Atomic force microscopy characterization of white and beige adipocyte differentiation.

Adipose tissue plays an essential role in systemic metabolism with white adipose tissue (WAT) making up most of the tissue and being involved in the regulation of energy homeostasis, and brown and beige adipose tissue (BAT) exhibiting thermogenic activity. There is promise in the conversion of white adipocytes into beige ones as a therapeutic potential to control and enhance systemic metabolism, but it is difficult to maintain this transformation in vivo because we do not fully understand the mechanism of conversion. In this study, we applied atomic force microscopy (AFM) to characterize beige or white adipocytes during the process of differentiation for morphology, roughness, adhesion, and elasticity at different time points. As cells differentiated to white and beige adipocytes, they exhibited morphological changes as they lipid loaded, transitioning from flattened elongated cells to a rounded shape indicating adipogenesis. While there was an initial decrease in elasticity for both beige and white adipocytes, white adipocytes exhibited a higher elasticity than beige adipocytes at all time points. Beige and white adipogenesis exhibited a decrease in adhesion energy compared to preadipocytes, yet at day 12, white adipocytes had a significant increase in adhesion energy compared to beige adipocytes. This work shows significant differences in the mechanical properties of white vs. beige adipocytes during differentiation. Results from this study contribute to a better understanding of the differentiation of adipocytes which are vital to the therapeutic induction, engineered models, and maintenance of beige adipocytes as a potential approach for enhancing systemic metabolism.

Interleukin 1ß and lipopolysaccharides induction dictate chondrocyte morphological properties and reduce cellular roughness and adhesion energy comparatively.

Osteoarthritis (OA) is a whole joint disease marked by the degradation of the articular cartilage (AC) tissue, chronic inflammation, and bone remodeling. Upon AC's injury, proinflammatory mediators including interleukin 1ß (IL1ß) and lipopolysaccharides (LPS) play major roles in the onset and progression of OA. The objective of this study was to mechanistically detect and compare the effects of IL1ß and LPS, separately, on the morphological and nanomechanical properties of bovine chondrocytes. Cells were seeded overnight in a full serum medium and the next day divided into three main groups: A negative control (NC) of a reduced serum medium and 10 ng/ml IL1ß or 10 ng/ml LPS-modified media. Cells were induced for 24 h. Nanomechanical properties (elastic modulus and adhesion energy) and roughness were quantified using atomic force microscopy. Nitric oxide, prostaglandin 2 (PGE2), and matrix metalloproteinases 3 (MMP3) contents; viability of cells; and extracellular matrix components were quantified. Our data revealed that viability of the cells was not affected by inflammatory induction and IL1ß induction increased PGE2. Elastic moduli of cells were similar among IL1ß and NC while LPS significantly decreased the elasticity compared to NC. IL1ß induction resulted in least cellular roughness while LPS induction resulted in least adhesion energy compared to NC. Our images suggest that IL1ß and LPS inflammation affect cellular morphology with cytoskeleton rearrangements and the presence of stress fibers. Finally, our results suggest that the two investigated inflammatory mediators modulated chondrocytes' immediate responses to inflammation in variable ways.

From Chondrocytes to Chondrons, Maintenance of Phenotype and Matrix Production in a Composite 3D Hydrogel Scaffold.

Osteoarthritis (OA) is a degenerative disease characterized by articular cartilage (AC) degradation that affects more than 30 million people in the USA. OA is managed with symptom-alleviating medications. Matrix-assisted autologous chondrocyte transplantation (MACT) is a tissue-engineered option, but current products are expensive and lack mechanical tunability or processability to match defect mechanical properties and anatomical shapes. Here, we explore the efficacy of a biocompatible hydrogel-based scaffold composed of sodium alginate, gelatin, and gum Arabic-referred to by SA-GEL-GA-to support bovine articular chondrocyte (bAChs) proliferation, pericellular matrix (PCM), and extracellular matrix (ECM) production. bAChs were grown for 45 days in SA-GEL-GA. Their viability, their live/dead status, histological staining, biochemical assays for glycosaminoglycans (GAGs) and collagen, atomic force microscopy (AFM) imaging, and immunofluorescence staining of collagen I, collagen II, aggrecan, and CD44 were assessed. We found that SA-GEL-GA was not cytotoxic, induced cellular proliferation by 6.1-fold while maintaining a round morphology, and supported ECM deposition by producing 3.9-fold more GAG compared to day 0. bAChs transformed into chondrons and produced a PCM enriched with collagen II (3.4-fold), aggrecan (1.7-fold), and CD44 (1.3-fold) compared to day 0. In summary, SA-GEL-GA supported the proliferation, ECM production, and PCM production of bAChs in vitro.

Sex-Specific Reduction in Inflammation of Osteoarthritic Human Chondrocytes and Nutraceutical-Dependent Extracellular Matrix Formation.

