad-spectrum antibacterial effect. Compared with clinical PRP gel, these bioactive fibrous hydrogels displayed a sustained release of GFs, reducing the administration frequency by 33 % availably during the wound treatment, but more prominent therapeutic effects such as effective reduced inflammation, in addition to promoting the growth of granulation tissue and angiogenesis, the formation of high-density hair follicles, and the generation of regular ordered and high-density collagen fiber network, which suggested great promise as exceptional candidates for treatment of diabetic foot ulcer in clinical settings.

Horizon Scanning in Tissue Engineering Using Citation Network Analysis.

BACKGROUND: Establishing a horizon scanning method is critical for identifying technologies that require new guidelines or regulations. We studied the application of bibliographic citation network analysis to horizon scanning. OBJECTIVE: The possibility of applying the proposed method to interdisciplinary fields was investigated with the emphasis on tissue engineering and its example, three-dimensional bio-printing. METHODOLOGY AND RESULTS: In all, 233,968 articles on tissue engineering, regenerative medicine, biofabrication, and additive manufacturing published between January 1, 1900 and November 3, 2021 were obtained from the Web of Science Core Collection. The citation network of the articles was analyzed for confirmation that the evolution of 3D bio-printing is reflected by tracking the key articles in the field. However, the results revealed that the major articles on the clinical application of 3D bio-printed products are located in clusters other than that of 3D bio-printers. We investigated the research trends in this field by analyzing the articles published between 2019 and 2021 and detected various basic technologies constituting tissue engineering, including microfluidics and scaffolds such as electrospinning and conductive polymers. The results suggested that the research trend of technologies required for product development and future clinical applications of the product are sometimes detected independently by bibliographic citation network analysis, particularly for interdisciplinary fields. CONCLUSION: This method can be applied to the horizon scanning of an interdisciplinary field. However, identifying basic technologies of the targeted field and following the progress of research and the integration process of each component of technology are critical.

Current Trends on Innovative Technologies in Topical Wound Care for Advanced Healing and Management.

OBJECTIVE: To investigate critically traditional and modern techniques for cutaneous wound healing and to provide comprehensive information on these novel techniques to encounter the challenges with the existing wound healing methods. SIGNIFICANCE: The financial burden and mortality associated with wounds is increasing, so managing wounds is essential. Traditional wound treatments include surgical and non-surgical methods, while modern techniques are advancing rapidly. This review examines the various traditional and modern techniques used for cutaneous wound healing. KEY FINDINGS: Traditional wound treatments include surgical techniques such as debridement, skin flaps, and grafts. Non-surgical treatments include skin replacements, topical formulations, scaffold-based skin grafts, and hydrogel-based skin dressings. More modern techniques include using nanoparticles, growth factors, and bioactive substances in wound dressings. Bioengineered skin substitutes using biomaterials, cells, and growth factors are also being developed. Other techniques include stem cell therapy, growth factor/cytokine therapy, vacuum-assisted wound closure, and 3D-printed/bio-printed wound dressings. CONCLUSION: Traditional wound treatments have been replaced by modern techniques such as stem cell therapy, growth factor/cytokine therapy, vacuum-assisted wound closure, and bioengineered skin substitutes. However, most of these strategies lack effectiveness and thorough evaluation. Therefore, further research is required to develop new techniques for cutaneous wound healing that are effective, cost-efficient, and appealing to patients.

The efficacy of a paeoniflorin-sodium alginate-gelatin skin scaffold for the treatment of diabetic wound: An in vivo study in a rat model.

OBJECTIVE: To investigate the efficacy of a paeoniflorin-sodium alginate (SA)-gelatin skin scaffold for treating diabetic wound in a rat model. METHODS: Bioinks were prepared using various percentages of paeoniflorin in the total weight of a solution containing SA and gelatin. Skin scaffolds containing 0%, 1%, 3%, 5%, and 10% paeoniflorin were printed using 3D bioprinting technology, and scaffold microstructure was observed with scanning electron microscopy. Skin scaffolds were then used in rats with diabetic wounds. H&E staining, Masson staining, and immunohistochemical staining for IL-1ß and CD31 were performed on days 7 and 14. RESULTS: All skin scaffolds had a mesh-like structure with uniform pore distribution. Wounds healed well in each group, with the 1% and 3% groups demonstrating the most complete healing. H&E staining showed that skin accessory organs had appeared in each group. On day 7, collagen deposition in the 3% group was higher than in the other groups (P＜0.05), and IL-1ß infiltration was lower in the 10% group than in the 3% group (P = 0.002). On day 14, IL-1ß infiltration was not significantly different between the 10% and 3% groups (P = 0.078). The CD31 level was higher in the 3% group than in the other groups on days 7 and 14 (P＜0.05). CONCLUSION: A 3% paeoniflorin-SA-gelatin skin scaffold promoted the healing of diabetic wounds in rats. This scaffold promoted collagen deposition and microvascular regeneration and demonstrated anti-inflammatory properties, suggesting that this scaffold type could be used to treat diabetic wounds.

