Interpenetrating network hydrogels for studying the role of matrix viscoelasticity in 3D osteocyte morphogenesis.

During bone formation, osteoblasts are embedded in a collagen-rich osteoid tissue and differentiate into an extensive 3D osteocyte network throughout the mineralizing matrix. However, how these cells dynamically remodel the matrix and undergo 3D morphogenesis remains poorly understood. Although previous reports investigated the impact of matrix stiffness in osteocyte morphogenesis, the role of matrix viscoelasticity is often overlooked. Here, we report a viscoelastic alginate-collagen interpenetrating network (IPN) hydrogel for 3D culture of murine osteocyte-like IDG-SW3 cells. The IPN hydrogels consist of an ionically crosslinked alginate network to tune stress relaxation as well as a permissive collagen network to promote cell adhesion and matrix remodeling. Two IPN hydrogels were developed with comparable stiffnesses (4.4-4.7 kPa) but varying stress relaxation times (t1/2, 1.5 s and 14.4 s). IDG-SW3 cells were pre-differentiated in 2D under osteogenic conditions for 14 days to drive osteoblast-to-osteocyte transition. Cellular mechanosensitivity to fluid shear stress (2 Pa) was confirmed by live-cell calcium imaging. After embedding in the IPN hydrogels, cells remained highly viable following 7 days of 3D culture. After 24 h, osteocytes in the fast-relaxing hydrogels showed the largest cell area and long dendritic processes. However, a significantly larger increase of some osteogenic markers (ALP, Dmp1, hydroxyapatite) as well as intercellular connections via gap junctions were observed in slow-relaxing hydrogels on day 14. Our results imply that fast-relaxing IPN hydrogels promote early cell spreading, whereas slow relaxation favors osteogenic differentiation. These findings may advance the development of 3D in vivo-like osteocyte models to better understand bone mechanobiology.

Guiding bone cell network formation in 3D via photosensitized two-photon ablation.

A long-standing challenge in skeletal tissue engineering is to reconstruct a three-dimensionally (3D) interconnected bone cell network in vitro that mimics the native bone microarchitecture. While conventional hydrogels are extensively used in studying bone cell behavior in vitro, current techniques lack the precision to manipulate the complex pericellular environment found in bone. The goal of this study is to guide single bone cells to form a 3D network in vitro via photosensitized two-photon ablation of microchannels in gelatin methacryloyl (GelMA) hydrogels. A water-soluble two-photon photosensitizer (P2CK) was added to soft GelMA hydrogels to enhance the ablation efficiency. Remarkably, adding 0.5 mM P2CK reduced the energy dosage threshold five-fold compared to untreated controls, enabling more cell-compatible ablation. By employing low-energy ablation (100 J/cm2) with a grid pattern of 1 µm wide and 30 µm deep microchannels, we induced dendritic outgrowth in human mesenchymal stem cells (hMSC). After 7 days, the cells successfully utilized the microchannels and formed a 3D network. Our findings reveal that cellular viability after low-energy ablation was comparable to unablated controls, whereas high-energy ablation (500 J/cm2) resulted in 42 % cell death. Low-energy grid ablation significantly promoted network formation and >40 µm long protrusion outgrowth. While the broad-spectrum matrix metalloproteinase inhibitor (GM6001) reduced cell spreading by inhibiting matrix degradation, cells invaded the microchannel grid with long protrusions. Collectively, these results emphasize the potential of photosensitized two-photon hydrogel ablation as a high-precision tool for laser-guided biofabrication of 3D cellular networks in vitro. STATEMENT OF SIGNIFICANCE: The inaccessible nature of osteocyte networks in bones renders fundamental research on skeletal biology a major challenge. This limit is partly due to the lack of high-resolution tools that can manipulate the pericellular environment in 3D cultures in vitro. To create bone-like cellular networks, we employ a two-photon laser in combination with a two-photon sensitizer to erode microchannels with low laser dosages into GelMA hydrogels. By providing a grid of microchannels, the cells self-organized into a 3D interconnected network within days. Laser-guided formation of 3D networks from single cells at micron-scale resolution is demonstrated for the first time. In future, we envisage in vitro generation of bone cell networks with user-dictated morphologies for both fundamental and translational bone research.

Magnetic PiezoBOTs: a microrobotic approach for targeted amyloid protein dissociation.

