Non-parenteral drug delivery systems using biomaterials have advantages over traditional parenteral strategies. For ocular and intranasal delivery, nanoparticulate systems must bind to and permeate through mucosal epithelium and other biological barriers. The incorporation of mucoadhesive and permeation-enhancing biomaterials such as chitosan facilitate this, but tend to increase the size and polydispersity of the nanoparticles, making practical optimization and implementation of mucoadhesive nanoparticle formulations a challenge. In this study, we adjusted key poly(lactic-co-glycolic) acid (PLGA) nanoparticle formulation parameters including the organic solvent and co-solvent, the concentration of polymer in the organic phase, the composition of the aqueous phase, the sonication amplitude, and the inclusion of chitosan in the aqueous phase. By doing so, we prepared four statistically unique size groups of PLGA NPs and equally-sized chitosan-PLGA NP counterparts. We loaded simvastatin, a candidate for novel ocular and intranasal delivery systems, into the nanoparticles to investigate the effects of size and surface modification on drug loading and release, and we quantified size- and surface-dependent changes in mucoadhesion in vitro. These methods and findings will contribute to the advancement of mucoadhesive nanoformulations for ocular and nose-to-brain drug delivery.

An accurate in vitro model of human adipose tissue could assist in the study of adipocyte function and allow for better tools for screening new therapeutic compounds. Cell culture models on two-dimensional surfaces fall short of mimicking the three-dimensional in vivo adipose environment, while three-dimensional culture models are often unable to support long-term cell culture due, in part, to insufficient mass transport. Microfluidic systems have been explored for adipose tissue models. However, current systems have primarily focused on 2D cultured adipocytes. In this work, a 3D human adipose microtissue was engineered within a microfluidic system. Human adipose-derived stem cells (ADSCs) were used as the cell source for generating differentiated adipocytes. The ADSCs differentiated within the microfluidic system formed a dense lipid-loaded mass with the expression of adipose tissue genetic markers. Engineered adipose tissue showed a decreased adiponectin secretion and increased free fatty acid secretion with increasing shear stress. Adipogenesis markers were downregulated with increasing shear stress. Overall, this microfluidic system enables the on-chip differentiation and development of a functional 3D human adipose microtissue supported by the interstitial flow. This system could potentially serve as a platform for in vitro drug testing for adipose tissue-related diseases.

Top-down functionalization of nanoparticles with cellular membranes imparts nanoparticles with enhanced bio-interfacing capabilities. Initial methods for membrane coating involved physical co-extrusion of nanoparticles and membrane vesicles through a porous membrane; however, recent works employ sonication as the disruptive force to reform membranes around the surface of nanoparticles. Although sonication is widely used, there remains a paucity of information on the effects of sonication variables on coating efficiency, leading to inconsistent membrane coating across studies. In this work, we present a systematic analysis of the sonication parameters that influence the membrane coating. The results showed that sonication amplitude, time, temperature, membrane ratio, sample volume, and density need to be considered in order to optimize membrane coating of polymeric nanoparticles.

Vascularization is critical for engineering mineralized tissues. It has been previously shown that biomaterials containing preformed endothelial networks anastomose to host vasculature following implantation. However, the networks alone may not increase regeneration. In addition, a clinically applicable source of cells for vascularization is needed. In this study, vascular networks were generated from endothelial cells (ECs) derived from human induced pluripotent stem cells (iPSCs). Network formation by iPSC-ECs within fibrin gels was investigated in a mesenchymal stem cells (MSCs) coculture spheroid model. Statistical design of experiments technique was evaluated for its predicting capability during the optimization of experimental parameters. The prevascularized units were combined with hydroxyapatite nanoparticles to develop a vascularized composite hydrogel that was implanted in a rodent critical-sized cranial defect model. Immunohistological staining for human-specific CD31 at week 1 indicated the presence and maintenance of the implanted vessels. At 8 weeks, the prevascularized systems resulted in higher vessel density over MSC-only scaffolds. The implanted vessels appeared to establish flow with host vasculature. While there was a slight increase in bone volume in the prevascularized bone construct compared to MSC-only bone constructs, there was not a profound increase in bone regeneration. These results show that scaffolds with network structures can be generated from ECs derived from iPSC and that the networks survive and inosculate with the host postimplantation in a bone model. Impact statement Vascularization is critical for engineering bone. Prevascularized scaffolds have been shown to improve postimplantation vascularization. Herein, vascularized networks were generated from induced pluripotent cells derived from endothelial cells. These vascularized units were combined with a fibrin/hydroxyapatite scaffold to develop a prevascularized construct for bone regeneration. Implantation of these scaffolds in a small animal cranial defect model resulted in network inosculation and increased vascularization, but exhibited only a limited effect on bone formation. This study provides insight into the challenges of generating vascularized bone.

