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Multilayer conformal coatings have been shown to provide a nanoscale barrier between cells and their environment with adequate stability, while regulating the diffusion of nutrition and waste across the cell membrane. The coating method aims to minimize capsule thickness and implant volume while reducing the need for immunosuppressive drugs, making it a promising approach for islet cell encapsulation in clinical islet transplantation for the treatment of Type 1 diabetes. This study introduces an immunoprotective nanocoating obtained through electrostatic interaction between quaternized phosphocholine-chitosan (PC-QCH) and tetrahydropyran triazole phenyl-alginate (TZ-AL) onto mouse ß-cell spheroids. First, successful synthesis of the proposed polyelectrolytes was confirmed with physico-chemical characterization. A coating with an average thickness of 540 nm was obtained with self-assembly of 4-bilayers of PC-QCH/TZ-AL onto MIN6 ß-cell spheroids. Surface coating of spheroids did not affect cell viability, metabolic activity, or insulin secretion, when compared to non-coated spheroids. The exposure of the polyelectrolytes to THP-1 monocyte-derived macrophages lead to a reduced level of TNF-α secretion and exposure of coated spheroids to RAW264.7 macrophages showed a decreasing trend in the secretion of TNF-α and IL-6. In addition, coated spheroids were able to establish normoglycemia when implanted into diabetic NOD-SCID mice, demonstrating in vivo biocompatibility and cellular function. These results demonstrate the ability of the PC-QCH/TZ-AL conformal coating to mitigate pro-inflammatory responses from macrophages, and thus can be a promising candidate towards nanoencapsulation for cell-based therapy, particularly in type 1 diabetes, where the insulin secreting ß-cells are subjected to inflammation and immune cell attack.

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[This corrects the article DOI: 10.3389/fcvm.2021.737934.].

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Human vocal folds (VF), a pair of small, soft tissues in the larynx, have a layered mucosal structure with unique mechanical strength to support high-level tissue deformation by phonation. Severe pathological changes to VF have causes including surgery, trauma, age-related atrophy, and radiation, and lead to partial or complete communication loss and difficulty in breathing and swallowing. VF glottal insufficiency requires injectable VF biomaterials such as hyaluronan, calcium hydroxyapatite, and autologous fat to augment VF functions. Although these biomaterials provide an effective short-term solution, significant variations in patient response and requirements of repeat reinjection remain notable challenges in clinical practice. Tissue engineering strategies have been actively explored in the search of an injectable biomaterial that possesses the capacity to match native tissue's material properties while promoting permanent tissue regeneration. This review aims to assess the current status of biomaterial development in VF tissue engineering. The focus will be on examining state-of-the-art techniques including modification with bioactive molecules, cell encapsulation, composite materials, as well as, in situ crosslinking with click chemistry. We will discuss potential opportunities that can further leverage these engineering techniques in the advancement of VF injectable biomaterials.

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Extracellular vesicles have gained wide momentum as potential therapeutics for osteoarthritis, a highly prevalent chronic disease that still lacks an approved treatment. The membrane-bound vesicles are secreted by all cells carrying different cargos that can serve as both disease biomarkers and disease modifiers. Nonetheless, despite a significant peak in research regarding EVs as OA therapeutics, clinical implementation seems distant. In addition to scalability and standardization challenges, researchers often omit to focus on and consider the proper tropism of the vesicles, the practicality and relevance of their source, their low native therapeutic efficacy, and whether they address the disease as a whole. These considerations are necessary to better understand EVs in a clinical light and have been comprehensively discussed and ultimately summarized in this review into a conceptualized framework termed the nanodiamond concept. Future perspectives are also discussed, and alternatives are presented to address some of the challenges and concerns.

