带植入物病理组织标本切磨片制作技术探讨

硬组织病理切磨技术主要是针对骨组织、带植入物的骨组织、埋置有坚硬植入物的其他组织标本,或在动物试验阶段进行了亲骨荧光素标记的不能进行脱钙的骨组织,通过脱水、浸润、包埋处理,由硬组织切磨系统完成的组织病理切片制作的技术。有文献报道骨组织及埋置坚硬植入物的组织进行病理组织切片制作,通常利用EXAKT硬组织切磨系统[1-3]。硬组织病理切磨技术最显著的特点是不破坏组织中的植入物,且保持了组织与植入物之间原有的组织结构形态,这对研究组织内植入物的长入情况具有重要意义。本文结合本实验室日常硬组织制片工作经验,分析硬组织病理切片制作的每个处理环节,以期能为其他技术人员提供参考。     

基于硬组织切片染色的颈椎钩椎关节的解剖研究

目的利用硬组织切片技术、苏木精—伊红染色及甲苯胺蓝染色方法对成人钩椎关节的组织形态及各层结构进行观察,并对钩椎关节的解剖结构进行探讨。方法选取成年男性全颈椎标本,截取C₂~C₆钩椎关节进行福尔马林固定,脱水,于光固化机中包埋聚合。利用硬组织切片机对标本进行切片处理,磨片机对标本进行磨片处理,并使用苏木精—伊红及甲苯胺蓝进行染色。观察染色后钩椎关节组织形态及结构特点。结果苏木精—伊红染色的钩椎关节骨切片可清晰见到椎体及椎间盘被钙盐沉积分开,上下椎体切面可见粉染骨小梁,骨小梁间可见骨髓组织。甲苯胺蓝染色的钩椎关节骨切片可见成骨细胞和破骨细胞的各种骨组织形态,椎体切面可见体积、宽度、分布均正常的蓝色骨小梁。结论硬组织切片技术可以较完整保留钩椎关节的固有组织形态,经苏木精—伊红染色、甲苯胺蓝染色均可清晰观察到钩椎关节各层结构及形态特征。     

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.     

针对组织工程多孔生物陶瓷的组织学技术优化探讨

目的 改进硬组织切片技术以适应生物陶瓷材料在骨组织工程中的研究.方法 探索硬组织切片的厚度、漂片温度、裱片方法、烤片温度和时间的最佳组合,针对阳离子防脱载玻片的使用条件进行反复比较,通过改进操作流程中的关键技术和需避免的问题,摸索出阳离子载玻片在硬组织切片裱片中的最佳应用条件,克服了硬组织切片制作技术中标本易破碎、切片易脱落及染色时染料容易吸附的缺点.结果 通过技术探索与改进,生物陶瓷支架材料体内植入后的类骨修复体标本的硬组织切片能保持其杂化后的组织结构与比较完整的材料结构,可进行Masson三色染色、苏木精-伊红(HE)及甲苯胺蓝染色.染色后镜下观察显示支架内杂化生长的组织结构完整、细胞形态清晰、切片质量好、生物陶瓷支架脱片少.荧光显微镜可观察到类骨修复体钙沉积现象完整.结论 改善了传统硬组织切片技术处理生物陶瓷材料时易于破坏组织-材料结构的缺点.改进的硬组织切片技术适应生物陶瓷材料在骨组织工程领域研究.     

