| BackgroundBone is a connective tissue composed mainly by mineral and type I collagen, whichdetermines the hardness and strength of bones. Bone is a load-bearing frame of the body.The bones are closely related to bone biomechanics in the process of growth,development, disease, injury and repair treatment. Therefore, bone bio-mechanicsresearch has a pivotal position in orthopedics. With the rapid development of bonemicro-mechanics, scholars both home and abroad have studied the mechanical propertiesof human bone gradually from macro-mechanical aspects to the micro-mechanicalaspects at micro and nano scales. Nanoindentation technique can not only accuratelymeasure the elastic modulus and hardness of the fine structure of the trabecular bone, butalso it can detect the fracture toughness of bone microstructure, thus having been widelyused by researchers in recent years. Because of its high accuracy, nanoindentationtechnique has become the forefront problem in the field of orthopedic research as to howto homogenize other factors in the testing process, as well as how to improve suchexperiments’ reproducibility and comparability. Using this technique’s recent findings onbones, it shows that the elastic modulus and hardness of human bone’s dried specimensare higher than the wet tissues, but the test specimens have only dry and wet states.Moreover, in such tests of the past, the specimen adopts dehydration embedding moreoften, making its collagen hardened and contracted, and the test results are difficult toobjectively reflect the real mechanical level of bones in the physiological environment of human body. Bones are viscoelastic biological tissues and factors that influence thetesting results also include the viscoelastic properties and surface roughness. Althoughthere are studies indicating that the appropriate load ratio and the peak load can reducethe effects of viscoelasticity and surface roughness, yet the testing results such as howthe variables interact with each other and how they finally decide human bones needfurther study.ObjectivesUse wet specimens which are not dehydrated to have measures on human cortical bonesand cancellous bones with different moisture content and under the change of selfmoisture content, to explore the dynamic changing process of cortical bones’ andcancellous bone’s mechanical properties as well as the effects on bone strength. Setdifferent loading methods to respectively have tests on dried and wet specimens, andclarify the effects of peak load and loading rate in human bone’s microscopic mechanicaltests.Materials and Methods1. Take the same corpse’s bilateral femur and lumber4vertebrae body specimens,adopting the “wet” method to make preparations of specimens. Cut a section of about5mm thickness of cortical bone and cancellous bone vertebrae flake from the uppermiddle section of famous and the cross-section direction of lumbar4vertebrae bodywith a diamond sawing machine.The surface of specimens was polished usingabrasive papers with progressively decreasing silicon carbide grit size (600,800,1000,1200，2000,4000grit) until the specimen surface showed a smooth, mirror likeunder continuous water irrigation. Then specimens were polished on microclothswith a0.05μm aluminum suspension, ultrasonic cleaning machine washed the debrisout of the surface of specimens with the deionized water flushing for10s finally. Thespecimens were soaked in the physiological saline, and put in a refrigerator at-20℃；Specimens were taken out of the refrigerator, and put under the normal temperaturenaturally. Test the cortical bone’s and cancellous bone’s variation of moisture contentrespectively in a lab environment, and use infrared trace moisture tester to prepare specimens with different moisture contents. Divide cortical bone into five groups andeach group is with moisture content of20‰,30‰,40‰,50‰, and60‰respectively. Divide cancellous bone into five groups and each group is with moisturecontent of5%,15%,25%,35%, and40%respectively.2. When testing cortical bone with different moisture contents, set the loading rate ofthe nano-indentation parameter as10nm/s, and peak load as500nm; whenunloaded to15%of the maximum load, the pressure head should be maintainedwithin1min, excluding the influence of thermal effects on transducers and capacitorsas well as the viscosity of bone tissues, and uploading at the same rate; the pressurepoint should be chosen in the symmetry plane between specimens, with6-8pointspressed for each group, and the pressure point region should be elected in the1/2position of the middle part (the intermediate region of the specimen’s symmetryplane) from cortical bone specimens’ center to the edge, and the distance between thepressure point should be maintained at20μm. Have nanoindentation pressure pointdetection under the surface of the microscope, record hardness and elastic modulus indifferent lower moisture contents, and use the one-way variance of analysis to havemultiple comparisons among groups.3. When testing cancellous bone with different moisture contents, set the loading rate ofthe nano-indentation parameter as333μN/s, and peak load as1000μN,then thepressure head should be maintained within5second, uploading at the same rate; thepressure point should be chosen in the the intermediate region of the specimen’ssymmetry plane, with6-8points pressed for each group, and the distance between thepressure point should be maintained at10μm. Have nanoindentation pressure pointdetection under the surface of the microscope, record hardness and elastic modulus indifferent lower moisture contents, and use the one-way variance of analysis to havemultiple comparisons among groups.4. Set three different loading modes: loading rate is6nm/s,8nm/s, and10nm/srespectively; the peak load is300nm,400nm, and500nm respectively; whenunloaded to15%of the maximum load, the pressure head maintains within1min, and then unload at the same rate, with the pressure point selection in the same method.Have tests on the cortical bones of20‰and60‰moisture respectively as to thethree modes, have multiple comparisons of one-way variance analysis among groups,and have comparison of the specimen data with two moisture contents；Set threedifferent loading modes: loading rate is200、250、333μN/s respectively; the peak loadis600μN、750μN and1000μN respectively; when loaded to the maximum, thepressure head maintains within5second, and then unload at the same rate, with thepressure point selection in the same method. Have tests on the cancellous bones of5%and40%moisture respectively as to the three modes, have multiple comparisonsof one-way variance analysis among groups, and have comparison of the specimendata with two moisture contents.Results1. Cortical bone’s elastic modulus and hardness values at different moisture content testsrange from3.89to18.81GPa GPa, and from0.15to0.56GPa, respectively. With theincrement of humidity, test values show a decline curve, and differences between eachhumidity have statistically significance (P <0.05).2. Cancellous bone’s elastic modulus and hardness values at different moisture contenttests range from3.89to18.81GPa, and from0.16to0.58GPa, respectively. With theincrement of humidity, test values show a decline curve, and differences between eachhumidity have statistically significance (P <0.05).3. Three loading modes have no significant effects on the test results of cortical boneswith the moisture of20‰，But they have statistical significance on the compassion ofspecimens with moisture content of60‰(P <0.05); with the increment of peak loadand loading rate, test value increases.4. Three loading modes have no significant effects on the test results of cancellous boneswith the moisture of5%，But they have statistical significance on the compassion ofspecimens with moisture content of40%(P <0.05); with the increment of peak loadand loading rate, test value increases.Conclusions With the increment of content samples’ moisture content, the test values of the humancortical bone’s and cancellous bone’s elastic modulus and hardness both become lower;using three load modes has no significant effects on the testing value of cortical bone withthe moisture content of20‰and elastic modulus and hardness with the moisture contentof5%, but has different results on specimen with the moisture content of60‰. Theelastic modulus and hardness varies with the changes of peak load and loading rate. Themoisture content of the sample itself will affect the test. When the test conditions change,the test results will be completely different according to different testing conditions of wetspecimens. When using nano-indentation technique, dehydrated embedded is usually usedto deal with specimens, having mechanical properties test on the drying specimens whosecollagen has been hardened and contracted, which is not comprehensive for us torecognize the human bone’s mechanical properties at physiological moist environment atthe micro level. |
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