A Computed Tomography-Based Fracture Prediction Model With Images of Vertebral Bones and Muscles by Employing Deep Learning: Development and Validation Study.

BACKGROUND: With the progressive increase in aging populations, the use of opportunistic computed tomography (CT) scanning is increasing, which could be a valuable method for acquiring information on both muscles and bones of aging populations. OBJECTIVE: The aim of this study was to develop and externally validate opportunistic CT-based fracture prediction models by using images of vertebral bones and paravertebral muscles. METHODS: The models were developed based on a retrospective longitudinal cohort study of 1214 patients with abdominal CT images between 2010 and 2019. The models were externally validated in 495 patients. The primary outcome of this study was defined as the predictive accuracy for identifying vertebral fracture events within a 5-year follow-up. The image models were developed using an attention convolutional neural network-recurrent neural network model from images of the vertebral bone and paravertebral muscles. RESULTS: The mean ages of the patients in the development and validation sets were 73 years and 68 years, and 69.1% (839/1214) and 78.8% (390/495) of them were females, respectively. The areas under the receiver operator curve (AUROCs) for predicting vertebral fractures were superior in images of the vertebral bone and paravertebral muscles than those in the bone-only images in the external validation cohort (0.827, 95% CI 0.821-0.833 vs 0.815, 95% CI 0.806-0.824, respectively; P<.001). The AUROCs of these image models were higher than those of the fracture risk assessment models (0.810 for major osteoporotic risk, 0.780 for hip fracture risk). For the clinical model using age, sex, BMI, use of steroids, smoking, possible secondary osteoporosis, type 2 diabetes mellitus, HIV, hepatitis C, and renal failure, the AUROC value in the external validation cohort was 0.749 (95% CI 0.736-0.762), which was lower than that of the image model using vertebral bones and muscles (P<.001). CONCLUSIONS: The model using the images of the vertebral bone and paravertebral muscle showed better performance than that using the images of the bone-only or clinical variables. Opportunistic CT screening may contribute to identifying patients with a high fracture risk in the future.

Prediction of treatment response after stereotactic radiosurgery of brain metastasis using deep learning and radiomics on longitudinal MRI data.

We developed artificial intelligence models to predict the brain metastasis (BM) treatment response after stereotactic radiosurgery (SRS) using longitudinal magnetic resonance imaging (MRI) data and evaluated prediction accuracy changes according to the number of sequential MRI scans. We included four sequential MRI scans for 194 patients with BM and 369 target lesions for the Developmental dataset. The data were randomly split (8:2 ratio) for training and testing. For external validation, 172 MRI scans from 43 patients with BM and 62 target lesions were additionally enrolled. The maximum axial diameter (Dmax), radiomics, and deep learning (DL) models were generated for comparison. We evaluated the simple convolutional neural network (CNN) model and a gated recurrent unit (Conv-GRU)-based CNN model in the DL arm. The Conv-GRU model performed superior to the simple CNN models. For both datasets, the area under the curve (AUC) was significantly higher for the two-dimensional (2D) Conv-GRU model than for the 3D Conv-GRU, Dmax, and radiomics models. The accuracy of the 2D Conv-GRU model increased with the number of follow-up studies. In conclusion, using longitudinal MRI data, the 2D Conv-GRU model outperformed all other models in predicting the treatment response after SRS of BM.

The Application of Fluorescence In Situ Hybridization in the Prescreening of Veronica Hybrids.

Fluorescence in situ hybridization (FISH), a molecular cytogenetic technique that enables the visualization and identification of specific DNA sequences within chromosomes, has emerged as a pivotal tool in plant breeding programs, particularly in the case of Veronica species. Veronica, a genus with a complex reproductive system, often poses challenges in accurately identifying hybrids because of its tendency to hybridize, which leads to intricate genetic variation. This study focused on the use of FISH as a prescreening method to identify true hybrids in Veronica breeding programs. FISH analysis was first performed on the parents to identify their 45S and 5S rDNA signals, along with their respective chromosome numbers. The signals were then compared with those of the twenty progenies with reference to their supposed parents. Five true hybrids, seven self-pollinated progenies, and eight false hybrids were identified through FISH. The findings highlight the significance of FISH as a screening method that contributes significantly to the efficiency of Veronica breeding programs by ensuring the preservation of desired genetic traits and minimizing the inadvertent inclusion of misidentified hybrids. To conclude, this study underscores the vital role of FISH in enhancing the precision and success of breeding programs and opens new avenues for improved breeding strategies and crop development.

