小鼠骨损伤模型在骨修复机制研究中的应用

骨折是常见多发病,建立标准的骨损伤动物模型是进行骨折实验研究的基础,由于小鼠具有和人类相似的遗传背景,并且可进行多种遗传修饰等优势,因此在骨折研究中的应用越来越广泛。     

骨的显微损伤及其对骨力学性能的影响

骨的显微损伤是由于反复受载所致的仅能在显微镜下观察到的骨基质的损伤,往往表现为各种形态的显微裂纹或弥散性损伤.通过特殊染色和标记可对显微损伤进行组织学观察和定量分析,并根据其形态和与骨单位的位置关系进行分类.显微损伤往往伴有骨强度的降低和骨脆性的增加,裂纹的长度及其位置是影响骨力学性能的决定因素.显微损伤的积累最终会导致骨的疲劳骨折.     

骨组织疲劳损伤的生物力学研究概述

骨组织是人体重要的承重器官,具有力学适应性,在疲劳载荷作用下会出现疲劳损伤,常见于运动员长跑、新兵训练以及老年人的日常活动,主要表现为骨组织显微裂纹的萌生、扩展,力学性能下降甚至是应力性骨折,危害很大.骨组织的疲劳损伤存在于包括超显微结构、显微结构和组织宏观的各个层面,皮质骨中的抗疲劳结构(骨单元)及内部的细胞成分(主要为骨细胞)对抵抗疲劳损伤、识别显微裂纹以及诱导后续定向骨重建过程具有重要作用.总结相关研究结果与结论有助于系统了解骨组织疲劳损伤行为及相应识别修复过程,同时也为后续骨组织疲劳损伤预防及治疗等临床研究工作的开展提供参考和方向.     

aFGF基因在大鼠脑皮质损伤神经元的表达

目的：为aFGF对成年大鼠脑损伤后脑内神经元的作用提供理论依据。方法：应用原位杂交和免疫组织化学方法观察了大鼠脑损伤后aFGF基因mRNA和蛋白在皮质神经元的表达变化。结果：脑损伤后15min及30min伤侧大脑皮质神经元aFGF mRNA和蛋白表达增加,aFGF mRNA表达于伤手6h达高峰,持续至伤后24h,以后表达逐步降低,于伤后72h恢复至正常水平。神经元aFGF蛋白表达于伤后1h达高峰,持续至伤后3h,伤后24h表达恢复正常。结论：脑损伤后皮质神经元aFGF基因表达增加与神经元抗损伤修复机制有关。     

利用有限元研究骨中显微损伤发展

目的 研究骨组织中矿物晶体与胶原纤维的相互作用对显微损伤扩展方式的影响。方法 在有限元模型的基础之上引入黏性单元,用拉伸-分离理论来模拟离子键、氢键、范德华力等的作用。结合随机场理论以及概率损伤分析方法,研究上述各种相互作用机制对骨组织中显微损伤扩展方式的影响。结果 当矿物晶体与胶原纤维通过离子键相结合时,他们之间的界面难以分离,因此骨组织中容易形成线性裂纹。而对于通过范德华力相结合的骨组织,其界面结合不稳定,因此显微损伤容易向着弥散损伤的方式发展。当矿物晶体与胶原纤维之间是通过氢键而相互作用时,发现在显微损伤积累的初始阶段,其发展方式倾向于线性裂纹,而随着显微损伤的逐渐积累,矿物晶体与胶原纤维之间的作用越来约弱,从而整个显微损伤的发展转变为了扩展损伤。结论 本研究的结果有助于理解骨组织中不同成分间的相互作用机制对骨后屈服变形中能量耗散过程的影响,从而进一步探索骨质疏松症以及老年性骨折的机理。     