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Lipid nanoparticles (LNPs) are biocompatible drug delivery systems that have found numerous applications in medicine. Their versatile nature enables the encapsulation and targeting of various types of medically relevant molecular cargo, including oligonucleotides, proteins, and small molecules for the treatment of diseases, such as cancer. Cancers that form solid tumors are particularly relevant for LNP-based therapeutics due to the enhanced permeation and retention effect that allows nanoparticles to accumulate within the tumor tissue. Additionally, LNPs can be formulated for both locoregional and systemic delivery depending on the tumor type and stage. To date, LNPs have been used extensively in the clinic to reduce systemic toxicity and improve outcomes in cancer patients by encapsulating chemotherapeutic drugs. Next-generation lipid nanoparticles are currently being developed to expand their use in gene therapy and immunotherapy, as well as to enable the co-encapsulation of multiple drugs in a single system. Other developments include the design of targeted LNPs to specific cells and tissues, and triggerable release systems to control cargo delivery at the tumor site. This review paper highlights recent developments in LNP drug delivery formulations and focuses on the treatment of solid tumors, while also discussing some of their current translational limitations and potential opportunities in the field.

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Bioactive surface coatings have retained the attention of researchers and physicians due to their versatility and range of applications in orthopedics, particularly in infection prevention. Antibacterial metal nanoparticles (mNPs) are a promising therapeutic, with vast application opportunities on orthopedic implants. The current research aimed to construct a polyelectrolyte multilayer on a highly porous titanium implant using alternating thin film coatings of chitosan and alginate via the layer-by-layer (LbL) self-assembly technique, along with the incorporation of silver nanoparticles (AgNPs) or titanium dioxide nanoparticles (TiO2NPs), for antibacterial and osteoconductive activity. These mNPs were characterized for their physicochemical properties using quartz crystal microgravimetry with a dissipation system, nanoparticle tracking analysis, scanning electron microscopy, and atomic force microscopy. Their cytotoxicity and osteogenic differentiation capabilities were assessed using AlamarBlue and alkaline phosphatase (ALP) activity assays, respectively. The antibiofilm efficacy of the mNPs was tested against Staphylococcus aureus. The LbL polyelectrolyte coating was successfully applied to the porous titanium substrate. A dose-dependent relationship between nanoparticle concentration and ALP as well as antibacterial effects was observed. TiO2NP samples were also less cytotoxic than their AgNP counterparts, although similarly antimicrobial. Together, these data serve as a proof-of-concept for a novel coating approach for orthopedic implants with antimicrobial and osteoconductive properties.

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Bone-mimicking scaffolds based on silk fibroin (SF) mixed with hydroxyapatite nanoparticles (HA NPs) and titanium oxide (TiO2) nanoparticles were created as materials for bone formation. Six scaffold groups were fabricated: S1 (SF), S2 (Silk + (HA: TiO2; 100: 0)), S3 (Silk, (HA: TiO2; 70: 30)), S4 (Silk + (HA NPs: TiO2; 50: 50)), S5 (Silk + (HA: TiO2; 30: 70)), and S6 (Silk + (HA NPs: TiO2; 0:100)). Scaffolds were characterized for molecular formation, structure, and morphology by Fourier transform infrared spectroscopy, element analysis, and X-ray diffraction. They were tested for physical swelling and compressive modulus. Scaffolds were cultured with MC3T3 and testedin vitroto evaluate their biological performance. The results showed that scaffolds with HA and TiO2demonstrated molecular interaction via amide I and phosphate groups. These scaffolds had smaller pore sizes than those without HA and TiO2. They showed more swelling and higher compressive modulus than the scaffolds without HA and TiO2. They exhibited better biological performance: cell adhesion, viability, proliferation, alkaline phosphatase activity, and calcium content than the scaffolds without HA and TiO2. Their porous walls acted as templates for cell aggregation and supported synthesis of calcium secreted from cells. S3 were the most suitable scaffolds. With their enhanced osteo-conductive function, they are promising for bone augmentation for oral and maxillofacial surgery.