Piezoelectric nanomaterials have become increasingly popular in the field of biomedical applications due to their high biocompatibility and ultrasound-mediated piezocatalytic properties. In addition, the ability of these nanomaterials to disaggregate amyloid proteins, which are responsible for a range of diseases resulting from the accumulation of these proteins in body tissues and organs, has recently gained considerable attention. However, the use of nanoparticles in biomedicine poses significant challenges, including targeting and uncontrolled aggregation. To address these limitations, our study proposes to load these functional nanomaterials on a multifunctional mobile microrobot (PiezoBOT). This microrobot is designed by coating magnetic and piezoelectric barium titanate nanoparticles on helical biotemplates, allowing for the combination of magnetic navigation and ultrasound-mediated piezoelectric effects to target amyloid disaggregation. Our findings demonstrate that acoustically actuated PiezoBOTs can effectively reduce the size of aggregated amyloid proteins by over 80% in less than 10 minutes by shortening and dissociating constituent amyloid fibrils. Moreover, the PiezoBOTs can be easily magnetically manipulated to actuate the piezocatalytic nanoparticles to specific amyloidosis-affected tissues or organs, minimizing side effects. These biocompatible PiezoBOTs offer a promising non-invasive therapeutic approach for amyloidosis diseases by targeting and breaking down protein aggregates at specific organ or tissue sites.

Synergizing Algorithmic Design, Photoclick Chemistry and Multi-Material Volumetric Printing for Accelerating Complex Shape Engineering.

The field of biomedical design and manufacturing has been rapidly evolving, with implants and grafts featuring complex 3D design constraints and materials distributions. By combining a new coding-based design and modeling approach with high-throughput volumetric printing, a new approach is demonstrated to transform the way complex shapes are designed and fabricated for biomedical applications. Here, an algorithmic voxel-based approach is used that can rapidly generate a large design library of porous structures, auxetic meshes and cylinders, or perfusable constructs. By deploying finite cell modeling within the algorithmic design framework, large arrays of selected auxetic designs can be computationally modeled. Finally, the design schemes are used in conjunction with new approaches for multi-material volumetric printing based on thiol-ene photoclick chemistry to rapidly fabricate complex heterogeneous shapes. Collectively, the new design, modeling and fabrication techniques can be used toward a wide spectrum of products such as actuators, biomedical implants and grafts, or tissue and disease models.

Analysis of the impact of peritoneal dialysis catheter tail-end design on catheter-related complications.

OBJECTIVE: Evaluate the impact of peritoneal dialysis catheter (PDC) tail-end design variations on PDC-related complications. METHOD: Effective data were extracted from databases. The literature was evaluated according to the Cochrane Handbook for Systematic Reviews of Interventions, and a meta-analysis was conducted. RESULTS: Analysis revealed that the straight-tailed catheter was superior to the curled-tailed catheter in minimizing catheter displacement and complication-induced catheter removal (RR = 1.73, 95%CI:1.18-2.53, p = 0.005). In terms of complication-induced PDC removal, the straight-tailed catheter was superior to the curled-tailed catheter (RR = 1.55, 95%CI: 1.15-2.08, p = 0.004). CONCLUSION: Curled-tail design of the catheter increased the risk of catheter displacement and complication-induced catheter removal, whereas the straight-tailed catheter was superior to the curled-tailed catheter in terms of reducing catheter displacement and complication-induced catheter removal. However, the analysis and comparison of factors such as leakage, peritonitis, exit-site infection, and tunnel infection did not reveal a statistically significant difference between the two designs.

Tomographic volumetric bioprinting of heterocellular bone-like tissues in seconds.

