| ObjectiveThis paper tend to investigate the effects of compressive strain on the biological activity of MC3T3-E1 cells under three-dimentinal culture condition by examining the proliferation and the expression of proteins and transcription factors known to be essential for osteogenic differentiation and functional maturation.Materials and Methods1. CS/HA/DBM scaffolds with sufficient structural integrity and high interconnected porosity were produced using different qualities ratios of CS/HA/DBM by the freeze-drying method. Both mechanical and biological properties of porous scaffolds were determined by local microarchitecture whose parameters were quantified based on micro computed tomography (Micro-CT) analysis. The mechanical properties of the same scaffolds were tested in the Instron Model 5865 mechanical testing machine using 100N sensor. In vitro, MC3T3-E1 preosteoblast cells were used to investigate cell attachment, spreading and proliferation on the scaffolds via hematoxyline and eosin (HE), scanning electron microscopy (SEM) and MTS assay in order to confirm the following experimental scaffolds and the optical loading time.2. Analyzed the behavior of the mechanical stimuli within 3D scaffolds in terms of stress and strain distributions in the solid material phase, by means of the finite element (FE) analysis, in order to optimize mechanical stimulation parameter of tissue-engineered bone. Cell-scaffold constructs were subjected to cyclical compressive strain under smart materials - piezoelectric ceramic.3. Being based on the results of biocompatibility of the scaffolds and preliminary laboratory, we selected once a day as the compression time, once 1h, on successive 6 days, and 1Hz as the frequency. We tended to investigate the proliferation of MC3T3-E1 cells using MTS method, and examine the expression of proteins and transcription factors known to be essential for osteogenic differentiation and functional maturation at the strain of 0με, 1200με, 2800με, 7000με. Results1. The freeze-drying process provided the well 3D scaffold with interconnected micropores. CS/HA/DBM scaffolds with sufficient structural integrity and high interconnected porosity were produced using different qualities ratios of CS/HA/DBM. Within porosity range of 48– 65%, the range of average compressive modulus and ultimate strength of the scaffolds was 3– 6 kPa and 11– 24 kPa, respectively. With the increasing of HA concentration at the equal weight of DBM, the average trabecular thickness and trabecular separation increased and bone surface/volume ratio decreased, with the result that the volume fraction increased, but the total porosity decreased.2. According to the physiological loading range, the actual experiment and the strain distribution in the scaffold calculated by FE, we considered 0με, 1200με, 2800με, 7000μεas the parameters of apparent average strain. More than 60% of internal strain is between 1000μεand 2000μεwhen subjected to apparent displacement of 1200με, but mainly concentrated on the 1000με; 60% between 1000μεand 2000μεwhile 2800μεapplied, but mainly concentrated on the 2000με; more than 60% above 5000μεwhen 7000μεapplied. There were stress concentration existed on local surface of trabacular bone within each bone column.3. Cell proliferation curve showed that excessive mechanical forces suppressed the proliferation of the preosteoblast MC3T3-E1 cells. Compared with static culture, the proliferation of the preosteoblast MC3T3-E1 cells increased at the strain of 1200μεand 2800με.4. Results from our experiment revealed that compressive strain on MC3T3-E1 cells at a magnitude occurring in physiologically loaded bone tissue increased Runx2, ALP, Col I and OCN mRNA release while BMP-2 release were increased. However, the higher magnitude inhibited their release.Conclusion1. Among the six scaffolds, the lowest quality HA scaffold had the best ideal porous structure with the majority of macrochanneled porous. The MC3T3-E1 cells were easy to adhere the scaffolds and proliferate which showed that the scaffolds had a good cytocompatibility. CS/HA/DBM scaffolds produced using 3:3:1.5 quality ratios were considered as the following experimental scaffolds according to the result of their microarchitectural parameters, mechanical properties and biocompatibility.2. Compressive strains were osteogenic and acted on bone cells in a magnitude-dependent manner and provided a possible molecular model for the increased bone strength observed in response to physical activity. The insight into the molecular mechanisms involved in the cellular responses to appropriate levels of biomechanical forces suggested that these forces were important to improve fracture healing. Additionally, these findings may provide a partial explanation that excessive mechanical forces suppressed the proliferation of the preosteoblast MC3T3-E1 cells.3. Such kind of studies combined with in vitro studies should contribute in the future to the understanding of the process of tissue differentiation within the scaffolds and therefore to optimize the mechanical stimulation. The appropriate compressive strains (1200με, 2800με) were osteogenic and the excessive mechanical forces (7000με) suppressed the proliferation of the preosteoblast MC3T3-E1 cells. The experimental results showed that the finite element analysis method wass a more effective way to explore micro-mechanical environment of bone tissue engineering. |
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