Amorphous calcium precipitations in human aortic valve.

Spheroidal, granular, and fibrillar particles were observed in human aortic valve calcifications. Spheroids were similar to amorphous calcium phosphate shown previously in syn thetic preparations and in bone. The frequent coexistence of needle shaped hydroxyapatite crystals with granular and fibrillar particles suggests that the latter are also amorphous calcium phosphate and may be precursors of hydroxyapatite. All three types of particles had a tendency to form laminated and spherular secondary and failed to give a crystalline pattern by electron diffraction.     

Matrix vesicles in newly synthesizing bone observed after ultracryotomy and ultramicroincineration.

Matrix vesicles were observed in femurs of 8-day-old chick embryos prepared by ultracryotomy. Some of the sections were subjected to ultramicroincineration. The unfixed tissues never came into contact with solutions, and thereby artifacts due to dissolution, redistribution, or rearrangement of the mineral constituents were avoided. In the osteoid, electron dense objects with the size and appearance of matrix vesicles were seen, although limiting membranes were not visible. After ultramicroincineration the vesicles were observed to contain small crystals and a less dense amorphous mineral material which may te the precursor of bone mineral. In addition, a ring of ash enclosed the crystalline and amorphous mineral and appeared to occupy the position of the vesicle membrane as seen in conventionally prepared material.     

Radiogenic free radicals as molecular probes in bone.

Exposure of bone mineral to X-rays generates free radicals. These are usually very labile, but can be stabilized at liquid nitrogen temperatures for study by electron spin resonance spectroscopy. The free radicals thus detected in the present study included one with resonances arising from an electron excess center and 2 species with electron-deficit centers: a phosphate anion radical and a radical associated with carbonate. Each of these radicals seemed to be located chiefly at the mineral surface and was sensitive to the surface environment. Presence of an organic phase, as in whole bone, modified free radical production in a manner that suggests interference with the formation of electron deficit centers. Comparison with other synthetic minerals suggests that precipitated carbonate-apatites are good models for bone mineral.     

Bone formation on synthetic precursors of hydroxyapatite

The aim of this study was to investigate the reaction of skeletal tissue to various synthetic calcium phosphate (Ca-P) compounds in vivo. Five synthetic Ca-P compounds were implanted into the subperiosteal area of the calvaria of 7-week-old BALB/c mice for one to 15 weeks. Synthetic compounds were dicalcium phosphate (DCP), octacalcium phosphate (OCP), amorphous calcium phosphate (ACP), Ca-deficient hydroxyapatite and hydroxyapatile (HA). Implanted DCP, OCP and ACP were found to be converted to apatitic phase by x-ray microdiffraction analysis using undecalcified specimens. Structure of bone was found out on all of Ca-P compounds eventually at late stage under the light microscope, but the rate of bone formation calculated from a number of experiments varied on respective synthetic Ca-P compound. It was high as 80% for DCP, OCP and ACP, but was low as 5.6% for Ca-deficient HA, and no reaction was found for HA at the stage of 3 weeks. Fine filaments and granular materials in the newly formed bone matrix were detected at 7 days around the remnants of OCP particles which already converted to apatitic phase by ultrastructural study of decalcified specimens. These structures were very similar to the components of bone nodules seen in intramembranous osteogenesis. It is postulated that the precursors of HA have an important role in intramembranous osteogenesis.     

Influence of electron beam irradiation doses on bone regeneration by octacalcium phosphate collagen composites

An octacalcium phosphate and collagen composite (OCP/Col) achieved efficient bone regeneration with excellent resorbability. After the confirmation of its safety and efficacy in preclinical animal studies, the present study investigated the influence of electron beam irradiation doses on bone regeneration by OCP/Col in order to secure its commercialization. OCP/Col was prepared as previously described and the packed OCP/Col was sterilized using different doses of electron beam irradiation. A standardized defect, 9 mm in diameter, was made in the rat calvarium. A disk of OCP/Col (diameter 9 mm, thickness 1.5 mm) sterilized using different doses of electron beam irradiation was then implanted into the defect. Five defects in each group were treated and fixed 4, 12 and 24 weeks after implantation. Specimens were decalcified and stained with haematoxylin and eosin. In a histomorphometrical analysis, the percentage of newly formed bone in the defect (n‐Bone%) was calculated. In OCP/Col 15 kGy, newly formed bone was enhanced and present throughout the defect with the resorption of OCP/Col. Furthermore, vigorous bone remodelling and bone maturation were observed. In OCP/Col 40 kGy, newly formed bone was not as prominent as that with OCP/Col 15 kGy. A histomorphometrical analysis using Student's t‐test at 24 weeks revealed that the n‐Bone% of OCP/Col 15 kGy (65.9 ± 7.14%) was significantly higher than that of OCP/Col 40 kGy (38.0 ± 10.2%). These results suggest that different electron beam irradiation doses influence bone regeneration by OCP/Col.     