Tomographic volumetric bioprinting (VBP) has recently emerged as a powerful tool for rapid solidification of cell-laden hydrogel constructs within seconds. However, its practical applications in tissue engineering requires a detailed understanding of how different printing parameters (concentration of resins, laser dose) affect cell activity and tissue formation. Herein, we explore a new application of VBP in bone tissue engineering by merging a soft gelatin methacryloyl (GelMA) bioresin (<5 kPa) with 3D endothelial co-culture to generate heterocellular bone-like constructs with enhanced functionality. To this, a series of bioresins with varying concentrations of GelMA and lithium Phenyl(2,4,6-trimethylbenzoyl)phosphinate (LAP) photoinitiator were formulated and characterized in terms of photo-reactivity, printability and cell-compatibility. A bioresin with 5% GelMA and 0.05% LAP was identified as the optimal formulation for VBP of complex perfusable constructs within 30 s at high cell viability (>90%). The fidelity was validated by micro-computed tomography and confocal microscopy. Compared to 10% GelMA, this bioresin provided a softer and more permissive environment for osteogenic differentiation of human mesenchymal stem cells (hMSCs). The expression of osteoblastic markers (collagen-I, ALP, osteocalcin) and osteocytic markers (podoplanin, Dmp1) was monitored for 42 days. After 21 days, early osteocytic markers were significantly increased in 3D co-cultures of hMSCs with human umbilical vein endothelial cells (HUVECs). Additionally, we demonstrate VBP of a perfusable, pre-vascularized model where HUVECs self-organized into an endothelium-lined channel. Altogether, this work leverages the benefits of VBP and 3D co-culture, offering a promising platform for fast scaled biofabrication of 3D bone-like tissues with unprecedented functionality. STATEMENT OF SIGNIFICANCE: This study explores new strategies for ultrafast bio-manufacturing of bone tissue models by leveraging the advantages of tomographic volumetric bioprinting (VBP) and endothelial co-culture. After screening the properties of a series of photocurable gelatin methacryloyl (GelMA) bioresins, a formulation with 5% GelMA was identified with optimal printability and permissiveness for osteogenic differentiation of human mesenchymal stem cells (hMSC). We then established 3D endothelial co-cultures to test if the heterocellular interactions may enhance the osteogenic differentiation in the printed environments. This hypothesis was evidenced by increased gene expression of early osteocytic markers in 3D co-cultures after 21 days. Finally, VBP of a perfusable cell-laden tissue construct is demonstrated for future applications in vascularized tissue engineering.

Dual carbon sequestration with photosynthetic living materials.

Natural ecosystems offer efficient pathways for carbon sequestration, serving as a resilient approach to remove CO2 from the atmosphere with minimal environmental impact. However, the control of living systems outside of their native environments is often challenging. Here, we engineered a photosynthetic living material for dual CO2 sequestration by immobilizing photosynthetic microorganisms within a printable polymeric network. The carbon concentrating mechanism of the cyanobacteria enabled accumulation of CO2 within the cell, resulting in biomass production. Additionally, the metabolic production of OH- ions in the surrounding medium created an environment for the formation of insoluble carbonates via microbially-induced calcium carbonate precipitation (MICP). Digital design and fabrication of the living material ensured sufficient access to light and nutrient transport of the encapsulated cyanobacteria, which were essential for long-term viability (more than one year) as well as efficient photosynthesis and carbon sequestration. The photosynthetic living materials sequestered approximately 2.5 mg of CO2 per gram of hydrogel material over 30 days via dual carbon sequestration, with 2.2 ± 0.9 mg stored as insoluble carbonates. Over an extended incubation period of 400 days, the living materials sequestered 26 ± 7 mg of CO2 per gram of hydrogel material in the form of stable minerals. These findings highlight the potential of photosynthetic living materials for scalable carbon sequestration, carbon-neutral infrastructure, and green building materials. The simplicity of maintenance, coupled with its scalability nature, suggests broad applications of photosynthetic living materials as a complementary strategy to mitigate CO2 emissions.

Overexpression miR-520a-3p inhibits acute myeloid leukemia progression via targeting MUC1.

BACKGROUND: Acute myeloid leukemia (AML) is one of the familiar malignant tumors in the hematological system. miR-520a-3p is reported to be involved in several cancers' progression. However, miR-520a-3p role in AML remains unclear. In this study, we aimed to clarify the role and potential mechanism of miR-520a-3p in AML. METHODS: Cell viability, proliferation, cycle and apoptosis were detected by MTT assay, colony formation assay, flow cytometry, respectively. The levels of PNCA, Bcl-2, Cleaved caspase 3, Cleaved caspase 9 and ß-catenin protein were detected by Western blot. Dual-luciferase reported assay was performed to detect the regulation between miR-520a-3p and MUC1. To verify the effect of miR-520a-3p on tumor proliferation in vivo, a non-homogenous transplant model of tumors was established. RESULTS: miR-520a-3p expression was down-regulated, and MUC1 expression was up-regulated in AML patients. miR-520a-3p overexpression suppressed THP-1 cell proliferation, induced cell cycle G0/G1 inhibition and promoted apoptosis. miR-520a-3p targeted MUC1 and negatively regulated its expression. MUC1 knockdown inhibited THP-1 cell proliferation and promoted apoptosis. miR-520a-3p overexpression inhibited AML tumors growth. CONCLUSION: Overexpression miR-520a-3p inhibited AML cell proliferation, and promoted apoptosis via inhibiting MUC1 expression and repressing Wnt/ß-catenin pathway activation.