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Decellularized extracellular matrix (dECM) is a promising material for tissue engineering applications. Tissue-specific dECM is often seen as a favorable material that recapitulates a native-like microenvironment for cellular remodeling. However, the minute quantity of dECM derivable from small organs like the vocal fold (VF) hampers manufacturing scalability. Small intestinal submucosa (SIS), a commercial product with proven regenerative capacity, may be a viable option for VF applications. This study aims to compare dECM hydrogels derived from SIS or VF tissue with respect to protein content and functionality using mass spectrometry-based proteomics and in vitro studies. Proteomic analysis reveals that VF and SIS dECM share 75% of core matrisome proteins. Although VF dECM proteins have greater overlap with native VF, SIS dECM shows less cross-sample variability. Following decellularization, significant reductions of soluble collagen (61%), elastin (81%), and hyaluronan (44%) are noted in VF dECM. SIS dECM contains comparable elastin and hyaluronan but 67% greater soluble collagen than VF dECM. Cells deposit more neo-collagen on SIS than VF-dECM hydrogels, whereas neo-elastin (~50 µg/scaffold) and neo-hyaluronan (~ 6 µg/scaffold) are comparable between the two hydrogels. Overall, SIS dECM possesses reasonably similar proteomic profile and regenerative capacity to VF dECM. SIS dECM is considered a promising alternative for dECM-derived biomaterials for VF regeneration.

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An increasing number of publications over the past ten years have focused on the development of chitosan-based cross-linked scaffolds to regenerate bone tissue. The design of biomaterials for bone tissue engineering applications relies heavily on the ideals set forth by a polytherapy approach called the "Diamond Concept". This methodology takes into consideration the mechanical environment, scaffold properties, osteogenic and angiogenic potential of cells, and benefits of osteoinductive mediator encapsulation. The following review presents a comprehensive summarization of recent trends in chitosan-based cross-linked scaffold development within the scope of the Diamond Concept, particularly for nonload-bearing bone repair. A standardized methodology for material characterization, along with assessment of in vitro and in vivo potential for bone regeneration, is presented based on approaches in the literature, and future directions of the field are discussed.

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Current delivery of chemotherapy, either intra-venous or intra-arterial, remains suboptimal for patients with head and neck tumors. The free form of chemotherapy drugs, such as docetaxel, has non-specific tissue targeting and poor solubility in blood that deters treatment efficacy. Upon reaching the tumors, these drugs can also be easily washed away by the interstitial fluids. Liposomes have been used as nanocarriers to enhance docetaxel bioavailability. However, they are affected by potential interstitial dislodging due to insufficient intratumoral permeability and retention capabilities. Here, we developed and characterized docetaxel-loaded anionic nanoliposomes coated with a layer of mucoadhesive chitosan (chitosomes) for the application of chemotherapy drug delivery. The anionic liposomes were 99.4 ± 1.5 nm in diameter with a zeta potential of -26 ± 2.0 mV. The chitosan coating increased the liposome size to 120 ± 2.2 nm and the surface charge to 24.8 ± 2.6 mV. Chitosome formation was confirmed via FTIR spectroscopy and mucoadhesive analysis with anionic mucin dispersions. Blank liposomes and chitosomes showed no cytotoxic effect on human laryngeal stromal and cancer cells. Chitosomes were also internalized into the cytoplasm of human laryngeal cancer cells, indicating effective nanocarrier delivery. A higher cytotoxicity (p < 0.05) of docetaxel-loaded chitosomes towards human laryngeal cancer cells was observed compared to human stromal cells and control treatments. No hemolytic effect was observed on human red blood cells after a 3 h exposure, proving the proposed intra-arterial administration. Our in vitro results supported the potential of docetaxel-loaded chitosomes for locoregional chemotherapy delivery to laryngeal cancer cells.

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Nanotherapeutics, on their path to the target tissues, face numerous physicochemical hindrances that affect their therapeutic efficacy. Physical barriers become more pronounced in pathological tissues, such as solid tumors, where they limit the penetration of nanocarriers into deeper regions, thereby preventing the efficient delivery of drug cargo. To address this challenge, we introduce a novel approach that employs surface acoustic wave (SAW) technology to sonoprint and enhance the delivery of nanoparticles onto and into cell spheroids. Our SAW platform is designed to generate focused and unidirectional acoustic waves for creating vigorous acoustic streaming while promoting Bjerknes forces. The effect of SAW excitation on cell viability, as well as the accumulation and penetration of nanoparticles on human breast cancer (MCF 7) and mouse melanoma (YUMM 1.7) cell spheroids were investigated. The high frequency, low input voltage, and contact-free nature of the proposed SAW system ensured over 92% cell viability for both cell lines after SAW exposure. SAW sonoprinting enhanced the accumulation of 100 nm polystyrene particles on the periphery of the spheroids to near four-fold, while the penetration of nanoparticles into the core regions of the spheroids was improved up to three times. To demonstrate the effectiveness of our SAW platform on the efficacy of nanotherapeutics, the platform was used to deliver nanoliposomes encapsulated with the anti-cancer metal compound copper diethyldithiocarbamate (CuET) to MCF 7 and YUMM 1.7 cell spheroids. A three-fold increase in the cytotoxic activity of the drug was observed in spheroids under the effect of SAW, compared to controls. The capacity of SAW-based devices to be manufactured as minuscule wearable patches can offer highly controllable, localized, and continuous acoustic waves to enhance drug delivery efficiency to target tissues.

