Micro- And Nano-Scale Structural And Mechanical Analyses Of Trabeculae In Ovariectomized Rat

Part one: The impacts of rotation center on the assessment oftrabecular bone densitometric and structural propertiesObject: To evaluate the influence of rotation center on the assessmentof trabecular bone densitometric and structural properties.Method: Twenty female Sprague-Dawley rats of 7-month-old wererandomly assigned to ovariectomized (OVX) and SHAM-operated(SHAM) groups. The left tibiae were harvested at 3 weekspostoperatively. High resolution micro-computed tomography (μCT) wasused to identify the densitometric and microstructural properties oftrabeculae in the proximal ends of tibia. After CT scanning, the bestartificial rotation center for each scan was obtained. Densitometric andmicrostructural analysis was peformed on the centers at different placesaway from the best center of±0.2,±0.5,±0.7,±1.0,±1.5 and±2.0 pixels,respectively.Results: In a variation of±2.0 pixels, the total bone mineral contentand apparent BMD were not changed by the rotation center displacement,but the tissue bone mineral content and BV/TV were increased withsimultaneous decease in tissue BMD by increasing of the center'sdisplacement. Tb.Th, Tb.N and Tb.Sp in OVX rats increased with thecenter's deflection. Structure model index (SMI) was unchanged in avariation of±2.0 pixels. Trabecualr area density (BS/BV), degree ofanisotropy (DA) and connectivity density (Conn.D) decreased with thecenter's deflection. The CV value of each parameter in OVX rats washigher than that in SHAM rats. The changing curves of bone parametersvs. centers could be well fitted by quadric regression models, by whichthe actual centers could be acquired. Moreover, there were somedifferences between the best artificial and actual center.Conclusions: The small displacements in the center of rotation wouldnot change the values of total bone mineral content, apparent BMD andSMI, while have some effects on the assement of Tb.Sp and Tb.N. TissueBMD, BV/TV, BS/BV, Tb.Th, DA and Conn.D would be significantly changed by displacement of the rotation center. There are some inevitabledifferences between the best artificial and actual centers. But the actualcenter could be obtained by quadric regression models, and thus theprecision of microCT analysis would be improved. Part two: Regional-specified compensation of trabecularhypertrophy or hypermineralization as a result of bone loss causedby estrogen deficiency in ratsObject: To characterize the regional variations in bone mineraldensity (BMD), microstructure and dynamic changes in OVX ratsMethod: 50 Female Sprague-Dawley rats of seven-month-old wereovariectomized (OVX), SHAM-operated (SHAM) or left unoperated(baseline control). The left tibiae were harvested at baseline, and 3 and 15weeks postoperatively. High resolution micro-computed tomography(μCT) was used to identify the densitometric and microstructuralproperties of trabeculae in the proximal ends (epiphysis and metaphysis)of the rat tibia. Blood samples were used for biochemical markersanalysis.Results: Apparent BMD and tissue BMD of the trabeculae at theepiphysis were significantly higher than those of metaphysis. Moreover,trabeculae at the epiphysis were thicker, fewer in number andconnectivity than those at the metaphysis which were more rod-like(P＜0.05). Serum ALP in OVX rats was significantly higher than that ofthe SHAM rats at 3 week post-operatively, while TRACP5b wassignificantly lower than that in SHAM rats at 15 weeks (P＜0.05). Therewere no differences in serum ALP at week 15 and TRACP5b at week 3between OVX and SHAM rats. Trabeculae at the metaphysis were moresusceptible to bone loss induced by estrogen-deprivation than at theepiphysis and this region was also greatly sensitive for adaptation inducedby bone loss. At the metaphysis, trabeculaer tissue BMD and thicknesswere unexpectedly higher at 15 weeks postoperatively than 3 weeks orbaseline (P＜0.05). In contrast, at the epiphysis, tissue BMD did notchange with time but trabecular thickness significantly increased at week15 compared to baseline and was also greater in OVX compared toSHAM rats (P＜0.05).Conclusion: Metaphyseal and epiphyseal trabeculae showregionally-specified variations in BMD and microstructure. Metaphyseal trabeculae are more susceptible to bone loss induced byestrogen-deficiency and would be compensated by either hypertrophy orhypermineralization, while epiphyseal trabeculae are mainly strengthenedby thickening. Such compemsatory behaviors may have partiallyovercome the detrimental effects of estrogen-deficiency on bone loss, andcould thus partially reverse the dramatic deterioration in bone mechanicalperformance. Part three: Micro and nano-scale structural and mechanical analysesof trabeculae in ovariectomized rat vertebraeObject: To evaluate trabecular micro-and nano-scale structural andmechanical integrity and 3D-and 2D-BMD in ovariectomized rats.Method: Twenty female Sprague-Dawley rats of 10-month-old wererandomly assigned to ovariectomy (OVX) and SHAM-operation (SHAM).Trabecular nano-mechanical properties like hardness and elastic modulusof the vertebral body were determined by nanoindentation. The surfacetopography was analyzed by atomic force microscopy (AFM). Trabecularvolumetric 3D BMD and microstructure were determined using micro CT.2D BMD of the lumbar vertebra was measured by DXA.Results: At 15 weeks post-surgery, volumetric BMD wassignificantly less in OVX than in SHAM, by-27.2 % at organ level and-12.0 % at tissue level. There were significantly less 3D trabecularBV/TV (-28.6%), Tb.Th (-13.8 %), and TB.N (-17.0 %) in OVX than inSHAM while Tb.Sp and the structure model index (SMI) weresignificantly greater by 19.2 % and 91.2 %, respectively. 2D DXA BMDof the sixth lumber vertebrae was significantly less in OVX than inSHAM, by-14.4 % with in vitro and-11.8 % with in vivo measurements.AFM images showed rough trabecular surfaces with many small nodulesclosely packed to each other in SHAM, and relatively smooth surfaceswith loosely packed large nodular structures in OVX. The inter-fibrillarspace were more compacted in SHAM than in OVX. The trabecularnanoindentation hardness (0.91±0.13 vs. 0.90±0.07 GPa, OVX vs.SHAM) and elastic modulus (21.0±2.5 vs. 22.0±2.4 GPa) werestatistically insignificant. AFM observation also showed that neither thenanoindentation depths of trabecular nor the residual shape caused byplastic deformation had significant difference between the two groups.Conclusions: Estrogen deprivation results in a dramatic deteriorationin trabecular micro-and nano-architectures, in 3D volumetric BMD atboth organ and tissue levels and in 2D DXA BMD, but not in the nano-mechanical properties of trabecular bone.