APL Bioengineering最新文献

Regenerative cartilage replacements are increasingly required in clinical settings for various defect repairs, including bronchial cartilage deficiency, articular cartilage injury, and microtia reconstruction. Poly (glycerol sebacate) (PGS) is a widely used bioelastomer that has been developed for various regenerative medicine applications because of its excellent elasticity, biodegradability, and biocompatibility. However, because of inadequate active groups, strong hydrophobicity, and limited ink extrusion accuracy, 3D printed PGS scaffolds may cause insufficient bioactivity, inefficient cell inoculation, and inconsistent cellular composition, which seriously hinders its further cartilage regenerative application. Here, we combined 3D printed PGS frameworks with an encapsulated gelatin hydrogel to fabricate a PGS@Gel composite scaffold. PGS@Gel scaffolds have a controllable porous microstructure, with suitable pore sizes and enhanced hydrophilia, which could significantly promote the cells' penetration and adhesion for efficient chondrocyte inoculation. Furthermore, the outstanding elasticity and fatigue durability of the PGS framework enabled the regenerated cartilage built by the PGS@Gel scaffolds to resist the dynamic in vivo environment and maintain its original morphology. Importantly, PGS@Gel scaffolds increased the rate of cartilage regeneration concurrent with scaffold degradation. The scaffold was gradually degraded and integrated to form uniform, dense, and mature regenerated cartilage tissue with little scaffold residue.

Drug-induced nephrotoxicity is a leading cause of drug attrition, partly due to the limited relevance of pre-clinical models of the proximal tubule. Culturing proximal tubule epithelial cells (PTECs) under fluid flow to mimic physiological shear stress has been shown to improve select phenotypes, but existing flow systems are expensive and difficult to implement by non-experts in microfluidics. Here, we designed and fabricated an accessible and modular flow system for culturing PTECs under physiological shear stress, which induced native-like cuboidal morphology, downregulated pathways associated with hypoxia, stress, and injury, and upregulated xenobiotic metabolism pathways. We also compared the expression profiles of shear-dependent genes in our in vitro PTEC tissues to that of ex vivo proximal tubules and observed stronger clustering between ex vivo proximal tubules and PTECs under physiological shear stress relative to PTECs under negligible shear stress. Together, these data illustrate the utility of our user-friendly flow system and highlight the role of shear stress in promoting native-like morphological and transcriptomic phenotypes in PTECs in vitro, which is critical for developing more relevant pre-clinical models of the proximal tubule for drug screening or disease modeling.

Accurately modeling oxygen transport and consumption is crucial to predict metabolic dynamics in cell cultures and optimize the design of tissue and organ models. We present a methodology to characterize the Michaelis-Menten oxygen consumption parameters in vitro, integrating novel experimental techniques and computational tools. The parameters were derived for hepatic cell cultures with different dimensionality (i.e., 2D and 3D) and with different surface and volumetric densities. To quantify cell packing regardless of the dimensionality of cultures, we devised an image-based metric, referred to as the proximity index. The Michaelis-Menten parameters were related to the proximity index through an uptake coefficient, analogous to a diffusion constant, enabling the quantitative analysis of oxygen dynamics across dimensions. Our results show that Michaelis-Menten parameters are not constant for a given cell type but change with dimensionality and cell density. The maximum consumption rate per cell decreases significantly with cell surface and volumetric density, while the Michaelis-Menten constant tends to increase. In addition, the dependency of the uptake coefficient on the proximity index suggests that the oxygen consumption rate of hepatic cells is superadaptive, as they modulate their oxygen utilization according to its local availability and to the proximity of other cells. We describe, for the first time, how cells consume oxygen as a function of cell proximity, through a quantitative index, which combines cell density and dimensionality. This study enhances our understanding of how cell-cell interaction affects oxygen dynamics and enables better prediction of aerobic metabolism in tissue models, improving their translational value.

