Osteogenic differentiation of adipose stem cells by endothelial cells co-culture within liquified capsules

Inspired by the native co-existence of multiple cell types and from the concept of deconstructing the stem cell niche, we propose a co-encapsulation strategy within liquified capsules. The present team has already proven the application of liquified capsules as bioencapsulation systems1. Here, we intend to use the optimized system towards osteogenic differentiation. Capsules encapsulating adipose stem cells alone (MONO-capsules) or in co-culture with endothelial cells (CO-capsules) were maintained in endothelial medium with or without osteogenic differentiation factors. The suitability of the capsules for living stem and endothelial cells encapsulation was demonstrated by MTS and DNA assays. The osteogenic differentiation was assessed by quantifying the deposition of calcium and the activity of ALP up to 21 days. CO capsules had an enhanced osteogenic differentiation, even when cultured in the absence of osteogenic factors. Furthermore, osteopontin and CD31 could be detected, which respectively indicate that osteogenic differentiation had occurred and endothelial cells maintained their phenotype. An enhanced osteogenic differentiation by co-encapsulation was also confirmed by the upregulation of osteogenic markers (BMP-2, RUNX2, BSP) while the expression of angiogenic markers (VEGF, vWF, CD31) revealed the presence of endothelial cells. The proposed capsules can also act as a growth factor release system upon implantation, as showed by VEGF and BMP-2 quantification. These findings demonstrate that the co-encapsulation of stem and endothelial cells within liquified injectable capsules provides a promising strategy for bone tissue engineering.  

Stromal vascular fraction cell sheets angiogenic potential for tissue engineering and regenerative medicine applications

One of the biggest concerns in the Tissue Engineering field is the correct vascularization of engineered constructs. Strategies involving the use of endothelial cells are promising but adequate cell sourcing and neo-vessels stability are enduring challenges. In this work, we propose the hypoxic pre-conditioning of the stromal vascular fraction (SVF) of human adipose tissue to obtain highly angiogenic cell sheets (CS). For that, SVF was isolated after enzymatic dissociation of adipose tissue and cultured until CS formation in normoxic (pO2=21%) and hypoxic (pO2=5%) conditions for 5 and 8 days, in basal medium. Immunocytochemistry against CD31 and CD146 revealed the presence of highly branched capillary-like structures, which were far more complex for hypoxia. ELISA quantification showed increased VEGF and TIMP-1 secretion in hypoxia for 8 days of culture. In a Matrigel assay, the formation of capillary-like structures by endothelial cells was more prominent when cultured in conditioned medium recovered from the cultures in hypoxia. The same conditioned medium increased the migration of adipose stromal cells in a scratch assay, when compared with the medium from normoxia. Histological analysis after implantation of 8 days normoxic- and hypoxic-conditioned SVF CS in a hindlimb ischemia murine model showed improved formation of neo-blood vessels. Furthermore, Laser Doppler results demonstrated that the blood perfusion of the injured limb after 30 days was enhanced for the hypoxic CS group. Overall, these results suggest that SVF CS created under hypoxia can be used as functional vascularization units for tissue engineering and regenerative medicine.

Skin-derived cell-sheets as powerful tools to engineer skin analogues

Cell/cell-extracellular matrix (ECM) dynamic interactions appear to have a major role in regulating communication through soluble signaling, directing cell binding and activating substrates that participate in the highly organized wound healing process. Moreover, these interactions are also crucial for in vitro mimicking cutaneous physiology. Herein we explore cell sheet (CS) engineering to create cellular constructs formed by keratinocytes (hKC), fibroblasts (hDFB) and dermal microvascular endothelial cells (hDMEC), to target skin wound healing but also the in vitro recreation of relevant models. Taking advantage of temperature-responsive culture surfaces, which allow harvesting cultured cells as intact sheets along with the deposited native ECM, varied combinations of homotypic and heterotypic three-dimensional (3-D) CS-based constructs were developed. Constructs combining one CS of keratinocytes as an epidermis-like layer plus a vascularized dermis composed by hDFB and hDMECs were assembled as skin analogues for advancing in vitro testing. Simultaneously both hKC and hDMEC were shown to significantly contribute to the re-epithelialization of full-thickness mice skin wounds by promoting an early epithelial coverage, while hDMEC significantly lead to increased vessels density, incorporating the neovasculature. Thus, although determined by the cellular nature of the constructs, these outcomes demonstrated that CS engineering appear as an unique technology that open the possibility to create numerous combinations of 3D constructs to target defective wound healing as well as the construction of in vitro models to further mimic cutaneous functions crucial for drug screening and cosmetic testing assays.