LncRNA GIHCG promoted the proliferation and migration of renal cell carcinoma through regulating miR-499a-5p/XIAP axis.

BACKGROUND: Our previous study demonstrated that lncRNA GIHCG is upregulated in renal cell carcinoma (RCC) and that knockdown of lncRNA GIHCG suppresses the proliferation and migration of RCC cells. However, the mechanism of lncRNA GIHCG in RCC needs further exploration. METHODS: The proliferation, cell cycle, migration, and apoptosis of RCC cells were tested using CCK-8, flow cytometry, wound healing and Annexin-V/-FITC/PI flow cytometry assays, respectively. Dual-luciferase reporter and RNA pull-down or RNA immunoprecipitation assays (RIPs) were performed to analyze the interactions among lncRNA GIHCG, miR-499a-5p and XIAP. A tumour xenograft study was conducted to verify the function of lncRNA GIHCG in RCC development in vivo. RESULTS: Knockdown of lncRNA GIHCG inhibited cell proliferation and migration and induced G0/G1 arrest while promoting apoptosis. Overexpression of lncRNA GIHCG led to the opposite results. LncRNA GIHCG sponged miR-499a-5p and downregulated its expression in RCC cells. MiR-499a-5p overexpression suppressed RCC cell growth. MiR-499a-5p targeted XIAP and inhibited its expression. LncRNA GIHCG knockdown reduced the growth of tumour xenografts in vivo and the expression of XIAP while increasing miR-499a-5p levels. CONCLUSION: LncRNA GIHCG accelerated the development of RCC by targeting miR-499a-5p and increasing XIAP levels.

Nano-3D-Printed Photochromic Micro-Objects.

Molecular photoswitches that can reversibly change color upon irradiation are promising materials for applications in molecular actuation and photoresponsive materials. However, the fabrication of photochromic devices is limited to conventional approaches such as mold casting and spin-coating, which cannot fabricate complex structures. Reported here is the first photoresist for direct laser writing of photochromic 3D micro-objects via two-photon polymerization. The integration of photochromism into thiol-ene photo-clickable resins enables rapid two-photon laser processing of highly complex microstructures and facile postmodification using a series of donor-acceptor Stenhouse adduct (DASA) photoswitches with different excitation wavelengths. The versatility of thiol-ene photo-click reactions allows fine-tuning of the network structure and physical properties as well as the type and concentration of DASA. When exposed to visible light, these microstructures exhibit excellent photoresponsiveness and undergo reversible color-changing via photoisomerization. It is demonstrated that the fluorescence variations of DASAs can be used as a reporter of photoswitching and thermal recovery, allowing the reading of DASA-containing sub-micrometric structures in 3D. This work delivers a new approach for custom microfabrication of 3D photochromic objects with molecularly engineered color and responsiveness.

3D bioprinting of graphene oxide-incorporated cell-laden bone mimicking scaffolds for promoting scaffold fidelity, osteogenic differentiation and mineralization.