基于损伤修复的骨重建数值模拟

目的 研究不同损伤情况下的骨重建行为.方法 提出一种疲劳机制作用下的骨重建模型,通过建立股骨近端三维有限元模型,并结合有限元法,分别模拟3种载荷工况下的骨重建情况,分析股骨近端的力学性能及密度变化.结果 通过增加载荷循环次数,使损伤不断增加.在不同损伤情况下,骨展现出不同的骨重建行为.骨重建作为一种修复机制,在一定范围内可以弥补由于疲劳损伤造成的骨量丢失.结论 提出的损伤自适应重建模型可以模拟不同损伤情况下的骨重建行为,以及由于载荷循环次数过大引起的过载吸收.研究疲劳损伤作用下的骨重建行为,可以为骨折预防以及术后康复治疗提供参考.     

大鼠额叶皮质损害的脑电功率谱和形态学研究

３５只ＳＤ大鼠随机分成三组：正常组、假损害组和损害组。采用机械抽吸法制作大鼠右侧额叶皮质损害模型。对各组大鼠分别进行脑电功率谱分析和比较．结果发现：正常大鼠和假损害大鼠两侧大脑半球δ、θ、α和β频段相对功率值相近似（Ｐ＞０．０５），ＤＴ／ＡＢ值（即δ＋θ／α＋β的绝对功率值之比）也相似（Ｐ＞０．０５），功率谱曲线两侧基本对称。额叶皮质损害大鼠损害侧δ、θ频段相对功率值较健例显著增高（Ｐ＜０．０５），α、β段相对功率值较健侧显著降低（Ｐ＜０．０５）；ＤＴ／ＡＢ值较健侧显著增加（Ｐ＜０．０５），功率谱曲线两侧明显不对称。额叶皮质损害后３天、２周、４用及８周功率谱分析结果无明显不同。形态学观察证实额叶皮质存在缺损区不因时间延长而自行修复．额叶皮质损害出现的脑功能障碍及损害区形态变化均不能自行修复，表明功能效应和形态结果相一致，提示脑电功率谱分析可作为临床和实验研究中评价脑功能的有效手段。     

自体块状皮质骨修复种植区颌骨缺损

背景:自体骨有骨传导、骨诱导及骨生成的特性,同时具有良好的生物相容性,移植后无排斥反应。但关于自体骨块移植后的骨块是完全被吸收替代?还是能保留细胞活性长期存留?仍存在争议。目的:观察皮质骨移植后改建再生过程中的组织学变化。方法:6只健康比格犬麻醉后拔除两侧上颌前磨牙,并去除颊侧宽约10 mm、长约15 mm、厚度约2 mm的骨板,建立上颌骨缺损模型,在双侧下颌骨体颊侧切取相应大小的块状皮质骨并修整边缘。在上颌骨缺损区移植皮质骨块,一侧为单纯块状皮质骨移植,另一侧在块状皮质骨移植同期植入种植体。分别于骨移植后3,6个月取材,进行大体观测和组织学观察,分析移植骨块的吸收率、移植骨中的细胞存活率。实验方案经大连医科大学动物实验伦理委员会批准。结果与结论:①大体观察可见移植骨块体积逐渐缩小,边缘圆钝,与基骨结合稳固;②6个月时全部种植体脱落;硬组织磨片可见移植骨块与基骨间存在新生骨连接;6个月时移植骨骨陷窝孔隙率明显低于3个月,移植骨吸收率显著高于3个月(均P<0.05);③结果说明,块状皮质骨移植后,能够与受植区基骨发生骨结合,其内部的骨细胞部分保持活力,随着愈合期延长,新生骨细胞的比例增加,移植骨块的体积逐渐变小。皮质骨移植同期种植后移植物吸收明显,种植体骨结合不良。     

