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Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for ...

University of Texas at San Antonio

Vascular networks provide nutrients, oxygen, and progenitor cells that are essential for bone function. It has been proposed that a preformed vascular network may enhance the performance of engineered bone. In this study vascular networks were generated from human umbilical vein endothelial cell and mesenchymal stem cell spheroids encapsulated in fibrin scaffolds, and the stability of preformed vascular networks and their effect on bone regeneration were assessed in an in vivo bone model. Under optimized culture conditions, extensive vessel-like networks formed throughout the scaffolds in vitro. After vascular network formation, the vascularized scaffolds were implanted in a critical sized calvarial defect in nude rats. Immunohistochemical staining for CD31 showed that the preformed vascular networks survived and anastomosed with host tissue within 1 week of implantation. The prevascularized scaffolds enhanced overall vascularization after 1 and 4 weeks. Early bone formation around the perimeter of the defect area was visible in X-ray images of samples after 4 weeks. Prevascularized scaffolds may be a promising strategy for engineering vascularized bone.

Preformed Vascular Networks Survive and Enhance Vascularization in Critical Sized Cranial Defects.

There is a need to overcome the donor-site morbidity and loss of volume over time that accompanies the current clinical approaches to treat soft tissue defects caused by disease and trauma. The development of bioactive constructs that can regenerate adipose tissue have made great progress toward addressing the limitations of current therapies, but their lack of vascularization and ability to meet the significant dimension requirements of tissue defects limit their clinical translatability. Microvascular fragments (MVFs) can form extensive vascular networks and contain resident cells that have the ability to differentiate into adipocytes. Therefore, the objective of this study was to determine if vascularized adipose tissue could be engineered using a fibrin-based hydrogel containing MVFs as the sole source of microvessels and adipocyte-forming cells. The potential for MVFs from different fat depots (epididymal, inguinal, and subcutaneous) to form microvascular networks and generate adipocytes when exposed to growth media (GM), adipogenic differentiation media (ADM), or when treated with GM before adipogenic induction (i.e., they were allowed to presprout before adipogenic induction) was evaluated. MVFs treated with adipogenic induction media, both with and without presprouting, contained lipid droplets, had an increase in expression levels of genes associated with adipogenesis (adiponectin and fatty acid synthase [FAS]), and had an increased rate of lipolysis. MVFs allowed to presprout before ADM treatment maintained their ability to form vascular networks while maintaining an elevated lipid content, adipogenic gene expression, and lipolysis rate. Collectively, these results support the contention that MVFs can serve as the sole source of biologic material for creating a vascularized adipose tissue scaffold. Impact statement Microvascular fragments have both the ability to form extensive vascular networks and function as a source of adipocytes. These phenomena were exploited as vascularized adipose tissue was generated by first allowing for a period of angiogenesis before the adipogenic induction. This strategy has the ability to provide a means of both improving soft tissue reconstruction while also serving as a model to better understand adipose tissue expansion.

A Straightforward Approach to Engineer Vascularized Adipose Tissue Using Microvascular Fragments.

The development of ectopic adipose tissue in skeletal muscle is associated with several skeletal muscle and metabolic pathologies, including Type II Diabetes Mellitus. The adipogenic differentiation of muscle precursor cells (MPCs) has been postulated to occur in skeletal muscle in vivo in a three-dimensional (3-D) configuration; therefore, it is appropriate to investigate this phenomenon using 3-D matrices in vitro. The capacity for MPC adipogenic differentiation in a 3-D environment was investigated in fibrin hydrogels by treating MPCs derived from healthy or diabetic animals with adipogenic induction medias that differed in their ability to increase lipid accumulation and activate the expression of genes associated with adipogenic differentiation (peroxisome proliferator-activated receptor gamma (PPARG), adiponectin (ADIPOQ), and fatty acid synthase (FAS)). The capacity for adipogenic differentiation was diminished, but not prevented, if myogenic differentiation preceded MPC exposure to adipogenic induction conditions. Conversely, adipogenic differentiation was greater in hydrogels containing MPCs from diabetic rats as compared to those derived from lean rats, as evidenced by an increase in lipid accumulation and adipogenic gene expression. Collectively, the data herein support a role for the MPCs in adipogenesis in a 3-D environment and that they may contribute to the ectopic accumulation of adipose tissue. The observation that the potential for adipogenic differentiation is maintained even after a period of myogenic differentiation alludes to the possibility that adipogenesis may occur during different phases of muscle development. Finally, the increase in adipogenic differentiation in hydrogels containing MPCs derived from diabetic animals provides strong evidence that a pathological environment in vivo increases their capacity for adipogenesis.