Three-dimensional bio-printing of decellularized extracellular matrix-based bio-inks for cartilage regeneration: a systematic review.

Cartilage injuries are common problems that increase with the population aging. Cartilage is an avascular tissue with a relatively low level of cellular mitotic activity, which makes it impossible to heal spontaneously. To compensate for this problem, three-dimensional bio-printing has attracted a great deal of attention in cartilage tissue engineering. This emerging technology aims to create three-dimensional functional scaffolds by accurately depositing layer-by-layer bio-inks composed of biomaterial and cells. As a novel bio-ink, a decellularized extracellular matrix can serve as an appropriate substrate that contains all the necessary biological cues for cellular interactions. Here, this review is intended to provide an overview of decellularized extracellular matrix-based bio-inks and their properties, sources, and preparation process. Following this, decellularized extracellular matrix-based bio-inks for cartilage tissue engineering are discussed, emphasizing cell behavior and in-vivo applications. Afterward, the current challenges and future outlook will be discussed to determine the conclusing remarks.

Printability of Double Network Alginate-Based Hydrogel for 3D Bio-Printed Complex Structures.

Three-dimensional (3D) bio-printing has recently emerged as a crucial technology in tissue engineering, yet there are still challenges in selecting materials to obtain good print quality. Therefore, it is essential to study the influence of the chosen material (i.e., bio-ink) and the printing parameters on the final result. The "printability" of a bio-ink indicates its suitability for bio-printing. Hydrogels are a great choice because of their biocompatibility, but their printability is crucial for exploiting their properties and ensuring high printing accuracy. However, the printing settings are seldom addressed when printing hydrogels. In this context, this study explored the printability of double network (DN) hydrogels, from printing lines (1D structures) to lattices (2D structures) and 3D tubular structures, with a focus on printing accuracy. The DN hydrogel has two entangled cross-linked networks and a balanced mechanical performance combining high strength, toughness, and biocompatibility. The combination of poly (ethylene glycol)-diacrylate (PEDGA) and sodium alginate (SA) enables the qualities mentioned earlier to be met, as well as the use of UV to prevent filament collapse under gravity. Critical correlations between the printability and settings, such as velocity and viscosity of the ink, were identified. PEGDA/alginate-based double network hydrogels were explored and prepared, and printing conditions were improved to achieve 3D complex architectures, such as tubular structures. The DN solution ink was found to be unsuitable for extrudability; hence, glycerol was added to enhance the process. Different glycerol concentrations and flow rates were investigated. The solution containing 25% glycerol and a flow rate of 2 mm/s yielded the best printing accuracy. Thanks to these parameters, a line width of 1 mm and an angle printing inaccuracy of less than 1° were achieved, indicating good shape accuracy. Once the optimal parameters were identified, a tubular structure was achieved with a high printing accuracy. This study demonstrated a 3D printing hydrogel structure using a commercial 3D bio-printer (REGEMAT 3D BIO V1) by synchronizing all parameters, serving as a reference for future more complex 3D structures.

NRAGE Confers Radiation Resistance in 2D and 3D Cell Culture and Poor Outcome in Patients With Esophageal Squamous Cell Carcinoma.

Objective: The purpose of the study is to explore the mechanism of NRAGE enhancing radioresistance of esophageal squamous cell carcinoma (ESCC) in 2D and 3D levels. Methods: Stably NRAGE-overexpressed ESCC cells and 3D-printing models for ESCC cells were established. Then, cellular malignancy indexes, such as cell morphology, proliferation, radioresistance, motility, apoptosis, cell cycle, and proteins of the Wnt/ß-catenin pathway, were compared between radioresistant and its parental cells in 2D and 3D levels. Additionally, 44 paraffin ESCC specimens with radical radiotherapy were selected to examine NRAGE and ß-catenin protein expression and analyze the clinical correlation. Results: Experiments in 2D culture showed that morphology of the Eca109/NRAGE cells was more irregular, elongated spindle-shaped and disappeared polarity. It obtained faster growth ability, stronger resistance to irradiation, enhanced motility, reduced apoptosis ratio and cell cycle rearrangement. Moreover, Western blot results showed ß-catenin, p-Gsk-3ß and CyclinD1 expressions were induced, while p-ß-catenin and Gsk-3ß expressions decreased in Eca109/NRAGE cells. Experiments in the 3D-printing model showed Eca109/NRAGE cell-laden 3D scaffolds had the advantage on growth and spheroiding according to the brightfield observation, scanning electron microscopy and Ki-67 IHC staining, and higher expression at the ß-catenin protein. Clinical analysis showed that NRAGE expression was higher in tumor tissues than in control tissues of ESCC patients from the Public DataBase. Compared with radiotherapy effective group, both NRAGE total and nuclear and ß-catenin nuclear expressions were significantly upregulated from ESCC specimens in invalid group. Further analysis showed a positive and linear correlation between NRAGE nuclear and ß-catenin nuclear expressions. Additionally, results from univariate and multivariate analyses revealed NRAGE nuclear expression could serve as a risk factor for ESCC patients receiving radical radiotherapy. Conclusion: ESCC cells with NRAGE nuclear accumulation demonstrated greater radioresistance, which may be related to the activation of the Wnt/ß-catenin signaling pathway. It indicated that NRAGE nuclear expression was a potential biomarker for monitoring radiotherapeutic response.

