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。     

Research on plastic embedding combined with tetracycline fluorescence labeling in undecalcified bone tissue

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.     

用Bouin液固定不脱钙制作胎儿指骨整体切片

组织学观察是骨组织研究的重要手段之一[1],由于骨组织致密、坚硬,并含有骨胶等成分,所以要制作一张高质量的切片(和软组织相比)有一定的困难[2].近年来出现的塑料包埋等[3,4]不脱钙骨切片及染色技术尚不够成熟和稳定[2].我们在教学过程中用胎儿指骨的整体切片做为观察软骨内成骨和骨组织组成及细胞结构的标本,制作时采用Bouin液做为固定液,用氯仿做为透明剂,不需脱钙制作出切片的组织和细胞的形态结构完整,染色鲜明、色泽深浅适当.而将皮肤和结缔组织等一起固定包埋制作骨切片的文献极少.现将我们的经验报道如下.     

不脱钙骨组织包埋技术的改进

在骨组织切片上同时进行组织形态学计量与免疫组化有较大难度。这是因为常用的包埋介质石蜡只能用于不含钙或含钙很少的组织，而骨组织富含钙盐，所以必须先脱钙。脱钙的骨组织可做免疫组化与原位杂交，但骨小梁的结构遭到了破坏，不能进行组织形态学计量，从而丢失了骨的生长与钙化方面的信息。塑料包埋的不脱钙骨组织虽能行组织形态学计量，却难以兼顾免疫组化与原位杂交。因此，建立一种可靠的、简便的且同时适应于三者的不脱钙骨组织包埋方法是骨病理学研究的关键。     

用Bouin液固定不脱钙制作胎儿指骨整体切片

组织学观察是骨组织研究的重要手段之一[1],通常由于骨组织致密,坚硬,并含有骨胶等成分,所以要切好一张高质量的切片(和软组织相比),有一定的困难[2].近年来推出了塑料包埋等[3,4]不脱钙骨切片及染色技术,但目前尚不够成熟稳定[2].我们在教学过程中用胎儿指骨的整体切片做为观察软骨内成骨和骨组织组成及细胞结构的标本,我们在制作此标本时,用Bouin液做为固定液,用氯仿做为透明剂,不需脱钙制作出切片的组织和细胞的形态结构完整,染色鲜明、色泽深浅适当,用于教学效果良好.而连着皮肤和结缔组织等一起固定包埋制作骨切片的文献极少.现将我们的经验报道如下(图1～4).     

实验动物骨折模型不脱钙切片的制作

实验动物骨折模型不脱钙切片的制作魏典，于顺禄我们在动物骨折愈合过程的骨计量学研究中，采用了甲基丙烯酸甲丁酯进行了骨标本不脱钙包埋、切片，从而保存了骨原有矿物质及标记的四环素。因而能在荧光显微镜下进行骨组织形态计量学观察与光镜下组织学观察。１材料和方法...     

塑料不脱钙、石蜡包埋、明胶浸泡法对骨组织切片的比较

骨组织内含有大量的钙盐和无机盐,如不脱钙切片,常给骨组织切片和染色造成非常大的困难,直接影响实验结果。作者根据研究的需要,对塑料包埋不脱钙、石蜡包埋、明胶浸泡３种方法切片及染色进行比较,以便找出一种最佳切片方法。１　材料与方法１－１　塑料包埋组　所用材料是我所研究生课题实验材料。新西兰大耳白兔１０只,体重２－２～２－５ｋｇ,♀、♂不限。取双侧尺桡骨１－５ｃｍ保留周围少量肌肉。１０％中性福尔马林固定。梯度乙醇脱水。甲液：甲基丙稀酸甲酯（ＭＭＡ）７５ｍｌ,邻苯二甲酸二丁酯（ＤＢＰ）２５ｍｌ。乙液：Ｍ…     