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For more than 70 years, acoustic waves have been used to screen, diagnose, and treat patients in hundreds of medical devices. The biocompatible nature of acoustic waves, their non-invasive and contactless operation, and their compatibility with wide visualization techniques are just a few of the many features that lead to the clinical success of sound-powered devices. The development of microelectromechanical systems and fabrication technologies in the past two decades reignited the spark of acoustics in the discovery of unique microscale bio applications. Acoustofluidics, the combination of acoustic waves and fluid mechanics in the nano and micro-realm, allowed researchers to access high-resolution and controllable manipulation and sensing tools for particle separation, isolation and enrichment, patterning of cells and bioparticles, fluid handling, and point of care biosensing strategies. This versatility and attractiveness of acoustofluidics have led to the rapid expansion of platforms and methods, making it also challenging for users to select the best acoustic technology. Depending on the setup, acoustic devices can offer a diverse level of biocompatibility, throughput, versatility, and sensitivity, where each of these considerations can become the design priority based on the application. In this paper, we aim to overview the recent advancements of acoustofluidics in the multifaceted fields of regenerative medicine, therapeutic development, and diagnosis and provide researchers with the necessary information needed to choose the best-suited acoustic technology for their application. Moreover, the effect of acoustofluidic systems on phenotypic behavior of living organisms are investigated. The review starts with a brief explanation of acoustofluidic principles, the different working mechanisms, and the advantages or challenges of commonly used platforms based on the state-of-the-art design features of acoustofluidic technologies. Finally, we present an outlook of potential trends, the areas to be explored, and the challenges that need to be overcome in developing acoustofluidic platforms that can echo the clinical success of conventional ultrasound-based devices.

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Decellularized porcine aortas are proposed as scaffolds for revolutionary active aortic grafts. A change in the static and dynamic mechanical properties, associated with the microstructure of elastin and collagen fibers, corresponds to alteration in the cyclic expansion and perfusion, in addition to possible graft damage. Therefore, the present study thoroughly investigates the mechanical response of the decellularized scaffolds of human and porcine origin to static and dynamic mechanical loads. The responses of the native human and porcine aortas are also compared; this is unavailable in the literature. Because the aorta is subjected to pulsatile blood pressure, dynamical responses to cyclic loads and their associated viscoelastic properties are particularly relevant for advanced graft design. In parallel, this study examines the microstructure of the decellularized aorta. The resulting data are compared to the analogous data obtained for the native human and porcine tissues. The results indicate that by using an optimized decellularization protocol - based on sodium dodecyl sulfate (SDS) and DNase - that minimizes mechanical and structural changes of the tissue, layered scaffolds with static and dynamic properties very similar to natural human aortas are obtained. In particular, a decellularized porcine aorta is non-inferior to a decellularized human aorta. STATEMENT OF SIGNIFICANCE: About 55,000 patients undergo abdominal aortic aneurysm repair annually in the USA. The currently implanted grafts present a large mechanical mismatch with the native tissue. This increases the pulsatile nature of the blood flow with negative consequences to the organ perfusion. For this reason, biomimetic and mechanically compatible grafts for aortic repair are urgently needed and they can be obtained through tissue engineering. In this study, scaffolds from porcine and human aortas are obtained from an optimized decellularization protocol. They are accurately compared to the native tissue and present the ideal static and dynamic mechanical properties for developing innovative aortic grafts.

