Short-term post-implantation dynamics of in vitro engineered human microvascularized adipose tissues.

Engineered adipose tissues are developed for their use as substitutes for tissue replacement in reconstructive surgery. To ensure a timely perfusion of the grafted substitutes, different strategies can be used such as the incorporation of an endothelial component. In this study, we engineered human adipose tissue substitutes comprising of functional adipocytes as well as a natural extracellular matrix using the self-assembly approach, without the use of exogenous scaffolding elements. Human microvascular endothelial cells (hMVECs) were incorporated during tissue production in vitro and we hypothesized that their presence would favor the early connection with the host vascular network translating into functional enhancement after implantation into nude mice in comparison to the substitutes that were not enriched in hMVECs. In vitro, no significant differences were observed between the substitutes in terms of histological aspects. After implantation, both groups presented numerous adipocytes and an abundant matrix in addition to the presence of host capillaries within the grafts. The substitutes thickness and volume were not significantly different between groups over the short-term time course of 14 days (d). For the microvascularized adipose tissues, human CD31 staining revealed a human capillary network connecting with the host microvasculature as early as 3 d after grafting. The detection of murine red blood cells within human CD31+ structures confirmed the functionality of the human capillary network. By analyzing the extent of the global vascularization achieved, a tendency towards increased total capillary network surface and volume was revealed for prevascularized tissues over 14 d. Therefore, applying this strategy on thicker reconstructed adipose tissues with rate-limiting oxygen diffusion might procure added benefits and prove useful to provide voluminous substitutes for patients suffering from adipose tissue loss or defects.

Impact of TNF and IL-1ß on capillary networks within engineered human adipose tissues.

Inflammation is a normal phase of the wound healing process, which likely occurs following tissue transplantation. For reconstructive surgery purposes, engineered adipose tissues represent promising alternatives to autologous fat grafts. It is therefore important to study the impact of an inflammatory microenvironment on the cellular functions of the different cell types comprised within matrix-rich reconstructed tissues. In this study, human reconstructed adipose tissues (hrATs) featuring a preformed capillary network formed by microvascular endothelial cells (hMVECs) were produced from adipose-derived stem/stromal cells (ASCs) by the self-assembly approach of tissue engineering. We hypothesized that a prolonged inflammatory context, mediated by tumor necrosis factor (TNF) and interleukin-1ß (IL-1ß), would impact hrATs' secretory profile and mediate detrimental effects on the microvascular network in vitro. Analysis of conditioned media established tissue responsiveness through the increased secretion of monocyte chemoattractant protein-1 (up to 23 fold), interleukin-6 (up to 69 fold) and angiopoietin-1 (up to 2.7 fold) after 3 and 6 days of cytokine exposure, along with a significant reduction in adiponectin secretion. Imaging of the preformed capillary network within the hrATs revealed increased disorganization in the presence of TNF/IL-1ß, featuring a less extended and less ramified network with apoptotic hMVECs in the remaining capillary structures. These results indicate that a prolonged inflammatory context can be deleterious to the capillary network featured by in vitro engineered tissues. Strategies aiming at preserving the integrity of the vascular network will help develop substitutes that are better suited to face inflammatory conditions upon grafting.

Creating capillary networks within human engineered tissues: impact of adipocytes and their secretory products.

The development of tissue-engineered substitutes of substantial volume is closely associated with the need to ensure rapid vascularization upon grafting. Strategies promoting angiogenesis include the in vitro formation of capillary-like networks within engineered substitutes. We generated both connective and adipose tissues based on a cell sheet technology using human adipose-derived stromal cells. This study evaluates the morphology and extent of the capillary networks that developed upon seeding of human microvascular endothelial cells during tissue production. We posited that adipocyte presence/secretory products could modulate the resulting capillary network when compared to connective substitutes. Analyses including confocal imaging of CD31-labeled capillary-like networks indicated slight differences in their morphological appearance. However, the total volume occupied by the networks as well as the frequency distribution of the structure's volumes were similar between connective and adipose tissues. The average diameter of the capillary structures tended to be 20% higher in reconstructed adipose tissues. Quantification of pro-angiogenic molecules in conditioned media showed greater amounts of leptin (15×), angiopoietin-1 (3.4×) and HGF (1.7×) secreted from adipose than connective tissues at the time of endothelial cell seeding. However, this difference was attenuated during the following coculture period in endothelial cell-containing media, correlating with the minor differences noted between the networks. Taken together, we developed a protocol allowing reconstruction of both connective and adipose tissues featuring well-developed capillary networks in vitro. We performed a detailed characterization of the network architecture within engineered tissues that is relevant for graft assessment before implantation as well as for in vitro screening of angiogenic modulators using three-dimensional models.

