Repurposing Decellularized Lung to Generate Vascularized Fat.

Conventional therapies to address critically sized defects in subcutaneous adipose tissue remain a reconstructive challenge for surgeons, largely due to the lack of graft pre-vascularization. Adipose tissue relies on a dense microvasculature network to deliver nutrients, oxygen, nonadipose tissue-derived growth factors, cytokines, and hormones, as well as transporting adipose tissue-derived endocrine signals to other organ systems. This chapter addresses these vascularization issues by combining decellularized lung matrices with a step-wise seeding of patient-specific adipose-derived stem cells and endothelial cells to develop large-volume, perfusable, and pre-vascularized adipose grafts.

Transplantation of committed pre-adipocytes from brown adipose tissue improves whole-body glucose homeostasis.

Obesity and its co-morbidities including type 2 diabetes are increasing at epidemic rates in the U.S. and worldwide. Brown adipose tissue (BAT) is a potential therapeutic to combat obesity and type 2 diabetes. Increasing BAT mass by transplantation improves metabolic health in rodents, but its clinical translation remains a challenge. Here, we investigated if transplantation of 2-4 million differentiated brown pre-adipocytes from mouse BAT stromal fraction (SVF) or human pluripotent stem cells (hPSCs) could improve metabolic health. Transplantation of differentiated brown pre-adipocytes, termed "committed pre-adipocytes" from BAT SVF from mice or derived from hPSCs improves glucose homeostasis and insulin sensitivity in recipient mice under conditions of diet-induced obesity, and this improvement is mediated through the collaborative actions of the liver transcriptome, tissue AKT signaling, and FGF21. These data demonstrate that transplantation of a small number of brown adipocytes has significant long-term translational and therapeutic potential to improve glucose metabolism.

Development of a More Environmentally Friendly Silk Fibroin Scaffold for Soft Tissue Applications.

A push for environmentally friendly approaches to biomaterials fabrication has emerged from growing conservational concerns in recent years. Different stages in silk fibroin scaffold production, including sodium carbonate (Na2CO3)-based degumming and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)-based fabrication, have drawn attention for their associated environmental concerns. Environmentally friendly alternatives have been proposed for each processing stage; however, an integrated green fibroin scaffold approach has not been characterized or used for soft tissue applications. Here, we show that the combination of sodium hydroxide (NaOH) as a substitute degumming agent with the popular "aqueous-based" alternative silk fibroin gelation method yields fibroin scaffolds with comparable properties to traditional Na2CO3-degummed aqueous-based scaffolds. The more environmentally friendly scaffolds were found to have comparable protein structure, morphology, compressive modulus, and degradation kinetics, with increased porosity and cell seeding density relative to traditional scaffolds. Human adipose-derived stem cells showed high viability after three days of culture while seeded in each scaffold type, with uniform cell attachment to pore walls. Adipocytes from human whole adipose tissue seeded into scaffolds were found to have similar levels of lipolytic and metabolic function between conditions, in addition to a healthy unilocular morphology. Results indicate that our more environmentally friendly methodology for silk scaffold production is a viable alternative and well suited to soft tissue applications.

Adipose Tissue Paracrine-, Autocrine-, and Matrix-Dependent Signaling during the Development and Progression of Obesity.

Obesity is an ever-increasing phenomenon, with 42% of Americans being considered obese (BMI ≥ 30) and 9.2% being considered morbidly obese (BMI ≥ 40) as of 2016. With obesity being characterized by an abundance of adipose tissue expansion, abnormal tissue remodeling is a typical consequence. Importantly, this pathological tissue expansion is associated with many alterations in the cellular populations and phenotypes within the tissue, lending to cellular, paracrine, mechanical, and metabolic alterations that have local and systemic effects, including diabetes and cardiovascular disease. In particular, vascular dynamics shift during the progression of obesity, providing signaling cues that drive metabolic dysfunction. In this review, paracrine-, autocrine-, and matrix-dependent signaling between adipocytes and endothelial cells is discussed in the context of the development and progression of obesity and its consequential diseases, including adipose fibrosis, diabetes, and cardiovascular disease.

Adipose Tissue Development Relies on Coordinated Extracellular Matrix Remodeling, Angiogenesis, and Adipogenesis.