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Effective treatments for critical size bone defects remain challenging. 6-Bromoindirubin-3'-Oxime (BIO), a glycogen synthase kinase 3ß inhibitor, is a promising alternative for treatment of these defects since it aids in promoting osteogenic differentiation. In this study, BIO is incorporated into a new formulation of the guanosine diphosphate cross-linked chitosan scaffold to promote osteogenic differentiation. BIO incorporation was confirmed with 13C NMR through a novel concentration dependent peak around 41 ppm. The rapid gelation rate was maintained along with the internal structure's stability. The 10 µM BIO dose supported the control scaffold's microstructure demonstrating a suitable porosity and a low closed pore percentage. While pore sizes of BIO incorporated scaffolds were slightly smaller, pore heterogeneity was maintained. A proof-of-concept study with C2C12 cells suggested a dose-dependent response of BIO on early stages of osteogenic differentiation within the scaffold. These results support future work to examine BIO's role on osteogenic differentiation and biomineralization of encapsulated cells in the scaffold for bone regeneration.

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In vitro drug screening for type 1 diabetes therapies has largely been conducted on human organ donor islets for proof of efficacy. While native islets are the ultimate target of these drugs (either in situ or for transplantation), significant benefit can be difficult to ascertain due to the highly heterogeneous nature of individual donors and the overall scarcity of human islets for research. We present an in vitro coculture model based on immortalized insulin-producing beta-cell lines with human endothelial cells in 3D spheroids that aims to recapitulate the islet morphology in an effort towards developing a standardized cell model for in vitro diabetes research. Human insulin-producing immortalized EndoC-ßH5 cells are cocultured with human endothelial cells in varying ratios to evaluate 3D cell culture models for type 1 diabetes research. Insulin secretion, metabolic activity, live cell fluorescence staining, and gene expression assays were used to compare the viability and functionality of spheroids composed of 100% beta-cells, 1 : 1 beta-cell/endothelial, and 1 : 3 beta-cell/endothelial. Monoculture and ßH5/HUVEC cocultures formed compact spheroids within 7 days, with average diameter ~140 µm. This pilot study indicated that stimulated insulin release from 0 to 20 mM glucose increased from ~8-fold for monoculture and 1 : 1 coculture spheroids to over 20-fold for 1 : 3 EndoC-ßH5/HUVEC spheroids. Metabolic activity was also ~12% higher in the 1 : 3 EndoC-ßH5/HUVEC group compared to other groups. Stimulating monoculture beta-cell spheroids with 20 mM glucose +1 µg/mL glycine-modified INGAP-P increased the insulin stimulation index ~2-fold compared to glucose alone. Considering their availability and consistent phenotype, EndoC-ßH5-based spheroids present a useful 3D cell model for in vitro testing and drug screening applications.

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Islet transplantation offers a long-term cure for Type 1 Diabetes (T1D), freeing patients from daily insulin injections. Therapeutic peptides have shown potential to increase the insulin output of pancreatic islets, maximizing the impact of grafted cells. The islet neogenesis-associated protein (INGAP), and its bioactive core (INGAP-P), stimulate beta-cell function and viability, offering the possibility for islet treatment prior to implant. However, dosing efficacy is limited by low circulation time and enzyme degradation. This proof-of-concept study presents the investigation of novel molecular variants of INGAP-P to find a more bioactive form. Custom-designed peptide variants of INGAP-P were synthesized and tested for their effect on the insulin secretion and gene expression of live human islets. We exposed the live islets of five donors to varying glucose concentrations with INGAP-P variants in solution. We identified four peptide variants (I9, I15Tyr, I19 and I15Cys) which displayed statistically significant enhancements over negative controls (representing a 1.6-2.8-fold increase in stimulation index). This is the first study that has assessed these INGAP-P variants in human islets. It highlights the potential for customized peptides for type 1 diabetes therapy and provides a foundation for future peptide-screening experiments.