Bioprinting is a promising technique for facilitating the fabrication of engineered bone tissues for patient-specific defect repair and for developing in vitro tissue/organ models for ex vivo tests. However, polymer-based ink materials often result in insufficient mechanical strength, low scaffold fidelity and loss of osteogenesis induction because of the intrinsic swelling/shrinking and bioinert properties of most polymeric hydrogels. Here, we developed a human mesenchymal stem cells (hMSCs)-laden graphene oxide (GO)/alginate/gelatin composite bioink to form 3D bone-mimicking scaffolds using a 3D bioprinting technique. Our results showed that the GO composite bioinks (0.5GO, 1GO, 2GO) with higher GO concentrations (0.5, 1 and 2 mg/ml) improved the bioprintability, scaffold fidelity, compressive modulus and cell viability at day 1. The higher GO concentration increased the cell body size and DNA content, but the 2GO group swelled and had the lowest compressive modulus at day 42. The 1GO group had the highest osteogenic differentiation of hMSC with the upregulation of osteogenic-related gene (ALPL, BGLAP, PHEX) expression. To mimic critical-sized calvarial bone defects in mice and prove scaffold fidelity, 3D cell-laden GO defect scaffolds with complex geometries were successfully bioprinted. 1GO maintained the best scaffold fidelity and had the highest mineral volume after culturing in the bioreactor for 42 days. In conclusion, GO composite bioinks had better bioprintability, scaffold fidelity, cell proliferation, osteogenic differentiation and ECM mineralization than the pure alginate/gelatin system. The optimal GO group was 1GO, which demonstrated the potential for 3D bioprinting of bone tissue models and tissue engineering applications.

Functionalized Bead Assay to Measure Three-dimensional Traction Forces during T-cell Activation.

When T-cells probe their environment for antigens, the bond between the T-cell receptor (TCR) and the peptide-loaded major histocompatibility complex (MHC) is put under tension, thereby influencing the antigen discrimination. Yet, the quantification of such forces in the context of T-cell signaling is technically challenging. Here, we developed a traction force microscopy platform which allows for quantifying the pulls and pushes exerted via T-cell microvilli, in both tangential and normal directions, during T-cell activation. We immobilized specific T-cell activating antibodies on the marker beads used to read out the hydrogel deformation. Microvilli targeted the functionalized beads, as confirmed by superresolution microscopy of the local actin organization. Moreover, we found that cellular components, such as actin, TCR, and CD45 reorganize upon interaction with the beads, such that actin forms a vortex-like ring structure around the beads and TCR is enriched at the bead surface, whereas CD45 is excluded from bead-microvilli contacts.

Biodegradable Metal-Organic Framework-Based Microrobots (MOFBOTs).

Microrobots and metal-organic frameworks (MOFs) have been identified as promising carriers for drug delivery applications. While clinical applications of microrobots are limited by their low drug loading efficiencies and the poor degradability of the materials used for their fabrication, MOFs lack motility and targeted drug delivery capabilities. The combination of these two fields marks the beginning of a new era; MOF-based small-scale robots (MOFBOTs) for biomedical applications. Yet, biodegradability is a major hurdle in the field of micro- and nanoswimmers including small-scale robots. Here, a highly integrated MOFBOT that is able to realize magnetic locomotion, drug delivery, and selective degradation in cell cultures is reported for the first time. The MOF used in the investigations does not only allow a superior loading of chemotherapeutic drugs and their controlled release via a pH-responsive degradation but it also enables the controlled locomotion of enzymatically biodegradable gelatin-based helical microrobots under magnetic fields. The degradation of the integrated MOFBOT is observed after two weeks, when all its components fully degrade. Additionally, drug delivery studies performed in cancer cell cultures show reduced viability upon delivery of Doxorubicin within short time frames. This MOFBOT system opens new avenues for highly integrated fully biodegradable small-scale robots.

Optimization of mechanical stiffness and cell density of 3D bioprinted cell-laden scaffolds improves extracellular matrix mineralization and cellular organization for bone tissue engineering.