Effect of phospholipids on the transformation of amorphous calcium phosphate to hydroxapatite in vitro.

The conversion of amorphous calcium phosphate (ACP) to crystalline hydroxyapatite (HA) was studied in vitro in the presence or absence of phosphatidyl serine (PS) and other phospholipids. ACP transformation and HA crystal growth were monitored by electron microscopy, selected-area electron diffraction and X-ray diffraction, and by measuring supernatant calcium and phosphate. PS, and other acidic phospholipids, had a significant stablizing effect on ACP. With performed ACP at ratios of only 1 lipid molecule per 30-50 Ca atoms, PS markedly delayed HA crystal formation. When PS was present during ACP precipitation, inhibition of conversion to HA was less pronounced, but crystal habit and aggregation were greatly altered resulting in stacks of thin, membrane-like sheets approximately 38-42 A thick. PS appeared to be most effective in blocking ACP to HA conversion when oriented primarily on the surface; it most affected subsequent crystal formation when distributed throughout the amorphous precursor. Phospholipids possessing anionic, and hence Ca-binding properties, were effective in stabilizing ACP; neutral zwitterion lipids, which have amphipathic properties but do not bind Ca, were not. In view of the presence of anionic lipids in matrix vesicles and their association with early mineral deposits, the current findings add further evidence that lipids may play a role in the control of normal mineralization in vivo.     

Electron microscopic characterization of lipoproteins from patients with familial type III hyperlipoproteinaemia.

The lipoproteins of very low density, low density and intermediate density were isolated by zonal ultracentrifugation from the plasma of subjects with familial type III hyperlipoproteinaemia and studied by electron microscopy. Each of these lipoproteins exhibited a spherical shape when free-standing. The very low density, low density and intermediate density lipoproteins ranged in diameter from 31 - 80, 17 - 23, .and 21 - 35 nm, respectively. These results are consistent with our previous findings that the intermediate density lipoprotein particles characteristic of plasma from type III subjects exhibit properties which are intermediate between those of very low density and low density lipoproteins.     

Electron Microscopic Characterization of Lipoproteins from Patients with Familial Type III Hyperlipoproteinaemia

The lipoproteins of very low density, low density and intermediate density were isolated by zonal ultracentrifugation from the plasma of subjects with familial type III hyperlipoproteinaemia and studied by electron microscopy. Each of these lipoproteins exhibited a spherical shape when free-standing. The very low density, low density and intermediate density lipoproteins ranged in diameter from 31 - 80, 17 - 23, and 21 - 35 nm, respectively. These results are consistent with, our previous findings that the intermediate density lipoprotein particles characteristic of plasma from type III subjects exhibit properties which are intermediate between those of very low density and low density lipoproteins.     

Particles of vaterite,a metastable CaCO3 polymorph,exhibit high biocompatibility for human osteoblasts and endothelial cells and may serve as a biomaterial for rapid bone regeneration

We have previously described a promising alternative to conventional synthetic bone biomaterials using vaterite, a metastable CaCO₃ polymorph that increases the local Ca²⁺ concentration in vitro and leads to an oversaturation of phosphate, the primary bone mineral. This stimulates a natural bone‐like mineralisation in a short period of time. In this study, sterile and endotoxin‐free vaterite particles were synthesised in a nearly quantitative yield. The 500–1,000 nm vaterite particles did not exhibit any cytotoxic effects as measured by MTS, lactate dehydrogenase, or crystal violet assays on the human osteoblast cell line (MG‐63) exposed to concentrations up to 500 μg/ml vaterite up to 72 hr. MG‐63, primary human osteoblasts or human umbilical vein endothelial cells in the presence of vaterite up to 500 μg/ml for 7 days exhibited typical growth patterns. Endothelial cells exhibited a normal induction of E‐selectin after exposure to LPS and MG‐63 cells in osteogenic differentiation medium showed an increased expression of alkaline phosphatase compared with the respective control cells without vaterite. MG‐63 cultured on a vaterite‐containing degradable poly(ethylene glycol)‐hydrogel exhibited strong adhesion and proliferation, similar to cells on cell culture plates. Cells did not attach to gels without vaterite. Our results demonstrate that vaterite particles are biocompatible, do not influence cell gene expression, and that vaterite in hydrogels may be able to serve for adhesion of osteoblasts and as a mineral substrate for natural bone formation by osteoblasts. These characteristics make vaterite particles a highly favourable compound for use in bone regeneration applications.     

