ULTRASTRUCTURAL INVESTIGATION ON EXPERIMENTAL FRACTURE HEALING VI. ELECTRON MICROSCOPIC OBSERVATION ON MATRIX VESICLES

In the course of healing of standardized radius fracture in rabbits, there is emergence of extracellular matrix vesicles with subsequent calcification priOr to the deposition of calcium salts in the collagen fibrils. The calcified matrix vesicles turn into flocculent cal cospherules which coalesce to form bone substance. The phenomenon of calcification of matrix vesicles takes place not only around the osteoblasts and chon drocytes but also around the fibroblasts. The same se- quential events about changes in matrix vesicle can be detected whether the fibroblasts are in an active se- creting state, and transform into osteocytes, or under- go degeneration, decease, and replacement by bone substance. Because deposition of calcium salt crystals takes place in the collagen fibrils abutting the fibro- blasts and the fibroblasts transform into osteocytes, the findings in our study show the importance of fibroblasts in osteogenesis in the course of fracture healing.     

ULTRASTRUCTURAL INVESTIGATION OF HEALING OF EXPERIMENTAL FRACTURE TREATED ACCOR.DING TO THE PRINCIPLE OF ACTIVATING CIRCULATION AND ALLEVIATING STAGNATION

Radix Salviae Miltiorrhizae, a drug activating the circulation and alleviating stagnation, was ad ministered t0 50 New Zealand rabbits that had re- ceived standardized fractures of the radius. Radio graphs of the fractures showed that callus formation appcared earlier and was denser than in 50 control rabbits studied earlier. Transmission electron micros copy revealed five favorable effects of the drug, the first three of which were cellular. First, the osteo genic cells (the progenitors of the csteob:asts and chondrocytes) increased in both number and sites of distribution. Second, the fibroblasts, in addition to changing in configuration, showed very active pro- tein synthesis, with exuberant extracellular coflagcn fibril formation. The normal process of fibroblast degeneration was also expedited. Thus, the replace ment of fibroblasts by bony tissue was promoted. Third, an increased number of osteoclasts emerged in different areas of fracture callus and thus remodel ing was facilitated. Fourth, there was exuberant collagen fibril formation, which promoted p*:oduc tion of organic matrix. Fifth, the swollen mito- chondria of the fibroblasts contained more dense cal cium granules and thus provided more calcium for the calcification of matrix vesicles and collagen fibrils.     

ELECTRON MICROSCOPIC OBSERVATION ON ENDOCHONDRAL OSSIFICATION WITH SPECIAL REFERENCE TO MATRIX VESICLES

The upper tibial ends of young rabbits are studied with transmission electron microsocpe. A sequence of events taking place in the chondrocytes of different zones in the cell columns of epiphyseal plate regarding multiplication, hypertrophy, degeneration, calcification and ossification has been depicted. The matrix vesicles play an important role in endochondral ossification. The matrix vesicles stem from two sources, deriving either from extrusion of intracytoplasmic dense bodies or from detachment of cell processes. Deposition of calcium salt crystals in the matrix vesicles produces calcospherules and conglomeration of the calcospherules yields bone tissues, In the course of metaphyseal remodelling the osteocytes in the lacunae undergo degenerative changes, including condensation, fragmentation and eventual absorption. The empty lacunae are filled up with matrix vesicles and collagen fibrils and progressive deposition of calcium salt crystals in them engenders bone tissues which fill up the lacunae.     

ULTRASTRUCTURAL INVESTIGATION. OF EXPERIMENTAL FRACTURE HEALING IV. ELECTRON MICROSCOPIC OBSERVATION ON TRANSFORMATION AND FATE OF FIBROBLASTS AND CHONDROCYTES

The results of electron microscopic observa- tion on transformation of fibroblasts and chond- rocytes in experimental fracture healing in 75 healthy male white rabbits are reported. It is found that in the course of fracture healing, fibroblasts either undergo degenerative changes leading to decease and replacement by bony tissue or transform without degeneration into osteocytes directly. Consequently they not only produce collagen fibrils and form fibrous callus, but also play an important role in elaborating bony callus and creating osteocytes. In endochondral ossification, chondrocytes pass through various degenerative changes to end in decease and replacement by bony tissue.     