生长因子复合支架材料在即刻种植周围骨缺损中的作用

背景:研究发现神经生长因子在骨的愈合过程中发挥重要作用,但关于生长因子复合支架材料在犬即刻种植周围骨缺损中作用的研究不多。目的:分析神经生长因子复合支架材料在犬即刻种植周围骨缺损中的作用。方法:制备神经生长因子复合锶磷灰石支架材料,建立犬下颌骨缺损动物模型,将犬下颌骨缺损种植体分为实验组、阳性对照组和空白对照组,实验组植入神经生长因子复合锶磷灰石;阳性对照组植入锶磷灰石;空白对照组不植入任何材料。观察3组犬下颌骨种植体周围骨缺损的CT三维图像重建情况和苏木精-伊红染色情况。结果与结论:1空白对照组犬骨缺损中央没有新骨生成,周围有少量新生骨生成;阳性对照组和实验组犬骨缺损处骨小梁从中间向四周扩散形成骨修复;2实验组犬下颌骨种植体周围骨小梁的骨密度、数量、厚度、种植体-骨结合率均高于阳性对照组和空白对照组(P0.05),阳性对照组4项指标高于空白对照组(P0.05);实验组犬下颌骨种植体周围骨小梁分离度低于阳性对照组和空白对照组(P0.05),阳性对照组低于空白对照组(P0.05);3苏木精-伊红染色显示,实验组犬下颌骨种植体支架网孔中有大量新骨生成,骨小梁排列整齐,周围骨质和新生组织连接紧密;阳性对照组犬下颌骨可见新生类骨质和骨小梁,以及少量死骨碎片;空白对照组骨缺损处有部分新骨生成,骨小梁排列不整齐,周围骨质和新生组织连接不紧密;4结果表明,神经生长因子复合支架材料能够促进犬即刻种植周围骨缺损的骨生成,是一种比较好的骨修复材料。     

电镜半薄切片应用于光镜研究的探讨

电镜半薄切片通常用于电镜超薄切片前的定位观察 ,我们在定位观察时发现 ,由于切片薄 ,结构显示清晰 ,可用于光镜研究 ,其观察结果优于石蜡切片。多年来 ,我们已将电镜半薄切片应用于实验研究和研究生课题实验。方法 :取材后将组织修为 1立方毫米左右 ,常规电镜样品处理 ,环氧树脂 6 18包埋 ,修块 ,L KBV型超薄切片机切片 ,切片厚度 1微米 ,脱树脂后 ,苏木精染色 ,H· E或甲苯胺兰染色 ,37℃温箱烤干 ,封片 ,观察。结果 :苏木精染色显示 ,细胞核呈紫兰色 ,胞浆淡兰色 ;H·E染色显示 ,细胞核为紫兰色 ,细胞质淡粉红色 ,但伊红染色较石蜡…     

膝关节腔内培养骨髓间充质干细胞复合脱钙骨的组织工程软骨

背景：如何更好地以组织工程学方法修复关节软骨缺损并达到良好的远期疗效目前尚无公识。 目的：创新性地在膝关节腔内培养兔骨髓间充质干细胞复合同种异体脱钙骨的组织工程软骨。  方法：采用全骨髓贴壁筛选法分离培养兔骨髓间充质干细胞,DMEM/F12完全培养基培养,成软骨诱导条件培养基诱导分化。取同种异体兔的髂骨和椎体骨制作成脱钙骨支架,诱导后的骨髓间充质干细胞种植于脱钙骨支架上,培养1 d后将细胞-支架复合物用筋膜包裹置于兔左膝关节腔内培养,单纯脱钙骨支架筋膜包裹置入右膝关节腔。于培养第4,8,12周分别取材,行大体观察并制成石蜡切片,采用苏木精-伊红染色、甲苯胺蓝染色,Ⅱ型胶原免疫组化染色方法进行组织学观察。 结果与结论：培养4,8周,细胞-支架组标本Ⅱ型胶原免疫组化的平均吸光度值(A)分别为0.263±0.031,0.340±0.052,单纯支架组标本分别为0.147±0.027,0.165±0.030,两组比较差异有显著性意义(P < 0.05);培养12周细胞-支架组标本Ⅱ型胶原免疫组化A值平均为0.362±0.037,标本类似正常软骨外观,Ⅱ型胶原免疫组化反应呈阳性;而单纯支架组脱钙骨支架降解。培养12周细胞-支架组苏木精-伊红染色结果显示细胞数量多,脱钙骨支架基本被吸收;而甲苯胺蓝染色结果显示有被染成紫红色的异染性基质形成。结果提示兔骨髓间充质干细胞复合同种异体脱钙骨可在兔膝关节腔内培养出组织工程软骨。     

