Biohybrid printing approaches for cardiac pathophysiological studies.

Bioengineered hearts, which include single cardiomyocytes, engineered heart tissue, and chamber-like models, generate various biosignals, such as contractility, electrophysiological, and volume-pressure dynamic signals. Monitoring changes in these signals is crucial for understanding the mechanisms of disease progression and developing potential treatments. However, current methodologies face challenges in the continuous monitoring of bioengineered hearts over extended periods and typically require sacrificing the sample post-experiment, thereby limiting in-depth analysis. Thus, a biohybrid system consisting of living and nonliving components was developed. This system primarily features heart tissue alongside nonliving elements designed to support or comprehend its functionality. Biohybrid printing technology has simplified the creation of such systems and facilitated the development of various functional biohybrid systems capable of measuring or even regulating multiple functions, such as pacemakers, which demonstrates its versatility and potential applications. The future of biohybrid printing appears promising, with the ongoing exploration of its capabilities and potential directions for advancement.

Bioprinting-Assisted Tissue Assembly for Structural and Functional Modulation of Engineered Heart Tissue Mimicking Left Ventricular Myocardial Fiber Orientation.

Left ventricular twist is influenced by the unique oriented structure of myocardial fibers. Replicating this intricate structural-functional relationship in an in vitro heart model remains challenging, mainly due to the difficulties in achieving a complex structure with synchrony between layers. This study introduces a novel approach through the utilization of bioprinting-assisted tissue assembly (BATA)-a synergistic integration of bioprinting and tissue assembly strategies. By flexibly manufacturing tissue modules and assembly platforms, BATA can create structures that traditional methods find difficult to achieve. This approach integrates engineered heart tissue (EHT) modules, each with intrinsic functional and structural characteristics, into a layered, multi-oriented tissue in a controlled manner. EHTs assembled in different orientations exhibit various contractile forces and electrical signal patterns. The BATA is capable of constructing complex myocardial fiber orientations within a chamber-like structure (MoCha). MoCha replicates the native cardiac architecture by exhibiting three layers and three alignment directions, and it reproduces the left ventricular twist by exhibiting synchronized contraction between layers and mimicking the native cardiac architecture. The potential of BATA extends to engineering tissues capable of constructing and functioning as complete organs on a large scale. This advancement holds the promise of realizing future organ-on-demand technology.

Tissue-specific gelatin bioink as a rheology modifier for high printability and adjustable tissue properties.

Decellularized extracellular matrix (dECM) has emerged as an exceptional biomaterial that effectively recapitulates the native tissue microenvironment for enhanced regenerative potential. Although various dECM bioinks derived from different tissues have shown promising results, challenges persist in achieving high-resolution printing of flexible tissue constructs because of the inherent limitations of dECM's weak mechanical properties and poor printability. Attempts to enhance mechanical rigidity through chemical modifications, photoinitiators, and nanomaterial reinforcement have often compromised the bioactivity of dECM and mismatched the desired mechanical properties of target tissues. In response, this study proposes a novel method involving a tissue-specific rheological modifier, gelatinized dECM. This modifier autonomously enhances bioink modulus pre-printing, ensuring immediate and precise shape formation upon extrusion. The hybrid bioink with GeldECM undergoes a triple crosslinking system-physical entanglement for pre-printing, visible light photocrosslinking during printing for increased efficiency, and thermal crosslinking post-printing during tissue culture. A meticulous gelatinization process preserves the dECM protein components, and optimal hybrid ratios modify the mechanical properties, tailoring them to specific tissues. The application of this sequential multiple crosslinking designs successfully yielded soft yet resilient tissue constructs capable of withstanding vigorous agitation with high shape fidelity. This innovative method, founded on mechanical modulation by GeldECM, holds promise for the fabrication of flexible tissues with high resilience.

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.

The Effect of 3-Dimensional-Printed Sequential Dual Drug-Releasing Patch on the Capsule Formation Around the Silicone Implant in a Rat Model.