Bioprinting is an emerging technology in which cell-laden biomaterials are precisely dispersed to engineer artificial tissues that mimic aspects of the anatomical and structural complexity of relatively soft tissues such as skin, vessels, and cartilage. However, reproducing the highly mineralized and cellular diversity of bone tissue is still not easily achievable and is yet to be demonstrated. Here, an extrusion-based 3D bioprinting strategy is utilized to fabricate 3D bone-like tissue constructs containing osteogenic cellular organization. A simple and low-cost bioink for 3D bioprinting of bone-like tissue is prepared based on two unmodified polymers (alginate and gelatin) and combined with human mesenchymal stem cells (hMSCs). To form 3D bone-like tissue and bone cell phenotype, the influence of different scaffold stiffness and cell density of 3D bioprinted cell-laden porous scaffolds on osteogenic differentiation and bone-like tissue formation was investigated over time. Our results showed that soft scaffolds (0.8%alg, 0.66 ± 0.08 kPa) had higher DNA content, enhanced ALP activity and stimulated osteogenic differentiation than stiff scaffolds (1.8%alg, 5.4 ± 1.2 kPa). At day 42, significantly more mineralized tissue was formed in soft scaffolds than in stiff scaffolds (43.5 ± 7.1 mm3 vs. 22.6 ± 6.0 mm3). Importantly, immunohistochemistry staining demonstrated more osteocalcin protein expression in high mineral compared to low mineral regions. Additionally, cells in soft scaffolds exhibited osteoblast- and early osteocyte-related gene expression and 3D cellular network within the mineralized matrix at day 42. Furthermore, the results showed that cell density in 15 M cells/ml can promote cell-cell connections at day 7 and mineral formation at day 14, while 5 M cells/ml had the significantly higher mineral formation rate than 15 M cells/ml from day 14 to day 21. In summary, this work reports the formation of 3D bioprinted bone-like tissue using a simple and low-cost cell-laden bioink, which was optimized for stiffness and cell density, showing great promise for bone tissue engineering applications. STATEMENT OF SIGNIFICANCE: In this study, we presented for the first time a framework combining 3D bioprinting, bioreactor system and time-lapsed micro-CT monitoring to provide in vitro scaffold fabrication, maturation, and mineral visualization for bone tissue engineering. 3D bone-like tissue constructs have been formed via optimizing scaffold stiffness and cell density. The soft scaffolds had higher cell proliferation, enhanced alkaline phosphatase activity and stimulated osteogenic differentiation with 3D cellular network foramtion than stiff scaffolds. Significantly more mineralized bone-like tissue was formed in soft scaffolds than stiff scaffolds at day 42. Meanwhile, cell density in 15 M cells/ml can promote cell-cell connections and mineral formation in 14 days, while the higher mineral formation rate was found in 5 M cells/ml from day 14 to day 21.

Substrate viscosity plays an important role in bacterial adhesion under fluid flow.

Many materials used in the medical settings such as catheters and contact lenses as well as most biological tissues are not purely elastic, but rather viscoelastic. While substrate elasticity has been investigated for its influence on bacterial adhesion, the impact of substrate viscosity has not been explored. Here, the importance of considering substrate viscosity is explored by using polydimethylsiloxane (PDMS) as the substrate material, whose mechanical properties can be tuned from predominantly elastic to viscous by varying cross-linking degree. Interfacial rheology and atomic force microscopy analysis prove that PDMS with a low cross-linking degree exhibits both low stiffness and high viscosity. This degree of viscoelasticity confers to PDMS a remarkable stress relaxation, a good capability to deform and an increased adhesive force. Bacterial adhesion assays were conducted under flow conditions to study the impact of substrate viscosity on Escherichia coli adhesion. The viscous PDMS not only enhanced E. coli adhesion but also conferred greater resistance to desorption against shear stress at air/liquid interface, compared to the PDMS with high crosslinking degree. These findings highlight the importance to consider substrate viscosity while studying bacterial adhesion. The current work provides new insights to an improved understanding of how bacteria interact with complex viscoelastic environments.

Bacterial Adhesion on Soft Materials: Passive Physicochemical Interactions or Active Bacterial Mechanosensing?

The influence of mechanical stiffness of biomaterials on bacterial adhesion is only sparsely studied and the mechanism behind this influence remains unclear. Here, bacterial adhesion on polydimethylsiloxane (PDMS) samples, having four different degrees of stiffness with Young's modulus ranging from 0.06 to 4.52 MPa, is investigated. Escherichia coli and Pseudomonas aeruginosa are found to adhere in greater numbers on soft PDMS (7- and 27-fold increase, respectively) than on stiff PDMS, whereas Staphylococcus aureus adheres in similar numbers on the four tested surfaces. To determine whether the observed adhesion behavior is caused by bacteria-specific mechanisms, abiotic polystyrene (PS) beads are employed as bacteria substitutes. Carboxylate-modified PS (PS-COOH) beads exhibit the same adhesion pattern as E. coli and P. aeruginosa with four times more adhered beads on soft PDMS than on stiff PDMS. In contrast, amine-modified PS (PS-NH2 ) beads adhere in similar numbers on all tested samples, reminiscent of S. aureus adhesion. This work demonstrates for the first time that the intrinsic physicochemical properties associated with PDMS substrates of different stiffness strongly influence bacterial adhesion and challenge the previously reported theory on active bacterial mechanosensing, which provides new insights into the design of antifouling surfaces.