Programming temporal stiffness cues within extracellular matrix hydrogels for modelling cancer niches.

Extracellular matrix (ECM) stiffening is a common occurrence during the progression of many diseases, such as breast cancer. To accurately mimic the pathophysiological context of disease within 3D in vitro models, there is high demand for smart biomaterials which replicate the dynamic and temporal mechanical cues of diseased states. This study describes a preclinical disease model, using breast cancer as an example, which replicates the dynamic plasticity of the tumour microenvironment by incorporating temporal (3-week progression) biomechanical cues within a tissue-specific hydrogel microenvironment. The composite hydrogel formulation, integrating adipose-derived decellularised ECM (AdECM) and silk fibroin, was initially crosslinked using a visible light-mediated system, and then progressively stiffened through spontaneous secondary structure interactions inherent between the polymer chains (∼10-15 kPa increase, with a final stiffness of 25 kPa). When encapsulated and cultured in vitro, MCF-7 breast cancer cells initially formed numerous, large spheroids (>1000 µm2 in area), however, with progressive temporal stiffening, cells demonstrated growth arrest and underwent phenotypic changes resulting in intratumoral heterogeneity. Unlike widely-investigated static mechanical models, this stiffening hydrogel allowed for progressive phenotypic changes to be observed, and fostered the development of mature organoid-like spheroids, which mimicked both the organisation and acinar-structures of mature breast epithelium. The spheroids contained a central population of cells which expressed aggressive cellular programs, evidenced by increased fibronectin expression and reduction of E-cadherin. The phenotypic heterogeneity observed using this model is more reflective of physiological tumours, demonstrating the importance of establishing temporal cues within preclinical models in future work. Overall, the developed model demonstrated a novel strategy to uncouple ECM biomechanical properties from the cellular complexities of the disease microenvironment and offers the potential for wide applicability in other 3D in vitro disease models through addition of tissue-specific dECM materials.

Restoring natural upper limb movement through a wrist prosthetic module for partial hand amputees.

BACKGROUND: Most partial hand amputees experience limited wrist movement. The limited rotational wrist movement deteriorates natural upper limb system related to hand use and the usability of the prosthetic hand, which may cause secondary damage to the musculoskeletal system due to overuse of the upper limb affected by repetitive compensatory movement patterns. Nevertheless, partial hand prosthetics, in common, have only been proposed without rotational wrist movement because patients have various hand shapes, and a prosthetic hand should be attached to a narrow space. METHODS: We hypothesized that partial hand amputees, when using a prosthetic hand with a wrist rotation module, would achieve natural upper limb movement muscle synergy and motion analysis comparable to a control group. To validate the proposed prototype design with the wrist rotation module and verify our hypothesis, we compared a control group with partial hand amputees wearing hand prostheses, both with and without the wrist rotation module prototype. The study contained muscle synergy analysis through non-negative matrix factorization (NMF) using surface electromyography (sEMG) and motion analyses employing a motion capture system during the reach-to-grasp task. Additionally, we assessed the usability of the prototype design for partial hand amputees using the Jebsen-Taylor hand function test (JHFT). RESULTS: The results showed that the number of muscle synergies identified through NMF remained consistent at 3 for both the control group and amputees using a hand prosthesis with a wrist rotation module. In the motion analysis, a statistically significant difference was observed between the control group and the prosthetic hand without the wrist rotation module, indicating the presence of compensatory movements when utilizing a prosthetic hand lacking this module. Furthermore, among the amputees, the JHFT demonstrated a greater improvement in total score when using the prosthetic hand equipped with a wrist rotation module compared to the prosthetic hand without this module. CONCLUSION: In conclusion, integrating a wrist rotation module in prosthetic hand designs for partial hand amputees restores natural upper limb movement patterns, reduces compensatory movements, and prevent the secondary musculoskeletal. This highlights the importance of this module in enhancing overall functionality and quality of life.

3D biofabrication of diseased human skin models in vitro.

Human skin is an organ located in the outermost part of the body; thus, it frequently exhibits visible signs of physiological health. Ethical concerns and genetic differences in conventional animal studies have increased the need for alternative in vitro platforms that mimic the structural and functional hallmarks of natural skin. Despite significant advances in in vitro skin modeling over the past few decades, different reproducible biofabrication strategies are required to reproduce the pathological features of diseased human skin compared to those used for healthy-skin models. To explain human skin modeling with pathological hallmarks, we first summarize the structural and functional characteristics of healthy human skin. We then provide an extensive overview of how to recreate diseased human skin models in vitro, including models for wounded, diabetic, skin-cancer, atopic, and other pathological skin types. We conclude with an outlook on diseased-skin modeling and its technical perspective for the further development of skin engineering.

