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.     

Bone adaptation to load: microdamage as a stimulus for bone remodelling

Mechanical loading in the proximal radius was increased by ulnar osteotomy (Group O), altered by Steinmann pinning (Group P) or unaltered in sham operated controls (Group C) in skeletally mature female sheep, aged 2-4 years. A series of intravenous fluorochromes were given to label bone formation and fuchsin-stained microdamage assessed at intervals of up to 24 weeks. Microcracks were present in all groups and were found in the original cortex near the periosteal surface. No microcracks were found in the new, fibrolamellar bone laid down at periosteal or endosteal surfaces. Mean microcrack length (49 microm, SD 10 microm) did not differ between groups or overtime. Microcrack numerical and surface densities and resorption cavity density peaked in all groups at 6 weeks, consistent with a regional acceleratory phenomenon (RAP), but the peaks were significantly greater in Group O. The density of refilling or secondary osteons peaked at 10 weeks and the mean time required for the formation of an osteon was 7.51 +/- 0.59 weeks. Fatigue-induced microdamage is normally present in bone and is increased due to repetitive loading of the mechanically overloaded radius. The location and timing of microcracks, resorption cavities and secondary osteons are consistent with the activation-resorption-formation remodelling cycle and suggest that microdamage is a stimulus for bone remodelling.     

大鼠皮质骨上机械性显微损伤的修复机制研究

目的研究大鼠皮质骨上植入物造成的机械性显微损伤的修复机制。方法 30只SD大鼠随机分为两组,一组行卵巢切除术,另一组行假手术。3个月后在右侧胫骨行钻孔术,分别于钻孔后1、2、4周后处死,处死前注射四环素和钙黄绿素进行标记。将含有钻孔的骨段(1 cm)应用大块碱性品红染色,甲基丙烯酸甲酯包埋后切成约50μm厚的切片。应用Bioquant图象分析系统对切片进行骨形态计量学分析。结果去卵巢大鼠和假手术大鼠都出现了与显微损伤有关的骨吸收腔,其中去卵巢组的孔隙率和骨吸收腔明显高于假手术组(P<0.05);随着术后时间的延长,吸收腔的数目逐渐增加,各时间点之间的差异有明显的统计学意义(P<0.05)。结论去卵巢大鼠的孔隙率和骨吸收腔数明显高于假手术大鼠,反映了在雌激素缺乏和较多裂纹形成两种因素刺激下,去卵巢大鼠皮质骨内的重建更为活跃,这会降低骨的强度从而增加骨折的危险性。     

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.     

Fluorescence-aided detection of microdamage in compact bone

En bloc staining with basic fuchsin is an established method for demonstrating microdamage in bone. Using transmitted light microscopy, variations in light intensity, depth of focus and magnification are necessary to distinguish fully-stained microcracks generated in vivo, from partially-stained or unstained artefactual cracks due to cutting and machining. This process is both difficult and time-consuming. In this study, 2 methods were used to examine fuchsin-stained microcracks in human rib sections, transmitted light and epifluorescence microscopy. No differences were found in crack number, density or length between the 2 methods indicating comparable accuracy. Using green epifluorescence, only microcracks containing fuchsin fluoresced orange against the darkfield background, enabling unstained, artefactual cracks to be screened out. Under UV epifluorescence, microcracks stained through the full 100 μm depth of the section fluoresced purple. Partially-stained artefactual cracks failed to fluoresce and were screened out. Epifluorescence is a simple, rapid and accurate screening method for differentiating fully-stained from artefactual microcracks in bone.     

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。     

Microstructural changes in cartilage and bone related to repetitive overloading in an equine athlete model