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Extracellular matrix is rich in biomolecules including structural proteins, glycosaminoglycans, and small molecules that are important for the maintenance and repair of tissue. Decellularized extracellular matrix (dECM) is expected to retain these key biomolecules and makes it a promising biomaterial candidate for regenerative medicine applications. To date, dECM-particle based biomaterials have been developed to engineer over 15 tissue types or organs, with the ultimate goal of mimicking specific biological and physical properties of the native tissue. The most common scaffold types are injectable hydrogels, electrospun scaffolds and bioprinted scaffolds. The purpose of this review paper is to highlight key challenges, fabrication methods and progress made for each tissue type, along with the discussion of other elements that are integral to push dECM biomaterials towards effective and specialized tissue repair.

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The 5-year overall survival rate remains approximately 50% for head and neck (H&N) cancer patients, even though new cancer drugs have been approved for clinical use since 2016. Cancer drug studies are now moving toward the use of three-dimensional culture models for better emulating the unique tumor microenvironment (TME) and better predicting in vivo response to cancer treatments. Distinctive TME features, such as tumor geometry, heterogenous cellularity, and hypoxic cues, notably affect tissue aggressiveness and drug resistance. However, these features have not been fully incorporated into in vitro H&N cancer models. This review paper aims to provide a scholarly assessment of the designs, contributions, and limitations of in vitro models in H&N cancer drug research. We first review the TME features of H&N cancer that are most relevant to in vitro drug evaluation. We then evaluate a selection of advanced culture models, namely, spheroids, organotypic models, and microfluidic chips, in their applications for H&N cancer drug research. Lastly, we propose future opportunities of in vitro H&N cancer research in the prospects of high-throughput drug screening and patient-specific drug evaluation.

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Acoustofluidics has shown great potential for label-free bioparticle patterning with excellent biocompatibility. Acoustofluidic patterning enables the induction of cell-cell interactions, which play fundamental roles in organogenesis and tissue development. One of the current challenges in tissue engineering is not only the control of the spatial arrangement of cells but also the preservation of cell patterns over time. In this work, we developed a standing surface acoustic wave-based platform and demonstrated its capability for the well-controlled and rapid cell patterning of adipose-derived mesenchymal stem cells in a high-density homogenous collagen hydrogel. This biocompatible hydrogel is easily UV crosslinked and can be retrieved within 3 min. Acoustic waves successfully guided the cells toward pressure nodal lines, creating a contactless alignment of cells in <5 s in culture media and <1 min in the hydrogel. The acoustically patterned cells in the hydrogel did not show a decrease in cell viability (>90%) 48 h after acoustic induction. Moreover, 45.53% and 30.85% increases in metabolic activity were observed in growth and differentiation media, respectively, on Day 7. On Day 14, a 32.03% change in metabolic activity was observed using growth media, and no significant difference was observed using differentiation media. The alkaline phosphatase activity showed an increase of 80.89% and 24.90% on Days 7 and 14, respectively, for the acoustically patterned cells in the hydrogel. These results confirm the preservation of cellular viability and improved cellular functionality using the proposed high-resolution acoustic patterning technique and introduce unique opportunities for the application of stem cell regenerative patches for the emerging field of tissue engineering.

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The metal complex copper diethyldithiocarbamate (CuET) induces cancer cell death by inhibiting protein degradation and induces proteotoxic stress, making CuET a promising cancer therapeutic. However, no clinical formulation of CuET exists to date as the drug is insoluble in water and exhibits poor bioavailability. To develop a scalable formulation, nanoliposomal (LP) CuET was synthesized using ethanol injection as a facile one-step method that is suitable for large-scale manufacturing. The nanoparticles are monodispersed, colloidally stable, and approximately 100 nm in diameter with an encapsulation efficiency of over 80%. LP-CuET demonstrates excellent stability in plasma, minimal size change, and little drug release after six-month storage at various temperatures. Additionally, melanoma cell lines exhibit significant sensitivity to LP-CuET and cellular uptake occurs predominantly through endocytosis in YUMM 1.7 cancer cells. Intracellular drug delivery is mediated by vesicle acidification with more nanoparticles being internalized by melanoma cells compared with RAW 264.7 macrophages. Additionally, the nanoparticles preferentially accumulate in YUMM 1.7 tumors where they induce cancer cell death in vivo. The development and characterization of a stable and scalable CuET formulation illustrated in this study fulfils the requirements needed for a potent clinical grade formulation.