Despite developing prenatally, the adipose tissue is unique in its ability to undergo drastic growth even after reaching its mature size. This development and subsequent maintenance rely on the proper coordination between the vascular niche and the adipose compartment. In this review, the process of adipose tissue development is broken down to explain (1) the ultrastructural matrix remodeling that is undertaken during simultaneous adipogenesis and angiogenesis, (2) the paracrine crosstalk involved during adipose development, (3) the mechanical regulators involved in adipose growth, and (4) the proteolytic and paracrine oversight for matrix remodeling during adipose development. It is crucial to gain a better understanding of the complex relationships that exist between adipose tissue and the vasculature during tissue development to provide insights into the pathological tissue expansion of obesity and to develop improved soft-tissue reconstruction techniques.

Engineering a 3D Vascularized Adipose Tissue Construct Using a Decellularized Lung Matrix.

Critically sized defects in subcutaneous white adipose tissue result in extensive disfigurement and dysfunction and remain a reconstructive challenge for surgeons; as larger defect sizes are correlated with higher rates of complications and failure due to insufficient vascularization following implantation. Our study demonstrates, for the first time, a method to engineer perfusable, pre-vascularized, high-density adipose grafts that combine patient-derived adipose cells with a decellularized lung matrix (DLM). The lung is one of the most vascularized organs with high flow, low resistance, and a large blood-alveolar interface separated by a thin basement membrane. For our work, the large volume capacity within the alveolar compartment was repurposed for high-density adipose cell filling, while the acellular vascular bed provided efficient graft perfusion throughout. Both adipocytes and hASCs were successfully delivered and remained in the alveolar space even after weeks of culture. While adipose-derived cells maintained their morphology and functionality in both static and perfusion DLM cultures, perfusion culture offered enhanced outcomes over static culture. Furthermore, we demonstrate that endothelial cells seamlessly integrate into the acellular vascular tree of the DLM with adipocytes. These results support that the DLM is a unique platform for creating vascularized adipose tissue grafts for large defect filling.

Silk Fibroin as a Green Material.

Silk fibroin has been explored as a suitable biomaterial due to its biocompatibility, tunable degradability, low toxicity, and mechanical properties. To harness silk fibroin's innate properties, it is purified from native silkworm cocoons by removing proteins and debris that have the potential to cause inflammatory responses. Typically, within the purification and fabrication steps, chemical solvents, energy-intensive equipment, and large quantities of water are used to reverse engineer silk fibroin into an aqueous solution and then process into the final material format. Gentler, green methods for extraction and fabrication have been developed that reduce or remove the need for harmful chemical additives and energy-inefficient equipment while still producing mechanically robust biomaterials. This review will focus on the alternative green processing and fabrication methods that have proven useful in creating silk fibroin materials for a range of applications including consumer and medical materials.

The Materiobiology of Silk: Exploring the Biophysical Influence of Silk Biomaterials on Directing Cellular Behaviors.

Biophysical properties of the extracellular environment dynamically regulate cellular fates. In this review, we highlight silk, an indispensable polymeric biomaterial, owing to its unique mechanical properties, bioactive component sequestration, degradability, well-defined architectures, and biocompatibility that can regulate temporospatial biochemical and biophysical responses. We explore how the materiobiology of silks, both mulberry and non-mulberry based, affect cell behaviors including cell adhesion, cell proliferation, cell migration, and cell differentiation. Keeping in mind the novel biophysical properties of silk in film, fiber, or sponge forms, coupled with facile chemical decoration, and its ability to match functional requirements for specific tissues, we survey the influence of composition, mechanical properties, topography, and 3D geometry in unlocking the body's inherent regenerative potential.

Therapeutic Ultrasound Triggered Silk Fibroin Scaffold Degradation.

A patient's capacity for tissue regeneration varies based on age, nutritional status, disease state, lifestyle, and gender. Because regeneration cannot be predicted prior to biomaterial implantation, there is a need for responsive biomaterials with adaptive, personalized degradation profiles to improve regenerative outcomes. This study reports a new approach to use therapeutic ultrasound as a means of altering the degradation profile of silk fibroin biomaterials noninvasively postimplantation. By evaluating changes in weight, porosity, surface morphology, compressive modulus, and chemical structure, it is concluded that therapeutic ultrasound can trigger enhanced degradation of silk fibroin scaffolds noninvasively. By removing microbubbles on the scaffold surface, it is found that acoustic cavitation is the mechanism responsible for changing the degradation profile. This method is proved to be safe for human cells with no negative effects on cell viability or metabolism. Sonication through human skin also effectively triggers scaffold degradation, increasing the clinical relevance of these results. These findings suggest that silk is an ultrasound-responsive biomaterial, where the degradation profile can be adjusted noninvasively to improve regenerative outcomes.