一种新型掺锶冻干骨材料的细胞相容性☆

背景：前期研究已成功制得掺锶冻干骨材料。目的：评价新型掺锶冻干骨材料的细胞相容性,并与普通冻干骨材料进行比较。方法：在掺锶冻干骨与普通冻干骨材料表面直接接种MC-3T3成骨细胞,评价成骨细胞的增殖活性、碱性磷酸酶活性及骨钙素活性,并用扫描电子显微镜观察细胞生长微观形态。结果与结论：掺锶冻干骨组成骨细胞生长正常,接种第3,5天细胞数量显著高于普通冻干骨组（P〈0.05）,细胞碱性磷酸酶活性始终高于普通冻干骨组（P〈0.05）。两组成骨细胞骨钙素水平差异无显著性意义（P〉0.05）。在掺锶冻干骨材料表面的成骨细胞伸展良好,细胞呈多角形或梭形,胞浆丰富,细胞伪足末端与材料表面紧密附着,细胞突起末端可以观察到一些细小的微突触结构。表明掺锶冻干骨材料具有优良的细胞相容性,其表面成骨细胞的生长状况优于普通冻干骨材料表面的成骨细胞。     

X-ray diffraction and electron microscope study of osteons during calcification

To obtain information on the changes in the inorganic bone fraction during calcification, low- and wide-angle X-ray diffraction techniques and electron microscopy have been applied to single osteon samples. The samples were cylindrically shaped and their axes corresponded to the axes of the Haversian canals. The selection was made according to the degree of calcification and the orientation of collagen bundles and inorganic particles. Osteons at both the initial and final stages of calcification were chosen. Arrangements of fiber bundles and inorganic particles in successive lamellae characteristic of three types of osteon were selected, that is, longitudinally structured osteons, transversely structured osteons, and alternately structured osteons. The results indicate that in osteonic lamellar bone there are two types of inorganic particles: (1) granules arranged in linear or needle-shapred entities with maximum width 40-45 A, which are regularly distributed at the level of the main band of the collagen fibrils where their maximum length reaches the length of the main band itself; that is , about 400 A; and (2) very long crystallites, with a diameter of 40-45 A, which grow with their crystallographic c-axis parallel to the collagen fibrils and cover much more than a major collagen period.     

脂质体介导pcDNA3/hVEGF165转染骨髓基质干细胞复合冻干骨的体内成骨和血管化

背景：以往的研究表明血管内皮生长因子、碱性成纤维细胞生长因子可以促进移植物的存活和体内生长。目的：观察脂质体介导的pcDNA3/hVEGF165转染骨髓基质干细胞后复合冻干松质骨在体内的成骨和血管化效果。方法：取同种异体新西兰大白兔的耾骨和股骨制备冻干骨,用脂质体将血管内皮生长因子转染入体外培养扩增新西兰大白兔骨髓基质干细胞中,使其附着于同种异体冻干松质骨。将新西兰大白兔分为3组,于兔竖脊肌分别植入单纯冻干骨、单纯骨髓间充质干细胞复合冻干骨组、转染有血管内皮生长因子的骨髓基质干细胞复合冻干松质骨。结果与结论：植入后8周时可见到转染血管内皮细胞生长因子的骨髓基质干细胞复合冻干松质骨标本的表面有软骨和骨质形成,有成骨和破骨细胞出现,并形成类骨质,在移植骨周围组织中有大量血管形成,其他两组仅有大量纤维包裹。说明,脂质体介导的pcDNA3/hVEGF165转染后骨髓基质干细胞复合冻干骨具有较好成骨活性,优于单纯骨髓基质干细胞复合冻干松质骨和单纯冻干骨。     