Engineering of Uniform Epidermal Layers via Sacrificial Gelatin Bioink-Assisted 3D Extrusion Bioprinting of Skin.

To reconstruct an ideal full-thickness skin model, basal keratinocytes must be distributed as a confluent monolayer on the dermis. However, the currently available extrusion bioprinting method for the skin is limited when producing an air-exposed cellular monolayer because the cells are encapsulated within a bioink. This is the first study to use sacrificial gelatin-assisted extrusion bioprinting to reproduce a uniform and stratified epidermal layer. Experimental analyses of the rheological properties, printability, cell viability, and initial keratinocyte adhesion shows that the optimal gelatin bioink concentration is 4 wt.%. The appropriate thickness of the bioprinted gelatin structure for achieving a confluent keratinocyte layer is determined to be 400 µm. The suggested strategy generates a uniform keratinocyte monolayer with tight junctions throughout the central and peripheral regions, whereas manual seeding generates non-uniform cellular aggregates and vacancies. These results influence gene expression, exhibiting a propensity for epidermal differentiation. Finally, the gelatin-assisted keratinocytes are bioprinted onto a dermis composed of gelatin methacryloyl and dermis-derived decellularized extracellular matrix to establish a full-thickness skin model. Thus, this strategy leads to significant improvements in epidermal differentiation/stratification. The findings demonstrate that the gelatin-assisted approach is advantageous for recreating reliable full-thickness skin models with significant consistency for mass production.

Blood-Lymphatic Integrated System with Heterogeneous Melanoma Spheroids via In-Bath Three-Dimensional Bioprinting for Modelling of Combinational Targeted Therapy.

Although metastatic melanoma can be managed with chemotherapy, its heterogeneity and resistance to therapy remain poorly understood. In addition to the spread of melanoma in the bloodstream, melanoma-stroma interaction and the lymphatic system play active roles in said heterogeneity and resistance, leading to its progression and metastasis. Reproducing the complexities of the melanoma microenvironment in vitro will help understanding its progression and enhance the translatability of potential cancer therapeutics. A blood-lymphatic integrated system with heterogeneous melanoma spheroids (BLISH) using the in-bath bioprinting process is developed. The process uniformly prints size-controllable metastatic melanoma spheroids along with biomimetic blood and lymphatic vessels (LVs). The system reproduces hallmark events of metastatic melanoma, such as tumor stroma interaction, melanoma invasion, and intravasation. The application of the system to investigate the anticancer effect of combinational targeted therapy suggests that it can be used to study the pathophysiology of melanoma and improve the accuracy of drug response monitoring in skin cancer.

Construction of Tissue-Level Cancer-Vascular Model with High-Precision Position Control via In Situ 3D Cell Printing.

During tumor progression, the size and location of the tumor are important factors closely associated with the metastatic potential of the cancer as they largely govern tumor hypoxia and angiogenesis. However, despite the achievements of previous studies, these critical factors are poorly studied, mainly due to the lack of a flexible technique that can readily control 3D tumor mimicking constructs and their spatial relations with vasculature. Here, a novel tissue-level platform consisting of a metastatic cancer unit (MCU) and a perfusable vascular endothelium system (VES) is presented using in situ 3D cell printing. Size-tunable and position-controllable 3D cancer spheroids (500-1000 µm) are directly printed within the established bath bioink with a self-driven perfusable vascular channel. The cancer-vascular interactions are generated through controlling the distance between MCU and VES to investigate metastasis-associated changes at adjacent and distal regions. The result shows that MCU in 600 µm diameter includes hypoxia, invasion, and angiogenetic signaling. The further observations demonstrate that the proximity of MCU to VES augments the epithelial-mesenchymal transition (EMT) in MCU and vascular dysfunction/inflammation in VES, corroborating the positional significance in tumor metastasis. The platform with the precise-positioning control enables the recapitulation of patient's detailed metastatic progression, opening the chance for precision cancer medicine.

HLA-G 14bp Ins/Del Polymorphism in the 3'UTR Region and Acute Rejection in Kidney Transplant Recipients: An Updated Meta-Analysis.