The palmar aspect of the third metacarpal (MC3) condyle of equine athletes is known to be subjected to repetitive overloading that can lead to the accumulation of joint tissue damage, degeneration, and stress fractures, some of which result in catastrophic failure. However, there is still a need to understand at a detailed microstructural level how this damage progresses in the context of the wider joint tissue complex, i.e. the articular surface, the hyaline and calcified cartilage, and the subchondral bone. MC3 bones from non‐fractured joints were obtained from the right forelimbs of 16 Thoroughbred racehorses varying in age between 3 and 8 years, with documented histories of active race training. Detailed microstructural analysis of two clinically important sites, the parasagittal grooves and the mid‐condylar regions, identified extensive levels of microdamage in the calcified cartilage and subchondral bone concealed beneath outwardly intact hyaline cartilage. The study shows a progression in microdamage severity, commencing with mild hard‐tissue microcracking in younger animals and escalating to severe subchondral bone collapse and lesion formation in the hyaline cartilage with increasing age and thus athletic activity. The presence of a clearly distinguishable fibrous tissue layer at the articular surface immediately above sites of severe subchondral collapse suggested a limited reparative response in the hyaline cartilage.     

骨微结构与微损伤检测方法的研究与进展

BACKGROUND: Bone fragility and poor bone quality due to osteoporosis are a major and increasing concern. Bone microarchitecture and microdamage, the important factors of bone quality, their detection technology and instrument have experienced a long development process.     

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。     

Microcracks in compact bone: a three-dimensional view

Microcracks have been implicated in the loss of bone quality for osteoporosis. In order to detect and monitor their growth, and to use these data to predict fractures, it is essential to obtain quantitative data regarding their shape in three dimensions. Beam-shaped bone samples from sheep radii were prepared and stained with fluorochrome dyes and tested in cyclical fatigue under four-point bending in a servo-hydraulic fatigue-testing machine. Samples were tested at a frequency of 30 Hz under load control at a stress range of 100 MPa. Holes were drilled into bone samples and used as reference points for reconstructions. A series of thin longitudinal sections were cut using a sledge macrotome. A two-dimensional image of each section was examined using an epifluorescence microscope and images transferred to a PC via a CCD low-light colour video camera. A three-dimensional image of each microcrack was reconstructed using computer software, and its dimensions measured. Cracks were elliptical in shape, longer in the longitudinal direction and with a mean aspect ratio of 5.5 +/- 1.05. The mean (+/- SD) length and width of labelled microcracks were 488 +/- 151 and 88 +/- 21 microm, respectively.     

Osteonal crack barriers in ovine compact bone

Bone is an anisotropic structure which can be compared to a composite material. Discontinuities within its microstructure may provide stress concentration sites for crack initiation, but act as a barrier to its propagation. This study looks specifically at the relationship between crack length and propagation in compact bone. Beam-shaped bone samples from sheep radii were prepared and stained with fluorochrome dyes and tested in cyclic fatigue under four-point bending in an INSTRON 1341 servo-hydraulic fatigue-testing machine. Samples were tested at a frequency of 30 Hz and stress range of 100 MPa under load control. Specimens were sectioned transversely using a diamond saw, slides prepared and examined using epifluorescence microscopy. Cracks in transverse sections were classified in terms of their location relative to cement lines surrounding secondary osteons. Mean crack length, crack numerical density and crack surface density were examined. Short microcracks (100 microm or less) were stopped at the cement lines surrounding osteons, microcracks of intermediate length (100-300 microm) were deflected as they hit the cement line, and microcracks that were able to penetrate through cement lines were longer (> 400 microm). These data show that bone microstructure allows the initiation of microcracks but acts as a barrier to crack propagation.     

Morphologic changes associated with functional adaptation of the navicular bone of horses

Failure of functional adaptation to protect the skeleton from damage is common and is often associated with targeted remodeling of bone microdamage. Horses provide a suitable model for studying loading-related skeletal disease because horses are physically active, their exercise is usually regulated, and adaptive failure of various skeletal sites is common. We performed a histologic study of the navicular bone of three groups of horses: (1) young racing Thoroughbreds (n = 10); (2) young unshod ponies (n = 10); and (3) older horses with navicular syndrome (n = 6). Navicular syndrome is a painful condition that is a common cause of lameness and is associated with extensive remodeling of the navicular bone; a sesamoid bone located within the hoof which articulates with the second and third phalanges dorsally. The following variables were quantified: volumetric bone mineral density; cortical thickness (Ct.Th); bone volume fraction, microcrack surface density; density of osteocytes and empty lacunae; and resorption space density. Birefringence of bone collagen was also determined using circularly polarized light microscopy and disruption of the lacunocanalicular network was examined using confocal microscopy. Remodeling of the navicular bone resulted in formation of transverse secondary osteons orientated in a lateral to medial direction; bone collagen was similarly orientated. In horses with navicular syndrome, remodeling often led to the formation of intracortical cysts and development of multiple tidemarks at the articular surface. These changes were associated with high microcrack surface density, low bone volume fraction, low density of osteocytes, and poor osteocyte connectivity. Empty lacunae were increased in Thoroughbreds. Resorption space density was not increased in horses with navicular syndrome. Taken together, these data suggest that the navicular bone may experience habitual bending across the sagittal plane. Consequences of cumulative cyclic loading in horses with navicular syndrome include arthritic degeneration of adjacent joints and adaptive failure of the navicular bone, with accumulation of microdamage and associated low bone mass, poor osteocyte connectivity, and low osteocyte density, but not formation of greater numbers of resorption spaces.     

