塑料包埋结合四环素荧光标记制作不脱钙骨组织切片的实验研究

目的探讨塑料包埋技术结合四环素荧光标记制作不脱钙骨组织切片的关键步骤及应用。方法选取四环素荧光标记的兔股骨进行塑料包埋,Leica SP 1600切片机切片,荧光显微镜观察后用苦味酸品红染色,并与常规石蜡包埋切片相比较。结果四环素荧光标记不脱钙骨组织经塑料包埋技术处理后,可清楚地观察类骨质形成、植入体及周围骨组织的整合程度,与常规石蜡包埋相比,染色层次分明,组织移位形变小。结论应用塑料包埋技术制作四环素荧光标记不脱钙骨组织切片,有利于行骨形态计量分析以及植入体与骨整合的研究。     

Enzyme and immunohistochemistry on undecalcified bone and bone marrow biopsies after embedding in plastic: A new embedding method for routine application

Summary A simplified method of low temperature methyl and butyl methacrylate embedding (up –20° to –15° C) is demonstrated using a proper redox system of benzoyl peroxide and aromatic amine. This method combines the morphological superiority of plastic-embedded bone tissue and bone marrow sections with the advantages of specific enzyme histochemical and immunochemical markers. The method permits good preservation of morphological details, the survival of antigenic determinants and the retention of enzyme activities. The specimens were fixed in 1.6% formaldehyde and 5% sucrose in 0.02 M phosphate buffer at pH 7.4, washed in 0.02 M phosphate buffer and 5% sucrose, dehydrated with acetone and impregnated with monomers of embedding medium. All these steps were carried out at +4° C. The method presented is especially suitable for enzyme histological and immunohistological diagnosis of primary and secondary bone tumours, soft tissue tumours, as well as myelo- and lymphoproliferative disorders of bone marrow biopsies. Examples are demonstrated with mono- and polyclonal antibodies and reaction products of hydrolytic enzymes.Dedicated to Prof. Dr. Gerhard Seifert on the occasion of his 70th birthday     

塑料包埋技术在骨组织研究中的应用

目的 研究塑料包埋技术制作的不脱钙硬组织切片,确定制作的关键步骤及用于骨组织研究的优点.方法 选取狗胫骨节段或带有金属种植体的骨材料塑料包埋,LeicaSP1600切片机(德国)切片,用苦味酸品红染色观察,并与常规石蜡包埋相比较.结果 不脱钙骨组织经塑料包埋技术处理后,可清楚地观察类骨质形成、多孔材料及种植体周围骨组织的整合程度.与常规石蜡包埋相比,染色层次分明,组织移位形变小.结论 应用规范塑料包埋技术制作不脱钙硬组织切片,更有利于行骨形态计量分析以及材料与骨整合的研究.     

塑料包埋技术在骨组织研究中的应用

目的研究塑料包埋技术制作的不脱钙硬组织切片,确定制作的关键步骤及用于骨组织研究的优点。方法选取狗胫骨节段或带有金属种植体的骨材料塑料包埋,LeicaSP1600切片机（德国）切片,用苦味酸品红染色观察,并与常规石蜡包埋相比较。结果不脱钙骨组织经塑料包埋技术处理后,可清楚地观察类骨质形成、多孔材料及种植体周围骨组织的整合程度。与常规石蜡包埋相比,染色层次分明,组织移位形变小。结论应用规范塑料包埋技术制作不脱钙硬组织切片,更有利于行骨形态计量分析以及材料与骨整合的研究。     

Single-dose tetracycline labeling for bone histomorphometry

A single oral dose of tetracycline deposits in bone and is readily identified with fluorescence microscopy. Two such time-spaced labels can be used to determine bone dynamic features. This is as accurate as conventional three-day labeling periods. The simplicity, improved compliance, and substantial reduction in time it takes to prepare a patient for bone biopsy all appear to be advantageous when compared with current recommendations.     

Plastic embedding procedure for 15 microns sections of large undecalcified tissue blocks.

