模拟失重状态下雌性大鼠骨质疏松模型骨结构及性能变化研究

A study on the changes of bone structure and properties in female rats under simulated weightlessness as osteoporosis models

目的研究模拟失重尾部悬吊雌性大鼠模型松质骨骨密度(bone mineral density,BMD)、骨小梁结构、骨组织形态、骨代谢生化指标及力学性能的改变,为女性航天员飞行后骨量变化及航天医学提供一定的理论支持。方法 3个月龄雌性SD大鼠20只,随机数表法分为两组,尾部悬吊4周组和空白对照组,每组10只。连续饲养4周,定期记录大鼠活动、饮食、排便、体重、有无脱毛、有无死亡或尾部脱落等一般情况。到期处死大鼠,双能X线吸收法(dual-energy X-ray absorption,DEXA)测定L4椎体、股骨踝部骨密度,Micro-CT进行骨小梁分析,改良丽春红染色法观察骨组织形态,ELISA法检测血清骨代谢生化标志物,并行生物力学检测。结果建模4周后,2组大鼠活动、饮食、排便均正常,体重增加量两组差异无统计学意义(P>0.05),2组均无脱毛、死亡或尾部脱落现象。悬吊组大鼠L4椎体、股骨髁部BMD均较对照组小,分别较对照组下降29%、30%(P<0.05)。悬吊组L4椎体、股骨髁部的骨体积分数(bone volume/total volume,BV/TV)、表面积体积比(bone surface/bone volume,BS/BV)、骨小梁厚度(trabecular thickness,Tb.Th)、骨小梁数目(trabecular number,Tb.N)分别较对照组小,L4椎体各指标较对照组分别下降40%、15%、30%、33%(P<0.05),股骨髁部各指标较对照组分别下降21%、25%、50%、19%(P<0.05);骨小梁间隙(trabecular separation,Tb.Sp)悬吊组较对照组大,且L4椎体、股骨髁部较对照组分别增加92%、33%(P<0.05)。腰椎松质骨ROI骨小梁三维重建发现,悬吊组大鼠腰椎椎体内骨小梁密集程度、骨小梁实质的体积、粗细程度及连续性均低于空白对照组,呈明显的"蜂窝样"改变,出现明显的骨质疏松和骨小梁三维结构破坏。50X显微镜下也观察到悬吊组大鼠椎体骨质疏松样改变明显,骨小梁不连续、变细、间隙增大明显。悬吊组大鼠血清骨碱性磷酸酶(human bone alkaline phosphatase,BALP)、抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)含量均较空白对照组高,为空白对照组的4.5和3.1倍(P<0.05)。悬吊组大鼠的腰椎最大压缩载荷、最大压缩应力、股骨的最大抗弯曲载荷均较对照组小,且较对照组分别下降了36%、33%、34%(P<0.05)。结论尾部悬吊雌性大鼠4周后出现严重的骨密度下降、骨小梁三维结构破坏、骨组织形态破坏、骨代谢失平衡、椎体及股骨生物力学显著下降,形成了明显的骨质疏松,理论上增加了骨折的风险。对女性航天员骨量丢失及骨折的风险应得到充分地认识。

Objective To study the changes of bone mineral density （ BMD ） of cancellous bone, trabecular bone structure, bone tissue morphology, bone metabolic biochemical markers and mechanical properties in tail-suspended female rats under simulated weightlessness, so as to provide certain theoretical support for the research on bone mass changes in female astronauts after the lfight and aerospace medicine.Methods Twenty 3-month-old female Sprague-Dawley （ SD ） rats were randomly divided into 2 groups. The tails of the rats in group A （n=10 ） were suspended for 4 weeks, and the other rates were taken as blank control group （n=10 ）. All the rates were continuously fed for 4 weeks, and their general conditions were recorded, including activities, eating and drinking,defecation, weight, unhairing or not, death and tail falling off. All the rats were sacrificed at predetermined time points. The BMD of the L4 vertebral body and femoral condyle was evaluated by dual-energy X-ray absorption （ DEXA ）. Micro computed tomography （ Micro-CT ） was applied to analyze the trabecular bone. Bone tissue slice staining was conducted by using ponceau. Serum bone metabolic biochemical markers were detected by Enzyme-linked immunosorbent assay （ ELISA ）, and biochemical properties were investigated.Results At 4 weeks after the establishment of osteoporosis models, the activities, eating and drinking and defecation of the rats in group A were normal, and the differences in weight gain were not statistically significant between the 2 groups （P〉0.05 ）. No unhairing, death or tail falling off was noticed in either group. When compared with that in blank control group, the BMD of the L4 vertebral body and femoral condyle in group A was reduced by 29% and 30% （P〈0.05 ）. The bone volume / total volume （ BV/TV ）, bone surface/bone volume （ BS/BV ）, trabecular thickness （ Tb.Th ） and trabecular number （ Tb.N ） of the L4 vertebral body in group A was reduced by 40%, 15%, 30% and 33% when compared with that in blank control group （P〈0.05 ）. The BV/TV, BS/BV, Tb.Th and Tb.N of the femoral condyle in group A was reduced by 21%, 25%, 50% and 19% when compared with that in blank control group （P〈0.05 ）. The trabecular separation （ Tb.Sp ） of the L4 vertebral body and femoral condyle in group A was increased by 92% and 33% respectively （P〈0.05 ） when compared with that in blank control group （P〈0.05 ）. The 3-dimensional （ 3 D ） reconstruction of the trabecular bone in region of interest （ ROI ） of lumbar cancellous bone showed group A was not as good as blank control group in the intensity, real volume, size and continuity of the trabecular bone in the lumbar vertebral body. Honeycomb-like structure was detected and obvious osteoporosis and 3D structural damage were noticed. The osteoporotic bone changes and incontinuity, diminution and enlarged space of the trabecular bone were observed in group A under the microscope （ 50X ）. The human bone alkaline phosphatase （ BALP ） and tartrate resistant acid phosphatase （ TRAP ） in group A was 4.5 and 3.1 times higher than that in blank control group （P〈0.05 ）. The maximal compressive load and maximal compressive stress of the lumbar vertebral body and the maximal lfexural load of the femur in group A were decreased by 36%, 33% and 34% when compared with that of blank control group （P〈0.05 ）.Conclusions The significant decrease of the BMD of cancellous bone, 3 D structural damage of the trabecular bone, morphological destruction of bone tissues, bone metabolism imbalance and obvious reduction of mechanical strength of the vertebral body and femur are detected in the tail-suspended rats at 4 weeks after the establishment of osteoporosis models. The visible signs of osteoporosis are observed, and the risk of fracture is increased theoretically. A full understanding of bone mass loss and fracture risk in female astronauts should be developed.