Divergent effects of myogenic differentiation and diabetes on the capacity for muscle precursor cell adipogenic differentiation in a fibrin matrix.

Skeletal muscle is a tissue that is directly involved in the progression and persistence of type 2 diabetes (T2D), a disease that is becoming increasingly common. Gaining better insight into the mechanisms that are affecting skeletal muscle dysfunction in the context of T2D has the potential to lead to novel treatments for a large number of patients. Through its ability to emulate skeletal muscle architecture while also incorporating aspects of disease, tissue-engineered skeletal muscle (TE-SkM) has the potential to provide a means for rapid high-throughput discovery of therapies to treat skeletal muscle dysfunction, to include that which occurs with T2D. Muscle precursor cells isolated from lean or obese male Zucker diabetic fatty rats were used to generate TE-SkM constructs. Some constructs were treated with adipogenic induction media to accentuate the presence of adipocytes that is a characteristic feature of T2D skeletal muscle. The maturity (compaction and creatine kinase activity), mechanical integrity (Young's modulus), organization (myotube orientation), and metabolic capacity (insulin-stimulated glucose uptake) were all reduced by diabetes. Treating constructs with adipogenic induction media increased the quantity of lipid within the diabetic TE-SkM constructs, and caused changes in construct compaction, cell orientation, and insulin-stimulated glucose uptake in both lean and diabetic samples. Collectively, the findings herein suggest that the recapitulation of structural and metabolic aspects of T2D can be accomplished by engineering skeletal muscle .

Diabetic Conditions Confer Metabolic and Structural Modifications to Tissue-Engineered Skeletal Muscle.

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.

Induced Pluripotent Stem Cell-Derived Endothelial Networks Accelerate Vascularization But Not Bone Regeneration.

Longitudinal in vivo monitoring is essential for the design and evaluation of biomaterials. An ideal method would provide three-dimensional quantitative information, high spatial resolution, deep tissue penetration, and contrast between tissue and material structures. Photoacoustic (PA) or optoacoustic imaging is a hybrid technique that allows three-dimensional imaging with high spatial resolution. In addition, photoacoustic imaging allows for imaging of vascularization based on the intrinsic contrast of hemoglobin. In this study, we investigated photoacoustic computed tomography (PACT) as a tool for longitudinal monitoring of an implanted hydrogel in a small animal model. Hydrogels were loaded with gold nanorods to enhance contrast and imaged weekly for 8 weeks. PACT allowed non-invasive three-dimensional, quantitative imaging of the hydrogels over the entire 8 weeks. Quantitative volume analysis was used to evaluate the in vivo degradation kinetics of the implants which deviated slightly from in vitro predictions. Multispectral imaging allowed for the simultaneous analysis of hydrogel degradation and local vascularization. These results provide support for the substantial potential of PACT as a tool for insight into biomaterial performance in vivo.

Gold nanorods enable noninvasive longitudinal monitoring of hydrogels in vivo with photoacoustic tomography.

Obesity and the metabolic disease epidemic has led to an increase in morbidity and mortality. A rise in adipose thermogenic capacity via activation of brown or beige fat is a potential treatment for metabolic diseases. However, an understanding of how local factors control adipocyte fate is limited. Mice with a null mutation in the laminin α4 (LAMA4) gene (KO) exhibit resistance to obesity and enhanced expression of thermogenic fat markers in white adipose tissue (WAT). In this study, changes in WAT extracellular matrix composition in the absence of LAMA4 were evaluated using liquid chromatography/tandem mass spectrometry. KO-mice showed lower levels of collagen 1A1 and 3A1, and integrins α7 (ITA7) and β1 (ITB1). ITA7-ITB1 and collagen 1A1-3A1 protein levels were lower in brown adipose tissue compared to WAT in wild-type mice. Immunohistochemical staining confirmed lower levels and different spatial distribution of ITA7 in KO-WAT. In culture studies, ITA7 and LAMA4 levels decreased following a 12-day differentiation of adipose-derived stem cells into beige fat, and knock-down of ITA7 during differentiation increased beiging. These results demonstrate that extracellular matrix interactions regulate adipocyte thermogenic capacity and that ITA7 plays a role in beige adipose formation. A better understanding of the mechanisms underlying these interactions can be used to improve systemic energy metabolism and glucose homeostasis.

