IntroductionSubcutaneous adipose tissue is an interesting source of autologous stem cells with a fundamental role in the pathophysiology of obesity, metabolic syndromes and insulin resistance. We hypothesize that obesity could alter the stromal-vascular fraction (SVF) and adipose stem cell (ASCs) functions, which could compromise its regenerative behavior. Furthermore, we aimed to evaluate whether ASCs derived from post bariatric surgery ex-obese women maintain their functions in a similar fashion as do those from individuals who have never been obese.MethodsThe SVF of subcutaneous adipose tissue from control (n = 6, body mass index – BMI - 27.5 ± 0.5 kg/m2), obese (n = 12, BMI 46.2 ± 5.1 kg/m2) and post bariatric surgery ex-obese (n = 7, initial BMI 47.8 ± 1.3 kg/m2; final BMI 28.1 ± 1.1 kg/m2) women were isolated and evaluated by flow cytometry. ASCs were tested for lipid accumulation by perilipin, adipose differentiation-related protein (ADRP) and Oil Red O staining after adipogenic stimulus. The cytokines secreted by the ASCs and after lipid accumulation induction were also evaluated.ResultsThe subcutaneous adipose tissue of obese and post bariatric surgery ex-obese women was enriched in pericytes (p = 0.0345). The number of supra-adventitial cells was not altered in the obese patients, but it was highly enriched in the post bariatric surgery ex-obese women (p = 0.0099). The ASCs of the post bariatric surgery ex-obese patients secreted more MCP-1 (monocyte chemoattractant protein-1; p = 0.0078). After lipid accumulation induction, the ASCs of the patients in all groups secreted less IL-6 than the ASCs with no adipogenic stimulus (p < 0.0001). Obese ASCs with lipid accumulation secreted the highest amount of IL-6 (p < 0.001) whereas the ASCs from the controls secreted the highest amount of adiponectin (p < 0.0001). The ASCs from the post bariatric surgery ex-obese patients showed the highest levels of lipid accumulation whereas those from the obese women had the lowest levels (p < 0.0001).ConclusionsSVF content and ASC behavior are altered in the subcutaneous adipose tissue of morbid obese women; these changes are not completely restored after bariatric surgery-induced weight loss. The cellular alterations described in this study could affect the regenerative effects of adipose stem cells. Further investigations are required to avoid jeopardizing the development of autologous stem cell-based therapies.

Adipose stem cells (ASCs) spheroids show enhanced regenerative effects compared to single cells. Also, spheroids have been recently introduced as building blocks in directed self-assembly strategy. Recent efforts aim to improve long-term cell retention and integration by the use of microencapsulation delivery systems that can rapidly integrate in the implantation site. Interlockable solid synthetic microscaffolds, so called lockyballs, were recently designed with hooks and loops to enhance cell retention and integration at the implantation site as well as to support spheroids aggregation after transplantation. Here we present an efficient methodology for human ASCs spheroids biofabrication and lockyballs cellularization using micro-molded non-adhesive agarose hydrogel. Lockyballs were produced using two-photon polymerization with an estimated mechanical strength. The Young’s modulus was calculated at level 0.1362 +/-0.009 MPa. Interlocking in vitro test demonstrates high level of loading induced interlockability of fabricated lockyballs. Diameter measurements and elongation coefficient calculation revealed that human ASCs spheroids biofabricated in resections of micro-molded non-adhesive hydrogel had a more regular size distribution and shape than spheroids biofabricated in hanging drops. Cellularization of lockyballs using human ASCs spheroids did not alter the level of cells viability (p › 0,999) and gene fold expression for SOX-9 and RUNX2 (p › 0,195). The biofabrication of ASCs spheroids into lockyballs represents an innovative strategy in regenerative medicine, which combines solid scaffold-based and directed self-assembly approaches, fostering opportunities for rapid in situ biofabrication of 3D building-blocks.

The discovery that adipose tissue represents an interesting source of multipotent stem cells has led to many studies exploring the clinical potential of these cells in cell-based therapies. Recent advances in understanding the secretory capacity of adipose tissue and the role of adipokines in the development of obesity and associated disorders have added a new dimension to the study of adipose tissue biology in normal and diseased states. Subcutaneous adipose tissue forms the interface between the clinical application of regenerative medicine and the establishment of the pathological condition of obesity. These two facets of adipose tissue should be understood as potentially related phenomena. Because of the functional characteristics of adipose stem cells, these cells represent a fundamental tool for understanding how these two facets are interconnected and could be important for therapeutic applications. In fact, adipose tissue stem cells have multiple functions in obesity related to adipogenic, angiogenic and secretory capacities. In addition, we have also previously described a predominance of larger blood vessels and an adipogenic memory in the subcutaneous adipose tissue after massive weight loss subsequent to bariatric surgery (ex-obese patients). Understanding the reversibility of the behavior of adipose stem cells in obeses and in weight loss is relevant to both physiological studies and the potential use of these cells in regenerative medicine.

The scaffold-free tissue engineering using spheroids is pointed out as an approach for optimizing the delivery system of cartilage construct. In this study, we aimed to evaluate the micromolded nonadhesive hydrogel (MicroTissues®) for spheroid compaction (2-day culture) and spontaneous chondrogenesis (21-day culture) using cartilage progenitors cells (CPCs) from human nasal septum without chondrogenic stimulus. CPC spheroids showed diameter stability (486 μm ± 65), high percentage of viable cells (88.1 ± 2.1), and low percentage of apoptotic cells (2.3%). After spheroid compaction, the synthesis of TGF-β1, TGF-β2, and TGF-β3 was significantly higher compared to monolayer (p < 0.005). Biomechanical assay revealed that the maximum forces applied to spheroids after chondrogenesis were 2.6 times higher than for those cultured for 2 days. After spontaneous chondrogenesis, CPC spheroids were entirely positive for N-cadherin, collagen type II and type VI, and aggrecan and chondroitin sulfate. Comparing to monolayer, the expression of SOX5 and SOX6 genes analyzed by qPCR was significantly upregulated (p < 0.01). Finally, we observed the capacity of CPC spheroids starting to fuse. To the best of our knowledge, this is the first time in the scientific literature that human CPC spheroids were formed by micromolded nonadhesive hydrogel, achieving a successful scaffold-free cartilage engineering without chondrogenic stimulus (low cost).