Tumor cells metastasize to distant organs mainly via hematogenous dissemination, in which circulating tumor cells (CTCs) are relatively vulnerable, and eliminating these cells has great potential to prevent metastasis. In vasculature, natural killer (NK) cells are the major effector lymphocytes for efficient killing of CTCs under fluid shear stress (FSS), which is an important mechanical cue in tumor metastasis. However, the influence of FSS on the cytotoxicity of NK cells against CTCs remains elusive. We report that the death rate of CTCs under both NK cells and FSS is much higher than the combined death induced by either NK cells or FSS, suggesting that FSS may enhance NK cell's cytotoxicity. This death increment is elicited by shear-induced NK activation and granzyme B entry into target cells rather than the death ligand TRAIL or secreted cytokines TNF-α and IFN-γ. When NK cells form conjugates with CTCs or adhere to MICA-coated substrates, NK cell activating receptor NKG2D can directly sense FSS to induce NK activation and degranulation. These findings reveal the promotive effect of FSS on NK cell's cytotoxicity toward CTCs, thus providing new insight into immune surveillance of CTCs within circulation.

Interfacing the human body with the next generation of electronics requires technological advancement in designing and producing bioelectronic circuits. These circuits must integrate electrical functionality while simultaneously addressing limitations in mechanical compliance and dynamics, biocompatibility, and consistent, scalable manufacturing. The combination of mechanically disparate materials ranging from elastomers to inorganic crystalline semiconductors calls for modular designs with reliable and scalable electromechanical connectors. Here, we report on a novel interconnection solution for soft-to-flexible bioelectronic interfaces using a patterned and machined flexible printed circuit board, which we term FlexComb, interfaced with soft transducing systems. Using a simple assembly process, arrays of protruding "fingers" bearing individual electrical terminals are laser-machined on a standard flexible printed circuit board to create a comb-like structure, namely, the FlexComb. A matching pattern is also machined in the soft system to host and interlock electromechanically the FlexComb connections via a soft electrically conducting composite. We examine the electrical and electromechanical properties of the interconnection and demonstrate the versatility and scalability of the method through various customized submillimetric designs. In a pilot in vivo study, we validate the stability and compatibility of the FlexComb technology in a subdural electrocorticography system implanted for 6 months on the auditory cortex of a minipig. The FlexComb provides a reliable and simple technique to bond and connect soft transducing systems with flexible or rigid electronic boards, which should find many implementations in soft robotics and wearable and implantable bioelectronics.

Untreated osteochondral defects are a leading cause of osteoarthritis, a condition that places a heavy burden on both patients and orthopedic surgeons. Although tissue engineering has shown promise for creating mechanically similar cartilage-like constructs, their integration with cartilage remains elusive. Therefore, a formulation of biodegradable, biocompatible biomaterial with sufficient mechanical and adhesive properties for cartilage repair is required. To accomplish this, we prepared biocompatible, photo-curable, mechanically robust, and highly adhesive GelMA-glycol chitosan (GelMA-GC) hydrogels. GelMA-GC hydrogels had a modulus of 283 kPa and provided a biocompatible environment (>70% viability of embedded chondrocytes) in long-term culture within a bovine cartilage ring. The adhesive strength of bovine chondrocyte-laden GelMA-GC hydrogel to bovine cartilage increased from 38 to 52 kPa over four weeks of culture. Moreover, intermittent uniaxial mechanical stimulation enhanced the adhesive strength to ∼60 kPa, indicating that the cartilage-hydrogel integration could remain secure and functional under dynamic loading conditions. Furthermore, gene expression data and immunofluorescence staining revealed the capacity of chondrocytes in GelMA-GC hydrogel to synthesize chondrogenic markers (COL2A1 and ACAN), suggesting the potential for tissue regeneration. The promising in vitro results of this work motivate further exploration of the potential of photo-curable GelMA-GC bioadhesive hydrogels for cartilage repair and regeneration.