Magnetic Resonance Imaging of Human Tissue-Engineered Adipose Substitutes.

Adipose tissue (AT) substitutes are being developed to answer the strong demand in reconstructive surgery. To facilitate the validation of their functional performance in vivo, and to avoid resorting to excessive number of animals, it is crucial at this stage to develop biomedical imaging methodologies, enabling the follow-up of reconstructed AT substitutes. Until now, biomedical imaging of AT substitutes has scarcely been reported in the literature. Therefore, the optimal parameters enabling good resolution, appropriate contrast, and graft delineation, as well as blood perfusion validation, must be studied and reported. In this study, human adipose substitutes produced from adipose-derived stem/stromal cells using the self-assembly approach of tissue engineering were implanted into athymic mice. The fate of the reconstructed AT substitutes implanted in vivo was successfully followed by magnetic resonance imaging (MRI), which is the imaging modality of choice for visualizing soft ATs. T1-weighted images allowed clear delineation of the grafts, followed by volume integration. The magnetic resonance (MR) signal of reconstructed AT was studied in vitro by proton nuclear magnetic resonance ((1)H-NMR). This confirmed the presence of a strong triglyceride peak of short longitudinal proton relaxation time (T1) values (200 ± 53 ms) in reconstructed AT substitutes (total T1=813 ± 76 ms), which establishes a clear signal difference between adjacent muscle, connective tissue, and native fat (total T1 ~300 ms). Graft volume retention was followed up to 6 weeks after implantation, revealing a gradual resorption rate averaging at 44% of initial substitute's volume. In addition, vascular perfusion measured by dynamic contrast-enhanced-MRI confirmed the graft's vascularization postimplantation (14 and 21 days after grafting). Histological analysis of the grafted tissues revealed the persistence of numerous adipocytes without evidence of cysts or tissue necrosis. This study describes the in vivo grafting of human adipose substitutes devoid of exogenous matrix components, and for the first time, the optimal parameters necessary to achieve efficient MRI visualization of grafted tissue-engineered adipose substitutes.

Characterization of In Vitro Engineered Human Adipose Tissues: Relevant Adipokine Secretion and Impact of TNF-α.

Representative modelling of human adipose tissue functions is central to metabolic research. Tridimensional models able to recreate human adipogenesis in a physiological tissue-like context in vitro are still scarce. We describe the engineering of white adipose tissues reconstructed from their cultured adipose-derived stromal precursor cells. We hypothesize that these reconstructed tissues can recapitulate key functions of AT under basal and pro-inflammatory conditions. These tissues, featuring human adipocytes surrounded by stroma, were stable and metabolically active in long-term cultures (at least 11 weeks). Secretion of major adipokines and growth factors by the reconstructed tissues was determined and compared to media conditioned by human native fat explants. Interestingly, the secretory profiles of the reconstructed adipose tissues indicated an abundant production of leptin, PAI-1 and angiopoietin-1 proteins, while higher HGF levels were detected for the human fat explants. We next demonstrated the responsiveness of the tissues to the pro-inflammatory stimulus TNF-α, as reflected by modulation of MCP-1, NGF and HGF secretion, while VEGF and leptin protein expression did not vary. TNF-α exposure induced changes in gene expression for adipocyte metabolism-associated mRNAs such as SLC2A4, FASN and LIPE, as well as for genes implicated in NF-κB activation. Finally, this model was customized to feature adipocytes representative of progressive stages of differentiation, thereby allowing investigations using newly differentiated or more mature adipocytes. In conclusion, we produced tridimensional tissues engineered in vitro that are able to recapitulate key characteristics of subcutaneous white adipose tissue. These tissues are produced from human cells and their neo-synthesized matrix elements without exogenous or synthetic biomaterials. Therefore, they represent unique tools to investigate the effects of pharmacologically active products on human stromal cells, extracellular matrix and differentiated adipocytes, in addition to compounds modulating adipogenesis from precursor cells.