胎盘组织液-同种异体冻干骨与同种异体冻干骨修复颌骨缺损的对照

背景:研究证实,冻干和辐照灭菌的同种异体骨是理想的骨移植材料,不仅具有良好的生物相容性、生物力学特性,而且还保留骨基质中的骨形态发生蛋白和构成形态发生蛋白所必须的一些酶类,具有一定的骨诱导能力.目的:观察胎盘组织液-同种异体冻干骨修复犬的人工颌骨缺损效果,以同种异体冻干骨为对照.设计、时间及地点:对比观察性实验,于2007-12/2008-09在哈尔滨医科大学动物实验中心完成.材料:健康杂种成年犬8只;同种异体冻干骨由湖北联结生物材料有限公司提供;胎盘组织液为广东珠海丽珠制药厂产品,2 mL/支.同种异体冻干骨:胎盘组织液=(4.0-5.0):1.方法:在犬上、下中切牙、尖牙、第一磨牙根尖相对应的颌骨处分别建立直径1.0 cm类半球型上下颌骨缺损模型共96个实验区,实验组选择大小合适的同种异体冻干骨及骨颗粒按比例浸泡在胎盘组织液呈饱和状,阳性对照组亦将大小合适的同种异体冻干骨及骨颗粒浸泡在生理盐水液呈饱和状,分别放入颌骨缺损处并添满,阴性对照组不植入任何材料.每只犬的实验区均由4个实验组材料、4个阳性对照组材料、4个阴性对照组材料组成.主要观察指标:术后2,4,8,12周取材进行放射线学和组织学观察.结果:实验组早期以软骨内化骨为主,晚期以膜内化骨为主,有明显的新骨形成,并与周围骨组织连接良好;阳性对照组可见新生骨,与周围骨组织连接欠佳;阴性对照组骨缺损区中央尚未被骨小梁充满.经组织学测定,术后2,4,8,12周实验组成骨量均高于其他两个对照组,差异有显著性意义(P<0.05,P<0.01).结论:胎盘组织液-同种异体冻干骨复合材料能够积极地促进颌骨缺损的修复并有效缩短修复时间.     

骨形态发生蛋白7诱导骨膜细胞体外构骨的超微结构

背景：骨形态发生蛋白被证实有成骨诱导作用,然而关于骨形态发生蛋白7诱导骨膜细胞成骨的超微结构研究尚未见报道。目的：观察骨膜细胞经成骨因子骨形态发生蛋白7诱导后的生物活性及超微结构。方法：实验取材于成人胫骨。分离出原代骨膜细胞后行常规体外培养,实验分为实验组和对照组。实验组加入骨形态发生蛋白7和细胞培养辅助剂,对照组仅仅加入了成骨细胞培养辅助剂。每组分别在第5,10,15天设3个时间点,每个时间点设3个样本,分别行钙结节的染色及采用相差显微镜观察大体形态结构,在透射电镜下观察超微形态结构。结果与结论：实验组和对照组形成的成骨细胞增殖良好,形态一致。细胞早期呈梭形,立体感强,饱满透明,分裂期呈短柱状或立方形,电镜下见成骨细胞内有大量的粗面内质网和高尔基复合体;后期细胞由长梭形逐渐变成宽梭和不规则形,后期射透电镜下可见细胞内有大量基质小泡,有膜包被,胞质内上有碱性磷酸酶及钙结合蛋白,泡内有钙盐结晶,成骨细胞的基底部和侧面出现突起与邻近的骨细胞突起相连。由骨形态发生蛋白7诱导的成骨细胞在体外增殖更快,细胞分裂及成骨速度更快。结果提示骨形态发生蛋白7在体外培养中具有明显增强成骨细胞增殖的能力。     

Ethylene oxide sterilisation of allogenic bone implants

We have evaluated the ability of ethylene oxide gas to penetrate bone matrices and subsequently kill a defined microbiological load. These experiments demonstrated that freeze-dried cancellous and cortical bone did not pose a barrier to the rapid diffusion of the gas. A normal cycle on a commercial ethylene oxide steriliser provided a sixfold overkill. Minor differences in the rate at which the microbiological load was killed (D-value) were seen between cortical and cancellous bone, processed and unprocessed bone, and bone of different thickness. These differences were negligible in comparison to the degree of overkill observed. Spore indicators placed freely in the steriliser chamber were shown to act as excellent monitors for the bone sterilisation process. Preliminary studies of ethylene oxide residuals indicated that processed, freeze-dried bone could be sterilised without leaving leachable toxic residuals in the matrix.     