BACKGROUND: Implant-based breast reconstruction is associated with increased risk of early infection and late-stage capsular contracture. OBJECTIVES: We evaluated the feasibility of a dual drug-releasing patch that enabled the controlled delivery of antibiotics and immunosuppressants in a temporally and spatially appropriate manner to the implant site. METHODS: The efficacy of a dual drug-releasing patch, which was 3-dimensional-printed (3D-printed) with tissue-derived biomaterial ink, was evaluated in rats with silicone implants. The groups included implant only (n = 10); implant plus bacterial inoculation (n = 14); implant, bacterial inoculation, and patch loaded with gentamycin placed on the ventral side of the implant (n = 10), and implant, bacterial inoculation, and patch loaded with gentamycin and triamcinolone acetonide (n = 9). Histologic and immunohistochemical analyses were performed 8 weeks after implantation. RESULTS: The 2 drugs were sequentially released from the dual drug-releasing patch and exhibited different release profiles. Compared to the animals with bacterial inoculation, those with the antibiotic-only and the dual drug-releasing patch exhibited thinner capsules and lower myofibroblast activity and inflammation, indicating better tissue integration and less foreign body response. These effects were more pronounced with the dual drug-releasing patch than with the antibiotic-only patch. CONCLUSIONS: The 3D-printed dual drug-releasing patch effectively reduced inflammation and capsule formation in a rat model of silicone breast reconstruction. The beneficial effect of the dual drug-releasing patch was better than that of the antibiotic-only patch, indicating its therapeutic potential as a novel approach to preventing capsular contracture while reducing concerns of systemic side effects.

Laminin-Augmented Decellularized Extracellular Matrix Ameliorating Neural Differentiation and Neuroinflammation in Human Mini-Brains.

Non-neural extracellular matrix (ECM) has limited application in humanized physiological neural modeling due to insufficient brain-specificity and safety concerns. Although brain-derived ECM contains enriched neural components, certain essential components are partially lost during the decellularization process, necessitating augmentation. Here, it is demonstrated that the laminin-augmented porcine brain-decellularized ECM (P-BdECM) is xenogeneic factor-depleted as well as favorable for the regulation of human neurons, astrocytes, and microglia. P-BdECM composition is comparable to human BdECM regarding brain-specificity through the matrisome and gene ontology-biological process analysis. As augmenting strategy, laminin 111 supplement promotes neural function by synergic effect with laminin 521 in P-BdECM. Annexin A1(ANXA1) and Peroxiredoxin(PRDX) in P-BdECM stabilized microglial and astrocytic behavior under normal while promoting active neuroinflammation in response to neuropathological factors. Further, supplementation of the brain-specific molecule to non-neural matrix also ameliorated glial cell inflammation as in P-BdECM. In conclusion, P-BdECM-augmentation strategy can be used to recapitulate humanized pathophysiological cerebral environments for neurological study.

Nanomaterials-incorporated hydrogels for 3D bioprinting technology.

In the field of tissue engineering and regenerative medicine, various hydrogels derived from the extracellular matrix have been utilized for creating engineered tissues and implantable scaffolds. While these hydrogels hold immense promise in the healthcare landscape, conventional bioinks based on ECM hydrogels face several challenges, particularly in terms of lacking the necessary mechanical properties required for 3D bioprinting process. To address these limitations, researchers are actively exploring novel nanomaterial-reinforced ECM hydrogels for both mechanical and functional aspects. In this review, we focused on discussing recent advancements in the fabrication of engineered tissues and monitoring systems using nanobioinks and nanomaterials via 3D bioprinting technology. We highlighted the synergistic benefits of combining numerous nanomaterials into ECM hydrogels and imposing geometrical effects by 3D bioprinting technology. Furthermore, we also elaborated on critical issues remaining at the moment, such as the inhomogeneous dispersion of nanomaterials and consequent technical and practical issues, in the fabrication of complex 3D structures with nanobioinks and nanomaterials. Finally, we elaborated on plausible outlooks for facilitating the use of nanomaterials in biofabrication and advancing the function of engineered tissues.

Contrast Agent-Free 3D Renal Ultrafast Doppler Imaging Reveals Vascular Dysfunction in Acute and Diabetic Kidney Diseases.