Gradients in mechanical properties, physical architecture and biochemical composition exist in a variety of complex tissues, yet 3D in vitro models that enable investigation of these cues on cellular processes, especially those contributing to vascularization of engineered tissues are limited. Here, a photopolymerization approach to create cell-laden hydrogel biomaterials with decoupled and combined gradients in modulus, immobilized cell adhesive peptide (RGD) concentration, and proteolytic degradation enabling spatial encapsulation of vascular spheroids is reported to elucidate their impact on vascular sprouting in 3D culture. Vascular spheroids encapsulated in these gradient scaffolds exhibit spatial variations in total sprout length. Scaffolds presenting an immobilized RGD gradient promote biased vascular sprouting toward increasing RGD concentration. Importantly, biased sprouting is found to be dependent on immobilized RGD gradient characteristics, including magnitude and slope, with increases in these factors contributing to significant enhancements in biased sprouting responses. Conversely, reduction in biased sprouting responses is observed in combined gradient scaffolds possessing opposing gradients in RGD and modulus. The presented work is the first to demonstrate the use of a cell-laden biomaterial platform to systematically investigate the role of multiple scaffold gradients as well as gradient slope, magnitude and orientation on vascular sprouting responses in 3D culture.

In vitro adipose tissue models can be used to provide insight into fundamental aspects of adipose physiology. These systems may serve as replacements for animal models, which are often poor predictors of obesity and metabolic diseases in humans. Adipose tissue consists of a rich vasculature that is essential to its function. However, the study of endothelial cell–adipocyte interactions has been challenging due to differences in culture conditions required for the survival and function of each cell type. To address this issue, we performed an extensive evaluation of the cell culture media composition to identify the conditions optimal for the co-culture of endothelial cells and adipocytes. The effects of individual media factors on cell survival, proliferation, and differentiation were systematically explored. Several media factors were determined to disrupt the co-culture system. Optimized culture conditions were identified and used to generate a vascularized human adipose microtissue. An interconnected vascular network was established within an adipose micro-tissue, and the networks were anastomosed with perfused channels to form a functional network. In conclusion, media conditions were identified that enabled endothelial cell–adipocyte co-culture and were used to support the formation of a vascularized adipose tissue within a microfluidic device.

Vascularization is critical for the survival of engineered tissues post implantation. It has been previously shown that biomaterials containing preformed networks can anastomose to host vasculature following implantation. However, the optimal source of cells for vascularization for clinical use remains elusive. In this study, vascular networks were generated from endothelial cells derived from human induced pluripotent stem cells (iPSCs). Network formation by iPSC-ECs within fibrin gels was investigated in a mesenchymal stem cell (MSC) co-culture spheroid model. Statistical design of experiments (DOE) techniques were applied to identify optimal conditions for vessel-like network formation. The prevascularized units were then combined with hydroxyapatite nanoparticles to develop a vascularized composite hydrogel that was implanted in a rodent critical sized cranial defect model. Immunohistological staining for human-specific CD31 at week 1 indicated the presence and maintenance of the implanted vessels. Erythrocytes in the vessel lumen further suggests anastomosis of vessels with host vasculature. At week 8, isolectin staining indicated functionality of the human implanted vessels. There was a slight increase in bone volume in prevascularized scaffolds compared to MSC-only scaffolds. However, a pronounced increased in bone regeneration with prevascularization was not observed. These results show that prevascularized scaffolds can be generated from ECs derived from iPSC and that the networks survive and inosculate with the host post implantation in a bone model.