Low-intensity pulsed ultrasound promotes cell viability and inhibits apoptosis of H9C2 cardiomyocytes in 3D bioprinting scaffolds via PI3K-Akt and ERK1/2 pathways.

The aim of this study was to investigate whether low-intensity pulsed ultrasound (LIPUS) promotes myocardial cell viability in three-dimensional (3D) cell-laden gelatin methacryloyl (GelMA) scaffolds. Cardiomyoblasts (H9C2s) were mixed in 6% (w/v) GelMA bio-inks and printed using an extrusion-based 3D bioprinter. These scaffolds were exposed to LIPUS with different parameters or sham-irradiated to optimize the LIPUS treatment. The viability of H9C2s was measured using Cell Counting Kit-8 (CCK8), cell cycle, and live and dead cell double-staining assays. Western blot analysis was performed to determine the protein expression levels. We successfully fabricated 3D bio-printed cell-laden GelMA scaffolds. CCK8 and live and dead cell double-staining assays indicated that the optimal conditions for LIPUS were a frequency of 0.5 MHz and an exposure time of 10 min. Cell cycle analysis showed that LIPUS promoted the entry of cells into the S and G2/M phases from the G0/G1 phase. Western blot analysis revealed that LIPUS promoted the phosphorylation and activation of ERK1/2 and PI3K-Akt. The ERK1/2 inhibitor (U0126) and PI3K inhibitor (LY294002) significantly reduced LIPUS-induced phosphorylation of ERK1/2 and PI3K-Akt, respectively, which in turn reduced the LIPUS-induced viability of H9C2s in 3D bio-printed cell-laden GelMA scaffolds. A frequency of 0.5 MHz and exposure time of 10 min for LIPUS exposure can be adapted to achieve optimized culture effects on myocardial cells in 3D bio-printed cell-laden GelMA scaffolds via the ERK1/2 and PI3K-Akt signaling pathways.

Impact of oxygen-calcium-generating and bone morphogenetic protein-2 nanoparticles on survival and differentiation of bone marrow-derived mesenchymal stem cells in the 3D bio-printed scaffold.

Although stem cell therapy is a major area of interest in tissue engineering, providing proper oxygen tension, good viability, and cell differentiation remain challenges in tissue-engineered scaffolds. In this study, an osteogenic scaffold was fabricated using the 3D bio-printing technique. The bio-ink contained alginate hydrogel, encapsulated human bone marrow-derived mesenchymal stem cells (hBM-MSCs), calcium peroxide nanoparticles (CPO NPs) as an oxygen generating biomaterial, and bone morphogenic protein-2 nanoparticles (BMP2 NPs) as an osteoinductive growth factor. CPO NPs were synthesized with the hydrolysis-precipitation method, and their concentrations in the bio-ink were optimized. Scaffolds containing CPO 3% (w/w) were preferred, because they generated sufficient oxygen gas for 20 days, increased mechanical strength after 20 days, and had sufficient stability. The CPO NPs effect on the viability of embedded hBM-MSCs under hypoxic conditions was analyzed. Live/Dead staining results represented a 22% improvement in CPO 3% scaffold viability on day 7. Therefore, CPO NPs constituted a promising survival factor. BMP2 NPs were prepared with the double emulsification technique. The incorporation of both BMP2 and CPO NPs resulted in the upregulation of Runt-related transcription factor 2, Collagen type I alpha 1, and the osteocalcin genes compared to internal references in osteogenic media. Overall, the proposed 3D bio-printed osteogenic scaffold in this study has moved scientific research one step forward toward successful stem cell therapy and helped improve host tissue healing by biological activity enhancement, especially for low oxygen pressure tissues.