A Preliminary Study on Factors That Drive Patient Variability in Human Subcutaneous Adipose Tissues.

Adipose tissue is a dynamic regulatory organ that has profound effects on the overall health of patients. Unfortunately, inconsistencies in human adipose tissues are extensive and multifactorial, including large variability in cellular sizes, lipid content, inflammation, extracellular matrix components, mechanics, and cytokines secreted. Given the high human variability, and since much of what is known about adipose tissue is from animal models, we sought to establish correlations and patterns between biological, mechanical, and epidemiological properties of human adipose tissues. To do this, twenty-six independent variables were cataloged for twenty patients, which included patient demographics and factors that drive health, obesity, and fibrosis. A factorial analysis for mixed data (FAMD) was used to analyze patterns in the dataset (with BMI > 25), and a correlation matrix was used to identify interactions between quantitative variables. Vascular endothelial growth factor A (VEGFA) and actin alpha 2, smooth muscle (ACTA2) gene expression were the highest loadings in the first two dimensions of the FAMD. The number of adipocytes was also a key driver of patient-related differences, where a decrease in the density of adipocytes was associated with aging. Aging was also correlated with a decrease in overall lipid percentage of subcutaneous tissue, with lipid deposition being favored extracellularly, an increase in transforming growth factor-ß1 (TGFß1), and an increase in M1 macrophage polarization. An important finding was that self-identified race contributed to variance between patients in this study, where Black patients had significantly lower gene expression levels of TGFß1 and ACTA2. This finding supports the urgent need to account for patient ancestry in biomedical research to develop better therapeutic strategies for all patients. Another important finding was that TGFß induced factor homeobox 1 (TGIF1), an understudied signaling molecule, which is highly correlated with leptin signaling, was correlated with metabolic inflammation. Furthermore, this study draws attention to what we define as "extracellular lipid droplets", which were consistently found in collagen-rich regions of the obese adipose tissues evaluated here. Reduced levels of TGIF1 were correlated with higher numbers of extracellular lipid droplets and an inability to suppress fibrotic changes in adipose tissue. Finally, this study indicated that M1 and M2 macrophage markers were correlated with each other and leptin in patients with a BMI > 25. This finding supports growing evidence that macrophage polarization in obesity involves a complex, interconnecting network system rather than a full switch in activation patterns from M2 to M1 with increasing body mass. Overall, this study reinforces key findings in animal studies and identifies important areas for future research, where human and animal studies are divergent. Understanding key drivers of human patient variability is required to unravel the complex metabolic health of unique patients.

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.

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.

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.

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.