| The number of injuries of the musculoskeletal system and bone tissue has been increasing in recent years and accounts for two-thirds of all injuries each year even in the developed parts of the world.This amounts to approximately 6.3 million bone fractures throughout a year.Tissue engineering and regenerative medicine are actively developing new and better ways to facilitate bone tissue recovery after injuries.However,progress in this area is limited by the increasing complexity of the available technology,application of biomaterials,and complexity associated with evaluating the results of these methods.Thus,there is a great need for a suitable investigative tool for the development and application of different methods in the field of bone tissue engineering and regenerative medicine.In recent decades,the electrical properties of biological tissues have been of great interest.This interest resulted from the possibility of performing non-invasive research using quick and easy measurement methods,as well as from benefitting from the absence of radiation exposure,employing a non-destructive research technique,and representing information in the form of numerical series data.In the early stages of the study of bone tissue,electric and dielectric properties of bone were detected which was later followed by the study of the relationship between composition and structure of bone tissue and its electric impedance(EI)characteristics.The electric impedance spectroscopy(EIS)has been wildly used to investigate cell behaviour and the environment in two and three-dimension(2D/3D).Nevertheless,until today,there is no accurate data on the application of EIS in monitoring and evaluation of bone healing process in real-time,including the study of bone cell 3D culture systems and evaluating cell secretion of extracellular matrix(ECM).In this study,firstly,we reproduced the models to mimic the dynamic deposition of the ECM in vitro,based on the biochemical changes in the fracture area and bone tissue content.Briefly,we studied the changes in EIS throughout the model using different concentrations of the main elements-60% inorganic calcium phosphate minerals(primarily hydroxyapatite [HAp]);30% organic materials(mainly collagen);and a general ECM component(hyaluronic acid [HA]),which is secreted by bone cells.The impedance of models and size of the pores in the scaffold tended to increase with increasing collagen content(2%,5%,13%,15%,20%,23% and 25%).The size of the pores in the samples also tended to increase with increasing amounts of HA in the scaffold(0.5%,1%,5% and 10%).The impedance values also demonstrated step-wise increases in proportion to HA content.The pore size of the HAp-alginate scaffold with different HAp concentrations(10%,15%,20% and 25%)did not undergo any major changes.Nevertheless,there was a gradual increase in impedance at concentrations of 10%,15%,and 20%,which increased rapidly at 25%.The results of the investigation showed that the impedances increased linearly with collagen and hyaluronan,but changed in a more complex manner with HAp.Moreover,the outcome shows a relationship between the amount of collagen,HA and HAp in the models with EIS and structure of the sample.In other words,changes in the amount of these elements in the systems can be detected by measuring the changes in EIS.In the next step,we created different bone-like 3D cell culture systems.Briefly,osteoblasts(OBs)with different densities(102,103,104,and 105 cells/m L)were encapsulated within a 3D scaffold(comprised of alginate hydrogel)and EIS resul ts were obtained in terms of change in capacitance.Cell behaviour was simultaneously examined;an adenovirus that can express the enhanced green fluorescent protein(EGFP)was introduced into the cells and detected using fluorescence microscopy.Our biosensing system was used to monitor different cell culture systems with OB,Raw 264.7 and 4T1 cell lines.It was observed that the capacitance depended on the cell line with the minimum value for Raw 264.7 cell line and the highest level for 4T1 cell line.Also,we observed higher capacitance in 4T1 cells that secret HA in comparison to control 4T1 cells after a 3-day culture.Moreover,we found significant advantages with using biosensor while investigating the electrical stimulation of 3D cell culture systems.Briefly,bone-like 3D cell culture systems(OB 105 cells/m L)was stimulated by using three different operating modes of electrical stimulation.The results illustrate that a self-developed,double-checking biosensor holds considerable potential as a non-invasive,real-time investigative tool to monitor the dynamically variable processes of a developing 3D culture system.In addition,we can control scaffold formation in 3D culture by EIS.Thereafter,we created two models that consisted of primary osteob last 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 increase in initial embedded OBs and also confirmed the cell proliferation over three days with the EGFP signal,as monitored by the fluorescent imaging component in our system.Interestingly,the change in capacitance was found to be associated with OB migration following stimulation.Also,it showed 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.In the last section,we present the study that used a rabbit bone critical-size in vivo model in real time,in detail,using non-invasive EIS measurements at 1,4,8,and 12 weeks after surgery for three groups(control,and two groups featuring biomaterials for the stimulation of osteoreparation).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 our examination.Our results illustrate that EIS holds considerable potential as a non-invasive tool for monitoring,in real time,the healing process of bone critical size defects(CSDs)by allowing to quantitatively characterize the changes of both HP and collagen.All in all,in this study,we investigated the possibility of applying EIS for bone healing.It showed that the bone healing process in vitro/in vivo and in bone-like 3D cell culture systems can be monitored and evaluated by EIS.Additionally,the scaffold formation process and the impact of forming factors ca n also be detected by EIS.These results have important implications with respect to understanding the bone healing process and new advances in biomedical engineering. |
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