A method is described that produces sections (15 microns) of large specimens (with dimensions up to 140 x 100 x 200 mm). They are suitable for comparison to CT images of that specimen and can be used as a basis for three-dimensional reconstructions. Frozen unfixed parts of the human body are embedded in polyurethane and scanned with CT. Undecalcified slices, 6 mm thick, of these specimens are cut with a bandsaw. These slices are successfully embedded in polyester resin by means of fixation, dehydration, and subsequent impregnation with this resin. Sections of 15 microns are obtained by cutting the trimmed and sandpapered polyester blocks with an LKB multirange microtome. They are collected on adhesive tape and stained according to Weigert-Azan, sealed between adhesive acetate sheets and photographed on colour slide film. As an example photographs of sections of the human elbow and lumbar spine region are presented.     

Effect of promethazine on double tetracycline bone labeling in old mice

Twenty-five-month-old female B6AF1 mice were injected intramuscularly (i.m.) with declomycin (75 mg/kg) 50 days and 2 days, or 20 days and 2 days, before sacrifice, and cross-sections of their femoral shafts were examined quantitatively for areas of tetracycline fluorescence. Two groups of mice received promethazine HCl in their drinking water (12 mg/dl) for 1 year, and the control groups were untreated. It was found that: the number of discrete areas of cortical and endosteal tetracycline deposition was increased slightly in the groups given promethazine; the length of the endosteal and cortical tetracycline deposits were 2-3 times greater, respectively, in the promethazine treated groups; and the distance between the cortical tetracycline deposits and the endosteum was 2.5 times greater in the promethazine groups. These results support the view that net bone deposition in osteopenic old mice is enhanced by promethazine.     

Plastic embedding procedure for 15 μm sections of large undecalcified tissue blocks

A method is described that produces sections (15 μm) of large specimens (with dimensions up to 140 × 100 × 200 mm). They are suitable for comparison to CT images of that specimen and can be used as a basis for three dimensional reconstructions. Frozen unfixed parts of the human body are embedded in polyurethane and scanned with CT. Undecalcified slices, 6 mm thick, of these specimens are cut with a bandsaw. These slices are successfully embedded in polyester resin by means of fixation, dehydration, and subsequent impregnation with this resin. Sections of 15 μm are obtained by cutting the trimmed and sandpapered polyester blocks with an LKB multirange microtome. They are collected on adhesive tape and stained according to Weigert-Azan, sealed between adhesive acetate sheets and photographed on colour slide film. As an example photographs of sections of the human elbow and lumbar spine region are presented. © 1992 Wiley-Liss, Inc.     

墨汁血管灌注和四环素荧光标记评价异体移植骨板活化状态

目的 探讨采用血管墨汁灌注和四环素荧光标记评价异体移植骨板活化状态的可行性。方法12只山羊右侧股骨移植深低温冷冻同种异体皮质骨板,分别在术后3、6、12、24周处死动物取材,通过血清骨钙素检测,大体观察、X线照片、移植骨板组织学检查、血管墨汁灌注和四环素荧光标记,对移植骨板再血管化以及成骨活性进行评价。结果术后3周血清骨钙素开始升高,12周达到最高水平,与术前比较,血清骨钙素差别有极显著性统计学意义(P<0.01)。术后3、6、12、24周移植骨板墨汁灌注血管面积比率和四环素荧光标记积分光密度值(IOD)升高,与术前比较,血管面积比率和四环素荧光标记积分光密度值差别有极显著性统计学意义(P<0.01),术后12周血管面积比率和四环素荧光标记累积光密度值达到顶峰,与术后3、6、24周比较(P<0.01)。结论深低温冷冻同种异体皮质骨板移植具有较强生物学活性,术后12周移植骨板完全再血管化,24周完成爬行替代和塑形改建。血管墨汁灌注和四环素荧光标记是判定移植骨板活化状态较好的方法之一。     

墨汁血管灌注和四环素荧光标记评价异体移植骨板活化状态

目的探讨采用血管墨汁灌注和四环素荧光标记评价异体移植骨板活化状态的可行性. 方法 12只山羊右侧股骨移植深低温冷冻同种异体皮质骨板,分别在术后3、6、12、24周处死动物取材,通过血清骨钙素检测,大体观察、X线照片、移植骨板组织学检查、血管墨汁灌注和四环素荧光标记,对移植骨板再血管化以及成骨活性进行评价.结果术后3周血清骨钙素开始升高,12周达到最高水平,与术前比较,血清骨钙素差别有极显著性统计学意义(P<0.01).术后3、6、12、24周移植骨板墨汁灌注血管面积比率和四环素荧光标记积分光密度值(IOD)升高,与术前比较,血管面积比率和四环素荧光标记积分光密度值差别有极显著性统计学意义(P<0.01),术后12周血管面积比率和四环素荧光标记累积光密度值达到顶峰,与术后3、6、24周比较(P<0.01).结论深低温冷冻同种异体皮质骨板移植具有较强生物学活性,术后12周移植骨板完全再血管化,24周完成爬行替代和塑形改建.血管墨汁灌注和四环素荧光标记是判定移植骨板活化状态较好的方法之一.     