To combat the irreversible decline in renal function associated with kidney disease, it is essential to establish non-invasive biomarkers for assessing renal microcirculation. However, the limited resolution and/or vascular sensitivity of existing diagnostic imaging techniques hinders the visualization of complex cortical vessels. Here, a 3D renal ultrafast Doppler (UFD) imaging system that uses a high ultrasound frequency (18 MHz) and ultrahigh frame rate (1 KHz per slice) to scan the entire volume of a rat's kidney in vivo is demonstrated. The system, which can visualize the full 3D renal vascular branching pyramid at a resolution of 167 µm without any contrast agent, is used to chronically and noninvasively monitor kidneys with acute kidney injury (AKI, 3 days) and diabetic kidney disease (DKD, 8 weeks). Multiparametric UFD analyses (e.g., vessel volume occupancy (VVO), fractional moving blood volume (FMBV), vessel number density (VND), and vessel tortuosity (VT)) describe rapid vascular rarefaction from AKI and long-term vascular degeneration from DKD, while the renal pathogeneses are validated by in vitro blood serum testing and stained histopathology. This work demonstrates the potential of 3D renal UFD to offer valuable insights into assessing kidney perfusion levels for future research in diabetes and kidney transplantation.

3D Cell Printing of Advanced Vascularized Proximal Tubule-on-a-Chip for Drug Induced Nephrotoxicity Advancement.

Renal dysfunction due to drug-induced nephrotoxicity (DIN) affects >20% of the adult population worldwide. The vascularized proximal tubule is a complex structure that is often the primary site of drug-induced kidney injury. Herein, a vascularized proximal tubule-on-a-chip (Vas-POAC) was fabricated, demonstrating improved physiological emulation over earlier single-cell proximal tubule models. A perfusable model of vascularized proximal tubules permits the growth and proliferation of renal proximal tubule cells and adjacent endothelial cells under various conditions. An in vitro Vas-POAC showed mature expressions of the tubule and endothelial cell markers in the mature epithelium and endothelium lumens after 7 days of culture. Expression in the mature proximal tubule epithelium resembled the polarized expression of sodium-glucose cotransporter-2 and the de novo synthesis of ECM proteins. These perfusable Vas-POACs display significantly improved functional properties relative to the proximal tubules-on-a-chip (POAC), which lacks vascular components. Furthermore, the developed Vas-POAC model evaluated the cisplatin-induced nephrotoxicity and revealed enhanced drug receptivity compared to POAC. We further evaluated the capability of the developed proximal tubule model to act as a functional platform that targets screening drug doses that can cause renal proximal tubule injury in adults. Thus, our cell-printed models may prove valuable for screening, thoughtful mechanistic investigations of DIN, and discovery of drugs that interfere with tubule formation.

Anthropogenic carbon dioxide origin tracing study in Anmyeon-do, South Korea: Based on STILT-footprint and emissions data.

Tracking the origin of greenhouse gases (GHGs) is critical for understanding regional GHG concentration variation and developing effective policies to reduce GHG emissions. This study provides quantitative information about the surface contribution to enhancement of carbon dioxide (CO2) concentration at Anmyeon-do (AMY), South Korea, using Stochastic Time-Inverted Lagrangian Transport (STILT) model and anthropogenic CO2 emission data. CO2 enhancement simulated by the STILT and emission data was positively correlated with measured CO2 anomalies at AMY with a correlation coefficient above 0.5. High and low CO2 days were selected using ground measurements of the CO2 mixing ratio at AMY during the winter season of 2018-2019. The surface contributions for the high and low CO2 days at AMY were compared quantitatively. When the high concentration was observed in AMY, the CO2 enhancements were dominated by domestic regions, especially from the metropolitan area in South Korea, due to the high footprint and large CO2 emissions. From the perspective of foreign regions, the surface contribution of eastern China regions (Shandong, Jiangsu-Shanghai) increased during high CO2 days compared to low CO2 days at AMY. During the high CO2 days, the ratio between CO2 and carbon monoxide, a co-emitted species, is large when the surface contribution of eastern China regions is relatively strong due to different regional combustion efficiency (i.e., high combustion efficiency in South Korea compared to that in China). The surface contribution based on STILT and emission data is useful for understanding the cause of high GHG concentration at the receptor (AMY in this study).

3D bioprinted vascularized lung cancer organoid models with underlying disease capable of more precise drug evaluation.

