Case Report: Formation of 3D Osteoblast Spheroid Under Magnetic Levitation for Bone Tissue Engineering

Abstract

Skeletal reconstruction is necessary in cases of bone defects created by tumors, trauma, and abnormalities. Regeneration of bone defects remains a critical problem, and current approaches are based on biocompatible scaffolds. Spheroids represent a simple 3D system since no supporting material is required for cell growth. Different techniques are used to generate spheroids, such as hanging drop, low-attachment plates, and magnetic nanoparticles. The idea of using magnetic nanoparticles is to cross-link through cell membrane overnight to create complex 3D cellular spheroid by using magnets to guide the cellular response. Herein, the current study aimed to achieve 3D human fetal osteoblast (hFOB) spheroid under magnetic levitation. Formation of 3D spheroid culture under magnetic levitation was evaluated by cell viability at 3, 7, and 14 days. Morphology of the 3D hFOB spheroid was analyzed by SEM and fluorescence microscopy and the differentiation towards mineralized lineage by ALP assay, qPCR, and alizarin red staining. The cell viability indicated that the 3D hFOB spheroid still viable after 14 days of culture. ALP assay, qPCR analysis expression of Col1, ALP, and Itg-β1 molecules, and calcium deposition with alizarin red showed a high level of bioactivity of the 3D hFOB spheroid. SEM images allowed the morphological analysis of the 3D microtissue-like spheroid with the presence of matrix deposition. These results indicate that magnetic levitation culture enables 3D stable osteoblast spheroids and could be a promising strategy for engineering application in the 3D construct in surgery regeneration of mineralized tissue.

Physiological Mineralization during In Vitro Osteogenesis in a Biomimetic Spheroid Culture Model

“…While 3D culture models of many other organs have already been routinely used for in vitro assays, research on osteoblasts and their progenitor cells is still largely based on conventional monolayer cultures. Yet, no generally accepted spheroid model for osteoblast(-like) cells has been established and major drawbacks of reported approaches were that biomineralization was either not described [ 21 , 22 , 23 , 24 ] or not (reliably) quantified [ 25 , 26 ]. Importantly, the ECM has been shown to alter drug responses, while its biochemical composition and mechanical properties are known to steer cell behavior differently in 2D and 3D, respectively [ 51 ].…”

“…Limited other research also provided evidence that the cellular response of osteoblast(-like) cells cultured in 3D substantially differs from that in 2D [ 21 , 22 ]. A major limitation of currently scarcely reported approaches is that mineralization was either not reported [ 21 , 22 , 23 , 24 ] or has not (reliably) been quantified [ 25 , 26 ]. Yet, no generally accepted spheroid model for osteoblast(-like) cells has been established so far.…”

Magnetic-Based Human Tissue 3D Cell Culture: A Systematic Review

“…Magnetic-based models allow us to effectively produce homogeneous 3D aggregates and with desirable compactness [ 1 , 17 ]. Of the twenty-five studies included in this review, nine studies used only magnetic levitation [ 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ], eleven studies used only the bioprinting method [ 14 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 ], and three studies used both methods [ 15 , 36 , 37 ]. Only two studies used the magnetic ring formation method [ 22 , 38 ].…”

“…The most recent m3D method, ring formation ( Figure 1 D), consists of firstly levitating the magnetized cells to allow for the formation of aggregates with ECM, followed by a second step to disintegrate these 3D cultures into dispersed cells, and, lastly, placing these plates atop a magnetic unit with ring-shaped neodymium magnets to induce cell aggregation in a toroidal shape [ 1 ]. These magnetic-based techniques are an easy procedure to implement and standardize using diverse cell types, allowing for a fast and consistent spheroid formation as well as a controlled cellular movement and aggregation [ 1 , 13 , 14 ] Moreover, since spheroids are able to produce their own endogenous ECM during their formation and aggregation process, there is no need to use an artificial matrix [ 1 , 13 ].…”

Biomedical Applications of Magnetic Levitation

“…[59] Moreover, MagLev is actively playing a role in the density measurement of illicit drugs and powders, as well as forensic samples, [14,60,61] 3D cell culturing, [62,63] and tissue engineering. [64][65][66][67] More recently, MagLev platforms have been integrated with smartphones [38,41,59] and fluorescent imaging [35] to further promote their versatility and portability in resource-limited settings.…”