Effect Of Microstructure Parameters Of Micro, NanoHA/CS Blend System On The Biological Characterastics Of MC3T3-E1

Background and objectiveAs the frame of body, bone tissue endlessly accommodates its quality, densityand internal structure in whole vital process. Osteoblast is the main mechanicalsensitively cell to response stress and strain signal. In the process of bone defectivereparation and engineered bone tissue construction, osteoblast adherent growth. Asthe main body to receive the external irritant, scaffold impart the irritant to theosteoblast. The proliferation and differentiation of the osteoblast will be changedbecause of the mechanical stimuli. So, it is very important to investigate the straindistributions in scaffolds for studying the cell proliferation and differentiation inscaffolds under mechanical stimuli. The Micro-CT technique allows acquiring,nondestructively, images of transversal sections of the scaffold. From these data, it ispossible to reconstruct the3D geometry and obtain a virtual model that represents thereal shape of the scaffold. Stress and strain distributions in a scaffold at a microscopiclevel can be studied using the finite element method. As bone transplantationmaterials and bone tissue engineering scaffolds, hydroxyapatite (HA) has beenutilized extensively because it can form strong chemical constitution with bone, butits brittleness and low fatigue resistance restrict its further application. Chitosan (CS)is considered as an appropriate functional material for tissue engineering scaffoldsbecause of high biocompatibility, biodegradability, and favourable process properties.Incorporation of HA into the chitosan matrix improved biocompatibility and tenacity,the HA/CS suggests their potential use for bone tissue engineering applications. Weaimed to detect the effect of mechanical stimuli on the biological characterastics ofMC3T3-E1cells on micro, nanoHA/CS composite membranes and scaffolds. Perhapsit is useful for the development of bone transplantation material and engineered boneconstruction.Methods(1)MicroHA was prepared by calcinating and milling degreased, deproteinedcancellous, and nanoHA was prepared by transonic and agitation method. HA/CS composite membranes were prepared by blending, and were tested for physical andchemical properties, biocompatibility. The proliferation and apoptosis of MC3T3-E1cells on composite membrane were tested for the best content of porous scaffolds.(2)Composite scaffolds were fabricated by the technique of compressionmolding and particulate leaching method with paraffin microsphere as porogen. Thecomposite scaffolds were tested for dynamic mechanical property, SEM observationand cell compatibility with MC3T3-E1cell seeding. The microstructure parameters ofscaffolds were obtained by being scanned using Micro-CT. The enzymaticdegradation was also investigated in vitro.(3)The Micro-CT technique allows acquiring, nondestructively, images oftransversal sections of the scaffold. From these data, it is possible to reconstruct the3D geometry and obtain a virtual model that represents the real shape of the scaffold.Stress and strain distributions in scaffold at a microscopic level were analyzed usingthe finite element method through the microstructure parameters to obtain the optimalapparent strain. Dynamic loading of sinusoidal wave,1Hz frequency,3000μεapparent strain was applied to cell/scaffold composite for10min per day. Cellsmorphology was observed by SEM. The proliferation and apoptosis of MC3T3-E1cells on composite scaffolds were tested. ALP protein level was detected by ELISA,BMP-2, Col I, OCN mRNA level were detected by Real-time PCR, BMP-2, Col I,OCN protein level were detected by Western blot.Results(1)MicroHA particle with relatively homogeneous particle size was preparedby calcinating and milling degreased, deproteined cancellous, and the D50was in therange of1.21～1.67. Rod-shape nanoHA particle with5nm diameter and50nmlength was prepared by transonic and agitation method. HA/CS composite membraneswere prepared by blending micro, nanoHA and CS. HA particle was distributeduniformly in the CS matrix and combined with CS tightly, so the HA particleflowability shortcoming was overcomed. The mechanical property of the compositemembrane was good. Comparing the effect of milling parameters and mass ratio onthe proliferation and morphology of MC3T3-E1cell, we optimized the parameters ofmembrane9as the scaffolds preparation parameters. No significant difference of theproliferation and apoptosis of MC3T3-E1cell between micro, nanoHA/CS compositemembrane. (2)Composite scaffolds were fabricated by the technique of compressionmolding and particulate leaching method with paraffin microsphere as porogen. Thepore connectivity was increased with the porogen content. The results showed that itwas more effective for pore connectivity using spherical porogen than cubic porogen.Dynamic mechanical results showed that scaffolds displayed viscoelastic property andthere was a hystersis effect between strain and stress. On the whole results ofmechanical and SEM, the ratio was optimized of50:70between composite solutionand paraffin volume for the next experiment. The microstructure parameters of microand nanoHA/CS composite scaffolds with same methods were uniform basically.3Dmodel in Mimics of composite scaffolds displayed satisfactory pore connectivity.MC3T3-E1cell could adhered and proliferation on the composite scaffolds, so bothmicro and nano composite scaffolds had good cytocompatibility. Micro andnanoHA/CS composite scaffolds were experienced degradation in the PBS solution oflysozyme. The degradation rate was slow before4weeks, but increased after4weeks,and the amount of reducing sugar in the supernatant was increased with thedegradation time.(3)Stress and strain distributions in the composite scaffold at a microscopiclevel were analyzed using the finite element method based Micro-CT, and the optimalapparent strain3000με which was benefit for cell growth was calculated. The finiteelement results also showed there was stress concentration in the scaffolds. After theapplication of dynamic loading on the cell/scaffolds composite, SEM observationrevealed MC3T3-E1cell could adhered and proliferation on the composite scaffolds,and the cell change morphology from fusiform to confluence. On day14,ColⅠmRNA and protein had high level than control group, but no significantdifference of OCN protein level. This results showed that the cells were notcompletely matured. ALP protein level increased on day7, but decreased on day14.The results showed that there was consistent between mRNA and protein level.Conclusions(1)MicroHA was prepared by milling and nanoHA was prepared by transonicand agitation method, but the scale effect was not significantly. There was notsignificant influence on the adheasion, proliferation and apoptosis of MC3T3-E1cell,and the reason perhaps was the agglomeration of HA particles.(2)Micro, nanoHA/CS composite scaffolds were fabricated by the technique of compression molding and particulate leaching method with paraffin microsphere asporogen. The pore connectivity was increased with the porogen content. Themicrostructure parameters of micro and nanoHA/CS composite scaffolds with samemethods were uniform basically. Micro and nanoHA/CS composite scaffolds wereexperienced degradation in the PBS solution of lysozyme. The degradation rate wasincreased after4weeks, and the amount of reducing sugar in the supernatant wasincreased with the degradation time.(3)Stress and strain distributions in the composite scaffold at a microscopiclevel could be analyzed using the finite element method based Micro-CT, and theoptimal apparent strain benefit for cell growth was calculated. Dynamic loadingpromoted the level of BMP-2, ColⅠmRNA and protein of MC3T3-E1cells on micro,nanoHA/CS composite scaffolds, but influenced OCN protein level not significantly.