不同组织来源的成纤维细胞的生物学特性比较

目的 探讨来自政治家皮肤、增生性瘢痕和瘢痕疙瘩组织的成纤维细胞的生物学特性。方法 对三种组织均采用组织块法进行细胞的原代培养,采用胰蛋白酶消化传代,分别利用绘制细胞生长曲线,^H-胸腺嘧啶掺入及^3H-脯氨酸掺入等方法观察细胞的增殖,DNA合成代谢及胶原合成等情况。并比较它们之间的差异。结果 瘢痕疙瘩成纤维细胞在细胞增殖、DNA合成代谢及胶原合成量等方面均明显高于正常皮肤及增生性瘢痕组织来源的成纤维细胞。后两者之间有一定区别但无统计学意义。结论 不同组织来源的成纤维细胞其生物学行为亦有所区别,因此,在进行实验研究时应有一定的针对性。     

EXPERIMENTAL STUDY ON INDUCTION OF OSTEOGENETIC POTENTIAL OF DERMAL FIBROBLASTS

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ULTRASTRUCTURE INVESTIGATION OF EXPERIMENTAL FRACTURE HEALING II.* ELECTRON MICROSCOPIC OBSP RTATION ON FIBRILLOGENESIS

Tnree different kinds of fibers are generated in the course of fracture healing: tibrin fibers, typical collagen fibrils produced by fibroblasts and ostcoblasts aud equally important atypical collagen fibrils produced by chondroblasts. In tho special situation of fracture repiiir mature collagen fibrils winh periodicity can be produced c7irectly in fibroblast CYtoplasZn.     

Ultrastructural investigation of granulation tissues on the osteoarthritic femoral head. A scanning electron microscopic and transmission electron microscopic study.

From the femoral heads of nine cases of advanced osteoarthritis of hip joint, three categories of granulation tissues with different colors (bright red, dark-red and white granulation tissues) and texture were obtained. After processing, the specimens were observed under scanning electron microscope and transmission electron microscope. With transformation of the granulation tissues from bright red to white, the collagen fibers therein contained gradually changed from fine to thick ones. Nevertheless, the cellular components in these three types of granulation tissues always possessed the characteristics of both fibroblasts and chondrocytes, i.e., fibroblastic configuration with pericellular Type I collagen fibrils bearing 64 nm periodicity; and chondrocytic scallop-shaped cytoplasmic membrane with intracytoplasmic fat droplets and glycogen granules and pericellular Type II collagen fibrils. All these indicated that in osteoarthritis of hip joints, the granulation tissues of the femoral heads transformed eventually into fibrocartilage, irrespective of their color and texture.

     

骨组织结构显微形态分析

目的 应用原子力显微镜和扫描电镜观察分析人长管状骨组织中的微观结构,以了解骨组织的超微结构以及胶原和矿物质的构成和形态。方法 取新鲜人尸体肱骨标本,固定脱水,分别制作骨磨片和切成薄片,分别在光学显微镜、原子力显微镜和扫描电镜分析骨组织矿化区的结构。结果 在光学显微镜下,骨陷窝呈环状排列在哈佛管周围,骨小管沟通骨陷窝与哈佛管和骨陷窝之间的联系。在原子力显微镜的观察下,大胶原蛋白的形态清楚显示;在矿化区胶原蛋白纤维增厚,呈小盘叠加形态,钙-磷晶体呈椭圆柱形状;可清晰观察到骨小管和骨陷窝的大小和形态及骨小管和骨陷窝的关系。在扫描电镜下,骨基质呈环形沿哈佛系统排列,钙-磷晶体之间相互呈规则排列。结论 原子力显微镜可分析胶原蛋白和基质中钙-磷晶体的三维结构形态、骨小管和骨陷窝的三维构成及大小;骨小管是交流营养物质和分子信号到骨陷窝中骨细胞的通道。     

骨组织结构显微形态分析

目的 应用原子力显微镜和扫捕电镜观察分析人长管状骨组织中的微观结构、以了解骨组织的超微结构以及胶原和矿物质的构成和形态。方法 取新鲜人尸体肱骨标本，固定脱水，分别制作骨磨片和切成薄片．分别在光学显微镜、原子力显微镜和扫描电镜分析骨组织矿化区的结构。结果 在光学显微镜下．骨陷窝呈环状排列在哈佛管周围，骨小管沟通骨陷窝与哈佛管和骨陷窝之间的联系。在原子力显微镜的观察下．大胶原蛋白的形态消楚显示；在矿化区胶顾蛋白纤维增厚．呈小盘叠加形态．钙-磷晶体呈椭圆柱形状；可清晰观察到骨小管和骨陷窝的大小和形念及骨小管和骨陷窝的关系，在扫描电镜下．骨基质呈环形沿哈佛系统排列．钙-磷晶体之间相互呈规则排列。结论 原子力显微镜可分析胶原蛋白和基质中钙-磷晶体的三维结构形态、骨小管和骨陷窝的三维构成及大小；骨小管是交流营养物质和分子信号到骨陷窝中骨细胞的通道。     