Background and Objectives: Acute kidney injury (AKI) affects the survival rate of kidney transplant organs and patients. Acute rejection (AR) due to AKI may lead to kidney transplantation failure. It is known that there is a relationship between human leukocyte antigen-G (HLA-G), which is involved in immune regulation, and AR in transplant patients. Moreover, 14-bp insertion/deletion polymorphism in the 3' untranslated region (UTR) region of the HLA-G gene is known to affect HLA-G expression. However, its relationship to AR is still controversial. The aim of this study was to investigate whether HLA-G 14-bp insertion/deletion polymorphism contributed to the development of AR in kidney transplant patients using a meta-analysis. Materials and Methods: To perform our meta-analysis, eligible studies about HLA-G 14-bp insertion/deletion polymorphism and AR were searched in electronic databases until 1 June 2021. Finally, a total of 336 patients with AR and 952 patients without AR in relation to kidney transplantation were analyzed from a total of nine studies. Results: In our results, the Del allele and Ins/Del+Del/Del and Del/Del genotypes significantly increased susceptibility of AR in Asian populations [odds ratio (OR) = 2.359, 95% confidence interval (CI) = 1.568-3.550, p = 3.8 × 10-5; OR = 3.357, 95% CI = 1.769-6.370, p = 0.002; OR = 2.750, 95% CI = 1.354-5.587, p = 0.0052 in each model, respectively]. Conclusions: Evidence of the present results indicate that HLA-G 14-bp insertion/deletion polymorphism is associated with susceptibility to AR in the Asian population.

3D Printing of Pharmaceutical Application: Drug Screening and Drug Delivery.

Advances in three-dimensional (3D) printing techniques and the development of tailored biomaterials have facilitated the precise fabrication of biological components and complex 3D geometrics over the past few decades. Moreover, the notable growth of 3D printing has facilitated pharmaceutical applications, enabling the development of customized drug screening and drug delivery systems for individual patients, breaking away from conventional approaches that primarily rely on transgenic animal experiments and mass production. This review provides an extensive overview of 3D printing research applied to drug screening and drug delivery systems that represent pharmaceutical applications. We classify several elements required by each application for advanced pharmaceutical techniques and briefly describe state-of-the-art 3D printing technology consisting of cells, bioinks, and printing strategies that satisfy requirements. Furthermore, we discuss the limitations of traditional approaches by providing concrete examples of drug screening (organoid, organ-on-a-chip, and tissue/organ equivalent) and drug delivery systems (oral/vaginal/rectal and transdermal/surgical drug delivery), followed by the introduction of recent pharmaceutical investigations using 3D printing-based strategies to overcome these challenges.

Engineering of diseased human skin equivalent using 3D cell printing for representing pathophysiological hallmarks of type 2 diabetes in vitro.

Despite many significant advances in 3D cell printing for skin, a disease model displaying the pathological processes present in the native skin has not been reported yet. Therefore, we were motivated for modeling a 3D diseased skin tissue with pathophysiological hallmarks of type 2 diabetes in vitro based on 3D cell printing technique. By stimulating epidermal-dermal intercellular crosstalk found in the native skin, it was hypothesized that normal keratinocytes would be differentiated as diabetic epidermis when interacting with the diabetic dermal compartment. To prove this, a novel wounded skin model was successfully devised during tissue maturation in vitro. Interestingly, the slow re-epithelization was observed in our diabetic model, which is a representative hallmark of diabetic skin. Using the versatility of 3D cell printing, the structural similarities and diabetic properties of the model were further augmented by addition of perfusable vascularized diabetic hypodermis. Insulin resistance, adipocyte hypertrophy, inflammatory reactions, and vascular dysfunction, as the typical hallmarks in diabetes, were found under hyperglycemia. Finally, the feasibility of this new disease model for drug development was successfully demonstrated through application of test drugs. We trust that this study provides a pioneering step towards 3D cell printing-based in vitro skin disease modeling.

Improved chondrogenic performance with protective tracheal design of Chitosan membrane surrounding 3D-printed trachea.