Mechanisms of action, chemical characteristics, and model systems of obesogens.

There is increasing evidence for the role of environmental endocrine disrupting contaminants, coined obesogens, in exacerbating the rising obesity epidemic. Obesogens can be found in everyday items ranging from pesticides to food packaging. Although research shows that obesogens can have effects on adipocyte size, phenotype, metabolic activity, and hormone levels, much remains unknown about these chemicals. This review will discuss what is currently known about the mechanisms of obesogens, including expression of the PPARs, hormone interference, and inflammation. Strategies for identifying obesogenic chemicals and their mechanisms through chemical characteristics and model systems will also be discussed. Ultimately, research should focus on improving models to discern precise mechanisms of obesogenic action and to test therapeutics targeting these mechanisms.

Adipose Tissue Fibrosis: Mechanisms, Models, and Importance.

Increases in adipocyte volume and tissue mass due to obesity can result in inflammation, further dysregulation in adipose tissue function, and eventually adipose tissue fibrosis. Like other fibrotic diseases, adipose tissue fibrosis is the accumulation and increased production of extracellular matrix (ECM) proteins. Adipose tissue fibrosis has been linked to decreased insulin sensitivity, poor bariatric surgery outcomes, and difficulty in weight loss. With the rising rates of obesity, it is important to create accurate models for adipose tissue fibrosis to gain mechanistic insights and develop targeted treatments. This article discusses recent research in modeling adipose tissue fibrosis using in vivo and in vitro (2D and 3D) methods with considerations for biomaterial selections. Additionally, this article outlines the importance of adipose tissue in treating other fibrotic diseases and methods used to detect and characterize adipose tissue fibrosis.

Human Adipose Derived Cells in Two- and Three-Dimensional Cultures: Functional Validation of an In Vitro Fat Construct.

Obesity, defined as a body mass index of 30 kg/m2 or above, has increased considerably in incidence and frequency within the United States and globally. Associated comorbidities including cardiovascular disease, type 2 diabetes mellitus, metabolic syndrome, and nonalcoholic fatty liver disease have led to a focus on the mechanisms promoting the prevention and treatment of obesity. Commonly utilized in vitro models employ human or mouse preadipocyte cell lines in a 2-dimensional (2D) format. Due to the structural, biochemical, and biological limitations of these models, increased attention has been placed on "organ on a chip" technologies for a 3-dimensional (3D) culture. Herein, we describe a method employing cryopreserved primary human stromal vascular fraction (SVF) cells and a human blood product-derived biological scaffold to create a 3D adipose depot in vitro. The "fat-on-chip" 3D cultures have been validated relative to 2D cultures based on proliferation, flow cytometry, adipogenic differentiation, confocal microscopy/immunofluorescence, and functional assays (adipokine secretion, glucose uptake, and lipolysis). Thus, the in vitro culture system demonstrates the critical characteristics required for a humanized 3D white adipose tissue (WAT) model.

Fat-On-A-Chip Models for Research and Discovery in Obesity and Its Metabolic Comorbidities.

The obesity epidemic and its associated comorbidities present a looming challenge to health care delivery throughout the world. Obesity is characterized as a sterile inflammatory process within adipose tissues leading to dysregulated secretion of bioactive adipokines such as adiponectin and leptin, as well as systemic metabolic dysfunction. The majority of current obesity research has focused primarily on preclinical animal models in vivo and two-dimensional cell culture models in vitro. Neither of these generalized approaches is optimal due to interspecies variability, insufficient accuracy with respect to predicting human outcomes, and failure to recapitulate the three-dimensional (3D) microenvironment. Consequently, there is a growing demand and need for more sophisticated microphysiological systems to reproduce more physiologically accurate human white and brown/beige adipose depots. To address this research need, human and murine cell lines and primary cultures are being combined with bioscaffolds to create functional 3D environments that are suitable for metabolically active adipose organoids in both static and perfusion bioreactor cultures. The development of these technologies will have considerable impact on the future pace of discovery for novel small molecules and biologics designed to prevent and treat metabolic syndrome and obesity in humans. Furthermore, when these adipose tissue models are integrated with other organ systems they will have applicability to obesity-related disorders such as diabetes, nonalcoholic fatty liver disease, and osteoarthritis. Impact statement The current review article summarizes the advances made within the organ-onchip field, as it pertains to adipose tissue models of obesity and obesity-related syndromes, such as diabetes, non-alcoholic fatty liver disease, and osteoarthritis. As humanized 3D adipose-derived constructs become more accessible to the research community, it is anticipated that they will accelerate and enhance the drug discovery pipeline for obesity, diabetes, and metabolic diseases by reducing the preclinical evaluation process and improving predictive accuracy. Such developments, applications, and usages of existing technologies can change the paradigm of personalized medicine and create substantial progress in our approach to modern medicine.

