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Reviews

Antibacterial Sonodynamic Therapy: Current Status and Future Perspectives
Jayishnu Roy - ,
Vijayalakshmi Pandey - ,
Iti Gupta - , and
Himanshu Shekhar *
Multidrug-resistant bacteria have emerged in both community and hospital settings, partly due to the misuse of antibiotics. The inventory of viable antibiotics is rapidly declining, and efforts toward discovering newer antibiotics are not yielding the desired outcomes. Therefore, alternate antibacterial therapies based on physical mechanisms such as light and ultrasound are being explored. Sonodynamic therapy (SDT) is an emerging therapeutic approach that involves exposing target tissues to a nontoxic sensitizing chemical and low-intensity ultrasound. SDT can enable site-specific cytotoxicity by producing reactive oxygen species (ROS) in response to ultrasound, which can be harnessed for treating bacterial infections. This approach can potentially be used for both superficial and deep-seated microbial infections. The majority of the sonosensitizers reported are nonpolar, exhibiting limited bioavailability and a high clearance rate in the body. Therefore, targeted delivery agents such as nanoparticle composites, liposomes, and microbubbles are being investigated. This article reviews recent developments in antibacterial sonodynamic therapy, emphasizing biophysical and chemical mechanisms, novel delivery agents, ultrasound exposure and image guidance strategies, and the challenges in the pathway to clinical translation.

Development and Advantages of Biodegradable PHA Polymers Based on Electrospun PHBV Fibers for Tissue Engineering and Other Biomedical Applications
Łukasz Kaniuk - and
Urszula Stachewicz *
This publication is Open Access under the license indicated. Learn More
Biodegradable polymeric biomaterials offer a significant advantage in disposable or fast-consuming products in medical applications. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is an example of a polyhydroxyalkanoate (PHA), i.e., one group of natural polyesters that are byproducts of reactions taking place in microorganisms in conditions with an excess carbon source. PHA polymers are a promising material for the production of everyday materials and biomedical applications. Due to the high number of monomers in the group, PHAs permit modifications enabling the production of copolymers of different compositions and with different proportions of individual monomers. In order to change and improve the properties of polymer fibers, PHAs are combined with either other natural and synthetic polymers or additives of inorganic phases. Importantly, electrospun PHBV fibers and mats showed an enormous potential in both the medical field (tissue engineering scaffolds, plasters, wound healing, drug delivery systems) and industrial applications (filter systems, food packaging). This Review summarizes the current state of the art in processing PHBV, especially by electrospinning, its degradation processes, and biocompatibility studies, starting from a general introduction to the PHA group of polymers.

Promising Graphene-Based Nanomaterials and Their Biomedical Applications and Potential Risks: A Comprehensive Review
Jie Li - ,
Huamin Zeng - ,
Zhaowu Zeng - ,
Yiying Zeng - , and
Tian Xie *
This publication is Open Access under the license indicated. Learn More
Graphene-based nanomaterials (GBNs) have been the subject of research focus in the scientific community because of their excellent physical, chemical, electrical, mechanical, thermal, and optical properties. Several studies have been conducted on GBNs, and they have provided a detailed review and summary of various applications. However, comprehensive comments on biomedical applications and potential risks and strategies to reduce toxicity are limited. In this review, we systematically summarized the following aspects of GBNs in order to fill the gaps: (1) the history, synthesis methods, structural characteristics, and surface modification; (2) the latest advances in biomedical applications (including drug/gene delivery, biosensors, bioimaging, tissue engineering, phototherapy, and antibacterial activity); and (3) biocompatibility, potential risks (toxicity in vivo/vitro and effects on human health and the environment), and strategies to reduce toxicity. Moreover, we have analyzed the challenges to be overcome in order to enhance application of GBNs in the biomedical field.

Bone Scaffolds: An Incorporation of Biomaterials, Cells, and Biofactors
Marjan Bahraminasab *- ,
Mahsa Janmohammadi - ,
Samaneh Arab - ,
Athar Talebi - ,
Vajihe Taghdiri Nooshabadi - ,
Parisa Koohsarian - , and
Mohammad Sadegh Nourbakhsh
Large injuries to bones are still one of the most challenging musculoskeletal problems. Tissue engineering can combine stem cells, scaffold biomaterials, and biofactors to aid in resolving this complication. Therefore, this review aims to provide information on the recent advances made to utilize the potential of biomaterials for making bone scaffolds and the assisted stem cell therapy and use of biofactors for bone tissue engineering. The requirements and different types of biomaterials used for making scaffolds are reviewed. Furthermore, the importance of stem cells and biofactors (growth factors and extracellular vesicles) in bone regeneration and their use in bone scaffolds and the key findings are discussed. Lastly, some of the main obstacles in bone tissue engineering and future trends are highlighted.

Iron Oxide Nanoparticles: Physicochemical Characteristics and Historical Developments to Commercialization for Potential Technological Applications
Hossein Etemadi - ,
Jenna K. Buchanan - ,
Nadia G. Kandile - , and
Paul G. Plieger *
Iron oxide nanoparticles (IONPs) have gained increasing attention in various biomedical and industrial sectors due to their physicochemical and magnetic properties. In the biomedical field, IONPs are being developed for enzyme/protein immobilization, magnetofection, cell labeling, DNA detection, and tissue engineering. However, in some established areas, such as magnetic resonance imaging (MRI), magnetic drug targeting (MDT), magnetic fluid hyperthermia (MFH), immunomagnetic separation (IMS), and magnetic particle imaging (MPI), IONPs have crossed from the research bench, received clinical approval, and have been commercialized. Additionally, in industrial sectors IONP-based fluids (ferrofluids) have been marketed in electronic and mechanical devices for some time. This review explores the historical evolution of IONPs to their current state in biomedical and industrial applications.

Stabilization of Salivary Biomarkers
Luciana d’Amone - ,
Giusy Matzeu - , and
Fiorenzo G. Omenetto *
The use of saliva as a diagnostic biofluid has been increasing in recent years, thanks to the identification and validation of new biomarkers and improvements in test accuracy, sensitivity, and precision that enable the development of new noninvasive and cost-effective devices. However, the lack of standardized methods for sample collection, treatment, and storage contribute to the overall variability and lack of reproducibility across analytical evaluations. Furthermore, the instability of salivary biomarkers after sample collection hinders their translation into commercially available technologies for noninvasive monitoring of saliva in home settings. The present review aims to highlight the status of research on the challenges of collecting and using diagnostic salivary samples, emphasizing the methodologies used to preserve relevant proteins, hormones, genomic, and transcriptomic biomarkers during sample handling and analysis.
Methods/Protocols

On-a-Chip-Based Sensitive Detection of Drug-Induced Apoptosis in Polarized Gastric Epithelial Cells
Liubov Bakhchova *- ,
Phatcharida Jantaree - ,
Anubhuti Gupta - ,
Berend Isermann - ,
Ulrike Steinmann - , and
Michael Naumann
ACS Editors' Choice® is a collection designed to feature scientific articles of broad public interest. Read the latest articles
Microfluidic devices for culturing cells have been successfully utilized for biomedical applications, including drug screening. Several cell lines could be cultivated in microengineered environments with promising results, but gastric cell lines have not yet been widely used or studied. Therefore, this study focuses on establishing a polarized gastric epithelial monolayer on-a-chip and describes a general-purpose methodology applicable for bonding any porous material to PDMS through an adhesive sublayer. The fully transparent microfluidic chip consists of two microfluidic channels separated by a collagen-coated porous membrane and lined by human polarized gastric epithelial (NCI-N87) cells. We present considerations on how to ensure continuous and stable flow through the channels. The continuous flow rate was achieved using a pressure-driven pump. Media flow at a constant rate (0.5 μL/min) rapidly led the gastric epithelial cells to develop into a polarized monolayer. The barrier integrity was assessed by the FITC-dextran test. The generation of a monolayer was faster than in the static Boyden chamber. Moreover, fluorescence microscopy was used to monitor the apoptotic cell death of gastric epithelial monolayers on-a-chip in response to camptothecin, a therapeutic gastric cancer drug.
Characterization, Synthesis, and Modifications

Rivet-Inspired Modification of Carbon Nanotubes by In Situ-Reduced Ag Nanoparticles To Enhance the Strength and Ductility of Zn Implants
Cijun Shuai - ,
Zhi Dong - ,
Wenjing Yang *- ,
Chongxian He - ,
Youwen Yang - , and
Shuping Peng *
Zinc shows promise for bone repair applications, while its strength and ductility require to be improved. Carbon nanotubes (CNTs) are exceptional reinforcements due to their superior strength, ultrahigh Young’s modulus, and large aspect ratio. However, their strong agglomeration and weak interfacial bonding with the matrix are key bottleneck problems restricting the reinforcing effect. In this study, Ag nanoparticles were in situ reduced on CNTs and then the CNT@Ag powders were used to prepare Zn-CNT@Ag implants by laser powder bed fusion. Results showed that Ag reacted with Zn to form a “knot”-like AgZn3 phase. It had the same lattice structure (HCP) with Zn, which indicated a good lattice matching with the matrix, thus improving the dispersion of CNTs. More significantly, the knot played a “rivet” role and enhanced the load transfer capacity, which advantaged the CNT strengthening effects by assisting in transferring the load. Moreover, it enhanced the heterogeneous nucleation effects during solidification, which weakened the texture strength of the matrix and thus increased the ductility by improving the sliding capacity. The compressive yield strength, ultimate tensile strength, and elongation of the Zn-CNT@Ag implant were increased by 22, 26, and 17% in comparison to Zn-CNTs. Moreover, the Zn-CNT@Ag implant exhibited favorable antibacterial activity with a bacterial inhibition rate of 87.79%. Additionally, it also exhibited a suitable degradation rate and acceptable biocompatibility.