化云宁对家兔角膜瘢痕微超结构的影响

用化云宁对家兔角膜瘢痕进行治疗,发现角膜基质层细胞数由多到少,功能由活跃到静止,胶原原纤维队排列紊乱到规则。推测药物对胶原形成细胞的增殖及胶原的合成功能有一定的抑制作用,因而混浊的角膜变得透明。用狄奥宁及碘化钠治疗未见类似变化。     

应用组织工程方法构建瘢痕疙瘩模型

目的 应用组织工程方法构建人瘢痕疙瘩动物模型,并探讨采用这一模型进行瘢痕疙瘩临床和实验室研究的可行性。方法 对人体瘢痕疙瘩组织中成纤维细胞进行原代培养,将体外培养第6~8代的成纤维细胞,接种到培养液预湿的聚乳酸-乙醇酸共聚物(PLGA)支架,形成体外复合体,将复合体转移至旋转式细胞培养系统容器内培养;1周后种植在20只雌性裸鼠皮下,同体两侧对照,第4、8、16、24周取材,对获得的瘢痕疙瘩组织进行组织学评价。结果 术后裸鼠全部成活。复合体移植24w后,在裸鼠皮下形成的瘢痕疙瘩保持了其原有的胶原形态,在电镜下可观察到植入物内同时存在纤维细胞和成纤维细胞,成纤维细胞内可见丰富的粗面内质网,细胞特性保持不变。结论 PLGA与瘢痕疙瘩成纤维细胞具有较好的亲和性,PLGA-瘢痕疙瘩成纤维细胞复合体在裸鼠体内可形成瘢痕疙瘩,值得进一步开发研制。     

Construction of animal models of keloid by tissue engineering]

OBJECTIVE: To construct animal model of keloid by using tissue engineering and to explore its potential clinical significance. METHOD: Fibroblasts from keloid were isolated and cultured. After being cultured to generation six or eight, fibroblasts were inoculated into scaffolds constructed from copolymers of polylactic acid (PLA) and polyglycolic acid (PGA), and then cultured in rotatory cell culture system for 1 week prior to subcutaneous transplantation into 20 athymic mice. After 4, 8, 16, 24 weeks, the specimens were cut out and examined histologically. RESULTS: All the mice survived after surgery. The collagen patterns of all the keloids were remained after 24 weeks. A lot of fibroblasts and their secreted collagen were observed under light microscope. Enlarged rough endoplasmic reticula inside the fibroblasts were revealed by transmission electron microscopy. The fibroblasts in specimens remained the capability of synthesizing and secreting collagen. CONCLUSIONS There is a good affinity between PLA/PGA and fibroblasts. Complex of PLA/PGA and fibroblasts could be cultured into keloids in athymic mice.     

The effects of early stage load-bearing on bone ingrowth of porous coated implant in rabbits]

To understand the effects of load-bearing on cementless fixation of porous coated implant, we inserted Co-Cr-Mo alloy implants into the distal femur of rabbits. Specimens were studied by means of histology, anti-collagen type I and III immunohistochemical staining, colloidal gold labeled immunoelectron microscopy, and computerized graphic analysis. The results revealed that stimulations of load-bearing benefited bone ingrowth, but the newly formed bone in the pores was absorbed and replaced by fibrous tissues after 12 weeks without stimulations of load-bearing. The fibrous tissues in the pores of the implant were primarily type III collagen after one week and type I collagen after two weeks. After 6 weeks, the residual tissue between ingrowth bone and metal bound antibodies was type I collagen. Fibroblasts participated in and possessed the action of osteogenesis.     

活性真皮基质的研制及生物学活性检测

目的构建含有成纤维细胞（Fb）-胶原-猪去细胞真皮基质（PADM）的活性真皮基质。研究培养液中转化生长因子-β1（TGF-β1）和碱性成纤维细胞生长因子（bFGF）的分泌情况。方法将Fb与胶原混合种植于PADM的表面,收集第2、4、6、8、10d培养液样本,双抗体夹心ELISA法测定其中TGF-β1、bFGF的含量。结果Fb在胶原内结构完整,与PADM形成复合真皮基质。TGF-β1和bFGF浓度培养第4天与第2天比较显著上升（P〈0．01）,并达到稳定水平。结论Fb-胶原-PADM真皮替代物可作为构建组织工程全层皮肤的真皮支架。     