骨形态计量学观察睾酮对雄性去势大鼠皮质骨的影响

目的通过骨形态计量方法观察雄激素替代疗法对去睾丸大鼠皮质骨代谢的影响。方法30只4月龄SD雄性大鼠,随机分成基础对照组(A组、实验开始时处死),年龄对照组(B组)、去睾丸组(C组)和去睾丸加睾丸酮组(D组),B组和C组生理盐水5ml·kg-1·d-1,D组甲基睾丸酮片1.8mg·kg-1·d-1,灌胃90d。实验结束,处死全部大鼠,取胫骨中段进行不脱钙骨制片,用计算机全自动图象分析系统进行骨组织形态计量学分析。结果去睾丸后皮质骨静态参数如截面总面积、髓腔面积等无明显变化,动态参数骨外膜骨形成降低(P<0.05),内膜骨形成和吸收均有增加趋势。睾酮则使去睾丸大鼠皮质骨的静态参数有增加趋势,促使骨外膜形成增加,减少内膜骨吸收(P<0.05),对内膜骨形成影响不大。结论睾酮补充治疗短期内能对抗去睾丸引起的大鼠皮质骨内外膜的代谢变化,维持正常的皮质骨结构。     

骨皮质切剥术在骨不愈合治疗中的应用

尽管骨愈合的能力很强,但仍有5％～10％的骨折愈合受到干扰,此时骨愈合的生物过程无法对抗骨创伤的局部病理生理学和生物学以及力学变化,导致延迟愈合或骨不连。我自2000年12月至2008年6月采用骨皮质切除术,自体松质骨或骨诱导活性植骨材料治疗骨不愈合患者10例,疗效满意,报道如下。     

骨微损伤、骨重建与代谢性骨病

骨微损伤能启动骨重建,骨重建障碍而导致微损伤积累可引发骨折。扫描电镜、同步加速器射线μ-CT和高分辨磁共振显像是研究骨微损伤的新方法,骨理化构成和年龄对微损伤发生和发展有重要影响,骨细胞在微损伤修复中起重要作用,骨微损伤研究有利于代谢性骨病防治。     

Multiscale imaging of bone microdamage

Abstract

Bone is a structural and hierarchical composite that exhibits remarkable ability to sustain complex mechanical loading and resist fracture. Bone quality encompasses various attributes of bone matrix from the quality of its material components (type-I collagen, mineral and non-collagenous matrix proteins) and cancellous microarchitecture, to the nature and extent of bone microdamage. Microdamage, produced during loading, manifests in multiple forms across the scales of hierarchy in bone and functions to dissipate energy and avert fracture. Microdamage formation is a key determinant of bone quality, and through a range of biological and physical mechanisms, accumulates with age and disease. Accumulated microdamage in bone decreases bone strength and increases bone’s propensity to fracture. Thus, a thorough assessment of microdamage, across the hierarchical levels of bone, is crucial to better understand bone quality and bone fracture. This review article details multiple imaging modalities that have been used to study and characterize microdamage; from bulk staining techniques originally developed by Harold Frost to assess linear microcracks, to atomic force microscopy, a modality that revealed mechanistic insights into the formation diffuse damage at the ultrastructural level in bone. New automated techniques using imaging modalities, such as microcomputed tomography are also presented for a comprehensive overview.     

Detecting microdamage in bone

Fatigue-induced microdamage in bone contributes to stress and fragility fractures and acts as a stimulus for bone remodelling. Detecting such microdamage is difficult as pre-existing microdamage sustained in vivo must be differentiated from artefactual damage incurred during specimen preparation. This was addressed by bulk staining specimens in alcohol-soluble basic fuchsin dye, but cutting and grinding them in an aqueous medium. Nonetheless, some artefactual cracks are partially stained and careful observation under transmitted light, or epifluorescence microscopy, is required. Fuchsin lodges in cracks, but is not site-specific. Cracks are discontinuities in the calcium-rich bone matrix and chelating agents, which bind calcium, can selectively label them. Oxytetracycline, alizarin complexone, calcein, calcein blue and xylenol orange all selectively bind microcracks and, as they fluoresce at different wavelengths and colours, can be used in sequence to label microcrack growth. New agents that only fluoresce when involved in a chelate are currently being developed--fluorescent photoinduced electron transfer (PET) sensors. Such agents enable microdamage to be quantified and crack growth to be measured and are useful histological tools in providing data for modelling the material behaviour of bone. However, a non-invasive method is needed to measure microdamage in patients. Micro-CT is being studied and initial work with iodine dyes linked to a chelating group has shown some promise. In the long term, it is hoped that repeated measurements can be made at critical sites and microdamage accumulation monitored. Quantification of microdamage, together with bone mass measurements, will help in predicting and preventing bone fracture failure in patients with osteoporosis.     