3D in vivo imaging techniques facilitate disease tracking and treatment, but bulky configurations and motion artifacts limit practical clinical applications. Compact light-field cameras with microlens arrays offer a feasible option for rapid volumetric imaging, yet their utilization in clinical practice necessitates an increased depth-of-field for handheld operation. Here, we report deep focus light-field camera (DF-LFC) with crosstalk-free solid immersion microlens arrays (siMLAs), allowing large depth-of-field and high-resolution imaging for handheld 3D intraoral scanning. The siMLAs consist of thin PDMS-coated microlens arrays and a metal-insulator-metal absorber to extend the focal length with low optical crosstalk and specular reflection. The experimental results show that the immersion of MLAs in PDMS increases the focal length by a factor of 2.7 and the transmittance by 5.6%-27%. Unlike conventional MLAs, the siMLAs exhibit exceptionally high f-numbers up to f/6, resulting in a large depth-of-field for light-field imaging. The siMLAs were fully integrated into an intraoral scanner to reconstruct a 3D dental phantom with a distance measurement error of 82 ± 41 μm during handheld operation. The DF-LFC offers a new direction not only for digital dental impressions with high accuracy, simplified workflow, reduced waste, and digital compatibility but also for assorted clinical endoscopy and microscopy.

During embryonic development, endothelial cells (ECs) undergo vasculogenesis to form a primitive plexus and assemble into networks comprised of mural cell-stabilized vessels with molecularly distinct artery and vein signatures. This organized vasculature is established prior to the initiation of blood flow and depends on a sequence of complex signaling events elucidated primarily in animal models, but less studied and understood in humans. Here, we have developed a simple vascular differentiation protocol for human pluripotent stem cells that generates ECs, pericytes, and smooth muscle cells simultaneously. When this protocol is applied in a 3D hydrogel, we demonstrate that it recapitulates the dynamic processes of early human vessel formation, including acquisition of distinct arterial and venous fates, resulting in a vasculogenesis angiogenesis model plexus (VAMP). The VAMP captures the major stages of vasculogenesis, angiogenesis, and vascular network formation and is a simple, rapid, scalable model system for studying early human vascular development in vitro.

Many different strategies can be found in the literature to model organ physiology, tissue functionality, and disease in vitro; however, most of these models lack the physiological fluid dynamics present in vivo. Here, we highlight the importance of fluid flow for tissue homeostasis, specifically in vessels, other lumen structures, and interstitium, to point out the need of perfusion in current 3D in vitro models. Importantly, the advantages and limitations of the different current experimental fluid-flow setups are discussed. Finally, we shed light on current challenges and future focus of fluid flow models applied to the newest bioengineering state-of-the-art platforms, such as organoids and organ-on-a-chip, as the most sophisticated and physiological preclinical platforms.

We present a microvascular model of fluid transport in the alveolar septa related to pulmonary edema. It consists of a two-dimensional capillary sheet coursing by several alveoli. The alveolar epithelial membrane runs parallel to the capillary endothelial membrane with an interstitial layer in between, making one long septal tract. A coupled system of equations uses lubrication theory for the capillary blood, Darcy flow for the porous media of the interstitium, a passive alveolus, and the Starling equation at both membranes. Case examples include normal physiology, cardiogenic pulmonary edema, acute respiratory distress syndrome (ARDS), hypoalbuminemia, and effects of PEEP. COVID-19 has dramatically increased ARDS in the world population, raising the urgency for such a model to create an analytical framework. Under normal conditions fluid exits the alveolus, crosses the interstitium, and enters the capillary. For edema, this crossflow is reversed with fluid leaving the capillary and entering the alveolus. Because both the interstitial and capillary pressures decrease downstream, the reversal can occur within a single septal tract, with edema upstream and clearance downstream. Clinically useful solution forms are provided allowing calculation of interstitial fluid pressure, crossflows, and critical capillary pressures. Overall, the interstitial pressures are found to be significantly more positive than values used in the traditional physiological literature. That creates steep gradients near the upstream and downstream end outlets, driving significant flows toward the distant lymphatics. This new physiological flow provides an explanation to the puzzle, noted since 1896, of how pulmonary lymphatics can function so far from the alveoli: the interstitium is self-clearing.