Enhanced Photothermal Performance by Carbon Dot-Chelated Polydopamine Nanoparticles
Qingfeng Shu - ,
Jie Liu - ,
Qing Chang - ,
Chenghao Liu - ,
Haifang Wang - ,
Yijun Xie *- , and
Xiaoyong Deng *
Polydopamine (PDA) has been widely used in biomedical applications including imaging contrast agents, antioxidants, UV protection, and photothermal therapy due to its biocompatibility, metal-ion chelation, free-radical scavenging, and wideband absorption, but its low photothermal efficiency still needs to be improved. In this study, we chelated near-infrared (NIR) sensitive carbon quantum dots on the surface of polydopamine (PDA-PEI@N,S-CQDs) to increase its near-infrared absorption. Surprisingly, although only 4% (w/w) of carbon quantum dots was conjugated on the PDA surface, it still increased the photothermal efficiency by 30%. Moreover, PDA-PEI@N,S-CQDs could also be used as the drug carrier for loading 60% (w/w) of the DOX and achieved stimuli-responsive drug release under lysosomal pH (pH 5.0) and 808 nm laser illumination. For in vitro therapeutic experiment, PDA-PEI@N,S-CQDs showed the remarkable therapeutic performance under 808 nm laser irradiation for killing 90% of cancer cells compared with 50% by pure PDA nanoparticles, and the efficacy was even higher after loading DOX owing to the synergistic effect by photothermal therapy and chemotherapy. This intelligent and effective therapeutic nanosystem based on PDA-PEI@N,S-CQDs showed enhanced photothermal behavior after chelating carbon dots and promoted the future development of a nanoplatform for stimuli-responsive photothermal/chemo therapy.

Characterization of Patterned Microbial Growth Dynamics in Aqueous Two-Phase Polymer Scaffolds
Andy J. Huang - ,
Andrew N. Clarke - ,
Naeimeh Jafari - , and
Brendan M. Leung *
This publication is Open Access under the license indicated. Learn More
Microbial growth confinement using liquid scaffolds based on an aqueous two-phase system (ATPS) is a promising technique to overcome the challenges in microbial–mammalian co-culture in vitro. To better understand the potential use of the ATPS in studying these complex interactions, the goal of this research was to characterize the effects of bacteria loading and biofilm maturation on the stability of a polyethylene glycol (PEG) and dextran (DEX) ATPS. Two ATPS formulations, consisting of 5% PEG/5% DEX and 10% PEG/10% DEX (w/v), were prepared. To test the containment limits of each ATPS formulation, Escherichia coli MG1655 overnight cultures were resuspended in DEX at optical densities (ODs) of 1, 0.3, 0.1, 0.03, and 0.01. Established E. coli colonies initially seeded at lower densities were contained within the DEX phase to a greater extent than E. coli colonies initially seeded at higher densities. Furthermore, the 10% PEG/10% DEX formulation demonstrated longer containment time of E. coli compared to the 5% PEG/5% DEX formulation. E. coli growth dynamics within the ATPS were found to be affected by the initial bacterial density, where colonies of lower initial seeding densities demonstrate more dynamic growth trends compared to colonies of higher initial seeding densities. However, the addition of DEX to the existing ATPS during the growth phase of the bacterial colony does not appear to disrupt the growth inertia of E. coli. We also observed that microbial growth can disrupt ATPS stability below the physical carrying capacity of the DEX droplets. In both E. coli and Streptococcus mutans UA159 colonies, the ATPS interfacial tensions are reduced, as suggested by the loss of fluorescein isothiocyanate (FITC)–DEX confinement and contact angel measurements, while the microbial colony remained well defined. In general, we observed that the stability of the ATPS microbial colony is proportional to polymer concentrations and inversely proportional to seeding density and culture time. These parameters can be combined as part of a toolset to control microbial growth in a heterotypic co-culture platform and should be considered in future work involving mammalian–microbial cell interactions.

Supramolecular Dual Drug Nanomicelles for Circumventing Multidrug Resistance
Muqiong Li - ,
Yang Bai - ,
Chengfei Liu *- ,
Li Fan *- , and
Wei Tian *
Overexpressed P-glycoprotein (P-gp), as the key factor in multidrug resistance (MDR), can greatly reduce intracellular drug levels as a result of chemotherapeutic drug efflux out of cancer cells. Although various P-gp antagonists have been developed to circumvent the MDR effect, the common strategies of physical entrapment or chemical conjugation of anticancer drugs may inevitably induce unstable circulation in vivo or poor response in cancer cells, greatly limiting the therapeutic efficacy. Herein a double-drug-based supramolecular complex (DDSC) was first constructed based on the host–guest interactions between two active drugs, curcumin (Cur)-bridged bis(β-cyclodextrin) and ferrocene-linked camptothecin (CPT). Supramolecular dual drug nanomicelles (SDDNMs) formed by the DDSC self-assembly are proposed to work against the MDR effect, in which Cur as a P-gp antagonist in combination with CPT realized stable transport in vivo and efficient stimuli-responsiveness in cells. In vitro and in vivo studies further confirmed that SDDNMs effectively circumvented the drug efflux induced by the MDR effect and remarkably enhanced the synergistic therapeutic effects.

Fabrication of Porous Crystalline PLGA-PEG Induced by Swelling during the Recrystallization Annealing Process
Jidong Dai - ,
Xin Tan - ,
Min Liang - ,
Dandan Wei - ,
Yinhua Tao - ,
Pengfei Ren - , and
Tianzhu Zhang *
Poly(lactide-co-glycolide) (PLGA) has been widely used as a scaffold material for tissue engineering owing to its biocompatibility, biodegradability, and biosafety. However, lactic acid (LA) produced during PLGA degradation is prone to inflammation, which is a shortcoming that must be avoided. To this end, crystalline PLGA-PEG was synthesized here for the first time. To make the crystalline PLGA-PEG more suitable for tissue engineering, porous crystalline PLGA-PEG was prepared via the swelling behavior during recrystallization annealing. The structure and properties of the porous crystalline PLGA-PEG were confirmed by SEM, POM, and XRD. Furthermore, the swelling behavior of different PEG molecular weights was studied, and the cell viability test and alkaline phosphatase activity test showed that PLGA-PEG has good biocompatibility. Such a porous crystalline PLGA-PEG will make PLGA have a broader application prospect in bone repair.

Fiber Formation and Mechanical Properties of Bombyx mori Silk Are Regulated by Vacuolar-Type ATPase
Xin Wang - ,
Xiaoyin Tan - ,
Qingsong Liu - ,
Yi Li - ,
Xinning Li - ,
Zhaoming Dong - ,
Haonan Dong - ,
Qingyou Xia - , and
Ping Zhao *
The mechanism of silk fiber formation in silkworms, Bombyx mori, is of particular scientific interest because it is closely related to the mechanical properties of silk fibers. However, there are still substantial knowledge gaps in understanding the details of this mechanism. Studies have found a pH gradient in the silk gland of silkworms. A vacuolar-type ATPase (V-ATPase) is thought to be involved in establishing this pH gradient. Although it is reported that the pH gradient plays a role in silk fibrillogenesis, the direct relationship between V-ATPase and silk mechanical properties is unclear. Thus, this study aims to clarify this relationship. We found that V-ATPase is highly and stably expressed in the anterior silk gland (ASG) and maintains the pH gradient and the fine structure of ASG. Inhibition of V-ATPase activity increased the β-sheet content and crystallinity of silk fibers. Tensile testing showed that the mechanical properties of silk fibers improved after inhibiting V-ATPase activity. All the data suggest that V-ATPase is a key factor in regulating silk fibrillogenesis and is related to the final mechanical properties of the silk fibers. V-ATPase is a potential target for silk mechanical property improvement.
Bio-interactions and Biocompatibility

Development of an Efficient Immunosensing Platform by Exploring Single-Walled Carbon Nanohorns (SWCNHs) and Nitrogen Doped Graphene Quantum Dot (N-GQD) Nanocomposite for Early Detection of Cancer Biomarker
Koushik Dutta - ,
Sriparna De - ,
Beauty Das - ,
Suman Bera - ,
Biswanath Guria - ,
Mir Sahidul Ali - , and
Dipankar Chattopadhyay *
In this work, a novel electrochemical immunosensor based on nitrogen doped graphene quantum dot (N-GQD) and single-walled carbon nanohorns (SWCNHs) was developed for the detection of α-fetoprotein (AFP), a cancer biomarker. Thus, to fabricate the platform of the immunosensor, nanocomposite architecture was developed by decorating N-GQD on the surface of the SWCNHs. The resulting hybrid architecture (N-GQD@SWCNHs) functioned as an exceptional base for the immobilization of antibody (Anti-AFP) through carbodiimide reaction with good stability and bioactivity. The immunosensor was prepared by evenly distributing the bioconjugates (N-GQD@SWCNHs/Anti-AFP) dispersion on the surface of the glassy carbon electrode, and subsequently blocking the remaining active sites by bovine serum albumin to prevent the nonspecific adsorption. Cyclic voltammetry and electrochemical impedance spectroscopy technique was employed to investigate the assembly process of the immunosensor. Under optimal conditions, the immunosensor exhibited a broad dynamic range in between 0.001 ng/mL to 200 ng/mL and a low detection limit of 0.25 pg/mL. Furthermore, the sensor showed high selectivity, desirable stability, and reproducibility. Measurements of AFP in human serum gave outstanding recovery within 99.2% and 102.1%. Thus, this investigation and the amplification strategy exhibited a potential role of the developed nanocomposite based sensor for early clinical screening of cancer biomarkers.