Despite encouraging progress in the development ofin vitrocancer models,in vitrocancer models that simultaneously recapitulate the complexity of the tumor microenvironment and its diverse cellular components and genetic properties remain lacking. Here, an advanced vascularized lung cancer (LC) model is proposed, which includes patient-derived LC organoids (LCOs), lung fibroblasts, and perfusable vessels using 3D bioprinting technology. To better recapitulate the biochemical composition of native lung tissues, a porcine lung-derived decellularized extracellular matrix (LudECM) hydrogel was produced to offer physical and biochemical cues to cells in the LC microenvironment. In particular, idiopathic pulmonary fibrosis-derived lung fibroblasts were used to implement fibrotic niches similar to actual human fibrosis. It was shown that they increased cell proliferation and the expression of drug resistance-related genes in LCOs with fibrosis. In addition, changes in resistance to sensitizing targeted anti-cancer drugs in LCOs with fibrosis were significantly greater in LudECM than in that Matrigel. Therefore, assessment of drug responsiveness in vascularized LC models that recapitulate lung fibrosis can help determine the appropriate therapy for LC patients accompanied by fibrosis. Furthermore, it is expected that this approach could be utilized for the development of targeted therapies or the identification of biomarkers for LC patients accompanied by fibrosis.

3D printable conductive composite inks for the fabrication of biocompatible electrodes in tissue engineering application.

Native tissues are affected by the microenvironment surrounding the tissue, including electrical activities. External electrical stimulation, which is used in replicating electrical activities and regulating cell behavior, is mainly applied in neural and cardiac tissues due to their electrophysiological properties. The in vitro cell culture platform with electrodes provides precise control of the stimulation property and eases the observation of the effects on the cells. The frequently used electrodes are metal or carbon rods, but their risk of damaging tissue and their mechanical properties that are largely different from those of native tissues hinder further applications. Biocompatible polymer reinforced with conductive fillers emerges as a potential solution to fabricate the complex structure of the platform and electrode. Conductive polymer can be used as an ink in the extrusion-based printing method, thus enabling the fabrication of volumetric structures. The filler simultaneously alters the electrical and rheological properties of the ink; therefore, the amount of additional compound should be precisely determined regarding printability and conductivity. This review provides an overview on the rheology and conductivity change relative to the concentration of conductive fillers and the applications of printed electrodes. Next, we discuss the future potential use of a cell culture platform with electrodes from in vitro and in vivo perspectives.

Coaxial cell printing of a human glomerular model: anin vitroglomerular filtration barrier and its pathophysiology.

Much effort has been expended in emulating the kidney's glomerular unit because of its limitless potential in the field of drug screening and nephrotoxicity testing in clinics. Herein, we fabricate a functional bilayer glomerular microvessel-on-a-chip that recapitulates the specific arrangement of the glomerular endothelial cell, podocyte layers, and the intervening glomerular basement membrane (GBM) in a single step. Our perfusable chip allows for the co-culture of monolayer glomerular endothelium and podocyte epithelium, which display mature functional markers of glomerular cells, and their proper interactions produce GBM proteins, which are the major components of the GBMin vivo. Furthermore, we test the selective permeability capacity, a representative hallmark function of the glomerular filtration barrier. Lastly, we evaluate the response of our glomerular model to Adriamycin- and hyperglycemia-induced injury to evaluate its applicability for drug screening and glomerular disease modeling.

Biohybrid 3D Printing of a Tissue-Sensor Platform for Wireless, Real-Time, and Continuous Monitoring of Drug-Induced Cardiotoxicity.

Drug-induced cardiotoxicity is regarded as a major hurdle in the early stages of drug development. Although there are various methods for preclinical cardiotoxicity tests, they cannot completely predict the cardiotoxic potential of a compound due to the lack of physiological relevance. Recently, 3D engineered heart tissue (EHT) has been used to investigate cardiac muscle functions as well as pharmacological effects by exhibiting physiological auxotonic contractions. However, there is still no adequate platform for continuous monitoring to test acute and chronic pharmacological effects in vitro. Here, a biohybrid 3D printing method for fabricating a tissue-sensor platform, composed of a bipillar-grafted strain gauge sensor and EHT, is first introduced. Two pillars are three-dimensionally printed as grafts onto a strain gauge-embedded substrate to promote the EHT contractility and guide the self-assembly of the EHTs along with the strain gauge. In addition, the integration of a wireless multi-channel electronic system allows for continuous monitoring of the EHT contractile force by the tissue-sensor platform and, ultimately, for the observation of the acute and chronic drug effects of cardiotoxicants. In summary, biohybrid 3D printing technology is expected to be a potential fabrication method to provide a next-generation tissue-sensor platform for an effective drug development process.