STUDIES OF TWO KINDS OF VIRUS PARTICLES WHICH COMPRISE INFLUENZA A2 VIRUS STRAINS : III. MORPHOLOGICAL CHARACTERISTICS: INDEPENDENCE OF MORPHOLOGICAL AND FUNCTIONAL TRAITS

Seven strains of influenza A2 virus were serially passed in the chick embryo, and morphological characteristics of the passages were examined in the electron microscope. With serial passage there was a change from a prominently filamentous appearance in early passages to an almost entirely spherical appearance in later passages. The number of passages required for the conversion to spherical morphology varied with different strains. The filament-sphere variation was found to be independent of the properties which differentiate "+" and "-" influenza A2 virus particles, and both highly filamentous and spherical populations of "+" and of "-" virus particles were obtained. The usefulness of these pairs of independent markers for genetic studies is discussed.     

Real time monitoring of screw insertion using acoustic emission can predict screw stripping in human cancellous bone

BackgroundTo develop experience, orthopaedic surgeons train their own proprioception to detect torque during screw insertion. This experience is acquired over time and when implanting conventional/non-locked screws in osteopenic cancellous bone the experienced surgeon still strips between 38 and 45%. Technology needs to be investigated to reduce stripping rates. Acoustic-Emission technology has the ability to detect stress wave energy transmitted through a screw during insertion into synthetic bone. Our hypothesis is Acoustic-Emission waves can be detected through standard orthopaedic screwdrivers while advancing screws through purchase and overtightening in cancellous human bone with different bone mineral densities replicating the clinical state.Methods77 non-locking 4 mm and 6.5 mm diameter cancellous bone screws were inserted through to stripping into the lateral condylar area of 6 pairs of embalmed distal femurs. Specimens had varying degrees of bone mineral density determined by quantitative CT. Acoustic-Emission energy and axial force were detected for each test.ResultsThe tests showed a significant high correlation between bone mineral density and Acoustic-Emission energy with R = 0.74. A linear regression model with the mean stripping load as the dependent variable and mean Acoustic-Emission energy, bone mineral densities and screw size as the independent variables resulted in r² = 0.94.InterpretationThis experiment succeeded in testing real time Acoustic-Emission monitoring of screw purchase and overtightening in human bone. Acoustic-Emission energy and axial compressive force have positive high correlation to bone mineral density. The purpose is to develop a known technology and apply it to improve the bone-metal construct strength by reducing human error of screw overtightening.     

In vitro evaluation of osteoblastic differentiation on amorphous calcium phosphate-decorated poly(lactic-co-glycolic acid) scaffolds

Calcium phosphate-decorated polyester matrices are promising scaffolds for bone tissue engineering that combine the tunable degradation of synthetic polymers and the osteoconductivity of calcium phosphate minerals. In this study, scaffolds decorated with stabilized amorphous calcium phosphate (ACP) minerals-which exhibit sustained dissolution and release of calcium and phosphate ions-were tested for their ability to support osteoblast proliferation and stimulate differentiation. The two ACPs tested were zirconia-hybridized ACP (ZrACP), which releases ions over a few days and converts in aqueous solution to hydroxyapatite (HAP), and zinc-hybridized ACP (ZnACP), which has a longer period of sustained ion release. MC3T3-E1 pre-osteoblasts were cultured in these scaffolds for up to 21 days, and cell number, alkaline phosphatase (ALP) activity and expression of osteogenic and bone-specific proteins were measured. Cell number, prostaglandin E(2) (PGE(2) ) synthesis and osteopontin (OPN) mRNA expression were elevated on calcium phosphate-decorated scaffolds relative to PLGA controls, while mRNA expression of osteocalcin (OCN), bone sialoprotein and bone morphogenetic protein (BMP)-4 were suppressed. Although MC3T3-E1 responses to the two ACPs were not statistically different, ZrACP-which converts more quickly to HAP-gave rise to slightly higher levels of mRNA expression for BMP-4, osterix, vascular endothelial growth factor (VEGF)-A, OCN and OPN, but slightly lower levels of PGE(2) synthesis, ALP activity and cell number. These results indicate that sintered PLGA microsphere scaffolds decorated with 0.5 wt% ZnACP or ZrACP support cell attachment and elicit a series of biological responses, but these responses do not appear to accelerate osteoblast differentiation.     

Presence of intracisternal type A particles in normal bone tissue from various mouse strains.

Intracisternal type A particles were detected by electron microscopy in cells of normal trabecular bone tissue from the mouse strains C57BL, NMRI, C3H, 101, and (C3H X 101)F1. The relatively small numbers of particles observed in all five strains were roughly equivalent. About 10% of the bone cell profiles examined in specimens from each animal contained a single particle. The intracisternal type A particles occurred predominantly in osteoblasts and rarely in osteoprogenitor cells and osteocytes. They were not found in osteoclasts.