Novel Biodegradable Zn Alloy with Exceptional Mechanical and In Vitro Corrosion Properties for Biomedical Applications
Ehsan Farabi *- ,
Julie Sharp - ,
Alireza Vahid - ,
Jiangting Wang - ,
Daniel M. Fabijanic - ,
Matthew R. Barnett - , and
Santiago Corujeira Gallo
A series of quaternary Zn-Al-Cu-Li alloys with different weight fractions of Cu, Al, and Li were developed and investigated for potential application in high load bearing bioresorbable implants. The developed alloys provided various fractions of binary and ternary intermetallic structures, which resulted in formation of multiphase microstructures containing a zinc-rich η-phase and LiZn4 and CuZn4 phases. The intermetallic phases promoted grain refinement and a good combination of mechanical properties. The developed Zn-2Al-4Cu-0.6Li alloy showed strength and ductility close to commercially pure Ti alloys with a UTS value of ∼535 MPa and elongation of 37%. The examination of in vitro corrosion behavior of the developed alloys in the modified Hanks’ solution revealed suitable corrosion rates (∼38.5 μm/year). The moderate corrosion rate was controlled by the formation of a homogeneous layer of stable corrosion products that protected the alloys from the corrosive environment, particularly in the late stages of immersion. The developed alloys with the most promising mechanical and corrosion properties appeared to be biocompatible to mouse fibroblast cells and human umbilical mesenchymal stem cells, making them suitable candidates for implant applications.

Correlating Corona Composition and Cell Uptake to Identify Proteins Affecting Nanoparticle Entry into Endothelial Cells
Aldy Aliyandi - ,
Catharina Reker-Smit - ,
Reinier Bron - ,
Inge S. Zuhorn *- , and
Anna Salvati *
This publication is Open Access under the license indicated. Learn More
The formation of the biomolecule corona on the surface of nanoparticles upon exposure to biological fluids critically influences nanocarrier performance in drug delivery. It has been shown that in some cases corona proteins can mediate specific nanoparticle interactions with cell receptors. Within this context, in order to identify corona proteins affecting nanoparticle uptake, in this work, correlation analysis is performed between the corona composition of a panel of silica nanoparticles of different sizes and surface functionalities and their uptake in four endothelial cell types derived from different organs. In this way, proteins that correlate with increased or decreased uptake were identified, and their effects were validated by studying the uptake of nanoparticles coated with a single protein corona and competition studies in brain and liver endothelium. The results showed that precoating nanoparticles with histidine-rich glycoprotein (HRG) alone strongly decreased uptake in both liver and brain endothelium. Furthermore, our results suggested the involvement of the transferrin receptor in nanoparticle uptake in liver endothelium and redirection of the nanoparticles to other receptors with higher uptake efficiency when the transferrin receptor was blocked by free transferrin. These data suggested that changes in the cell microenvironment can also affect nanoparticle uptake and may lead to a different interaction site with nanoparticles, affecting their uptake efficiency. Overall, correlating the composition of the protein corona and nanoparticle uptake by cells allows for the identification of corona molecules that can be used to increase as well as to reduce nanoparticle uptake by cells.

Dual Mode Sensing of Binding and Blocking of Cancer Exosomes to Biomimetic Human Primary Stem Cell Surfaces
Johana Uribe - ,
Walther C. Traberg - ,
Adel Hama - ,
Victor Druet - ,
Zeinab Mohamed - ,
Amanda Ooi - ,
Anna-Maria Pappa - ,
Miriam Huerta - ,
Sahika Inal - ,
Róisín M. Owens - , and
Susan Daniel *
Cancer-derived exosomes (cEXOs) facilitate transfer of information between tumor and human primary stromal cells, favoring cancer progression. Although the mechanisms used during this information exchange are still not completely understood, it is known that binding is the initial contact established between cEXOs and cells. Hence, studying binding and finding strategies to block it are of great therapeutic value. However, such studies are challenging for a variety of reasons, including the need for human primary cell culture, the difficulty in decoupling and isolating binding from internalization and cargo delivery, and the lack of techniques to detect these specific interactions. In this work, we created a supported biomimetic stem cell membrane incorporating membrane components from human primary adipose-derived stem cells (ADSCs). We formed the supported membrane on glass and on multielectrode arrays to offer the dual option of optical or electrical detection of cEXO binding to the membrane surface. Using our platform, we show that cEXOs bind to the stem cell membrane and that binding is blocked when an antibody to integrin β1, a component of ADSC surface, is exposed to the membrane surface prior to cEXOs. To test the biological outcome of blocking this interaction, we first confirm that adding cEXOs to cultured ADSCs leads to the upregulation of vascular endothelial growth factor, a measure of proangiogenic activity. Next, when ADSCs are first blocked with anti-integrin β1 and then exposed to cEXOs, the upregulation of proangiogenic activity and cell proliferation are significantly reduced. This biomimetic membrane platform is the first cell-free label-free in vitro platform for the recapitulation and study of cEXO binding to human primary stem cells with potential for therapeutic molecule screening as it is compatible with scale-up and multiplexing.

Implantable and Degradable Thermoplastic Elastomer
Allison Siehr - ,
Craig Flory - ,
Trenton Callaway - ,
Robert J. Schumacher - ,
Ronald A. Siegel - , and
Wei Shen *
Biodegradable and implantable materials having elastomeric properties are highly desirable for many biomedical applications. Here, we report that poly(lactide)-co-poly(β-methyl-δ-valerolactone)-co-poly(lactide) (PLA-PβMδVL-PLA), a thermoplastic triblock poly(α-ester), has combined favorable properties of elasticity, biodegradability, and biocompatibility. This material exhibits excellent elastomeric properties in both dry and aqueous environments. The elongation at break is approximately 1000%, and stretched specimens completely recover to their original shape after force is removed. The material is degradable both in vitro and in vivo; it degrades more slowly than poly(glycerol sebacate) and more rapidly than poly(caprolactone) in vivo. Both the polymer and its degradation product show high cytocompatibility in vitro. The histopathological analysis of PLA-PβMδVL-PLA specimens implanted in the gluteal muscle of rats for 1, 4, and 8 weeks revealed similar tissue responses as compared with poly(glycerol sebacate) and poly(caprolactone) controls, two widely accepted implantable polymers, suggesting that PLA-PβMδVL-PLA can potentially be used as an implantable material with favorable in vivo biocompatibility. The thermoplastic nature allows this elastomer to be readily processed, as demonstrated by the facile fabrication of the substrates with topographical cues to enhance muscle cell alignment. These properties collectively make this polymer potentially highly valuable for applications such as medical devices and tissue engineering scaffolds.

Imparting Immunomodulatory Activity to Scaffolds via Biotin–Avidin Interactions
Emily B. Lurier - ,
Victoria A. Nash - ,
Hannah S. Abee - ,
Tamar B. Wissing - ,
Carlijn V.C. Bouten - ,
Anthal I.P.M. Smits *- , and
Kara L. Spiller *
Biotin–avidin interactions have been explored for decades as a technique to functionalize biomaterials, as well as for in vivo targeting, but whether changes in these interactions can be leveraged for immunomodulation remain unknown. The goal of this study was to investigate how biotin density and avidin variant can be used to deliver the immunomodulatory cytokine, interleukin 4 (IL4), from a porous gelatin scaffold, Gelfoam, to primary human macrophages in vitro. Here, we demonstrate that the degree of scaffold biotinylation controlled the binding of two different avidin variants, streptavidin and CaptAvidin. Biotinylated scaffolds were also loaded with streptavidin and biotinylated IL4 under flow, suggesting a potential use for targeting this biomaterial in vivo. While biotin–avidin interactions did not appear to influence the protein release in this system, increasing degrees of biotinylation did lead to increased M2-like polarization of primary human macrophages over time in vitro, highlighting the capability to leverage biotin–avidin interactions to modulate the macrophage phenotype. These results demonstrate a versatile and modular strategy to impart immunomodulatory activity to biomaterials.
Controlled Release and Delivery Systems