Better experimental models are needed to enhance our understanding of metabolic regulation which is seen in obesity and metabolic disorders, such as type 2 diabetes. In vitro models based on microfluidics enable physiological representations of tissues with several advantages over conventional culture systems, such as perfused flow to better mimic the physiological environment. Although cell lines such as 3T3-L1 have been incorporated in microfluidic devices, murine primary preadipocytes have not been differentiated and maintained for long-term monitoring in these culture systems. We describe the differentiation of these cells into white adipose depots on a perfused microfluidic chip. We compare the effects of shear flow on these cells, and show with a direct comparison of high/low shear conditions that direct shear is detrimental to the viability of preadipocytes. We further develop a dual-chamber microfluidic chip that enables perfusion while at the same time protects the cells from direct fluidic shear. We show that the dual-layer microfluidic device enables long-term culture of cells and allows stimulation of cells through perfusion-we can culture, differentiate, and maintain the differentiated adipose tissue for over multiple weeks in the device. Both triglycerides and lipolytic glycerol production increased significantly by several folds during differentiation. After successful differentiation, the adipocytes had upregulated expression of leptin and adiponectin, which are important makers of the final stage of adipogenic differentiation. In conclusion, the dual-layer microfluidic device incorporated with primary adipocytes improves the understanding of adipose differentiation under dynamic conditions and is positioned to serve as a disease model for studying obesity and other metabolic disorders.

The growing problem of obesity has reached epidemic proportion worldwide and with over 1.5 billion adults aged 20 years and older characterized as being overweight [1]. Being overweight or obese remarkably increases the risks of developing other metabolic diseases such as diabetes. Adipose tissue is a key player in metabolic disorders including obesity and diabetes. Recent studies highlight the critical role adipose plays in maintaining metabolic homeostasis [2, 3]. Development of in vitro models of adipose tissue is highly desirable to improve our understanding of disease biology of obesity and metabolic diseases. Microfluidic on‐chip in vitro models offer advantages compared to traditional static culture system in terms of mimicking the complex physiological environment for culturing, differentiating, and stimulating adipocytes in a small‐volume perfused cell culture system [4, 5]. Here, we aim to establish a model for differentiation of pre‐adipocytes into mature adipose depots on a microfluidic chip. Mouse primary pre‐adipocytes isolated from subcutaneous white adipose tissue (WAT) were differentiated into mature white adipocytes in a microfluidic chip under perfused flow of 5 μ L/h. Our unique microfluidic chip enables for long‐term culture of cells and allows stimulation of cells through perfusion – we can culture, differentiate, and maintain the differentiated adipose tissue for over 21 days in the device. Upon differentiation, the cells increasingly accumulated triglycerides, which was quantified using fluorescence microscopy. Further confirmation of the differentiation was assessed by measuring the gene expression of markers including leptin and adiponectin. In addition, we collected and analyzed the perfusate for adipose secretions including adiponectin. Our results indicate an improved understanding of adipose differentiation under dynamic conditions and can serve as disease models for obesity and other metabolic disorders. Support or Funding Information This work was supported by NIH grant DK095984. [1] Ahima RS. Digging deeper into obesity. Journal of Clinical Investigation 2011;121:2076–9.[2] Clark AM, Sousa KM, Chisolm CN, MacDougald OA, Kennedy RT. Reversibly sealed multilayer microfluidic device for integrated cell perfusion and on‐line chemical analysis of cultured adipocyte secretions. Analytical and Bioanalytical Chemistry 2010;397:2939–47.[3] Lee PL, Tang Y, Li H, Guertin DA. Raptor/mTORC1 loss in adipocytes causes progressive lipodystrophy and fatty liver disease. Molecular Metabolism 2016;5:422–32.[4] Engler AJ, Hemmingsen M, Vedel S, Skafte‐Pedersen P, Sabourin D, Collas P, et al. The Role of Paracrine and Autocrine Signaling in the Early Phase of Adipogenic Differentiation of Adipose‐derived Stem Cells. PLoS ONE 2013;8:e63638.[5] Godwin LA, Brooks JC, Hoepfner LD, Wanders D, Judd RL, Easley CJ. A microfluidic interface for the culture and sampling of adiponectin from primary adipocytes. The Analyst 2015;140:1019–25.