INTRODUCTION: The aim of this study was to investigate the ability of osteoarthritic human chondrocytes to produce articular cartilage (AC) tissues with a reduced inflammatory environment in response to 4 anti-inflammatory nutraceuticals: alpha-tocopherol (Alpha), gallic acid (G), ascorbic acid (AA), and catechin hydrate (C). METHODS: Chondrocytes isolated from patients who underwent total knee arthroplasty surgeries were divided into groups (9 male; mean age, 66.2 ± 3.5 years and 11 female; mean age, 64.2 ± 3.1 years). Cells were cultured based on sex and supplemented with either a negative control (NC) medium or NC plus one of the nutraceuticals at a concentration of 50 µM. At day 21, cultures were characterized histologically, biochemically, and for gene expression of vital markers. RESULTS: At day 21, 62.3% and 66.2% reduction in nitric oxide (NO) content was evident for female and male cells, respectively. G-treatment of female cells resulted in the lowest expression of nitric oxide synthase-2 (NOS2), matrix metalloproteinase-13 (MMP13), and collagen type-10 (COL10). Alpha-treatment of male cells resulted in the lowest expression of NOS2, bone morphogenic protein-2, MMP13, COL10 and tumor necrosis factor alpha induced protein-6 (TNFAIP6) relative to NC. AA and Alpha treatment resulted in the highest glycosaminoglycan (GAG) content for female and male cultures, respectively. CONCLUSION: A sex-dependent response of osteoarthritic chondrocytes to nutraceutical treatment was evident. Our results suggest the use of G for female cells and Alpha for male cells in OA applications seems to be favorable in reducing inflammation and enhancing chondrocytes' ability to form AC tissues.

In vitro effects of nutraceutical treatment on human osteoarthritic chondrocytes of females of different age and weight groups.

The in vitro effects of four nutraceuticals, catechin hydrate, gallic acid, α-tocopherol and ascorbic acid, on the ability of human osteoarthritic chondrocytes of two female obese groups to form articular cartilage (AC) tissues and to reduce inflammation were investigated. Group 1 represented thirteen females in the 50-69 years old range, an average weight of 100 kg and an average body mass index (BMI) of 34⋅06 kg/m2. Group 2 was constituted of three females in the 70-80 years old range, an average weight of 75 kg and an average BMI of 31⋅43 kg/m2. The efficacy of nutraceuticals was assessed in monolayer cultures using histological, colorimetric and mRNA gene expression analyses. AC engineered tissues of group 1 produced less total collagen and COL2A1 (38-fold), and higher COL10A1 (2⋅7-fold), MMP13 (50-fold) and NOS2 (15-fold) mRNA levels than those of group 2. In comparison, engineered tissues of group 1 had a significant decrease in NO levels from day 1 to day 21 (2⋅6-fold), as well as higher mRNA levels of FOXO1 (2-fold) and TNFAIP6 (16-fold) compared to group 2. Catechin hydrate decreased NO levels significantly in group 1 (1⋅5-fold) while increasing NO levels significantly in group 2 (3⋅8-fold). No differences from the negative control were observed in the presence of other nutraceuticals for either group. In conclusion, engineered tissues of the younger but heavier patients responded better to nutraceuticals than those from the older but leaner study participants. Finally, cells of group 2 formed better AC tissues with less inflammation and better extracellular matrix than cells of group 1.

Enhanced matrix production by cocultivated human stem cells and chondrocytes under concurrent mechanical strain.

Conventional treatments of osteoarthritis have failed to re-build functional articular cartilage. Tissue engineering clinical treatments for osteoarthritis, including autologous chondrocyte implantation, provides an alternative approach by injecting a cell suspension to fill lesions within the cartilage in osteoarthritic knees. The success of chondrocyte implantation relies on the availability of chondrogenic cell lines, and their resilience to high mechanical loading. We hypothesize we can reduce the numbers of human articular chondrocytes necessary for a treatment by supplementing cultures with human adipose-derived stem cells, in which stem cells will have protective and stimulatory effects on mixed cultures when exposed to high mechanical loads, and in which coculture will enhance production of requisite extracellular matrix proteins over those produced by stretched chondrocytes alone. In this work, adipose-derived stem cells and articular chondrocytes were cultured separately or cocultivated at ratios of 3:1, 1:1, and 1:3 in static plates or under excessive cyclic tensile strain of 10% and results were compared to culturing of both cell types alone with and without cyclic strain. Results indicate 75% of chondrocytes in engineered articular cartilage can be replaced with stem cells with enhanced collagen over all culture conditions and glycosaminoglycan content over stretched cultures of chondrocytes. This can be done without observing adverse effects on cell viability. Collagen and glycosaminoglycan secretion, when compared to chondrocyte alone under 10% strain, was enhanced 6.1- and 2-fold, respectively, by chondrocytes cocultivated with stem cells at a ratio of 1:3.