Robust Antigen-Specific T Cell Activation within Injectable 3D Synthetic Nanovaccine Depots
Jorieke Weiden - ,
Marjolein Schluck - ,
Melina Ioannidis - ,
Eric A. W. van Dinther - ,
Mahboobeh Rezaeeyazdi - ,
Fawad Omar - ,
Juulke Steuten - ,
Dion Voerman - ,
Jurjen Tel - ,
Mustafa Diken - ,
Sidi A. Bencherif - ,
Carl G. Figdor - , and
Martijn Verdoes *
This publication is Open Access under the license indicated. Learn More
Synthetic cancer vaccines may boost anticancer immune responses by co-delivering tumor antigens and adjuvants to dendritic cells (DCs). The accessibility of cancer vaccines to DCs and thereby the delivery efficiency of antigenic material greatly depends on the vaccine platform that is used. Three-dimensional scaffolds have been developed to deliver antigens and adjuvants locally in an immunostimulatory environment to DCs to enable sustained availability. However, current systems have little control over the release profiles of the cargo that is incorporated and are often characterized by an initial high-burst release. Here, an alternative system is designed that co-delivers antigens and adjuvants to DCs through cargo-loaded nanoparticles (NPs) incorporated within biomaterial-based scaffolds. This creates a programmable system with the potential for controlled delivery of their cargo to DCs. Cargo-loaded poly(d,l-lactic-co-glycolic acid) NPs are entrapped within the polymer walls of alginate cryogels with high efficiency while retaining the favorable physical properties of cryogels, including syringe injection. DCs cultured within these NP-loaded scaffolds acquire strong antigen-specific T cell-activating capabilities. These findings demonstrate that introduction of NPs into the walls of macroporous alginate cryogels creates a fully synthetic immunostimulatory niche that stimulates DCs and evokes strong antigen-specific T cell responses.

Hafnium Oxide-Based Nanoplatform for Combined Chemoradiotherapy
Anastasiia A. Sherstiuk - ,
Sergey A. Tsymbal - ,
Anna F. Fakhardo - ,
Vladimir N. Morozov - ,
Elena F. Krivoshapkina - ,
Evamarie Hey-Hawkins - , and
Pavel V. Krivoshapkin *
Recently, the combined therapy has become one of the main approaches in cancer treatment. Combining different approaches may provide a significant outcome by triggering several death mechanisms or causing increased damage of tumor cells without hurting healthy ones. The supramolecular nanoplatform based on a high-Z metal reported here is a suitable system for the targeted delivery of chemotherapeutic compounds, imaging, and an enhanced radiotherapy outcome. HfO2 nanoparticles coated with oleic acid and a monomethoxypoly(ethylene glycol)-poly(ε-caprolactone) copolymer shell (nanoplatform) are able to accumulate inside cancer cells and release doxorubicin (DOX) under specific conditions. Neither uncoated nor coated nanoparticles show any cytotoxicity in vitro. DOX loaded onto a nanoplatform demonstrates a lower IC50 value than pure DOX. X-ray irradiation of cancer cells loaded with a nanoplatform shows a higher death rate than that for cells without nanoparticles. These results provide an important foundation for the development of complex nanoscale systems for combined cancer treatment.

Visualization and Evaluation of Chemoembolization on a 3D Decellularized Organ Scaffold
Xu Gao - ,
Zijian Chen - ,
Zhengchang Chen - ,
Xiaoya Liu - ,
Yucheng Luo - ,
Jingyu Xiao - ,
Yanan Gao - ,
Yutao Ma - ,
Chuang Liu - ,
Hwa Liang Leo - ,
Hanry Yu - , and
Qiongyu Guo *
Transarterial chemoembolization (TACE) has emerged as the mainstay treatment for patients suffering from unresectable intermediate hepatocellular carcinoma and also holds the potential to treat other types of hypervascular cancers such as renal cell carcinoma. However, an in vitro model for evaluating both embolic performance and drug-release kinetics of the TACE embolic agents is still lacking since the current models greatly simplified the in vivo vascular systems as well as the extracellular matrices (ECM) in the organs. Here, we developed a decellularized organ model with preserved ECM and vasculatures as well as a translucent appearance to investigate chemoembolization performances of a clinically widely used embolic agent, i.e., a doxorubicin-loaded ethiodised oil (EO)-based emulsion. We, for the first time, utilized an ex vivo model to evaluate the liquid-based embolic agent in two organs, i.e., liver and kidneys. We found that the EO-based emulsion with enhanced stability by incorporating an emulsifier, i.e., hydrogenated castor oil-40 (HCO), showed an enhanced occlusion level and presented sustained drug release in the ex vivo liver model, suggesting an advantageous therapeutic effect for TACE treatment of hepatocellular carcinoma. In contrast, we observed that drug-release burst happened when applying the same therapy in the kidney model even with the HCO emulsifier, which may be explained by the presence of the specific renal vasculature and calyceal systems, indicating an unfavorable effect in the renal tumor treatment. Such an ex vivo model presents a promising template for chemoembolization evaluation before in vivo experiments for the development of novel embolic agents.

Miniatured Fluidics-Mediated Modular Self-Assembly of Anticancer Drug–Amino Acid Composite Microbowls for Combined Chemo-Photodynamic Therapy in Glioma
Sonika Chibh - ,
Vibhav Katoch - ,
Manish Singh - ,
Bhanu Prakash *- , and
Jiban Jyoti Panda *
A particulate carrier with the ability to load a combination of therapeutic molecules acting via diverse modes to initiate cancer cell ablation would help heighten anticancer therapeutic outcomes and mitigate harmful side effects due to high doses of mono drug therapy. Moving a step closer, herein, we have developed doxorubicin–curcumin–amino acid-based composite microbowls (CMBs) following miniaturized fluid flow-based self-assembly. The CMBs were further exploited as dual chemo-photodynamic therapeutic agents in C6 glioma cells cultured in both two-dimensional (2D) monolayer and as three-dimensional (3D) spheroids. These CMBs showed synergistic and visible (blue)-light-sensitive cell-killing effects in both C6 cells and 3D spheroids. Furthermore, these bowl-shaped structures also demonstrated good stability and excellent in vitro cytocompatibility in C6 glioma cells. Our results indicated that CMBs with asymmetric cavities could potentially be used as a combinatorial drug carrier enabling simultaneous chemo- and phototherapy for effective cancer treatment. The use of blue light, from the visible part of the electromagnetic system, to generate the phototherapeutic effect further advocates for the ease and widespread applicability of the systems.

Targeted Delivery of Chloroquine to Antigen-Presenting Cells Enhances Inhibition of the Type I Interferon Response
Marilyn E. Allen - ,
Amit Golding - ,
Violeta Rus - ,
Nicholas B. Karabin - ,
Sophia Li - ,
Chamille J. Lescott - ,
Sharan Bobbala - ,
Evan A. Scott - , and
Gregory L. Szeto *
Systemic lupus erythematosus (SLE) causes damaging inflammation in multiple organs via the accumulation of immune complexes. These complexes activate plasmacytoid dendritic cells (pDCs) via toll-like receptors (TLRs), contributing to disease pathogenesis by driving the secretion of inflammatory type I interferons (IFNs). Antimalarial drugs, such as chloroquine (CQ), are TLR antagonists used to alleviate inflammation in SLE. However, they require ∼3 months of continuous use before achieving therapeutic efficacy and can accumulate in the retinal pigment epithelium with chronic use, resulting in retinopathy. We hypothesized that poly(ethylene glycol)-b-poly(propylene sulfide) filamentous nanocarriers, filomicelles (FMs), could directly deliver CQ to pDCs via passive, morphology-based targeting to concentrate drug delivery to specific immune cells, improve drug activity by increased inhibition of type I IFN, and enhance efficacy per dose. Healthy human peripheral blood mononuclear cells were treated with soluble CQ or CQ-loaded FMs, stimulated with TLR agonists or SLE patient sera, and type I IFN secretion was quantified via multi-subtype IFN-α ELISA and MX1 gene expression using real-time reverse transcription-quantitative polymerase chain reaction. Our results showed that 50 μg CQ/mg FM decreased MX1 expression and IFN-α production after TLR activation with either synthetic nucleic acid agonists or immune complex-rich sera from SLE patients. Cellular uptake and biodistribution studies showed that FMs preferentially accumulate in human pDCs and monocytes in vitro and in tissues frequently damaged in SLE patients (i.e., kidneys), while sparing the eye in vivo. These results showed that nanocarrier morphology enables drug delivery, and CQ-FMs may be equally effective and more targeted than soluble CQ at inhibiting SLE-relevant pathways.

Zinc(II)-Cyclen Multifunctional Complex Module-Mediated Polycation-Based High-Performance pDNA Vectors
Zhaoming Chen - ,
Shuai Liu - ,
Weijie Liu - ,
Peng Jiang - ,
Lan Wang - ,
Jiaqi Li - ,
Hao Zhou *- , and
Tianying Guo *
A kind of novel multifunctional modules based on zinc(II)-coordinative cyclen has been developed, which is utilized to modify low molecular weight polyethylenimine (LMWPEI) obtaining high-performance DNA vectors. A series of in vitro experiments were carried out to explore the performance of the module in improving the key process of gene transfection, such as DNA condensation, serum resistance, cellular uptake, and endosomal escape. The results demonstrate that there is a significant synergistic effect between the functional module and PEI2.5k in the process of breaking through the key barriers of gene transfection. The optimal Zn-PCD mediates 160-fold higher gluciferase activity than commercial transfection reagents PEI25k in ADSC stem cells with more than 90% cell viability and achieves excellent transfection efficiency in diverse cell types, for instance, HepG2 cells, 293T cells, and 293F suspension cells.

