*3.2. Three-Dimensional Models*

Biomaterials Used for 3D Adipose Tissue Culture

Due to the decreased physiological relevance of culturing adipocytes using 2D techniques, 3D scaffolds are often used to grow and model adipose tissue. Several synthetic and natural biomaterials have been used to successfully culture ASCs and whole adipose tissue ranging from synthetic to natural polymers (Table 1). While some progress has been made modeling fibrotic adipose tissue using 3D in vitro platforms, this area of research is still at early stages of development with large potential for growth.

Diabetic patients have higher degrees of adipose tissue fibrosis [79]. To better understand the link between the ECM and adipocyte metabolic regulation, researchers used human decellularized adipose tissue from patients with and without type II diabetes mellitus as a model system. Combining ECM from a nondiabetic patient and adipocytes from a diabetic patient resulted in adipocytes that had fully restored basal lipolysis and insulin stimulated glucose uptake. Conversely, combining ECM from a diabetic patient and adipocytes from a nondiabetic patient decreased insulin sensitivity, but had no effect on lipolysis [80]. This suggests that dysfunctional fibrotic cues are found in the matrix and are not an intrinsic property of the cells.

The interaction of matrix stiffness and architecture with adipocytes was also tested in an in vitro model. Researchers incorporated ethylene glycol-bis-succinic acid N-hydroxysuccinimide ester (PEGDS) into a collagen hydrogel system. The addition of PEGDS created a more organized and interconnected ECM by establishing intrafibrillar and interfibrillar crosslinks. This was used to investigate the effect mechanical changes, like those that occur during adipose tissue fibrosis, have on adipocyte morphology and function. In stiffer hydrogels, adipocytes experienced increased profibrotic gene expression and ECM deposition. By inhibiting actin contractility, the researchers determined that the adipocyte dysfunction was regulated by actin cytoskeletal stress fibers that registered the mechanical properties of the surrounding environment [81]. This research indicates that if fibrosis could be reversed cells could be rescued from the fibrotic phenotype. Additionally, this research further supports that fibrotic cues are found in the extracellular matrix.

To mimic in vivo fibrosis in another system, researchers created hypoxic clusters of ASCs that grew into 3D cell masses comprised of ASCs, alpha smooth muscle actin positive (αSMA-positive) cells, necrotic cells, and collagen [82]. Researchers compared these 3D cell masses to 2D cultures and found that there was a significant increase in TGFβ1 secretion, hydroxyproline concentration, and lactate dehydrogenase secretion. These changes indicate an increase in expression of the fibrotic phenotype, ECM production, and cell death which align with physiological changes that occur during adipose tissue fibrosis development [82].


#### **Table 1.** Biomaterials that have been used in vitro to culture adipose tissue.

#### *3.3. Biomaterial Considerations for In Vitro Modeling*

When designing a biomaterial for modeling adipose tissue fibrosis, evaluating biomaterials that successfully grow healthy adipose tissue, as well as models that are used for other fibrotic tissues, can be a good starting point. Important biomaterial characteristics that should be considered follow (Figure 2).

• *Sti*ff*ness*: Stiffness is an intrinsic property of a material and is defined as the resistance of an elastic material to deformation by an applied force. Adipocytes and ASCs are mechanosensitive and mechanoresponsive [100,101]. In the body, mechanical forces are balanced internally by the cytoskeleton and externally through the ECM. Scaffolding materials act as the ECM in in vitro models, therefore, the stiffness should be carefully considered as it will influence the cellular cytoskeleton and phenotype [100]. Scaffold stiffness can be controlled by adding other polymers or additives [81]. A recent study found that stiffer biomaterials triggered fibrotic traits in adipocytes, such as increased profibrotic gene expression and ECM deposition [81]. Additionally, stiffer matrices were found to promote osteogenic di fferentiation of ASCs, while softer matrices promoted adipogenic commitment [101].


#### *Int. J. Mol. Sci.* **2020**, *21*, 6030


**Figure 2.** Schematic illustrating important biomaterial properties that should be considered when designing an in vitro fibrotic adipose tissue model.

#### **4. Current Methods of Modeling Fibrotic Adipose Tissue In Vivo**