Non-destructive characterization of microdamage in cortical bone using low field pulsed NMR

The microcracking and damage accumulation process in human cortical bone was characterized by performing cyclic loading under four-point bending at ambient temperature. A non-destructive nuclear magnetic resonance (NMR) spin-spin (T(2)) relaxation technique was applied to quantify the apparent changes in bone porosity as a function of cyclic loading and prior damage accumulation, first to unloaded cortical bone to quantify the initial porosity and then to fatigued cortical bone that was subjected to cyclic loading to various levels of modulus degradation and microdamage in the form of microcracks. The NMR T(2) relaxation time and amplitude data of the fatigued bone were compared against the undamaged state. The difference in the T(2) relaxation time data was taken as a measure of the increase in pore size, bone porosity or microcrack density due to microdamage induced by cyclic loading. A procedure was developed to deduce the number and size distributions of microcracks formed in cortical bone. Serial sectioning of the fatigued bone showed the formation of microcracks along the cement lines or within the interstitial tissue. The results on the evolution of microdamage derived from NMR measurements were verified by independent experimental measurements of microcrack density using histological characterization techniques. The size distribution and population of the microcracks were then utilized in conjunction with an analytical model to predict the degradation of the elastic modulus of cortical bone as a function of damage accumulation.     

兔骨髓基质细胞诱导成骨细胞复合同种异体生物衍生骨实验研究

目的探讨体外培养的骨髓基质细胞与自体来源的生物衍生骨复合后的生长特性，为进一步研究骨髓基质细胞作为种子细胞，以及探索一种良好的支架材料提供实验依据。方法分离纯化兔骨髓基质细胞并诱导成成骨细胞后与同种异体来源的生物衍生骨复合后体外培养，并在相差显微镜和扫描电镜下观察其生长情况。结果骨髓基质细胞在生物衍生骨上贴附生长、增殖良好。结论骨髓基质细胞可作为骨组织工程的理想种子细胞，与生物衍生骨复合后可作为骨组织工程的载体。     

Characterization of eight different tetracyclines: advances in fluorescence bone labeling

Polychrome sequential labeling with fluorochromes is a standard technique for the investigation of bone formation and regeneration processes in vivo. However, for human application, only tetracycline and its derivates are approved as fluorochromes. Therefore, the aim of this study was to determine the fluorescence characteristics of the different tetracycline derivates to assess the feasibility of sequential in vivo bone labeling using distinguishable fluorochromes. Eight different tetracycline derivates were injected subcutaneously into growing rats as a single dose or sequentially in different combinations. After preparation of resin-embedded undecalcified bone sections, the fluorescence properties of the tetracycline derivates in bone were analyzed using conventional fluorescence microscopy, spectral image analysis and confocal laser scanning microscopy. Each tetracycline derivate exhibited a characteristic fluorescence spectrum, but the differences between them were small. Chlortetracycline could be discriminated reliably from all other derivates and could therefore be combined with any other tetracycline derivate for reliably distinguishable double labeling. Tetracycline itself exhibited the brightest fluorescence of all the investigated derivates. Interestingly, in conventional microscopy the same tetracycline derivative can appear in different colours to the human eye, even if spectral analysis confirmed identical emission peaks. In conclusion, the data suggest that fluorescence double labeling of bone is feasible using appropriate tetracycline derivates in combination with spectral imaging modalities.     