Stromal Barrier-Dismantled Nanodrill-Like and Cancer Cell-Targeted pH-Responsive Polymeric Micelles for Further Enhancing the Anticancer Efficacy of Doxorubicin
Chuhang Zhou - ,
Xinping Hu - ,
Qi Liu - ,
Leqi Wang - ,
Yuanhang Zhou - ,
Yao Jin - ,
Yining Ma - , and
Yan Liu *
Cancer-associated fibroblasts (CAFs) were believed to establish a tight physical barrier and a dense scaffold for tumor cells to make them maintain immunosuppression and drug resistance, strongly hindering nanoparticles to penetrate into the core of tumor tissues and limiting the performance of tumor cell-targeted nanoparticles. Here, we fabricated the substrate Z-Gly-Pro of fibroblast activation protein α (FAPα) and folic acid-codecorated pH-responsive polymeric micelles (dual ligand-modified PEOz-PLA polymeric micelles, DL-PP-PMs) that possessed nanodrill and tumor cell-targeted functions based on Z-Gly-pro-conjugated poly(2-ethyl-2-oxazoline)-poly(D,l-lactide) (ZGP-PEOz-PLA), folic acid (FA)-conjugated PEOz-PLA (FA-PEOz-PLA), and PEOz-PLA for cancer therapy. The micelles with about 40 nm particle size and a narrow distribution exhibited favorable pH-activated endo/lysosome escape induced by their pH responsibility. In addition, the enhancement of in vitro cellular uptake and cytotoxicity to folate receptors or FAPα-positive cells for doxorubicin (DOX)/DL-PP-PMs compared with DOX/PP-PMs evidenced the dual target ability of DOX/DL-PP-PMs, which was further supported by in vivo biodistribution results. As expected, in the human oral epidermal carcinoma (KB) cells xenograft nude mice model, the remarkable enhancement of antitumor efficacy for DOX/DL-PP-PMs with low toxicity was observed compared with DOX/FA-PP-PMs and DOX/ZGP-PP-PMs. The possible mechanism was elucidated to be the dismantling of the stromal barrier by nanodrill-like DOX/DL-PP-PMs via the deletion of CAFs evidenced by the downregulation of α-SMA and inhibition of their functions proved by the decrease in the microvascular density labeled with CD31 and the reduction in the extracellular matrix detected by the collagen content, thereby promoting tumor penetration and enhancing their uptake by tumor cells. The present research offered an alternative approach integrating anticancer and antifibrosis effects in one delivery system to enhance the delivery efficiency and therapeutic efficacy of anticancer drugs.

Injectable Erythrocyte Gel Loaded with Bulleyaconitine A for the Treatment of Rheumatoid Arthritis
Hao Chen - ,
Bo Tian - ,
Xiyao Fang - ,
Jinyu Bai *- ,
Qingle Ma - ,
Yue Zhang - ,
Jialu Xu - ,
Beilei Wang - ,
Qin Fan - ,
Ziying Fei - ,
Huaxing Dai - ,
Huajian Shan - ,
Xiang Gao - ,
Qirong Dong - ,
Chao Wang *- , and
Xiaozhong Zhou *
Rheumatoid arthritis (RA) is a systemic autoimmune disease with clinical manifestations including joint cartilage, synovitis, and bone damage. Here we developed an injectable erythrocyte gel loaded with Bulleyaconitine A (BLA) for the treatment of RA and demonstrated its anti-inflammatory effects in vivo and in vitro. In vitro experiments showed that BLA could effectively down-regulate the expression of pro-inflammatory factor in activated macrophages through the nuclear factor-κB (NF-κB) pathway. In vivo experiments have shown that the injection of BLA@RBCs in the inflammatory joints of CIA mice increases the local concentration of BLA in a long time. Improved therapeutic outcomes and reduced toxicity of BLA are demonstrated in our work. Together, the developed BLA@RBCs drug delivery system provides an alternative strategy to treat RA joints and shows high potential in clinical RA treatment.

Sodium Alginate Hydrogel-Mediated Cancer Immunotherapy for Postoperative In Situ Recurrence and Metastasis
Yingying Zhang - ,
Tiange Wang - ,
Yinping Zhuang - ,
Tiandi He - ,
Xiaoli Wu - ,
Lin Su - ,
Jun Kang *- ,
Jin Chang *- , and
Hanjie Wang *
With the development of technology, adjuvant immunotherapy has become a promising strategy for prevention of postoperative tumor regression and metastasis by stimulating the host immune response. However, the therapeutic effects are still unsatisfactory due to the lack of synergy between different methods. In this study, an efficient synergistic immunotherapy system based on injectable sodium alginate hydrogels was designed to inhibit in situ recurrence and metastasis at the same time. On the one hand, an injectable sodium alginate (SA) hydrogel microsystem loaded with toll-like receptor (TLR) agonists (CpG ODNs) was synthesized for inhibiting in situ recurrence, and then carcinoembryonic antigen (CEA) probe was also added to detect CEA based on fluorescence resonance energy transfer (FRET) technology to monitor the occurrence and development of tumor recurrence. On the other hand, an anti-programmed cell death 1 ligand 1 antibody (anti-PD-L1)-modified SA nanogel loaded with indocyanine green (ICG@SA-anti-PD-L1 nanogel) was prepared for diagnosing and inhibiting lung metastasis by assisting orthotopic tumor therapy. In vitro and in vivo results demonstrated that this SA micro/nanosystem could monitor and inhibit postoperative recurrence and metastasis. We hope that this micro/nano-synergistic system will become an effective strategy for postoperative adjuvant immunotherapy.
Tissue Engineering and Regenerative Medicine

Prefabricated 3D-Printed Tissue-Engineered Bone for Mandibular Reconstruction: A Preclinical Translational Study in Primate
Shuai-shuai Cao - ,
Shu-yi Li - ,
Yuan-ming Geng - ,
Kausik Kapat - ,
Shang-bin Liu - ,
Fidel Hugo Perera - ,
Qian Li - ,
Hendrik Terheyden - ,
Gang Wu - ,
Yue-juan Che *- ,
Pedro Miranda *- , and
Miao Zhou *
This publication is Open Access under the license indicated. Learn More
The advent of three dimensionally (3D) printed customized bone grafts using different biomaterials has enabled repairs of complex bone defects in various in vivo models. However, studies related to their clinical translations are truly limited. Herein, 3D printed poly(lactic-co-glycolic acid)/β-tricalcium phosphate (PLGA/TCP) and TCP scaffolds with or without recombinant bone morphogenetic protein −2 (rhBMP-2) coating were utilized to repair primate’s large-volume mandibular defects and compared efficacy of prefabricated tissue-engineered bone (PTEB) over direct implantation (without prefabrication). 18F-FDG PET/CT was explored for real-time monitoring of bone regeneration and vascularization. After 3-month’s prefabrication, the original 3D-architecture of the PLGA/TCP-BMP scaffold was found to be completely lost, while it was properly maintained in TCP-BMP scaffolds. Besides, there was a remarkable decrease in the PLGA/TCP-BMP scaffold density and increase in TCP-BMP scaffolds density during ectopic (within latissimus dorsi muscle) and orthotopic (within mandibular defect) implantation, indicating regular bone formation with TCP-BMP scaffolds. Notably, PTEB based on TCP-BMP scaffold was successfully fabricated with pronounced effects on bone regeneration and vascularization based on radiographic, 18F-FDG PET/CT, and histological evaluation, suggesting a promising approach toward clinical translation.

Three-Dimensional Tendon Scaffold Loaded with TGF-β1 Gene Silencing Plasmid Prevents Tendon Adhesion and Promotes Tendon Repair
Genbin Wu - ,
Binbin Sun - ,
Chunfeng Zhao - ,
Zhuoying Wang - ,
Songsong Teng - ,
Mengkai Yang - ,
Zhi Cui - ,
Guoliang Zhu *- , and
Yinxian Yu *
Tendon adhesion formation is associated with the aberrant expression of many genes, and interfering with the expression of these genes can prevent adhesion and promote tendon repair. Recent studies have found that silencing the transforming growth factor β-1 (TGF-β1) gene can reduce the occurrence of tendon adhesions. The development of tissue engineering and three-dimensional (3D) printing technology have provided new solutions for tendon repair. In this study, TGF-β1 gene silencing microRNA (miRNA) based RNAi plasmid was loaded on a 3D tendon scaffold using 3D printing technology. In vitro experiments confirmed the sustained release of plasmid and the good biocompatibility of the printed tendon scaffold. Subsequently, the TGF-β1 gene silencing plasmid loaded tendon scaffold was implanted in a chicken tendon defect model to evaluate the effect of the scaffold in vivo. The results from biomechanical tests and histological examinations showed that the scaffold not only promoted tendon regeneration but also prevented tendon adhesion, which was conducive to the recovery of biofunction. Evaluation of protein expression showed that the loaded plasmids prevented tendon adhesion and promoted tendon functional repair via silencing of the TGF-β1 gene.

