Scalable Generation of Pre-Vascularized and Functional Human Beige Adipose Organoids.

Obesity and type 2 diabetes are becoming a global sociobiomedical burden. Beige adipocytes are emerging as key inducible actors and putative relevant therapeutic targets for improving metabolic health. However, in vitro models of human beige adipose tissue are currently lacking and hinder research into this cell type and biotherapy development. Unlike traditional bottom-up engineering approaches that aim to generate building blocks, here a scalable system is proposed to generate pre-vascularized and functional human beige adipose tissue organoids using the human stromal vascular fraction of white adipose tissue as a source of adipose and endothelial progenitors. This engineered method uses a defined biomechanical and chemical environment using tumor growth factor ß (TGFß) pathway inhibition and specific gelatin methacryloyl (GelMA) embedding parameters to promote the self-organization of spheroids in GelMA hydrogel, facilitating beige adipogenesis and vascularization. The resulting vascularized organoids display key features of native beige adipose tissue including inducible Uncoupling Protein-1 (UCP1) expression, increased uncoupled mitochondrial respiration, and batokines secretion. The controlled assembly of spheroids allows to translate organoid morphogenesis to a macroscopic scale, generating vascularized centimeter-scale beige adipose micro-tissues. This approach represents a significant advancement in developing in vitro human beige adipose tissue models and facilitates broad applications ranging from basic research to biotherapies.

Cell Aggregate Assembly through Microengineering for Functional Tissue Emergence.

Compared to cell suspensions or monolayers, 3D cell aggregates provide cellular interactions organized in space and heterogeneity that better resume the real organization of native tissues. They represent powerful tools to narrow down the gap between in vitro and in vivo models, thanks to their self-evolving capabilities. Recent strategies have demonstrated their potential as building blocks to generate microtissues. Developing specific methodologies capable of organizing these cell aggregates into 3D architectures and environments has become essential to convert them into functional microtissues adapted for regenerative medicine or pharmaceutical screening purposes. Although the techniques for producing individual cell aggregates have been on the market for over a decade, the methodology for engineering functional tissues starting from them is still a young and quickly evolving field of research. In this review, we first present a panorama of emerging cell aggregates microfabrication and assembly technologies. We further discuss the perspectives opened in the establishment of functional tissues with a specific focus on controlled architecture and heterogeneity to favor cell differentiation and proliferation.

An estradiol releasing, proangiogenic hydrogel as a candidate material for use in soft tissue interposition.

INTRODUCTION AND OBJECTIVES: Soft tissue interposition (STI) using local and/or regional flaps is often necessary in urogenital reconstruction to stimulate wound healing and prevent recurrence. Harvesting STI flaps can cause donor site morbidity and may not be available in some patients. In this study, we designed estradiol (E2) releasing hydrogel that could be used as an alternative to a STI flap and to investigate its ability to stimulate tissue production and angiogenesis. MATERIALS AND METHODS: A hydrogel was constructed by crosslinking a solution of estradiol, methacrylated gelatin (15%, w/v), and methacrylated hyaluronic acid (1%, w/v). The release of estradiol was measured using a UV-spectrophotometer (λmax = 220 nm). Angiogenesis was evaluated by an ex ovo chicken embryo chorioallantoic membrane (CAM) assay. RESULTS: Estradiol was gradually released from the hydrogel over 21 days. The hydrogels could be easily manipulated with surgical forceps without any deformation. The hydrogels significantly increased collagen production of human dermal fibroblasts (HDFs). Scanning electron microscopic examination demonstrated that HDFs produced significantly more extracellular matrix (ECM) on estradiol releasing hydrogels compared with the controls. Estradiol releasing hydrogels doubled the number of blood vessels growing toward the hydrogel compared with the controls (vasculogenic index, 59.6 [±6.4] and 25.6 [±4.0], respectively; [P < 0.05]). CONCLUSION: We present a proangiogenic, degradable hydrogel that can be used as an off-the-shelf available substitute to traditional STI flaps. This is achieved by using estradiol as a potent stimulator of new tissue production and new blood vessel formation.

Nuclear targeting peptide-modified, DOX-loaded, PHBV nanoparticles enhance drug efficacy by targeting to Saos-2 cell nuclear membranes.

The aim of this study was to target nano sized (266 ± 25 nm diameter) poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) particles carrying Doxorubicin (DOX), an anticancer agent, to human osteosarcoma cells (Saos-2). A nuclear targeting molecule (Nuclear Localization Signal, NLS), a 17 a.a. peptide, was attached onto the doxorubicin loaded nanoparticles. NLS conjugated nanoparticles surrounded the cell nuclei, but did not penetrate them. Free doxorubicin and doxorubicin loaded nanoparticles entered the cytoplasm and were evenly distributed within the cytoplasm. The localization of the NLS-targeted particles around the nuclear membrane caused a significantly higher decrease in the cancer cell numbers due to apoptosis or necrosis than the untargeted and free doxorubicin formulations showing the importance of targeting the nanoparticles to the nuclear membrane in the treatment of cancer.

Development of a UV crosslinked biodegradable hydrogel containing adipose derived stem cells to promote vascularization for skin wounds and tissue engineering.

The aim of this study was to design a dermal substitute containing adipose derived stem cells (ADSC) that can be used to improve the regeneration of skin on difficult wound beds by stimulating rapid neovascularization. This was achieved by first synthesizing methacrylated gelatin (GelMA) and methacrylated hyaluronic acid (HAMA) precursors which could be stored at -80 oC after lyophilisation. Polymer precursors were then dissolved in media (in 15:1 ratio), ADSCs added together with the photoinitiator and crosslinked with 40 s of UV. Hydrogels degraded by 50% over 3 weeks in an in vitro environment. ADSC loaded hydrogels could be easily handled with forceps (compressive modulus was 6 kPa). Transparency of the gel would allow a full field-of-view of a wound site. The hydrogels provided a suitable microenvironment for ADSC proliferation as shown by the filopodia observed in confocal micrographs. In vivo studies demonstrated that stem cell loaded hydrogels increased vascularization by up to 3 fold compared to their cell free counterparts. In conclusion, GelMA/HAMA hydrogels loaded with ADSC showed the desired proliferative and angiogenic properties essential to promote angiogenesis for wound healing and improving survival of tissue engineered skin.