Laminin α4 (LAMA4) is located in the extracellular basement membrane that surrounds each individual adipocyte. Here we show that LAMA4 null (Lama4−/−) mice exhibit significantly higher energy expenditure (EE) relative to wild-type (WT) mice at room temperature and when exposed to a cold challenge, despite similar levels of food intake and locomotor activity. The Lama4−/− mice are resistant to age- and diet-induced obesity. Expression of uncoupling protein 1 is higher in subcutaneous white adipose tissue of Lama4−/− mice relative to WT animals on either a chow diet or a high-fat diet. In contrast, uncoupling protein 1 expression was not increased in brown adipose tissue. Lama4−/− mice exhibit significantly improved insulin sensitivity compared with WT mice, suggesting improved metabolic function. Overall, these data provide critical evidence for a role of the basement membrane in EE, weight gain, and systemic insulin sensitivity.

Peptide nucleic acid (PNA) is a mimic of DNA that shows a high chemical stability and can survive the enzymatic degradation of nucleases and proteases. The superior binding properties of PNA enable the formation of PNA/DNA or PNA/RNA duplex with excellent thermal stability and unique ionic strength effect. The introduction of microarray makes it possible to achieve accurate, high throughput parallel analysis of DNA or RNA with a highly integrated and low reagents consuming device. This powerful tool expands the applications of PNA in genotyping based on single nucleotide polymorphism (SNP) detection, the monitoring of disease-related miRNA expression and pathogen detection. This review paper discusses the fabrications of PNA microarrays through in situ synthesis strategy or spotting method by automatic devices, the various detection methods for the microarray-based hybridization and the current applications of PNA microarrays.

Peptide nucleic acids (PNAs) are a class of nucleic acid mimics that can bind to the complementary DNA or RNA with high specificity and sensitivity. PNA-based microarrays have distinct characteristics and have improved performance in many aspects compared to DNA microarrays. A new set of PNA monomers has been synthesized and used as the building blocks for the preparation of high density PNA microarrays. These monomers have their backbones protected by the photolabile group 2-(2-nitrophenyl)propyloxy carbonyl (NPPOC), and their exocyclic amino groups protected by amide carbonyl groups. A light-directed synthesis system was designed and applied to the in situ synthesis of a PNA microarray with a density of over 10,000 probes per square centimeter. This PNA microarray was able to detect single and multiple base-mismatches correctly with a high discrimination ratio.

Brown and beige adipose tissues have a significant capacity for energy expenditure that may be exploited as a treatment for obesity and metabolic disease. However, the limited volumes of these tissues in adults hinders realization of this potential. Engineering beige adipose tissue may provide an alternative source of this tissue. In this paper we describe the preparation of poly(ethylene glycol) (PEGDA) hydrogels with mechanical properties similar to native adipose tissue. Adipose derived stem cells (ASC) were cultured in hydrogels without adhesive sequences or degradable monomers. Cells were able to differentiate, independent of scaffold properties and were maintained as a viable and functioning adipose tissue mass. The cells expressed their own basement membrane proteins consistent with the composition of adipose tissue. The ASCs could be induced to express uncoupling protein-1 (UCP-1) and cIDEA, makers of beige adipocytes with expression level varying with hydrogel stiffness. This hydrogel-based culture system serves as a first step in engineering beige adipose tissue.

Accelerating the photodeprotection rate of photolabile protecting group is conducive to a light-directed chemical reaction, especially for the in situ synthesis of a biochip. Herein, a photosensitizer pyrene was applied to the photodeprotection of 2-(2-nitrophenyl)propyloxycarbony (NPPOC) groups on self-assembled monolayers (SAMs). It was found that the addition of pyrene could largely improve photodeprotection rate, and effectively prevent molecule damage that are often encountered by the photosensitizer 2-isopropyl thioxanthone (ITX). The most likely explanation for this result is that the whole photodeprotection process involves three joint actions, including ultraviolet light irradiation, triplet energy transfer by pyrene, and singlet fluorescence irradiation from pyrene. The joint actions enable the transfer of over-absorbed energy from pyrene to protecting groups in terms of fluorescence rather than free radicals produced by ITX that are detrimental to the molecules modified on glass substrates. Pyrene dissolved in an optimized combination of mixed solvent of dimethylacetamide (DMAC), ethanol, and dioxane with a volume ratio of 1:1:1 was tested to produce a complete photodeprotection of NPPOC groups within 6 min under 365 nm ultraviolet with an intensity of 10.8 mW/cm2. Meanwhile, tens to hundreds of cycles of photodeprotection could be conducted at a high efficiency. This research will shed light on the deprotection of photolabile groups with weak ultraviolet using a fluorescent sensitizer.