RGDSP-Decorated Hyaluronate Hydrogels Facilitate Rapid 3D Expansion of Amylase-Expressing Salivary Gland Progenitor Cells
Eric W. Fowler - ,
Anitha Ravikrishnan - ,
Robert L. Witt - ,
Swati Pradhan-Bhatt - , and
Xinqiao Jia *
In vitro engineering of salivary glands relies on the availability of synthetic matrices presenting essential cell-instructive signals to guide tissue growth. Here, we describe a biomimetic, hyaluronic acid (HA)-based hydrogel platform containing covalently immobilized bioactive peptides derived from perlecan domain IV (TWSKV), laminin-111 (YIGSR, IKVAV), and fibronectin (RGDSP). The HA network was established by the thiol/acrylate reaction, and bioactive peptides were conjugated to the network with high efficiency without significantly altering the mechanical property of the matrix. When encapsulated as single cells in peptide-modified HA hydrogels, human salivary gland stem/progenitor cells (hS/PCs) spontaneously organized into multicellular spheroids with close cell–cell contacts. Conjugation of RGDSP and TWSKV signals in HA gels significantly accelerated cell proliferation, with the largest spheroids observed in RGDSP-tagged gels. Peptide conjugation did not significantly alter the expression of acinar (AMY1), ductal (TFCP2L1), and progenitor (KRT14) markers at the mRNA level. Characterization of three-dimensional (3D) cultures by immunocytochemistry showed positive staining for keratin-5 (K5), keratin-14 (K14), integrin-β1, and α-amylase under all culture conditions, confirming the maintenance of the secretory progenitor cell population. Two-dimensional (2D) adhesion studies revealed that integrin-β1 played a key role in facilitating cell–matrix interaction in gels with RGDSP, IKVAV, and TWSKV signals. Overall, conjugation of the RGDSP peptide to HA gels improved cell viability, accelerated the formation of epithelial spheroids, and promoted the expansion of the progenitor cell population in 3D. This work represents an essential first step toward the development of an engineered salivary gland.

Measuring the Effects of Cytokines on the Modification of Pericellular Rheology by Human Mesenchymal Stem Cells
Maryam Daviran - ,
John A. McGlynn - ,
Jenna A. Catalano - ,
Hannah E. Knudsen - ,
Kilian J. Druggan - ,
Kiera J. Croland - ,
Amanda Stratton - , and
Kelly M. Schultz *
Implantable hydrogels are designed to treat wounds by providing structure and delivering additional cells to damaged tissue. These materials must consider how aspects of the native wound, including environmental chemical cues, affect and instruct delivered cells. One cell type researchers are interested in delivering are human mesenchymal stem cells (hMSCs) due to their importance in healing. Wound healing involves recruiting and coordinating a variety of cells to resolve a wound. hMSCs coordinate the cellular response and are signaled to the wound by cytokines, including transforming growth factor-β (TGF-β) and tumor necrosis factor-α (TNF-α), present in vivo. These cytokines change hMSC secretions, regulating material remodeling. TGF-β, present from inflammation through remodeling, directs hMSCs to reorganize collagen, increasing extracellular matrix (ECM) structure. TNF-α, present primarily during inflammation, cues hMSCs to clear debris and degrade ECM. Because cytokines change how hMSCs degrade their microenvironment and are naturally present in the wound, they also affect how hMSCs migrate out of the scaffold to conduct healing. Therefore, the effects of cytokines on hMSC remodeling are important when designing materials for cell delivery. In this work, we encapsulate hMSCs in a polymer–peptide hydrogel and incubate the scaffolds in media with TGF-β or TNF-α at concentrations similar to those in wounds. Multiple particle tracking microrheology (MPT) measures hMSC-mediated scaffold degradation in response to these cytokines, which mimics aspects of the in vivo microenvironment post-implantation. MPT uses video microscopy to measure Brownian motion of particles in a material, quantifying structure and rheology. Using MPT, we measure increased hMSC-mediated remodeling when cells are exposed to TNF-α and decreased remodeling after exposure to TGF-β when compared to untreated hMSCs. This agrees with previous studies that measure: (1) TNF-α encourages matrix reorganization and (2) TGF-β signals the formation of new matrix. These results enable material design that anticipates changes in remodeling after implantation, improving control over hMSC delivery and healing.

Highly Structured 3D Electrospun Conical Scaffold: A Tool for Dental Pulp Regeneration
Lisa Terranova *- ,
Aurélien Louvrier - ,
Anne Hébraud - ,
Christophe Meyer - ,
Gwenaël Rolin - ,
Guy Schlatter - , and
Florent Meyer
New procedures envisioned for dental pulp regeneration after pulpectomy include cell homing strategy. It involves host endogenous stem cell recruitment and activation. To meet this cell-free approach, we need to design a relevant scaffold to support cell migration from tissues surrounding the dental root canal. A composite membrane made of electrospun poly(lactic acid) nanofibers and electrosprayed polycaprolactone with tannic acid (TA) microparticles which mimics the architecture of the extracellular matrix was first fabricated. After rolling the membrane in the form of a 3D conical scaffold and subsequently coating it with gelatin, it can be directly inserted into the root canal. The porous morphology of the construct was characterized by SEM at different length scales. It was shown that TA was released from the 3D conical scaffold after 2 days in PBS at 37 °C. Biocompatibility studies were first assessed by seeding human dental pulp stem cells (DPSCs) on planar membranes coated or not coated with gelatin to compare the surfaces. After 24 h, the results highlighted that the gelatin-coating increased the membrane biocompatibility and cell viability. Similar DPSC morphology and proliferation on both membrane surfaces were observed. The culture of DPSCs on conical scaffolds showed cell colonization in the whole cone volume, proving that the architecture of the conical scaffold was suitable for cell migration.

Silk Fibroin/Collagen Blended Membrane Fabricated via a Green Papermaking Method for Potential Guided Bone Regeneration Application: In Vitro and In Vivo Evaluation
Dandan Luo - ,
Chenxue Yao - ,
Rui Zhang - ,
Ruibo Zhao *- ,
M. Zubair Iqbal - ,
Asim Mushtaq - ,
In-Seop Lee - , and
Xiangdong Kong *
Guided bone regeneration (GBR) technology is a commonly used surgical procedure for the repair of damaged periodontal tissues. Poor mechanical property and rapid degradation rate are the major reasons for GBR membrane failure in clinical applications. Herein, we applied a green papermaking method to fabricate silk fibroin (SF) membranes blended with collagen and tested their performance. The results showed that the blended SF75 (SF and collagen in a weight ratio of 75:25) membranes are biocompatible with good mechanical properties in the wet condition and appropriate biodegradation rate. MC3T3-E1 osteoblast cell adhesion and proliferation on the membranes were improved by the hybrid biological functions of SF and collagen. Subcutaneous implantation in rats for 9 weeks demonstrated that the membranes induced a less severe inflammatory response. The biodegradation time of the SF75 membranes was appropriate for tissue regeneration. This research, for the first time, reports a blended membrane prepared from silk fibroin and collagen with an ecofriendly method, which shows promise for application in guided bone regeneration.

Mechanical Integrity in a Dynamic Interpenetrating Hydrogel Network of Supramolecular Peptide–Polysaccharide Supports Enhanced Chondrogenesis
Jijo Thomas - ,
Nidhi Gupta - ,
Jojo P. Joseph - ,
Vianni Chopra - ,
Asish Pal *- , and
Deepa Ghosh *
Tissue engineering demands intelligently designed scaffolds that encompass the properties of the target tissues in terms of mechanical and bioactive properties. An ideal scaffold for engineering a cartilage tissue should provide the chondrocytes with a favorable 3D microarchitecture apart from possessing optimal mechanical characteristics such as compressibility, energy dissipation, strain stiffening, etc. Herein, we used a unique design approach to develop a hydrogel having a dynamic interpenetrating network to serve as a framework to support chondrocyte growth and differentiation. An amyloid-inspired peptide amphiphile (1) was self-assembled to furnish kinetically controlled nanofibers and incorporated in a dynamic covalently cross-linked polysaccharide network of carboxymethyl cellulose dialdehyde (CMC-D) and carboxymethyl chitosan (CMCh) using Schiff base chemistry. The dynamic noncovalent interaction played a pivotal role in providing the desired modulation in the structure and mechanical properties of the double-network hydrogels that are imperative for cartilage scaffold design. The adaptable nature supported shear-induced extrusion of the hydrogel and facilitated various cellular functions while maintaining its integrity. The potential of the as-developed hydrogels to support in vitro chondrogenesis was explored using human chondrocytes. Evidence of improved cell growth and cartilage-specific ECM production confirmed the potential of the hydrogel to support cartilage tissue engineering while reaffirming the significance of mimicking the biophysical microenvironment to induce optimal tissue regeneration.