Fracture toughening mechanism of cortical bone: an experimental and numerical approach

In this investigation, the crack propagation mechanisms contributing to the toughness of cortical bone were studied using a combination of experimental and numerical approaches. Compact tension (CT) specimens were prepared from bovine cortical bones to achieve crack propagation in the longitudinal and transverse directions. Stable crack extension experiments were conducted to distinguish the crack growth resistance curves, and virtual multidimensional internal bond (VMIB) modeling was adopted to simulate the fracture responses. Results from experiments indicated that cortical bone exhibited rising resistance curves (R-curves) for crack extension parallel and perpendicular to the bone axis; the transverse fracture toughness was significantly larger, indicating that the fracture properties of cortical bone are substantially anisotropic. Microscopic observations showed that the toughening mechanisms in the longitudinal and transverse directions were different. When the crack grew in the transverse direction, the crack deflected significantly, and crack bifurcations were found at the crack wake, while, in the longitudinal direction, the crack was straight and uncracked ligaments were observed. Numerical simulations also revealed that the fracture resistance in the transverse direction was greater than that in the longitudinal direction.     

Relationships between in vivo microdamage and the remarkable regional material and strain heterogeneity of cortical bone of adult deer, elk, sheep and horse calcanei

Natural loading of the calcanei of deer, elk, sheep and horses produces marked regional differences in prevalent/predominant strain modes: compression in the dorsal cortex, shear in medial-lateral cortices, and tension/shear in the plantar cortex. This consistent non-uniform strain distribution is useful for investigating mechanisms that mediate the development of the remarkable regional material variations of these bones (e.g. collagen orientation, mineralization, remodeling rates and secondary osteon morphotypes, size and population density). Regional differences in strain-mode-specific microdamage prevalence and/or morphology might evoke and sustain the remodeling that produces this material heterogeneity in accordance with local strain characteristics. Adult calcanei from 11 animals of each species (deer, elk, sheep and horses) were transversely sectioned and examined using light and confocal microscopy. With light microscopy, 20 linear microcracks were identified (deer: 10; elk: six; horse: four; sheep: none), and with confocal microscopy substantially more microdamage with typically non-linear morphology was identified (deer: 45; elk: 24; horse: 15; sheep: none). No clear regional patterns of strain-mode-specific microdamage were found in the three species with microdamage. In these species, the highest overall concentrations occurred in the plantar cortex. This might reflect increased susceptibility of microdamage in habitual tension/shear. Absence of detectable microdamage in sheep calcanei may represent the (presumably) relatively greater physical activity of deer, elk and horses. Absence of differences in microdamage prevalence/morphology between dorsal, medial and lateral cortices of these bones, and the general absence of spatial patterns of strain-mode-specific microdamage, might reflect the prior emergence of non-uniform osteon-mediated adaptations that reduce deleterious concentrations of microdamage by the adult stage of bone development.     