Cell-Membrane-Inspired Silicone Interfaces that Mitigate Proinflammatory Macrophage Activation and Bacterial Adhesion.

Biofouling on silicone implants causes serious complications such as fibrotic encapsulation, bacterial infection, and implant failure. Here we report the development of antifouling, antibacterial silicones through covalent grafting with a cell-membrane-inspired zwitterionic gel layer composed of 2-methacryolyl phosphorylcholine (MPC). To investigate how substrate properties influence cell adhesion, we cultured human-blood-derived macrophages and Escherichia coli on poly(dimethylsiloxane) (PDMS) and MPC gel surfaces with a range of 0.5-50 kPa in stiffness. Cells attach to glass, tissue culture polystyrene, and PDMS surfaces, but they fail to form stable adhesions on MPC gel surfaces due to their superhydrophilicity and resistance to biofouling. Cytokine secretion assays confirm that MPC gels have a much lower potential to trigger proinflammatory macrophage activation than PDMS. Finally, modification of the PDMS surface with a long-term stable hydrogel layer was achieved by the surface-initiated atom-transfer radical polymerization (SI-ATRP) of MPC and confirmed by the decrease in contact angle from 110 to 20° and the >70% decrease in the attachment of macrophages and bacteria. This study provides new insights into the design of antifouling and antibacterial interfaces to improve the long-term biocompatibility of medical implants.

Near-Infrared Light-Sensitive Polyvinyl Alcohol Hydrogel Photoresist for Spatiotemporal Control of Cell-Instructive 3D Microenvironments.

Advanced hydrogel systems that allow precise control of cells and their 3D microenvironments are needed in tissue engineering, disease modeling, and drug screening. Multiphoton lithography (MPL) allows true 3D microfabrication of complex objects, but its biological application requires a cell-compatible hydrogel resist that is sufficiently photosensitive, cell-degradable, and permissive to support 3D cell growth. Here, an extremely photosensitive cell-responsive hydrogel composed of peptide-crosslinked polyvinyl alcohol (PVA) is designed to expand the biological applications of MPL. PVA hydrogels are formed rapidly by ultraviolet light within 1 min in the presence of cells, providing fully synthetic matrices that are instructive for cell-matrix remodeling, multicellular morphogenesis, and protease-mediated cell invasion. By focusing a multiphoton laser into a cell-laden PVA hydrogel, cell-instructive extracellular cues are site-specifically attached to the PVA matrix. Cell invasion is thus precisely guided in 3D with micrometer-scale spatial resolution. This robust hydrogel enables, for the first time, ultrafast MPL of cell-responsive synthetic matrices at writing speeds up to 50 mm s-1 . This approach should enable facile photochemical construction and manipulation of 3D cellular microenvironments with unprecedented flexibility and precision.

Laser photofabrication of cell-containing hydrogel constructs.

The two-photon polymerization (2PP) of photosensitive gelatin in the presence of living cells is reported. The 2PP technique is based on the localized cross-linking of photopolymers induced by femtosecond laser pulses. The availability of water-soluble photoinitiators (PI) suitable for 2PP is crucial for applying this method to cell-containing materials. Novel PIs developed by our group allow 2PP of formulations with up to 80% cell culture medium. The cytocompatibility of these PIs was evaluated by an MTT assay. The results of cell encapsulation by 2PP show the occurrence of cell damage within the laser-exposed regions. However, some cells located in the immediate vicinity and even within the 2PP-produced structures remain viable and can further proliferate. The control experiments demonstrate that the laser radiation itself does not damage the cells at the parameters used for 2PP. On the basis of these findings and the reports by other groups, we conclude that such localized cell damage is of a chemical origin and can be attributed to reactive species generated during 2PP. The viable cells trapped within the 2PP structures but not exposed to laser radiation continued to proliferate. The live/dead staining after 3 weeks revealed viable cells occupying most of the space available within the 3D hydrogel constructs. While some of the questions raised by this study remain open, the presented results indicate the general practicability of 2PP for 3D processing of cell-containing materials. The potential applications of this highly versatile approach span from precise engineering of 3D tissue models to the fabrication of cellular microarrays.