烧骨DNA提取与检测技术的实验研究

目的　探索烧骨DNA提取与检测方法。 方法　常压下以200℃分别对30例成人股骨焚烧1h后,生物冷冻研磨,EDTA脱钙,改良酚－氯仿提取DNA,光度计检测DNA浓度和纯度;5色荧光标记,PCR复合扩增,基因分析仪毛细管电泳,软件收集数据和分析。 结果　改良酚-氯仿法提取烧骨DNA浓度为(28.5±1.7 )ng/μl,16个基因位点基本齐全,相对荧光单位（RFU）＞500;传统酚-氯仿法提取烧骨DNA浓度为(4.7±0.8 )ng/μl,基因位点检出不全,RFU＜500。 结论　改良酚-氯仿法、PCR复合扩增、基因分析能对烧骨DNA进行有效提取和检测,但操作时要严格规程。     

Joint anatomy,design, and arthroses: Insights of the Utah paradigm

This model of joint design argues 1) that excessive fatigue damage (MDx) in articular cartilage collagen can be the “final cause” of an arthrosis; 2) that known responses of a growing joint's anatomy and geometry, and modeling and maintenance activities, to mechanical loads minimize that cause and thus arthroses; 3) and many biomechanical, biochemical, cell‐biologic, genetic and traumatic “first causes” of arthroses could lead to that final cause. The model depends partly on the following facts (marked by a single asterisk) and ideas (marked by a double asterisk). A) During growth a joint's total loads can increase over 20 times* without causing an arthrosis,* yet in adults an equal loading increase would cause one.* B) Fatigue damage (MDx) occurs in joint tissues,* larger strains increase it,* and minimizing strains reduces it.* C) Bone can repair amounts of MDx below an “MDx threshold,”* but larger amounts can escape repair and accumulate.* The model assumes articular cartilage has similar features.** D) Bone modeling makes bones strong enough to keep their strains below bone's MDx threshold and minimize MDx.* Chondral modeling shapes and sizes joints during growth;* that would keep articular cartilage strains below the chondral MDx threshold** to minimize chondral MDx and arthroses.** Normal chondral modeling nearly stops in adults,* which might explain point A above.** E) Throughout life maintenance activities preserve optimal physical, chemical and biologic properties of a joint's tissues.* To past emphases on the biochemical, genetic, cellular and molecular biologic features of adult joint physiology, this model adds organ‐level, tissue‐level and vital‐biomechanical features of growing joints that invite study and understanding at lower levels of biologic organization. Anat Rec 255:162–174, 1999. © 1999 Wiley‐Liss, Inc.     

骨水泥成形术治疗骨转移瘤的临床观察

目的 观察应用微创技术治疗骨转移瘤引起疼痛的效果.方法 17例骨转移患者,59处骨转移病灶(55处在椎体,3处在四肢骨,1处在骨盆),其中男性5例,女性12例,年龄30～78岁,平均年龄58.9岁,影像学检查均证实有骨转移瘤(X线片、CT及MRI).临床伴有明显持续性骨痛,症状持续时间为2～18个月,平均5.7个月.17例患者59处骨转移灶均给予骨水泥成形术治疗.观察治疗前后患者的疼痛强度的变化情况、镇痛药使用评分并行SF-12健康状况调查.结果 17例59处病灶手术顺利完成.患者的疼痛评分由治疗前的5.0～9.5(平均7.9±1.2)减少为治疗后的3.1～6.8(平均5.1±1.0)(P＜0.01).患者的止痛药使用评分由治疗前的3～4(平均3.6±0.6),减少为治疗后的1～3(平均2.4±1.1)(P＜0.01).SF-12健康调查评分治疗前为-17～-33,平均-30.00±4.03,治疗后为-18～-33,平均-26.00±3.94(P＜0.05).术后随访6～17个月,平均12.3个月,患者疼痛均较术前减轻,17例患者中1例在治疗期间出现1个椎体新发的压缩性骨折.结论 应用骨水泥成形术治疗骨转移瘤可有效地缓解疼痛,改善患者后期生活质量.