犬平滑肌样细胞和胶原包埋PGA支架组织的相容性

目的 研究骨髓间充质干细胞所诱导的犬平滑肌样细胞和胶原包埋聚羟基乙酸（PGA）支架的组织相容性。方法 胶原包埋PGA构建复合支架,犬骨髓间充质干细胞诱导血管平滑肌样细胞,评价组织相容性。结果 HE染色见胶原包埋PGA组有平滑肌样细胞生长;甲苯胺蓝染色见平滑肌样细胞被染成浅蓝色,胶原包埋PGA组较单纯PGA组明显增多;电镜观察,胶原包埋PGA组可见到细胞在支架上贴附和生长良好。结论 细胞和胶原包埋PGA的支架组织相容性良好。     

四种脱钙液对骨和骨髓组织及结构影响的比较

背景：由于骨和骨髓组织结构非常特殊,在常规的病理制片过程中,要同时显示坚韧质硬的骨组织和含有多种幼稚娇嫩造血细胞的骨髓组织是非常困难的。目的：选择一种既能完全除去骨组织中的钙质,又能保护骨髓组织及细胞结构不受破坏的最佳脱钙液。方法：将含骨髓组织的犬长骨组织随机分4组,同等条件下,14%硝酸甲醛生理盐水溶液;14%硝酸水溶液;20%甲盐酸甲醛生理盐水溶液和20%甲盐酸水溶液4种不同脱钙液脱钙,记录脱钙时间,常规脱水、切片、苏木精-伊红染色,镜下观察,对比4种脱钙液对骨和骨髓组织常规病理切片质量和其苏木精-伊红染色效果。结果与结论：14%硝酸甲醛生理盐水溶液组脱钙能力最强,脱钙最均匀,耗时最短,既能有效完全除去骨皮质中的钙质,又能保护骨髓组织及细胞的形态完好,切片质量和苏木精-伊红染色的效果最佳;14%硝酸水溶液组脱钙液,使骨组织疏松发泡,组织变黄,对组织的损伤大,脱钙不均匀,组织切片不完整,制片质量和苏木精-伊红染色的效果最差;20%甲盐酸甲醛生理盐水溶液组对组织的脱钙能力、损伤程度、制片质量和苏木精-伊红染色效果都比14%硝酸甲醛生理盐水溶液组稍差;20%甲盐酸水溶液组对骨和骨髓组织的损伤小,脱钙效果好,切片质量和苏木精-伊红染色的效果佳,介于20%甲盐酸甲醛生理盐水溶液组和14%硝酸水溶液组之间。结果表明,4种脱钙液对骨和骨髓组织都具有良好的脱钙能力,制片质量及苏木精-伊红染色效果比较,14%硝酸甲醛生理盐水溶液> 20%甲盐酸甲醛生理盐水溶液> 20%甲盐酸水溶液> 14%硝酸水溶液;14%硝酸甲醛生理盐水溶液最适用于临床常规病理制片中骨和骨髓组织的脱钙。中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Measurement of fracture callus material properties via nanoindentation

In bone fracture healing, the extent to which the injured bone regains stability and strength depends on the mechanical properties of the tissues that are formed during healing. While many techniques have been used to quantify the overall mechanical behavior of fracture calluses, few data exist on the material properties of individual callus tissues. The overall goal of this study was to quantify these material properties. Nanoindentation was performed at multiple locations across thin (200mum), longitudinal sections of rat fracture callus at 35 days post fracture. Following indentation, sections were stained with alizarin red S and alcian blue to obtain semi-quantitative estimates of tissue mineral content and proteoglycan content, respectively. Indentation moduli varied over three orders of magnitude (0.61-1010MPa) throughout the callus. Much of this variation was due to the presence of multiple tissue types: the indentation moduli of granulation tissue, chondroid tissue and woven bone ranged 0.61-1.27MPa (median=0.99MPa), 1.39-4.42MPa (median=2.89MPa) and 26.92-1010.00MPa (median=132.00MPa), respectively. In regions of alizarin red staining, the indentation modulus was correlated (r=0.62, P=0.04) with stain intensity, suggesting a positive correlation between modulus and mineral content in woven bone. In addition, the indentation modulus of woven bone along the periosteal aspect of the cortex increased with distance from the fracture gap (P=0.004). These results demonstrate the usefulness of nanoindentation in characterizing the elastic properties of the heterogeneous mixture of tissues present in bone fracture callus.     