Nanocomposite Conductive Bioinks Based on Low-Concentration GelMA and MXene Nanosheets/Gold Nanoparticles Providing Enhanced Printability of Functional Skeletal Muscle Tissues
Selwa Boularaoui - ,
Aya Shanti - ,
Michele Lanotte - ,
Shaohong Luo - ,
Sarah Bawazir - ,
Sungmun Lee - ,
Nicolas Christoforou - ,
Kamran A. Khan *- , and
Cesare Stefanini *
This publication is Open Access under the license indicated. Learn More
There is a growing need to develop novel well-characterized biological inks (bioinks) that are customizable for three-dimensional (3D) bioprinting of specific tissue types. Gelatin methacryloyl (GelMA) is one such candidate bioink due to its biocompatibility and tunable mechanical properties. Currently, only low-concentration GelMA hydrogels (≤5% w/v) are suitable as cell-laden bioinks, allowing high cell viability, elongation, and migration. Yet, they offer poor printability. Herein, we optimize GelMA bioinks in terms of concentration and cross-linking time for improved skeletal muscle C2C12 cell spreading in 3D, and we augment these by adding gold nanoparticles (AuNPs) or a two-dimensional (2D) transition metal carbide (MXene nanosheets) for enhanced printability and biological properties. AuNP and MXene addition endowed GelMA with increased conductivity (up to 0.8 ± 0.07 and 0.9 ± 0.12 S/m, respectively, compared to 0.3 ± 0.06 S/m for pure GelMA). Furthermore, it resulted in an improvement of rheological properties and printability, specifically at 10 °C. Improvements in electrical and rheological properties led to enhanced differentiation of encapsulated myoblasts and allowed for printing highly viable (97%) stable constructs. Taken together, these results constitute a significant step toward fabrication of 3D conductive tissue constructs with physiological relevance.

Characterization and Chromatographic Isolation of Platelet Extracellular Vesicles from Human Platelet Lysates for Applications in Neuroregenerative Medicine
Ariunjargal Nyam-Erdene - ,
Ouada Nebie - ,
Liling Delila - ,
Luc Buée - ,
David Devos - ,
Szu-Yi Chou - ,
David Blum - , and
Thierry Burnouf *
Human platelet lysates (HPLs) made from clinical-grade platelet concentrates are currently evaluated in the preclinical models of Parkinson’s disease, Alzheimer’s disease, traumatic brain injury, and others, as a new polyvalent neuroprotective biotherapy of the central nervous system. However, the presence and content of extracellular vesicles (EVs) in HPLs and their potential contribution to the neuroprotective and neurorestorative activities of HPLs are still unknown. We, therefore, characterized the EVs present in four different HPL preparations and after purification by size-exclusion chromatography. We then tested the effect of the isolated EVs on neuronal cell repair. We identified that all four HPLs contained a high and similar amount of EVs (1011 to 1012/mL) with a mean size ranging from ca. 50 to 300 nm and a negative zeta potential as determined by nanoparticle tracking analysis and dynamic light scattering. Western blot analysis revealed that the EVs present in HPLs expressed the clusters of differentiation 41 (CD41) and 61 (CD61) characteristic of platelets. These EVs were efficiently isolated from HPL proteins by Sepharose CL-2B size-exclusion column chromatography as confirmed by total protein determination and protein profile by sodium dodecyl sulfate polyacrylamide gel electrophoresis, with 73–85% recovery and maintenance of their size, negative zeta potential, and CD41 and CD61 expression. Interestingly, the EVs purified from the four HPLs exhibited a differential capacity to promote cell growth and migration in a wound-healing assay using SH-SY5Y neuronal cells, and one EV preparation stimulated network formation in primary neuronal cultures. These data indicated that the EVs present in HPLs have different neuroregenerative capacities and that some EV preparations may have interesting applications as a stand-alone therapy for usage in neuroregenerative medicine.

Three-Dimensional-Printed Flexible Scaffolds Have Tunable Biomimetic Mechanical Properties for Intervertebral Disc Tissue Engineering
Samantha L. Marshall - ,
Timothy D. Jacobsen - ,
Erik Emsbo - ,
Archana Murali - ,
Kevin Anton - ,
Jessica Z. Liu - ,
Helen H. Lu - , and
Nadeen O. Chahine *
The intervertebral disc (IVD) exhibits complex structure and biomechanical function, which supports the weight of the body and permits motion. Surgical treatments for IVD degeneration (e.g., lumbar fusion, disc replacement) often disrupt the mechanical environment of the spine which lead to adjacent segment disease. Alternatively, disc tissue engineering strategies, where cell-seeded hydrogels or fibrous biomaterials are cultured in vitro to promote matrix deposition, do not recapitulate the complex IVD mechanical properties. In this study, we use 3D printing of flexible polylactic acid (FPLA) to fabricate a viscoelastic scaffold with tunable biomimetic mechanics for whole spine motion segment applications. We optimized the mechanical properties of the scaffolds for equilibrium and dynamic moduli in compression and tension by varying fiber spacing or porosity, generating scaffolds with de novo mechanical properties within the physiological range of spine motion segments. The biodegradation analysis of the 3D printed scaffolds showed that FPLA exhibits lower degradation rate and thus has longer mechanical stability than standard PLA. FPLA scaffolds were biocompatible, supporting viability of nucleus pulposus (NP) cells in 2D and in FPLA+hydrogel composites. Composite scaffolds cultured with NP cells maintained baseline physiological mechanical properties and promoted matrix deposition up to 8 weeks in culture. Mesenchymal stromal cells (MSCs) cultured on FPLA adhered to the scaffold and exhibited fibrocartilaginous differentiation. These results demonstrate for the first time that 3D printed FPLA scaffolds have de novo viscoelastic mechanical properties that match the native IVD motion segment in both tension and compression and have the potential to be used as a mechanically stable and biocompatible biomaterial for engineered disc replacement.
Imaging and Diagnostics

Generation of Nanoyeast Single-Chain Variable Fragments as High-Avidity Biomaterials for Dengue Virus Detection
Fahimeh Farokhinejad - ,
Rebecca E. Lane - ,
Richard J. Lobb - ,
Selvakumar Edwardraja - ,
Alain Wuethrich *- ,
Christopher B. Howard *- , and
Matt Trau *
Bioengineered yeast bio-nanomaterials termed nanoyeasts displaying antibody single-chain variable fragments (scFvs) against diagnostic targets are a promising alternative to monoclonal antibodies (mAbs). A potential limitation for translating nanoyeasts into diagnostic tools is batch-to-batch variability. Herein, we demonstrate a systematic approach for cost-efficient production of highly specific nanoyeasts that enabled accurate dengue virus (DENV) detection by immunoassay (2.5% CV). Yeasts bioengineered to surface express DENV-specific scFvs (up to 66% of the total cell population) were fragmented into nanoyeast fractions trialing sonication, bead beating, and high-pressure disruption methods. Nanoyeast fractions from sonication had optimal target binding, uniform particle size (±89 nm), were stable, and retained diagnostic activity for 7 days at 37 °C compared to traditional mAbs that lost activity after 1 day at 37 °C. We engineered a panel of nanoyeast scFvs targeting DENV nonstructural protein 1 (NS1): (i) specific for serotyping DENV 1–4 and (ii) cross-reactive anti-DENV scFvs that are suitable for “yes/no” diagnostic applications. We demonstrate highly specific nanoyeast scFvs for serotyping DENV. We show that nanoyeast scFvs specifically detect NS1 in simulated patient plasma with a limit of detection of 250 ng/mL, the concentration found in infected patients.
Applications and Health

Anticorrosion and Cytocompatibility Assessment of Graphene-Doped Hybrid Silica and Plasma Electrolytic Oxidation Coatings for Biomedical Applications
Juan P. Fernández-Hernán *- ,
Antonio J. López - ,
Belén Torres - ,
Enrique Martínez-Campos - ,
Endzhe Matykina - , and
Joaquín Rams
This publication is Open Access under the license indicated. Learn More
Magnesium AZ31 alloy substrates were coated with different coatings, including sol–gel silica-reinforced with graphene nanoplatelets, sol–gel silica, plasma electrolytic oxidation (PEO), and combinations of them, to improve cytocompatibility and control the corrosion rate. Electrochemical corrosion tests, as well as hydrogen evolution tests, were carried out using Hanks’ solution as the electrolyte to assess the anticorrosion behavior of the different coating systems in a simulated body fluid. Preliminary cytocompatibility assessment of the different coating systems was carried out by measuring the metabolic activity, deoxyribonucleic acid quantification, and the cell growth of premyoblastic C2C12-GFP cell cultures on the surface of the different coating systems. Anticorrosion behavior and cytocompatibility were improved with the application of the different coating systems. The use of combined PEO + SG and PEO + SG/GNP coatings significantly decreased the degradation of the specimens. The monolayer sol–gel coatings, with and without GNPs, presented the best cytocompatibility improvement.