EXAKT切磨系统在带金属种植体软硬组织切片中的应用

背景：目前常用的Polycut硬组织切片机难以制作皮质骨或含坚硬植入物组织的薄层切片,近年来这些切片多用EXAKT硬组织切磨系统进行制作。 目的：实验采用EXAKT切磨系统制作含金属内植物的软、硬组织薄层切片。 方法：取大鼠股骨远端、小鼠股骨干骨折部、带有钛合金种植体的犬下颌骨和带有钴铬支架的猪冠状动脉标本,经多聚甲醛固定和丙酮脱水后用有机玻璃(甲基丙烯酸甲酯)浸透包埋。组织包埋块用EXAKT系统作厚度约300 μm的切片,然后磨片至厚5~10 μm的切片。不同切片用苏木精-伊红和甲苯胺蓝染色后在光学显微镜下观察形态。 结果与结论：甲苯胺蓝染色显示,大鼠股骨远端骨组织切片中干骺端矿化骨小梁呈浅蓝色、皮质骨结构完整,无碎裂。含带有钛合金种植体的犬下颌骨切片浅蓝色宿主骨与黑色金属种植体密切结合,形成牢固的界面。四环素标记的小鼠股骨干骨折部骨痂切片,在荧光显微镜(紫外光)下显示明显的双层荧光带。苏木精-伊红染色的包裹钴铬合金支架材料的猪冠状动脉组织切片显示,支架材料维持在原位,与血管壁无脱离或游移现象,组织无皱缩或折叠。结果提示,采用EXAKT切磨系统技术能制作出高质量的软、硬组织薄层切片,可清晰显示各种组织结构和人工植入物。     

骨组织不脱钙大切片制作技术

正骨组织制片是常规病理学技术制片中的难点。骨组织富含钙盐和无机盐,必须先进行组织脱钙才能进行石蜡包埋、切片。并且脱钙过程中经常存在脱钙不彻底或过分脱钙的情况,导致无法制片或常规染色不佳。骨组织在制片过程中原有组织形态通常会发生改变,不能准确的评价骨的动态重建过程[1],如新骨的形成、骨样组织的矿化等。常规人体股骨头大标本制片,只是切取病变部位,无法整体观察骨小梁生长的结构、方向性等构筑学参数。为解决上述问题,本     

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.     

Immunohistochemical staining of plastic embedded bone marrow trephine biopsy specimens after microwave heating.

AIMS--To investigate (1) whether adequate immunohistochemical staining can be achieved on sections cut from plastic embedded bone marrow trephine biopsy specimens after microwave heating in citrate buffer; and (2) whether this immunohistochemical staining is comparable with that achieved on routine sections cut from paraffin wax embedded trephine biopsy specimens after decalcification procedures. METHODS--Sixty five consecutive bone marrow trephine biopsy specimens of more than 1 cm in length were divided transversely into two equal parts. One part was processed in paraffin wax followed by decalcification. The other part was embedded in the epoxyresin Polarbed 812 followed by the cutting of 1 micron sections. Both parts underwent immunohistochemical staining by an identical panel of antibodies. With Polarbed 812 plastic embedded sections, microwave heating in citrate buffer was undertaken before the application of antisera. RESULTS--On sections cut from plastic embedded material, immunohistochemical staining was generally satisfactory, easy to interpret and comparable with that achieved with paraffin wax embedded material. Exceptions were antibodies to neutrophil elastase and CD61 where immunostaining was consistently negative on plastic embedded sections. Immunohistochemical staining for CD20 was consistently more reliable on plastic embedded sections. CONCLUSIONS--The results provide evidence that, with few exceptions, satisfactory immunohistochemical staining is possible on plastic embedded bone marrow trephine biopsy specimens after microwave heating in citrate buffer. This, combined with the advantage of superior cellular morphology with semi-thin (1 micron) sections of plastic embedded material, make such embedding procedures the preferred method for the processing of bone marrow trephine biopsy specimens.     

Is it necessary to embed bone marrow biopsies in plastic for haematological diagnosis?

With the increase in the use of bone marrow trephines for diagnosis have come numerous reports that traditional methods of preparation (by decalcification and embedding in paraffin wax) should be replaced by plastic embedding to avoid decalcification. It has been argued that only by this means can the high quality preparations needed for accurate haematopathological diagnosis be achieved. The present study challenges this viewpoint and argues that with a little extra care and attention conventional paraffin embedding techniques can give equally high quality preparations. Sections prepared in this way meet the diagnostic needs of the haematologist, without requiring a separate technique to be established in the pathology laboratory solely for bone marrow trephines.     