大鼠颅骨成骨细胞体外培养形成钙化结节的光镜电镜研究

自新生大鼠颅骨分离出的成骨细胞在含５０ｍｇ／Ｌ维生素Ｃ和１０ｍｍｏｌ／Ｌβ－甘油磷酸钠的ＤＭＥＭ＋１０％ＦＣＳ培养液的逐渐出现聚集重叠而形成结节，ＶｏｎＫｏｓｓａ染色证实结节内有钙盐沉积，细胞的碱性磷酸酶染色显示结节的钙化与高活性酶的活动有关，电镜示结节内有大量的钙盐沉积在胶原纤维上，一些类似骨细胞的细胞被包埋在钙化骨质内，整个由细胞，胶原纤维和钙盐组成的三维结构似于体内膜内成骨。实验结果提示：体     

定向诱导骨髓间充质干细胞-藻酸盐复合物构建软骨组织

目的研究骨髓间充质干细胞(M SC s)经向软骨细胞定向诱导后,与藻酸盐复合,构建组织工程软骨的可能性。方法取大鼠股骨骨髓,经梯度离心法和贴壁法分离M SC s,经鉴定后用含转化生长因子1β10 ng/m l、地塞米松1-0 7m o l/L、转铁蛋白3 m g/m l、胰岛素2 m g/m l的诱导因子培养基对M SC s进行诱导,14 d后免疫组化检测Ⅱ型胶原分泌情况,将诱导后和未经诱导的M SC s以1×106/m l细胞密度与藻酸钙复合接种于裸鼠,分别于4周、8周后行组织学检测。结果经诱导的M SC sⅡ型胶原免疫组化阳性,与藻酸盐复合4周,HE染色可见软骨样细胞、软骨陷窝形成,阿辛蓝染色和M asson染色可见新生的软骨细胞和细胞外胶原,8周后软骨细胞明显增多,软骨细胞分泌胶原丰富,而未经诱导的M SC s无明显软骨细胞生成。结论M SC s经定向诱导后,可与藻酸钙复合在体内形成软骨样组织,可能发展为临床修补软骨缺损的一条有效途径。     

贯叶连翘提取物对TGF—β1诱导肺成纤维细胞胶原合成影响的体外研究

目的研究贯叶连翘提取物对TGF-β1诱导离体肺成纤维细胞胶原合成的影响。方法离体成纤维细胞分别加入TGF-β1（10ng／ml）、贯叶连翘提取物（250mg／L）以及TGF-β1＋贯叶连翘提取物共培养48h,设为TGF-β1组,贯叶连翘组,TGF-β1贯叶连翘组,取DMEM培养液为对照组。应用SP免疫细胞化学法,免疫荧光细胞化学法及平均光密度（MOD）分析,观察在TGF-β1作用下,肺成纤维细胞α-SMA、Ⅰ、Ⅲ型胶原蛋白的表达及贯叶连翘提取物的干预。结果TGF-β1诱导肺成纤维细胞α-SMA、Ⅰ、Ⅲ型胶原阳性染色表达均显著高于对照组。贯叶连翘提取物干预48小时后,α-SMA、Ⅰ、Ⅲ型胶原阳性染色表达明显下降,仍高于空白对照组。结论贯叶连翘提取物可抑制TGF-β1：诱导肺成纤维细胞转分化及Ⅰ、Ⅲ型胶原蛋白的合成。     

骨折愈合过程中大鼠体内SOD活力变化及外源性SOD对骨痂形成的影响

为探讨超氧化物对实验性骨折愈合的影响，本文检测了实验性骨折前后大鼠体内超氧化物歧化酶活力的变化，并观察了使用外源性超氧化物歧化酶以后，骨折部位骨痂组织细胞的形态学变化。结果表明：骨折后大鼠体内超氧化物歧化酶活力明显低于骨折前（Ｐ〈０．０５）；外源性超氧化物歧化酶可使早期骨痂内毛细血管含量增加，软骨细胞，成纤维细胞功能活跃，基质钙化迅速，完全，骨性骨痂发生早。认为超氧化物歧化酶有促进骨折愈合的作用。     

SURFACE ULTRASTRUCTURAL INVESTIGATION OF EXPERIMENTAL FRACTURE HEALING WITH SCANNING ELECTRON MICROSCOPE

The healing process of a standardized radial fracture was studied under scanning electron microscope (SEM). The active cellular activities of all main kinds of "osteogenesis cells", the collagen fibrils they produce and the three dimensional configurations of various calli are {lescribed. In the process of calcification, calcium salt moves from intracellular to the intercellular milieu throughout the whole course of fracture healing. In the early stage, calcium s.alt crystals deposited within the collagen fibrils in an un- homogeneous manner. As union is attained, the callus turns into finer homogeneous and becomes denser and more compact. It is concluded that the SEM is of value in the study of fracture healing mechanism.