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.     

Plastic embedding in routine histology I: Preparation of semi-thin sections of undecalcified marrow cores

The preparation of sections of bone marrow cores in a routine histology laboratory requires decalcification and paraffin embedding, which produces shrinkage and considerable loss of cellular detail. This may be avoided by using plastic embedding procedures. This report describes a simplified routine procedure for using methylmethacrylate as a plastic embedding medium for the preparation of semi-thin sections of undecalcified bone marrow cores. A modification of the May-Grunwald-Giemsa stain is also given which provides good colour differentiation of various haematopoietic cells in the marrow. The method is simple, reproducible, requires no expensive equipment, and is suitable for routine processing of bone marrow biopsy cores in any histopathology laboratory.     

Simultaneous presentation of soft tissue and bone tissue in computed tomography with combined window

OBJECTIVES: To examine whether in computed tomography (CT) soft tissue and bone tissue can be simultaneously presented with a combined window without loss of diagnostic information compared to the separate presentations with soft tissue and bone tissue windows. MATERIALS AND METHODS: Forty-seven CT examinations from different patients with pathological changes at the soft tissue or bone tissue after an accident or due to malignant tumour were evaluated. The CT data were transformed into grey level data with three different windows: (1) soft tissue window; (2) bone tissue window to show spongiosa and bone cortex; and (3) special window to show ethmoidal sinus and mastoidal cells. The images were then weighted with a weighting factor of 2, 3 and 1, respectively, resulting in one image with combined window. This image was compared with the conventional soft tissue and bone tissue images. RESULTS: All diagnostic information could be obtained and anatomical details be recognized on the image with combined window. In some cases soft tissue structures could be delineated from each other or from adjacent bone better on this image than on the soft tissue image. CONCLUSIONS: Combined window could enable a reduction of film consumption or digital storage because soft tissue and bone tissue are presented on the same image and not separately. The risk for overlooking important pathological changes might be reduced as both tissues are always presented.     

骨组织工程的研究进展

目的：了解组织工程骨的相关概念及其特点,以及在组织工程骨研究中种子细胞、支架材料、生长因子、组织工程骨的构建及其应用的最新研究进展。方法：查阅相关中外文献,了解组织工程骨最新的发展动态及临床应用进展。结果：组织工程骨对骨缺损的修复是积极有效的。结论：随着研究的深入,骨组织工程技术最终会解决骨缺损的治疗这一骨外科领域的难题。     

Growth and turnover of rat otoconia as revealed by labeling with tetracycline

Background: The formation process of otoconia remains controversial, and the turnover rate of mammalian otoconia has not been determined. Methods: Tetracycline was administered as a tracer for calcium, and the growth and turnover of rat otoconia were examined by fluoresence microscopy. Results: Exposure of rats to tetracycline from 15.5 gestational day to 3-day postpartum resulted in incorporation of the drug into central portions of all otoconia, in both maculae sacculi and utriculi. When postnatal rats were injected subcutaneously with tetracycline, uptake of the drug into otoconia depended on the age of rats at injection. Apparent fluorescence was emitted from the periphery of all otoconia when tetracycline was injected into 7-day-old or younger rats. However, very little or no fluorescence was observed when this reagent was administered 10 days after birth. No fluorescence was detected when rats of 12 days of age or older were given this antibiotic. Otoconia that had been labeled with tetracycline during gestation were monitored during subsequent development, and it was found that all otoconia retained labelling in their central portions for at least 12 months. No otoconia that were not labeled with tetracycline were found. Conclusions: These findings indicate that: (1) all otoconia grow synchronously during late gestation and the neonatal period (up to about 10 days after birth) by accretion, (2) no new otoconia are formed subsequently, and (3) essentially no turnover of otoconia occurs and probably no turnover of calcium takes place under normal conditions once otoconia have formed. © 1995 Wiley-Liss, Inc.