3D printing with silk: considerations and applications.

Silk is a natural polymer sourced mainly from spiders and silkworms. Due to its biocompatibility, biodegradability, and mechanical properties, it has been heavily investigated for biomedical applications. It can be processed into a number of formats, such as scaffolds, films, and nanoparticles. Common methods of production create constructs with limited complexity. 3D printing allows silk to be printed into more intricate designs, increasing its potential applications. Extrusion and inkjet printing are the primary ways silk has been 3D printed, though other methods are beginning to be investigated. Silk has been integrated into bioink with other polymers, both natural and synthetic. The addition of silk is primarily done to offer more desirable viscosity characteristics and mechanical properties for printing. Silk-based bioinks have been used to fabricate medical devices and tissues. This article discusses recent research and printing parameters important for 3D printing with silk.

3D biomaterial matrix to support long term, full thickness, immuno-competent human skin equivalents with nervous system components.

Current commercially available human skin equivalents (HSEs) are used for relatively short term studies (∼1 week) due in part to the time-dependent contraction of the collagen gel-based matrix and the limited cell types and skin tissue components utilized. In contrast, here we describe a new matrix consisting of a silk-collagen composite system that provides long term, stable cultivation with reduced contraction and degradation over time. This matrix supports full thickness skin equivalents which include nerves. The unique silk-collagen composite system preserves cell-binding domains of collagen while maintaining the stability and mechanics of the skin system for long-term culture with silk. The utility of this new composite protein-based biomaterial was demonstrated by bioengineering full thickness human skin systems using primary cells, including nerves and immune cells to establish an HSE with a neuro-immuno-cutaneous system. The HSEs with neurons and hypodermis, compared to in vitro skin-only HSEs controls, demonstrated higher secretion of pro-inflammatory cytokines. Proteomics analysis confirmed the presence of several proteins associated with inflammation across all sample groups, but HSEs with neurons had the highest amount of detected protein due to the complexity of the model. This improved, in vitro full thickness HSE model system utilizes cross-linked silk-collagen as the biomaterial and allows reduced reliance on animal models and provides a new in vitro tissue system for the assessment of chronic responses related to skin diseases and drug discovery.

Microscopic considerations for optimizing silk biomaterials.

Silk is an especially appealing biomaterial due to its adaptable mechanical properties, allowing it to be used in a wide range of tissue engineering applications. However, processing conditions play a critical role in determining silk's mechanical properties, biodegradability, and biocompatibility. While bulk properties of silk have been widely explored, focusing on microscopic features is becoming increasingly important, as modifications at this scale largely affect the resulting regenerative properties of the biomaterial. Structural changes caused by the silk source, extraction, and processing should be carefully considered, as they will affect the biocompatibility and degradability of silk fibroin. Processing techniques and physical properties of silk that make it an ideal material for many biomedical applications will be explored. This article is categorized under: Implantable Materials and Surgical Technologies > Nanotechnology in Tissue Repair and Replacement Implantable Materials and Surgical Technologies > Nanomaterials and Implants.

Modeling Diabetic Corneal Neuropathy in a 3D In Vitro Cornea System.

