A dual-transduction-integrated biosensing system to examine the 3D cell-culture for bone regeneration.

Three-dimensional (3D) cell cultures developed with living cells and scaffolds have demonstrated outstanding potential for tissue engineering and regenerative medicine applications. However, no suitable tools are available to monitor dynamically variable cell behavior in such a complex microenvironment. In particular, simultaneously assessing cell behavior, cell secretion, and the general state of a 3D culture system is of a really challenging task. This paper presents our development of a dual-transduction-integrated biosensing system that assesses electrical impedance in conjunction with imaging techniques to simultaneously investigate the 3D cell-culture for bone regeneration. First, we created models to mimic the dynamic deposition of the extracellular matrix (ECM) in 3D culture, which underwent osteogenesis by incorporating different amounts of bone-ECM components (collagen, hydroxyapatite [HAp], and hyaluronic acid [HA]) into alginate-based hydrogels. The formed models were investigated by means of electrical impedance spectroscopy (EIS), with the results showing that the impedances increased linearly with collagen and hyaluronan, but changed in a more complex manner with HAp. Thereafter, we created two models that consisted of primary osteoblast cells (OBs), which expressed the enhanced green fluorescent protein (EGFP), and 4T1 cells, which secreted the EGFP-HA, in the alginate hydrogel. We found the capacitance (associated with impedance and measured by EIS) increased with the increases in initial embedded OBs, and also confirmed the cell proliferation over 3 days with the EGFP signal as monitored by the fluorescent imaging component in our system. Interestingly, the change in capacitance is found to be associated with OB migration following stimulation. Also, we show higher capacitance in 4T1 cells that secret HA when compared to control 4T1 cells after a 3-day culture. Taken together, we demonstrate that our biosensing system is able to investigate the dynamic process of 3D culture in a non-invasive and real-time manner.

Electrical impedance spectroscopy - a potential method for the study and monitoring of a bone critical-size defect healing process treated with bone tissue engineering and regenerative medicine approaches.

The development of strategies of bone tissue engineering and regenerative medicine has been drawing considerable attention to treat bone critical-size defects (CSDs). Notably, new strategies and/or treatment approaches always require appropriate tools to track the healing process so as to evaluate their success. In this paper, we present the development of a novel approach for the non-invasive, yet real-time, monitoring and assessment of bone CSDs treated with biomaterials and biomedical approaches. For this, we employed the technique of electrical impedance spectroscopy (EIS) to quantitatively monitor and assess the changes in electrical impedance, and thus the regeneration process. In our in vitro tests, we examined the biochemical changes of the fracture area and investigated the influence of collagen and hydroxyapatite on the changes in electrical impedance by EIS, thus inferring the changes in bone regeneration and structure. Based on this success, we further demonstrated, in real time, the process of regeneration of the traumatic area in an in vivo rabbit model. Our electrical-impedance data of the experiment groups, i.e., the ones treated with natural coral and bone morphogenetic protein-2 (BMP-2), revealed that each group has its unique impedance graph characteristics, which are directly associated with the degree of regeneration. For comparison, we also employed radiography, gross anatomy, and histological analyses in examination. Our results illustrate that EIS holds considerable potential as a non-invasive tool for monitoring, in real time, the healing of bone CSDs by allowing for quantitatively characterizing the changes of both hydroxyapatite and collagen.