Titanium metals form direct bonding to bone after alkali and heat treatments

In this article we evaluated the bone-bonding strengths of titanium and titanium alloy implants with and without alkali and heat treatments using the conventional canine femur push-out model. Four kinds of smooth cylindrical implants, made of pure titanium or three titanium alloys, were prepared with and without alkali and heat treatments. The implants were inserted hemitranscortically into canine femora. The bone-bonding shear strengths of the implants were measured using push-out test. At 4 weeks all types of the alkali- and heat-treated implants showed significantly higher bonding strength (2.4-4.5 MPa) than their untreated counterparts (0.3-0.6 MPa). At 12 weeks the bonding strengths of the treated implants showed no further increase, while those of the untreated implants had increased to 0.6-1.2MPa. Histologically, alkali- and heat-treated implants showed direct bonding to bony tissue without intervening fibrous tissue. On the other hand, untreated implants usually had intervening fibrous tissue at the interface between bone and the implant. The early and strong bonding to bone of alkali- and heat-treated titanium and its alloys without intervening fibrous tissue may be useful in establishing cementless stable fixation of orthopedic implants.     

Plastic Slides in the Preparation of Implant and Tissue for Interface Analysis

Abstract

Advances in ground section histology of hard tissues and implants have been made with the use of plastic slides in combination with a cyanoacrylate glue. This paper describes the techniques used to prepare ground sections containing implant and bone for microradiography, light microscopy, and scanning electron microsopic analysis. The use of plastic slides can reduce specimen loss and provide flat, thin sections for backscattered electron microscopic and histological analysis. (The J Histotechnol 12:307, 1989)     

A clinical case report: interface analysis of a successful well-functioning transmandibular implant from a cadaver mandible

Transmandibular implants (TMI) are indicated both for functional reconstruction of the severely atrophic mandible and when routine augmentation is unpredictable. This study investigates the interface of bone around a TMI, retrieved from the cadaver. The TMI had successfully functioned for 7 years. The mandible was immersed in 10% formaldehyde and sectioned into nine appropriate pieces. Samples were embedded in polymethylmethacrylate, and cut around the transosseous posts and cortical screws in both vertical and horizontal sections. Samples were analyzed at 400 MHz (nominal lateral resolution, 2.5 microm) using a UH3 Scanning Acoustic Microscope (Olympus, Tokyo, Japan). The middle of implant specimens 1-4 were cut to 50 microm, and stained by toluidine blue for light microscopy. Dental X-rays showed no bone resorption around any implant. On a 2-mm lateral scan, almost uniform interface space was seen between bone and implant surface in cortical screws. There are wider spaces around the transmandibular posts in the superior area. Histology revealed the small area of direct contact. There is bone marrow space in the interface, with no significant fibrous tissue. We interpret these results at the interface to be because of adaptation for stress distribution.     

Macroporous calcium phosphate bioceramics in dog femora: a histological study of interface and biodegradation

This study examines the biodegradation behaviour of calcium phosphate macroporous bioceramics consisting of hydroxylapatite or beta-whitlockite implants. The implantations were performed in hard tissue of femora and muscular tissue of dogs for periods of 6 and 12 month. With microradiography and light microscopy, respectively, of ground and thin sections it was shown that hydroxylapatite remained unchanged and beta-whitlockite degraded within 6 month in bone tissue. In the muscular tissue, the biodegradation of beta-whitlockite was slower. In addition, beta-whitlockite gave rise to a cellular response of lymphocytes and plasma cells rather than hydroxylapatite in the soft tissue region. For tissue response hydroxylapatite is more suitable as an implant material than beta-whitlockite.     

