Dynamic microfactories co-encapsulating osteoblastic and adipose-derived stromal cells for the biofabrication of bone units

Abstract

Cells with differentiation potential into mesodermal types are the focus of emerging bone tissue engineering (TE) strategies as an alternative autologous source. When the source of cells is extremely limited or not readily accessible, such as in severe injuries, a tissue biopsy may not yield the required number of viable cells. In line, adipose-derived stromal cells (ASCs) quickly became attractive for bone TE, since they can be easily and repeatably harvested using minimally invasive techniques with low morbidity. Inspired by the multiphenotypic cellular environment of bone, we propose the co-encapsulation of ASCs and osteoblasts (OBs) in self-regulated liquefied and multilayered microcapsules. We explore the unique architecture of such hybrid units to provide a dynamic environment using a simple culture in spinner flasks. Results show that microtissues were successfully obtained inside the proposed microcapsules with an appropriate diffusion of essential molecules for cell survival and signaling. Remarkably, microcapsules cultured in the absence of supplemental osteogenic differentiation factors presented osteopontin immunofluorescence, evidencing that the combined effect of the dynamic environment, and the paracrine signaling between ASCs and OBs may prompt the development of bone-like microtissues. Furthermore, microcapsules cultured under dynamic environment presented an enhanced mineralized matrix and a more organized extracellular matrix ultrastructure compared to static cultures used as control. Altogether, data in this study unveil an effective engineered bioencapsulation strategy for the in vitro production of bone-like microtissues in a more realistic and cost-effective manner. Accordingly, we intend to use the proposed system as hybrid devices implantable by minimally invasive procedures for bone TE applications.

Geometrically Controlled Liquefied Capsules for Modular Tissue Engineering Strategies

“…In fact, the fluid flow drove cells to adhere to the surface of the microparticles, to proliferate, and to deposit a complex collagen‐rich extracellular matrix in such a way that microparticles were assembled in 3D micro‐constructs. [ 17 ]…”

“…The liquefied core also allows dynamic stimuli by simply using stirring flaks, as we recently demonstrated. [ 17 ] Moreover, the liquefied core confers the ability to adapt to structures with irregular shapes, such as tissue defects, while also allowing minimally implantation procedures by injection. After implantation, the presence of the multilayered membrane avoids the dispersion of the core contents to peripheral regions of the body.…”

“…drove cells to adhere to the surface of the microparticles, to proliferate, and to deposit a complex collagen-rich extracellular matrix in such a way that microparticles were assembled in 3D micro-constructs. [17] Herein, we intend to take advantage of the key features that liquefied and multilayered capsules bring to cell encapsulation systems aiming tissue repair, by proposing millimeter-sized geometrically shaped modular systems. For that, a versatile technique was used for the high-throughput fabrication of freestanding microgels encapsulating cells and μPCL.…”

“…[15] Furthermore, molecular factors such as vascular endothelial growth factor and osteopontin released by encapsulated cells were measured and detected in the culture medium, and thus indicating that cytokines released by the encapsulated cells were able to cross the multilayered membrane of capsules to the outside. [17] After polymeric sequential deposition on the surface of a sacrificial template, the core was eliminated. [28,29] Therefore, to obtain a liquefied core where cells can co-assemble and build their own tissue-like 3D microenvironment, microgels surrounded by the multilayered membrane were immersed in EDTA solution.…”

“…In fact, we already had verified the superior bioperformance of such cell encapsulation systems compared to classical hydrogels. [12,[15][16][17] These reports evidence the ability of liquefied capsules for long-term cell survival, a major challenge for TE strategies aiming cell encapsulation. Such results were confirmed for the encapsulation of different phenotypes of cells, such as L929, human mesenchymal-derived stromal cells, human endothelial cells, human osteoblasts, among others.…”