The reaction chamber is important due to its wide applications. Based on the idea of the bionics, a novel spindle-shaped chamber (S-chamber) for microfluidics was designed to provide uniform flow and eliminate stagnant corners for microchannels. The computational fluid dynamics simulation results demonstrate that these S-chambers have a better performance compared to the conventional diamond-shaped chambers. An S-chamber with the optimized shape, which consists of a rectangle reaction region interfaced to the inlet/outlet channel through an expansion region with smooth arc edges, was fabricated by micromolding of polydimethylsiloxane. This S-chamber was fixed into a microreactor and mounted horizontally on a synthesizer for biochemical reactions. Solvent perfusion experiments and synthetic optimization experiments for in situ synthesis of peptide nucleic acids microarray were performed. The experimental results indicate that the newly designed and fabricated S-chamber provides excellent homogeneous perfusion flows. This type of S-chamber is designed for the most convenient fixation in the horizontal direction, without the need to consider the complicated effect caused by other housing directions. It has a wide application for cell culturing, microarray synthesis, gene hybridization, and many other microfluidic system-based techniques requiring uniform flow conditions.

Peptide nucleic acids (PNA) microarray assembled with hundreds of unique PNA oligomers has been regarded as a new and mighty competitor of DNA chip in gene analyzing. However, PNA microarray is still a luxury art due to the difficult and laborious chemical synthesis. Herein, we have developed a fully-automated synthesizer for PNA microarray through photolithography. A preactivation mixer was designed and integrated into the synthesizer in order to get rid of the annoying manual process and increase the coupling efficiency of PNA monomers. The PNA patterning model was carried out to check the performance of the automated synthesizer, revealing that an exposure time of 3 min was sufficient for the complete removal of o-nitroveratryloxycarbonyl (NVOC) groups from the synthetic sites with the help of photosensitizer isopropylthioxanthone and the stepwise yield was measured to be about 98.0%, which is comparable with that from conventional fluorenyl-methyloxycarbonyl (FMOC) chemistry. Those results have definitely demonstrated the possibility and capability of this fully-automated synthesizer to fabricate high-quality PNA microarrays.

DNA microarrays have become one of the most powerful tools in modern biology. Expanding demand for mass production of microarrays has created considerable interest in improving the cost effectiveness of microarray fabrication techniques. Several algorithms dedicated to reducing the number of synthesis cycles for the in situ synthesis of high density DNA microarrays were presented in this paper. Meanwhile, an operation that reverses the probes on a microarray based on those algorithms was introduced and evaluated. The results indicate a possibility for a further reduction of the synthesis cycles.

Cu pattern on 3-mercaptopropyltrimethoxysilane self-assembled monolayers (MPTS-SAMs) modified glass substrate was achieved by a combination of hydrophobic treatment through microcontact printing, activation and electroless plating. The MPTS-SAMs modified glass substrate was selectively deactivated by microcontact printing 1-hexadecanethiol ethanol solution. X-ray photoelectron spectroscopy (XPS) and water contact angle measurements confirmed that a selectively deactivated pattern was obtained, which was attributed to the formation of disulfide linkages between MPTS and 1-hexadecanethiol. The substrate was selectively activated by dipping into Ag colloids solution and then applied for electroless Cu plating. XPS spectra suggested an ideal catalytic pattern on the substrate due to the deposition of Ag particles on the MPTS and the formation of S-Ag bonds. SEM showed that the microstructure of Cu pattern on MPTS-SAMs was in good agreement with the corresponding silicon master with a resolution of 10 microm. The average electrical resistivity was about 1.8 x 10(-6) omegacm, measured by four-point probe technique. The results suggested that microcontact printing deactivating reagents on SAMs is a potential technique for Cu patterns preparation.