Abstract
Modular tissue engineering (MTE) is a novel “bottom-up” approach to create engineered biological tissues from microscale repeating units. Our aim was to obtain microtissue constructs, based on polymer microspheres (MSs) populated with cells, which can be further assembled into larger tissue blocks and used in bone MTE. Poly(L-lactide-co-glycolide) MS of 165 ± 47 µm in diameter were produced by oil-in-water emulsification and treated with 0.1 M NaOH. To improve cell adhesion, MSs were coated with poly-L-lysine (PLL) or human recombinant collagen type I (COL). The presence of oxygenated functionalities and PLL/COL coating on MS was confirmed by X-ray photoelectron spectroscopy (XPS). To assess the influence of medium composition on adhesion, proliferation, and osteogenic differentiation, preosteoblast MC3T3-E1 cells were cultured on MS in minimal essential medium (MEM) and osteogenic differentiation medium (OSG). Moreover, to assess the potential osteoblast–osteoclast cross-talk phenomenon and the influence of signaling molecules released by osteoclasts on osteoblast cell culture, a medium obtained from osteoclast culture (OSC) was also used. To impel the cells to adhere and grow on the MS, anti-adhesive cell culture plates were utilized. The results show that MS coated with PLL and COL significantly favor the adhesion and growth of MC3T3-E1 cells on days 1 and 7, respectively, in all experimental conditions tested. On day 7, three-dimensional MS/cell/extracellular matrix constructs were created owing to auto-assembly. The cells grown in such constructs exhibited high activity of early osteogenic differentiation marker, namely, alkaline phosphatase. Superior cell growth on PLL- and COL-coated MS on day 14 was observed in the OSG medium. Interestingly, deposition of extracellular matrix and its mineralization was particularly enhanced on COL-coated MS in OSG medium on day 14. In our study, we developed a method of spontaneous formation of organoid-like MS-based cell/ECM constructs with a few millimeters in size. Such constructs may be regarded as building blocks in bone MTE.
Highlights
One of the main issues in traditional tissue engineering (TTE) is limited diffusion of nutrients and waste removal, which are impeded in the center of the scaffolds [1]
As shown in the literature, cells cultured on MSs in 3D conditions have a lower potential to dedifferentiate [8,9]. We hypothesize that it may work in an opposite way: cells cultured in a medium without differentiating factors (i.e., MEM) have the potential for osteogenic differentiation, even without differentiating factors, but only owing to the signals provided by the 3D structure of the we investigated the impact of medium composition, i.e., the presence of osteogenic differentiation factors and addition of medium from osteoclast cell cultures (OSCs)
PLGA MSs coated with PLL and COL enhanced adhesion and proliferation of preosteoblast MC3T3-E1 cells, as shown by cell metabolic activity test and microscopic observation after fluorescence staining of the cytoskeleton and nuclei
Summary
One of the main issues in traditional tissue engineering (TTE) is limited diffusion of nutrients and waste removal, which are impeded in the center of the scaffolds [1]. Modular tissue engineering (MTE) is a novel concept that differs from the TTE approach based on centimeter size scaffolds, cells, and signaling molecules. MTE uses small building blocks, which can be organized into more complex and functional tissue-like constructs, sometimes made of different types of cells, e.g., those involved in osteogenesis and neovascularization [2,3]. The MTE approach may provide conditions allowing the cells to build and remodel all components, i.e., microscaffolds, extracellular matrix (ECM), and other cells, because cells may arbitrarily organize their surroundings, as happens in vivo. In MTE, it is not a porosity of the material that determines where cells grow but the cells themselves [4]. The fact that natural organs are made of small, three-dimensional (3D), well-organized, repeating units makes the MTE approach more biomimetic than
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