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.

3D bioprinted multilayered cerebrovascular conduits to study cancer extravasation mechanism related with vascular geometry.

Cerebral vessels are composed of highly complex structures that facilitate blood perfusion necessary for meeting the high energy demands of the brain. Their geometrical complexities alter the biophysical behavior of circulating tumor cells in the brain, thereby influencing brain metastasis. However, recapitulation of the native cerebrovascular microenvironment that shows continuities between vascular geometry and metastatic cancer development has not been accomplished. Here, we apply an in-bath 3D triaxial bioprinting technique and a brain-specific hybrid bioink containing an ionically crosslinkable hydrogel to generate a mature three-layered cerebrovascular conduit with varying curvatures to investigate the physical and molecular mechanisms of cancer extravasation in vitro. We show that more tumor cells adhere at larger vascular curvature regions, suggesting that prolongation of tumor residence time under low velocity and wall shear stress accelerates the molecular signatures of metastatic potential, including endothelial barrier disruption, epithelial-mesenchymal transition, inflammatory response, and tumorigenesis. These findings provide insights into the underlying mechanisms driving brain metastases and facilitate future advances in pharmaceutical and medical research.

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.

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.

3D bioprinting of a trachea-mimetic cellular construct of a clinically relevant size.

Despite notable advances in extrusion-based 3D bioprinting, it remains a challenge to create a clinically-sized cellular construct using extrusion-based 3D printing due to long printing times adversely affecting cell viability and functionality. Here, we present an advanced extrusion-based 3D bioprinting strategy composed of a two-step printing process to facilitate creation of a trachea-mimetic cellular construct of clinically relevant size. A porous bellows framework is first printed using typical extrusion-based 3D printing. Selective printing of cellular components, such as cartilage rings and epithelium lining, is then performed on the outer grooves and inner surface of the bellows framework by a rotational printing process. With this strategy, 3D bioprinting of a trachea-mimetic cellular construct of clinically relevant size is achieved in significantly less total printing time compared to a typical extrusion-based 3D bioprinting strategy which requires printing of an additional sacrificial material. Tracheal cartilage formation was successfully demonstrated in a nude mouse model through a subcutaneous implantation study of trachea-mimetic cellular constructs wrapped with a sinusoidal-patterned tubular mesh preventing rapid resorption of cartilage rings in vivo. This two-step 3D bioprinting for a trachea-mimetic cellular construct of clinically relevant size can provide a fundamental step towards clinical translation of 3D bioprinting based tracheal reconstruction.

Neural stem cell delivery using brain-derived tissue-specific bioink for recovering from traumatic brain injury.

Traumatic brain injury is one of the leading causes of accidental death and disability. The loss of parts in a severely injured brain induces edema, neuronal apoptosis, and neuroinflammation. Recently, stem cell transplantation demonstrated regenerative efficacy in an injured brain. However, the efficacy of current stem cell therapy needs improvement to resolve issues such as low survival of implanted stem cells and low efficacy of differentiation into respective cells. We developed brain-derived decellularized extracellular matrix (BdECM) bioink that is printable and has native brain-like stiffness. This study aimed to fabricate injured cavity-fit scaffold with BdECM bioink and assessed the utility of BdECM bioink for stem cell delivery to a traumatically injured brain. Our BdECM bioink had shear thinning property for three-dimensional (3D)-cell-printing and physical properties and fiber structures comparable to those of the native brain, which is important for tissue integration after implantation. The human neural stem cells (NSCs) (F3 cells) laden with BdECM bioink were found to be fully differentiated to neurons; the levels of markers for mature differentiated neurons were higher than those observed with collagen bioinkin vitro. Moreover, the BdECM bioink demonstrated potential in defect-fit carrier fabrication with 3D cell-printing, based on the rheological properties and shape fidelity of the material. As F3 cell-laden BdECM bioink was transplanted into the motor cortex of a rat brain, high efficacy of differentiation into mature neurons was observed in the transplanted NSCs; notably increased level of MAP2, a marker of neuronal differentiation, was observed. Furthermore, the transplanted-cell bioink suppressed reactive astrogliosis and microglial activation that may impede regeneration of the injured brain. The brain-specific material reported here is favorable for NSC differentiation and suppression of neuroinflammation and is expected to successfully support regeneration of a traumatically injured brain.

Tissue-Specific Decellularized Extracellular Matrix Bioinks for Musculoskeletal Tissue Regeneration and Modeling Using 3D Bioprinting Technology.

The musculoskeletal system is a vital body system that protects internal organs, supports locomotion, and maintains homeostatic function. Unfortunately, musculoskeletal disorders are the leading cause of disability worldwide. Although implant surgeries using autografts, allografts, and xenografts have been conducted, several adverse effects, including donor site morbidity and immunoreaction, exist. To overcome these limitations, various biomedical engineering approaches have been proposed based on an understanding of the complexity of human musculoskeletal tissue. In this review, the leading edge of musculoskeletal tissue engineering using 3D bioprinting technology and musculoskeletal tissue-derived decellularized extracellular matrix bioink is described. In particular, studies on in vivo regeneration and in vitro modeling of musculoskeletal tissue have been focused on. Lastly, the current breakthroughs, limitations, and future perspectives are described.

3D Cell-Printed Hypoxic Cancer-on-a-Chip for Recapitulating Pathologic Progression of Solid Cancer.

Cancer microenvironment has a significant impact on the progression of the disease. In particular, hypoxia is the key driver of cancer survival, invasion, and chemoresistance. Although several in vitro models have been developed to study hypoxia-related cancer pathology, the complex interplay of the cancer microenvironment observed in vivo has not been reproduced yet owing to the lack of precise spatial control. Instead, 3D biofabrication approaches have been proposed to create microphysiological systems for better emulation of cancer ecology and accurate anticancer treatment evaluation. Herein, we propose a 3D cell-printing approach to fabricate a hypoxic cancer-on-a-chip. The hypoxia-inducing components in the chip were determined based on a computer simulation of the oxygen distribution. Cancer-stroma concentric rings were printed using bioinks containing glioblastoma cells and endothelial cells to recapitulate a type of solid cancer. The resulting chip realized central hypoxia and aggravated malignancy in cancer with the formation of representative pathophysiological markers. Overall, the proposed approach for creating a solid-cancer-mimetic microphysiological system is expected to bridge the gap between in vivo and in vitro models for cancer research.

Comparison of epidemiology, emergency care, and outcomes of acute ischemic stroke between young adults and elderly in Korean population: a multicenter observational study.

Stroke in young adults has been known to show a lower incidence and a better prognosis. Only a few studies have examined the epidemiology and outcomes of ischemic stroke in young adults and compared them with the elderly in Korean population. All consecutive patients with ischemic stroke visiting 29 participating emergency departments were enrolled from November 2007 to October 2009. Patients with less than 15 yr of age and unknown information on age and confirmed diagnosis were excluded. We categorized the patients into young adults (15 to 45 yr) and elderly (46 yr and older) groups. Of 39,156 enrolled all stroke patients, 25,818 with ischemic stroke were included and analyzed (young adult; n=1,431, 5.5%). Young adult patients showed lower prevalence of most chronic diseases but significantly higher prevalence in exercise, current smoking, and alcohol consumption. Hospital mortality was significantly lower in young adults than elderly (1.1% vs. 3.1%, P<0.001). Higher number of patients in elderly group (68.1%) showed worsening change of modified Rankin Scale than young adults (65.2%). Young adults ischemic stroke showed favorable hospital outcomes than the elderly in Korean population.