Interfacial analysis of fiber-reinforced bioactive glass dental root implants

Bioactive glass can form an effective bond with bone. Essential for this connection are the interfacial reactions which lead to the development of a Si-rich film covered by a CaP-rich film. The presence of these layers can be demonstrated clearly by EDX analysis of fiber-reinforced bioactive glass and bulk bioactive glass implants installed for 4 and 16 months in the partial edentulous jaws of beagle dogs. EDX analysis reveals three types of microchemical interface. The first type develops when the implant is bonded to bone. Here, a smooth transition of the CaP profile can be observed between bioactive glass and the bone, thus providing for a compositional gradient between the implant and the surrounding tissues. The second type is seen when the implant surface is surrounded by fibrous tissue. This causes a discontinuity in the CaP profile. The third type is characterized by a gradual decrease in the Ca and P concentrations across the interface, caused by the presence of Ca and P in the fibrous tissue near the implant surface. This suggests that the interface is dynamic in time and transforms to a functionally better optimized interface. EDX analysis does not reveal any metal fiber ion contamination of the outer glass rim of the implant. When stainless-steel or wrought Co-Cr alloy is exposed to the surrounding fluids, the interfacial osteogenesis is disturbed, possibly by a synergistic effect of glass ions and metal ions. Exposure of titanium does not interfere with this osteogenesis. The bone bonding can also be influenced by surgical trauma. However, with precise implantation techniques, an enhancement of bone growth by osteoconductivity can be measured.     

Callus formation and remodeling at titanium implants

Titanium is biocompatible with bone tissue, and during the healing process bone makes intimate contact with the implant surface. Although much is known about the long-term healing of implants, less is known about the callus formation at implants. In this study, the histology of bone healing was studied during the period between 4 and 14 days. Incisions were made in rat tibia. Some incisions were simply left to heal, while in others titanium discs were implanted. Smooth implants as well as implants with different porosities were used. After 4, 7, and 14 days of healing, the sites of bone incisions were retrieved, decalcified, sectioned, and stained. The aim was to compare normal fracture healing with implant healing and to see whether implant properties influenced the short-term healing process. Similarities between fracture healing and implant healing were evident. In both cases, inflammation, soft and hard callus formation, and remodeling had taken place during the period investigated. Between 7 and 14 days substantial bone resorption occurred around the implants. While after 14 days the marrow was almost completely reconstituted during normal wound healing, at the implants a thin layer of bone remained in close contact with the surface. Results from bone-implant contact measurements indicate that the surface properties of the implants do not have a significant influence on the early bone formation, since there were no significant differences between the smooth surface and any of the porous surfaces.     

Calcium and magnesium content in hard tissues of rats under condition of subchronic lead intoxication.

Lead manifests toxic effects in almost all organs and tissues, especially in: the nervous system, hematopoietic system, kidney and liver. This metal has a special affinity for deposition in hard tissue, i.e., bones and teeth. It is generally believed that the main mechanism of its toxicity relies on its interaction with bioelements, especially with Ca and Mg. This article analyses the influence of Pb poisoning on Ca and Mg content in hard tissues, (mandible, femur, teeth and skull) of female and young rats. Experiments were carried out on 60 female rats, AO breed, and on 80 of their young rats (offspring). Female rats were divided into three groups: the first one was a control group, the second one received 100 mg/kg Pb2+ kg b.wt. per day in drinking water, the third one received 30 mg/kg Pb(2+) kg b.wt. per day in drinking water. Young rats (offspring) were divided into the same respective three groups. Lead, calcium and magnesium content in hard tissues (mandible, femur, teeth-incisors and skull) was determined by flame atomic absorption spectrophotometry in mineralized samples. There was a statistically significant Pb deposition in all analyzed female and young rat hard tissues. Ca and Mg contents were significantly reduced in all female and young rat hard tissues. These results show that Pb poisoning causes a significant reduction in Ca and Mg content in animal hard tissues, which is probably the consequence of competitive antagonism between Pb and Ca and Mg.