In recent tracheal tissue engineering, limitations in cartilage reconstruction, caused by immature delivery of chondrocyte-laden components, have been reported beyond the complete epithelialization and integration of the tracheal substitutes with the host tissue. In an attempt to overcome such limitations, this article introduces a protective design of tissue-engineered trachea (TraCHIM) composed of a chitosan-based nanofiber membrane (CHIM) and a 3D-printed biotracheal construct. The CHIM was created from chitosan and polycaprolactone (PCL) using an electrospinning process. Upon addition of chitosan to PCL, the diameter of electrospun fibers became thinner, allowing them to be stacked more closely, thereby improving its mechanical properties. Chitosan also enhances the hydrophilicity of the membranes, preventing them from slipping and delaminating over the cell-laden bioink of the biotracheal graft, as well as protecting the construct. Two weeks after implantation in Sprague-Dawley male rats, the group with the TraCHIM exhibited a higher number of chondrocytes, with enhanced chondrogenic performance, than the control group without the membrane. This study successfully demonstrates enhanced chondrogenic performance of TraCHIM in vivo. The protective design of TraCHIM opens a new avenue in engineered tissue research, which requires faster tissue formation from 3D biodegradable materials, to achieve complete replacement of diseased tissue.

Flexible Adipose-Vascular Tissue Assembly Using Combinational 3D Printing for Volume-Stable Soft Tissue Reconstruction.

A new concept, assembling cell-laden tissue modules, is for the first time proposed for soft tissue engineering. Adipose-vascular tissue modules composed of a synthetic polymer-based substructure and customized bioinks using planar 3D cell printing are engineered. Such tissue modules are systematically assembled into a synthetic polymer-based module holder fabricated with rotational 3D printing, resulting in the development of a flexible and volumetric tissue assembly. Whereas most of the previous studies about the construction of adipose tissue are limited to hypoxia, poor vascularization, rapid resorption, and mismatch in mechanical properties, it is aimed to realize the construction of nonhypoxic, flexible, and volume-stable tissue assembly in this study. The significance of engineered tissue assembly is proven through various in vitro and in vivo evaluations. In particular, stable volume and remarkable neovascularization/adipogenesis are observed in the implanted assembly over four weeks. Interestingly, the size of newly formed lipid droplets and the remodeled morphology in the assembly are comparable to those in native adipose tissue. As far as it is known, this work is a first report suggesting a cell printing-based tissue assembly for functional reconstruction of soft tissue.

Intracorneal injection of a detachable hybrid microneedle for sustained drug delivery.

There are increasing demands for long-term and controlled corneal drug delivery to treat various ocular diseases. Although biodegradable ocular inserts or contact lenses have been developed, the invasiveness and inefficiency of the approaches still need to be improved. Microneedle (MN) technology can deliver therapeutic molecules to the eye in a minimally invasive manner. However, the current ocular MN technology is limited to either short-term corneal drug delivery or retinal drug delivery by suprachoroidal injection. For long-term and minimally invasive corneal drug delivery, we have developed a detachable biodegradable MN that can be delivered to the inside of the cornea for sustained drug release. The detachable and biodegradable MN is a hybrid MN consisting of a drug-loaded biodegradable tip and a supporting base. The hybrid MN can be applied to the cornea by impact insertion, and it leaves only the drug-loaded biodegradable tip within the corneal tissue so that it can release the drug for a certain period. By concentration-controlled molding, the dimension of drug-loaded MN tips was precisely controlled and their detachability was optimized. The detachable tip and a supporting base were assembled to form a hybrid MN by pressure-assisted transfer molding. We carefully optimized the dimension of the drug-tip, injection dwell time, and insertion depth to achieve effective intracorneal injection of the drug-tip. The detachable hybrid MN was applied to an Acanthamoeba keratitis model wherein a biodegradable drug-tip was successfully delivered to the inside of the mouse cornea in vivo. Follow-up of the MN-treated cases for 7 days confirmed the therapeutic efficacy of the detachable biodegradable MN tips. STATEMENT OF SIGNIFICANCE: For the treatment of infectious diseases in the cornea, such as keratitis, eye drops need to be applied topically every hour for a couple of days. This is extremely uncomfortable, and poor compliance to such tightly scheduled drug administration can result in permanent scar formation in the cornea. In this work, we demonstrate a simple and rapid injection of biodegradable microneedle tips in the corneal tissue wherein the tips can deliver antibacterial drugs for 4 days to treat keratitis. Unlike other patch-style microneedle technologies, this approach allows for insertion depth-controlled and highly localized injection of detachable individual microneedle tips to the diseased tissue for sustained drug delivery. This overcomes the limitations of patch-style microneedles such as short-term drug delivery and unnecessary blockage of tissue.