3D printed multi-growth factor delivery patches fabricated using dual-crosslinked decellularized extracellular matrix-based hybrid inks to promote cerebral angiogenesis.

Generally, brain angiogenesis is a tightly regulated process, which scarcely occurred in the absence of specific pathological conditions. Delivery of exogenous angiogenic factors enables the induction of desired angiogenesis by stimulating neovasculature formation. However, effective strategies of mimicking the angiogenesis process with exogenous factors have not yet been fully explored. Herein, we develop a 3D printed spatiotemporally compartmentalized cerebral angiogenesis inducing (SCAI) hydrogel patch, releasing dual angiogenic growth factors (GFs), using extracellular matrix-based hybrid inks. We introduce a new hybrid biomaterial-based ink for printing patches through dual crosslinking mechanisms: Chemical crosslinking with aza-Michael addition reaction with combining methacrylated hyaluronic acid (HAMA) and vascular-tissue-derived decellularized extracellular matrix (VdECM), and thermal crosslinking of VdECM. 3D printing technology, a useful approach with fabrication versatility with customizable systems and multiple biomaterials, is adopted to print three-layered hydrogel patch with spatially separated dual GFs as outer- and inner-layers that provide tunable release profiles of multiple GFs and fabrication versatility. Consequently, these layers of the patch spatiotemporally separated with dual GFs induce excellent neovascularization in the brain area, monitored by label-free photoacoustic microscopy in vivo. The developed multi-GFs releasing patch may offer a promising therapeutic approach of spatiotemporal drugs releasing such as cerebral ischemia, ischemic heart diseases, diabetes, and even use as vaccines. STATEMENT OF SIGNIFICANCE: Effective strategies of mimicking the angiogenesis process with exogenous factors have not yet been fully explored. In this study, we develop a 3D printed spatiotemporally compartmentalized cerebral angiogenesis inducing (SCAI) hydrogel patch, releasing dual angiogenic growth factors (GFs) using extracellular matrix-based hybrid inks. We introduce a new hybrid biomaterial-based ink through dual crosslinking mechanisms: Chemical crosslinking with aza-Michael addition, and thermal crosslinking. 3D printing technology is adopted to print three-layered hydrogel patch with spatially separated dual GFs as outer- and inner-layers that provide tunable release profiles of multiple GFs and fabrication versatility. Consequently, these layers of the patch spatiotemporally separated with dual GFs induce excellent neovascularization in the brain area, monitored by photoacoustic microscopy in vivo.

Extracellular matrix-based sticky sealants for scar-free corneal tissue reconstruction.

Regenerative medicine requires both tissue restoration and ease of compliance for clinical application. Considering this, sticky tissue sealants have been shown to have great potentials over surgical suturing and wound treatment. However, tissue sealants currently used pose challenges such as uncontrollable adhesion formation, mechanical mismatch, and lack of tissue restoration. A new sticky sealant based on gelatinized cornea-derived extracellular matrix (GelCodE) with a visible light-activating system is firstly being introduced in this study. De novo tissue regeneration relies on the matrisome in charge of tissue-organization and development within GelCodE while visible light-based photopolymerization with ruthenium/sodium persulfate rapidly induces covalent bonds with the adjacent tissues. The ease of not only in vivo application, biocompatibility, and biointegration, but also exceptional de novo tissue formation is demonstrated in this study. Interestingly, newly regenerated tissues were shown to have normal tissue-like matrices with little scar formation. Hence, this work presents a promising strategy to meet clinical demands for scar-free tissue recovery with superior ease of clinical application.

Bioprinting-assisted tissue assembly to generate organ substitutes at scale.

Various external cues can guide cellular behavior and maturation during developmental processes. Recent studies on bioprinting-assisted tissue engineering have considered this a practical, versatile, and flexible way to provide external cues to developing engineered tissues. An ensemble of multiple external cues can improve the speed and capability of morphogenesis. In this review, we discuss how bioprinting and biomaterials provide multiple guidance to generate micro-sized building blocks with specific shapes and also highlight their applications in tissue assembly toward volumetric tissue and organ generation. Furthermore, we discuss our perspectives on the future translation of bioprinting technologies integrated with artificial intelligence (AI) and robot-assisted apparatus to promote automation, standardization, and clinical translation of bioprinted tissues.