Skeletal alterations in hypophysectomized rats: II. A histomorphometric study on tibial cortical bone

Background: Pituitary hormones play an important role in bone growth, modeling, and remodeling. The purpose of this study is to examine the effect of hypophysectomy (HX) on tibial cortical bone with histomorphometry. Methods: Forty-Five female Sprague-Dawiey rats, at 3 months of age, were hypophysectomized or served as intact controls. They were sacrificed at 0, 2, and 5 weeks after the surgery. Cortical bone histomorphometry was performed on double-fluorescent-labeled 30-mcm-thick sections of the tibial shaft. Results: The dry weight and density of tibial diaphysis and the cortical bone area of the tibial shaft in the HX rats were significantly lower (P<0.05) than that of the age-matched intact rats, but did not differ between the HX and basal control rats. The dynamic data show that the bone formation parameters (labeled surface, mineral apposition rate, and bone formation rate) were profoundly decreased (P<0.01) on both the periosteal and endocortical surfaces in the HX rats as compared with the age-matched intact rats at the 2 and 5 weeks. However, the decrease in the labeled surface was much less on the endocortical envelope than on the periosteal envelope in the HX rats. Although no significant change was detected in the medullar size between the HX and age-matched intact rats, the eroded surface on the endocortical surface was greater (P<0.05) in the HX rats than in the intact rats at either time point. Conclusions: Hypophysectomy-suppressed, radial growth-dependent bone gain without a bone loss in the tibial shaft of the young rat. This is associated with decreased modeling-dependent bone formation. A greater eroded surface on the endosteum did not affect the marrow size at 5 weeks after hypophysectomy. © 1995 Wiley-Liss, Inc.     

双膦酸盐药物长期应用对显微损伤和骨力学性能的影响

双膦酸盐类药物作为治疗骨质疏松的一线药物,可以通过降低骨转换来增加骨密度,防止骨折的发生,在临床已经有较长期应用。最近研究表明,双膦酸盐类药物在抑制骨重建的同时,会影响显微损伤的修复,导致显微损伤的积聚和骨质量的下降,进而降低骨的韧性,削弱骨的力学性能。已有临床报告指出,骨质疏松病人使用双膦酸盐后有可能发生非创伤性骨折。本文综述了双膦酸盐类药物对骨显微损伤和骨力学性能的影响。     

Effect of aging on the transverse toughness of human cortical bone: evaluation by R-curves

The age-related deterioration in the quality (e.g., strength and fracture resistance) and quantity (e.g., bone-mineral density) of human bone, together with increased life expectancy, is responsible for increasing incidence of bone fracture in the elderly. The present study describes ex vivo fracture experiments to quantitatively assess the effect of aging on the fracture toughness properties of human cortical bone specifically in the transverse (breaking) orientation. Because bone exhibits rising crack-growth resistance with crack extension, the aging-related transverse toughness is evaluated in terms of resistance-curve (R-curve) behavior, measured for bone taken from a wide range of age groups (25–74 years). Using this approach, both the ex vivo crack-initiation and crack-growth toughness are determined and are found to deteriorate with age; however, the effect is far smaller than that reported for the longitudinal toughness of cortical bone. Whereas the longitudinal crack-growth toughness has been reported to be reduced by almost an order of magnitude for human cortical bone over this age range, the corresponding age-related decrease in transverse toughness is merely ∼14%. Similar to that reported for X-ray irradiated bone, with aging cracks in the transverse direction are subjected to an increasing incidence of crack deflection, principally along the cement lines, but the deflections are smaller and result in a generally less tortuous crack path.     

Quantitative peripheral computed tomodensitometric study of cortical and trabecular bone mass in relation with menopause

Changes in total, cortical and trabecular bone mass were studied using quantitative peripheral computed tomodensitometry on the forearm of 58 normal eugonadal premenopausal women and 116 normal postmenopausal women to evaluate the evolution of bone components with age. In premenopausal women, no changes were seen in any bone component. In postmenopausal women, only trabecular bone mass diminished in the first 5 years after menopause (P < 0.05). It continued to decrease in the next 5 years (P < 0.05), but not later. Cortical bone mass experienced a significant loss 6–10 years after menopause (P < 0.001), and more than 15 years after menopause (P < 0.0005). These results are similar to those obtained with other techniques, and document the differing behavior of the cortical and trabecular bone components with years of menopause.