Diabetes mellitus is a disease caused by innate or acquired insulin deficiency, resulting in altered glucose metabolism and high blood glucose levels. Chronic hyperglycemia is linked to development of several ocular pathologies affecting the anterior segment, including diabetic corneal neuropathy and keratopathy, neovascular glaucoma, edema, and cataracts leading to significant visual defects. Due to increasing disease prevalence, related medical care costs, and visual impairment resulting from diabetes, a need has arisen to devise alternative systems to study molecular mechanisms involved in disease onset and progression. In our current study, we applied a novel 3D in vitro model of the human cornea comprising of epithelial, stromal, and neuronal components cultured in silk scaffolds to study the pathological effects of hyperglycemia on development of diabetic corneal neuropathy. Specifically, exposure to sustained levels of high glucose, ranging from 35 mM to 45 mM, were applied to determine concentration-dependent effects on nerve morphology, length and density of axons, and expression of metabolic enzymes involved in glucose metabolism. By comparing these metrics to in vivo studies, we have developed a functional 3D in vitro model for diabetic corneal neuropathy as a means to investigate corneal pathophysiology resulting from prolonged exposure to hyperglycemia.

Variability in responses observed in human white adipose tissue models.

Obesity is a risk factor for a myriad of diseases including diabetes, cardiovascular dysfunction, cirrhosis, and cancer, and there is a need for new systems to study how excess adipose tissue relates to the onset of disease processes. This study provides proof-of-concept patient-specific tissue models of human white adipose tissue to accommodate the variability in human samples. Our 3D tissue engineering approach established lipolytic responses and changes in insulin-stimulated glucose uptake from small volumes of human lipoaspirate, making this methodology useful for patient specific sample source assessments of treatment strategies, drug responses, disease mechanisms, and other responses that vary between patients. Mature unilocular cells were maintained ex vivo in silk porous scaffolds for up to a month of culture and imaged non-invasively with coherent anti-Stokes Raman scattering. Interestingly, differences in responsiveness between tissues were observed in terms of magnitude of lipolysis, ability to suppress lipolysis, differences in glucose uptake, and lipid droplet size. Body mass index was not a factor in determining tissue responsiveness; rather, it is speculated that other unknown variables in the backgrounds of different patients (ethnicity, athleticism, disease history, lifestyle choices, etc.) likely had a more significant effect on the observed differences. This study reinforces the need to account for the variability in backgrounds and genetics within the human population to determine adipose tissue responsiveness. In the future, this tissue system could be used to inform individualized care strategies-enhancing therapeutic precision, improving patient outcomes, and reducing clinical costs.

Development of a Three-Dimensional Adipose Tissue Model for Studying Embryonic Exposures to Obesogenic Chemicals.

Obesity is a rising issue especially in the United States that can lead to heart problems, type II diabetes, and respiratory problems. Since the 1970s, obesity rates in the United States have more than doubled in adults and children. Recent evidence suggests that exposure to certain chemicals, termed "obesogens," in utero may alter metabolic processes, predisposing individuals to weight gain. There is a need to develop a three-dimensional human tissue system that is able to model the effects of obesogens in vitro in order to better understand the impact of obesogens on early development. Human embryonic-derived stem cells in three-dimensional collagen embedded silk scaffolds were exposed to three different obesogens: Bisphenol A (BPA), Bisphenol S (BPS), and Tributyltin (TBT). The exposed tissues accumulated triglycerides and increased expression of adipogenic genes (Perilipin (PLIN1), peroxisome proliferator-activated receptor gamma (PPARy), fatty acid binding protein 4 (FABP4)) compared to equivalent control cultures with no obesogen exposure. These cultures were also compared to human adult stem cell cultures, which did not respond the same upon addition of obesogens. These results demonstrate the successful development of a representative tissue model of in utero obesogen exposures. This tissue system could be used to determine mechanisms of action of current obesogens and to screen other potential obesogens.

Expandable and Rapidly Differentiating Human Induced Neural Stem Cell Lines for Multiple Tissue Engineering Applications.

Limited availability of human neurons poses a significant barrier to progress in biological and preclinical studies of the human nervous system. Current stem cell-based approaches of neuron generation are still hindered by prolonged culture requirements, protocol complexity, and variability in neuronal differentiation. Here we establish stable human induced neural stem cell (hiNSC) lines through the direct reprogramming of neonatal fibroblasts and adult adipose-derived stem cells. These hiNSCs can be passaged indefinitely and cryopreserved as colonies. Independently of media composition, hiNSCs robustly differentiate into TUJ1-positive neurons within 4 days, making them ideal for innervated co-cultures. In vivo, hiNSCs migrate, engraft, and contribute to both central and peripheral nervous systems. Lastly, we demonstrate utility of hiNSCs in a 3D human brain model. This method provides a valuable interdisciplinary tool that could be used to develop drug screening applications as well as patient-specific disease models related to disorders of innervation and the brain.