Integrins and extracellular matrix proteins modulate adipocyte thermogenic capacity.

Advances in the engineering of comprehensive skeletal muscle models  will improve drug screening platforms and can lead to better therapeutic approaches for the treatment of skeletal muscle injuries. To this end, a vascularized tissue-engineered skeletal muscle (TE-SkM) model that includes adipocytes was developed to better emulate the intramuscular adipose tissue that is observed in skeletal muscles of patients with diseases such as diabetes. Muscle precursor cells cultured with and without microvessels derived from adipose tissue (microvascular fragments) were used to generate TE-SkM constructs, with and without a microvasculature, respectively. TE-SkM constructs were treated with adipogenic induction media to induce varying levels of adipogenesis. With a delayed addition of induction media to allow for angiogenesis, a robust microvasculature in conjunction with an increased content of adipocytes was achieved. The augmentation of vascularized TE-SkM constructs with adipocytes caused a reduction in maturation (compaction), mechanical integrity (Young's modulus), and myotube and vessel alignment. An increase in basal glucose uptake was observed in both levels of adipogenic induction, and a diminished insulin-stimulated glucose uptake was associated with the higher level of adipogenic differentiation and the greater number of adipocytes.

Adipogenic Differentiation Alters Properties of Vascularized Tissue-Engineered Skeletal Muscle.

We introduce cytoNet, a cloud-based tool to characterize cell populations from microscopy images. cytoNet quantifies spatial topology and functional relationships in cell communities using principles of network science. Capturing multicellular dynamics through graph features, cytoNet also evaluates the effect of cell-cell interactions on individual cell phenotypes. We demonstrate cytoNet's capabilities in four case studies: 1) characterizing the temporal dynamics of neural progenitor cell communities during neural differentiation, 2) identifying communities of pain-sensing neurons in vivo, 3) capturing the effect of cell community on endothelial cell morphology, and 4) investigating the effect of laminin α4 on perivascular niches in adipose tissue. The analytical framework introduced here can be used to study the dynamics of complex cell communities in a quantitative manner, leading to a deeper understanding of environmental effects on cellular behavior. The versatile, cloud-based format of cytoNet makes the image analysis framework accessible to researchers across domains.

cytoNet: Spatiotemporal network analysis of cell communities.

Engineered beige adipose tissues could be used for screening therapeutic strategies or as a direct treatment for obesity and metabolic disease. Microvascular fragments are vessel structures that can be directly isolated from adipose tissue and may contain cells capable of differentiation into thermogenic, or beige, adipocytes. In this study, culture conditions were investigated to engineer three-dimensional, vascularized functional beige adipose tissue using microvascular fragments isolated from both healthy animals and a model of type II diabetes (T2D). Vascularized beige adipose tissues were engineered and exhibited increased expression of beige adipose markers, enhanced function, and improved cellular respiration. While microvascular fragments isolated from both lean and diabetic models were able to generate functional tissues, differences were observed in regard to vessel assembly and tissue function. This study introduces an approach that could be employed to engineer vascularized beige adipose tissues from a single, potentially autologous source of cells.

Engineering Functional Vascularized Beige Adipose Tissue from Microvascular Fragments of Models of Healthy and Type II Diabetes Conditions.

In this study, we described a method for generating functional, beige (thermogenic) adipose microtissues from human microvascular fragments (MVFs). The MVFs were isolated from adipose tissue acquired from adults over 50 years of age. The tissues express thermogenic gene markers and reproduce functions essential for the potential therapeutic impact of beige adipose tissues such as enhanced lipid metabolism and increased mitochondrial respiration. MVFs serve as a potential single, autologous source of cells that can be isolated from adult patients, induced to recreate functional aspects of beige adipose tissue and enable rapid vascularization post-transplantation. This approach has the potential to be used as an autologous therapy for metabolic diseases or as a model for the development of a personalized approach to high-throughput drug development/screening for adipose tissue.

Katerina Stojkova

Department of Biomedical Engineering and Chemical Engineering

University of Texas at San Antonio

Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments

Katerina Stojkova

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biomedical Engineering and Chemical Engineering

University of Texas at San Antonio

Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments

Department of Biomedical Engineering and Chemical Engineering