Microwaveable Toluidine Blue Stain for Surface Staining of Undecalcified Bone Sections

Abstract

Toluidine blue stain has been widely used for staining undecalcified bone sections, but previous techniques required removal of embedding plastic or surface etching with both alcohol and acid solutions. In this study, solutions of toluidine blue at different pHs were used to stain unetched and acidetched sections. A group of the test staining solutions were heated in an effort to enhance section staining. Acid-etching prior to staining was found to be necessary to achieve bone matrix staining, but methanol etching could be eliminated if staining solutions were heated to 55–60°C. Sections stained with the heated solutions buffered to pH 7.0 or 8.0 had optimum morphology. (The J Histotechnol 17:357, 1994)     

Comparative evaluation of bone marrow aspirate particle smears, imprints and biopsy sections

Comparative evaluation of bone marrow aspirate particle smears, imprints and biopsy sections was done on 30 haematological problems. Core needle biopsy of the bone marrow is a safe and useful procedure. It is a valuable diagnostic aid for measurement of marrow cellularity, metastatic tumours and fibrosis. It should not be taken as a substitute for examination of the marrow by aspiration smear but is a complementary procedure which affords several advantages. Bone marrow biopsy was of maximum utility in myelofibrosis which was diagnosed on biopsy alone. There were three additional cases with normal bone marrow aspiration in which specific diagnosis could only be made from bone marrow biopsy sections. New methodologies i.e. plastic embedding and semi thin sections of undecalcified bone marrow, can be expected to improve the cytological details of tissue obtained by biopsy. Imprint preparations obtained from biopsy can be useful in patients of malignancy but we have found them to be of limited value except in cases of dry tap.     

A novel methodology for imaging new bone formation around bioceramic bone substitutes

In this study, Frost's bulk-staining in combination with confocal laser scanning microscopy (CLSM) was used to image and characterize ground sections of undecalcified rat bone tissue with in situ bioceramic implants. This was addressed by bulk staining specimens in alcohol-soluble basic fuchsin dye. The ground sections were imaged using CLSM in the confocal fluorescence mode. Confocal images revealed that the newly formed bone could be clearly distinguished from bone marrow and cortical bone, as well as from the implant material.     

甲基丙烯酸羟乙酯塑料包埋切片技术在小鼠胎脑组织学分析中的应用

目的 探索塑料包埋切片法在小鼠胎脑组织学分析中的应用。 方法 取胚胎期13.5d（E13.5）小鼠,4% 多聚甲醛4℃固定过夜,分离胎鼠头部,甲基丙烯酸羟乙酯(HEMA)塑料包埋组织并切片;对切片进行HE 染色。 结果 与石蜡包埋相比,采用HEMA塑料包埋的方法进行胎鼠脑组织学分析,形态结构保存较完整、HE染色效果较清晰。 结论 利用塑料包埋技术进行小鼠胎脑组织学分析,优于石蜡包埋方法。     

Undecalcified preparation of bone tissue: Report of technical experience and development of new methods

Summary For reliable quantitative and qualitative analysis of bone specimens undecalcified preparation is essential. The conventional technique for this purpose is embedding in methylmethacrylate. Larger bone specimens, highly sclerotic specimens, cortical bone or bone implants consisting of metals or ceramics require modifications of this technique or completely new methods. We report our experience with the undecalcified preparation of 47700 bone specimens. New techniques such as the cutting of large area sections up to a size of 5×6 cm and grinding procedures for completely artefact-free preparation which are applied in special cases are also described. A new technique of combinded two- and three-dimensional analysis of bone specimens is presented. In our experience these methods are fundamental for morphological investigation of bone.     

How we process trephine biopsy specimens: epoxy resin embedded bone marrow biopsies

Improved cytomorphology of semithin resin sections over paraffin wax embedded sections may be important in diagnostic haematopathology. However, resin embedding can make immunohistochemical antigen detection or DNA isolation for clonal gene rearrangement assays difficult. This review describes the processing of bone marrow biopsies using buffered formaldehyde based fixation and epoxy resin embedding, with or without EDTA decalcification. Traditional semithin resin sections are completely rehydrated after etching in home made sodium methoxide solution. Resin elimination allows high resolution staining of tissue components with common histological stains. Efficient antigen retrieval and the Envision-HRP system permit the immunohistological detection of many antigens of diagnostic relevance, with retention of high quality cytomorphology. Furthermore, DNA can be extracted for clonality analysis. The technique can be completed within a similar time period to that of paraffin wax processing with only approximately 30% increase in cost. This technique has been used for diagnosis in over 4000 bone marrow biopsies over the past 14 years. By meeting traditional and contemporary demands on the haematopathologist, it offers a powerful alternative to paraffin wax processing for diagnosis and research.