Molecular Response to Nanopatterned Implants in the Human Jaw Bone
Dimitrios Karazisis - ,
Omar Omar - ,
Sarunas Petronis - ,
Peter Thomsen *- , and
Lars Rasmusson
This publication is Open Access under the license indicated. Learn More
Implant surface modification by nanopatterning is an interesting route for enhancing osseointegration in humans. Herein, the molecular response to an intentional, controlled nanotopography pattern superimposed on screw-shaped titanium implants is investigated in human bone. When clinical implants are installed, additional two mini-implants, one with a machined surface (M) and one with a machined surface superimposed with a hemispherical nanopattern (MN), are installed in the posterior maxilla. In the second-stage surgery, after 6–8 weeks, the mini-implants are retrieved by unscrewing, and the implant-adherent cells are subjected to gene expression analysis using quantitative polymerase chain reaction (qPCR). Compared to those adherent to the machined (M) implants, the cells adherent to the nanopatterned (MN) implants demonstrate significant upregulation (1.8- to 2-fold) of bone-related genes (RUNX2, ALP, and OC). No significant differences are observed in the expression of the analyzed inflammatory and remodeling genes. Correlation analysis reveals that older patient age is associated with increased expression of proinflammatory cytokines (TNF-α and MCP-1) on the machined implants and decreased expression of pro-osteogenic factor (BMP-2) on the nanopatterned implants. Controlled nanotopography, in the form of hemispherical 60 nm protrusions, promotes gene expressions related to early osteogenic differentiation and osteoblastic activity in implant-adherent cells in the human jaw bone.

Localized Photothermal Ablation Therapy of Obstructive Rectal Cancer Using a Nanofunctionalized Stent in a Mouse Model
Hong-Tao Hu - ,
Jung-Hoon Park - ,
Zhe Wang - ,
Nader Bakheet - ,
Shi-Jun Xu - ,
Eun Ji Lee - ,
Dong-Hyun Kim - ,
Song Hee Kim - ,
Ho-Young Song - ,
Jae Yong Jeon *- , and
Suhwan Chang *
The self-expanding metal stent (SEMS) is a versatile, palliative treatment method for unresectable, malignant, non-vascular strictures. Colorectal cancer (CRC) is one of the candidates for the application of the SEMS, in combination with the photothermal ablation (PTA) technique that enhances its therapeutic efficacy. The objective of this study was to investigate the efficacy of stent-mediated PTA therapy in an endoscopy-guided, orthotopic rectal cancer model. A total of 30 of 40 mice with the tumor size of grade 4 were included and were divided into three groups of 10 mice each. Group A underwent a gold nanoparticle (AuNP)-coated SEMS but no near-infrared (NIR) irradiation, group B received an uncoated control SEMS with NIR irradiation, and group C received a AuNP-coated SEMS and NIR irradiation together. Colonoscopy and in vivo imaging, immunohistochemical analysis, and quantitative reverse-transcription polymerase chain reaction of major tumor markers were performed. Stent placement and PTA were technically successful using colonoscopy. The tumor grade reduction after PTA is significant in group C, compared with groups A or B (p < 0.001). Molecular analysis validated this observation with a significantly reduced Mapk1 proliferation marker or increased Jnk expression. Histological analysis confirmed the localized PTA therapy using AuNP-coated SEMS profoundly ablated tumor outgrowth through the stent. Our results indicate that this novel strategy of localized PTA therapy could be a promising option for palliative treatment of CRC and to support prolonged stent patency with a decreased tumor volume.

Antibacterial and Antibiofouling Activities of Antimicrobial Peptide-Functionalized Graphene–Silver Nanocomposites for the Inhibition and Disruption of Staphylococcus aureus Biofilms
Thanusu Parandhaman - ,
Priyadarshani Choudhary - ,
Baskaran Ramalingam - ,
Michael Schmidt - ,
Sridevi Janardhanam - , and
Sujoy K. Das *
Owing to the emergence of antibiotic-resistant strains, bacterial infection and biofilm formation are growing concerns in healthcare management. Herein, we report an eco-benign strategy for the synthesis and functionalization of graphene–silver (rGOAg) nanocomposites with an antimicrobial peptide (AMP) for the treatment of Staphylococcus aureus infection. The synthesis of rGOAg nanocomposites was carried out by simple microwave reduction, and the as-synthesized rGOAg was covalently functionalized with an AMP. As a natural AMP, poly-l-lysine (PLL) functionalization of rGOAg enhanced the antibacterial efficacy and target specificity against the S. aureus biofilm. The robust bactericidal efficiency and biofilm disruption by AMP-functionalized rGOAg (designated as GAAP) occurred through the “contact–kill–release” mode of action, where the electrostatic interaction with bacterial cells together with intracellular ROS generation induced physical disruption to the cell membrane. The internalization of GAAP into the cytoplasm through the damaged cell membrane caused an outburst of intracellular proteins and DNA. Crystal violet staining along with fluorescence and confocal microscopic images showed an effective inhibition and disruption of the S. aureus biofilm upon treatment with GAAP. PLL functionalization also prevented the dissolution of Ag+ ions and thereby minimized the in vitro toxicity of GAAP to the 3 T6 fibroblast and human red blood cells. The ex vivo rat skin disinfection model further demonstrated the potency of GAAP in eliminating the biofilm formation and disruption of the S. aureus biofilm. The obtained results demonstrated a general approach for designing a functional nanocomposite material to disrupt the mature biofilm and provided a promising strategy for treating bacterial infection.
Manufacturing, Technology, and Devices

Directional Transportation in a Self-Pumping Dressing Based on a Melt Electrospinning Hydrophobic Mesh
Zungui Shao - ,
Junyu Chen - ,
Ling-jie Ke - ,
Qingfeng Wang - ,
Xiang Wang - ,
Wenwang Li - , and
Gaofeng Zheng *
Self-pumping wound dressings with directional water transport ability have been widely studied for their function of directional extraction of excessive biofluid from wounds while keeping the wound in a moderately humid environment to realize rapid wound healing. However, the existing solutions have not paid close attention to the fabrication of a nonirritating hydrophobic layer facing the wounds, which may cause irritation to wounds and thereby further worsen inflammation. Herein, a flexible and elastic thermoplastic polyurethane (TPU) hydrophobic microfiber mesh (TPU-HMM) produced by melt electrospinning (MES) is reported. The TPU-HMM was compounded to a hydrophilic nanofiber membrane, which was fabricated by blending with polyamide 6 and poly(ethylene glycol) (PA6-PEG) to form a composite self-pumping dressing, for which the breakthrough pressure in a reverse direction was 12.8 times than that in a positive direction and the forward water transmission rate was increased by 700%. It shows good directional water transport ability and is expected to absorb excessive biofluid of the wounds. This solvent-free and easy-process TPU-HMM provides a new strategy for the development of functional self-pumping textiles, and the solvent-free fabrication method for fibers, which eliminates the potential toxicity brought by solvent residues, offers more possibilities for its applications in biomedicine.

Polymer-Coated Magnetic Microspheres Conjugated with Growth Factor Receptor Binding Peptides Enable Cell Sorting
John D. Krutty - ,
Jian Sun - ,
Kevin Koesser - ,
William L. Murphy - , and
Padma Gopalan *
The separation and sorting of human cells is an important step in the bioprocessing of cell-based therapeutics. Heterogeneous mixtures of cells must be sorted to isolate the desired cell type and purify the final product. This process is often achieved by antibody-based sorting techniques. In this work, we demonstrate that magnetic microspheres may be functionalized with peptides that selectively bind to cells on the basis of their relative concentration of specific surface proteins. Five-micrometer-magnetic microspheres were coated with the synthetic copolymer PVG (poly(poly(ethylene glycol)methyl ether methacrylate-ran-vinyl dimethyl azlactone-ran-glycidyl methacrylate) and functionalized with the vascular endothelial growth factor receptor binding peptide (VRBP), which binds to the vascular endothelial growth factor receptor (VEGFR). These microspheres exhibited low cytotoxicity and bind to cells depending on their relative surface protein expression. Finally, coated, magnetic microspheres were used to separate heterogeneous populations of cells dependent on their VEGFR expression through magnetic-assisted cell sorting (MACS), demonstrating that peptide-based cell sorting mechanisms may be useful in the bioprocessing of human-cell-based products.
Emerging Trends

Designer DNA Hydrogels Stimulate 3D Cell Invasion by Enhanced Receptor Expression and Membrane Endocytosis
Shanka Walia - ,
Vinod Morya - ,
Ankit Gangrade - ,
Supriyo Naskar - ,
Aditya Guduru Teja - ,
Sameer Dalvi - ,
Prabal K Maiti - ,
Chinmay Ghoroi - , and
Dhiraj Bhatia *
DNA has emerged as one of the smartest biopolymers to bridge the gap between chemical science and biology to design scaffolds like hydrogels by physical entanglement or chemical bonding with remarkable properties. We present here a completely new application of DNA-based hydrogels in terms of their capacity to stimulate membrane endocytosis, leading to enhanced cell spreading and invasion for cells in ex vivo 3D spheroids models. Multiscale simulation studies along with DLS data showed that the hydrogel formation was enhanced at lower temperature and it converts to liquid with increase in temperature. DNA hydrogels induced cell spreading as observed by the increase in cellular area by almost two-fold followed by an increase in the receptor expression, the endocytosis, and the 3D invasion potential of migrating cells. Our first results lay the foundation for upcoming diverse applications of hydrogels to probe and program various cellular and physiological processes that can have lasting applications in stem cell programming and regenerative therapeutics.
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