Electroconductive, Adhesive, Non-Swelling, and Viscoelastic Hydrogels for Bioelectronics.

As a new class of materials, implantable flexible electrical conductors have recently been developed and applied to bioelectronics. An ideal electrical conductor requires high conductivity, tissue-like mechanical properties, low toxicity, reliable adhesion to biological tissues, and the ability to maintain its shape in wet physiological environments. Despite significant advances, electrical conductors that satisfy all these requirements are insufficient. Herein, a facile method for manufacturing a new conductive hydrogels through the simultaneous exfoliation of graphite and polymerization of zwitterionic monomers triggered by microwave irradiation is introduced. The mechanical properties of the obtained conductive hydrogel are similar to those of living tissue, which is ideal as a bionic adhesive for minimizing contact damage due to mechanical mismatches between hard electronics and soft tissues. Furthermore, it exhibits excellent adhesion performance, electrical conductivity, non-swelling, and high conformability in water. Excellent biocompatibility of the hydrogel is confirmed through a cytotoxicity test using C2C12 cells, a biocompatibility test on rat tissues, and their histological analysis. The hydrogel is then implanted into the sciatic nerve of a rat and neuromodulation is demonstrated through low-current electrical stimulation. This hydrogel demonstrates a tissue-like extraneuronal electrode, which possesses high conformability to improve the tissue-electronics interfaces, promising next-generation bioelectronics applications.

3D cell-printing of gradient multi-tissue interfaces for rotator cuff regeneration.

Owing to the prevalence of rotator cuff (RC) injuries and suboptimal healing outcome, rapid and functional regeneration of the tendon-bone interface (TBI) after RC repair continues to be a major clinical challenge. Given the essential role of the RC in shoulder movement, the engineering of biomimetic multi-tissue constructs presents an opportunity for complex TBI reconstruction after RC repair. Here, we propose a gradient cell-laden multi-tissue construct combined with compositional gradient TBI-specific bioinks via 3D cell-printing technology. In vitro studies demonstrated the capability of a gradient scaffold system in zone-specific inducibility and multi-tissue formation mimicking TBI. The regenerative performance of the gradient scaffold on RC regeneration was determined using a rat RC repair model. In particular, we adopted nondestructive, consecutive, and tissue-targeted near-infrared fluorescence imaging to visualize the direct anatomical change and the intricate RC regeneration progression in real time in vivo. Furthermore, the 3D cell-printed implant promotes effective restoration of shoulder locomotion function and accelerates TBI healing in vivo. In summary, this study identifies the therapeutic contribution of cell-printed constructs towards functional RC regeneration, demonstrating the translational potential of biomimetic gradient constructs for the clinical repair of multi-tissue interfaces.

Predicting multipotency of human adult stem cells derived from various donors through deep learning.

Adult stem cell-based therapeutic approaches have great potential in regenerative medicine because of their immunoregulatory properties and multidifferentiation capacity. Nevertheless, the outcomes of stem cell­based therapies to date have shown inconsistent efficacy owing to donor variation, thwarting the expectation of clinical effects. However, such donor dependency has been elucidated by biological consequences that current research could not predict. Here, we introduce cellular morphology-based prediction to determine the multipotency rate of human nasal turbinate stem cells (hNTSCs), aiming to predict the differentiation rate of keratocyte progenitors. We characterized the overall genes and morphologies of hNTSCs from five donors and compared stemness-related properties, including multipotency and specific lineages, using mRNA sequencing. It was demonstrated that transformation factors affecting the principal components were highly related to cell morphology. We then performed a convolutional neural network-based analysis, which enabled us to assess the multipotency level of each cell group based on their morphologies with 85.98% accuracy. Surprisingly, the trend in expression levels after ex vivo differentiation matched well with the deep learning prediction. These results suggest that AI­assisted cellular behavioral prediction can be utilized to perform quantitative, non-invasive, single-cell, and multimarker characterizations of live stem cells for improved quality control in clinical cell therapies.