骨唾液酸蛋白(BSP)的结构与功能

骨唾液酸蛋白 (Bonesialoprotein ,简称BSP)是细胞外基质中的一种糖蛋白 ,主要分布在矿化组织中 ,参与骨代谢 ,但近年研究发现BSP在癌细胞的骨转移中发挥作用并对血管生成有促进作用。本文对BSP的结构与功能及研究状况作一介绍。     

Method for Histological Preparation of Bone Sections Containing Titanium Implants

A thin sectioning technique involving hand grinding has been developed to produce 20—40-μn-thick sections of bone-titanium implant sites. Components include: 1) surface staining of sections prior to mounting on slides so bone labels (oxytetracycline-HCI and 2,4-bis(N,N-dicarbometnyl) aminomethylfluorescein (DCAF)) can be seen in sections viewed with transmitted light, 2) a pneumatic sample press for bonding sections to slides with a thin, uniform glue line and without trapped air bubbles, and 3) bonding methyl methacrylate embedded sections to clear acrylic slides with methyl methacrylate monomer to provide enhanced bond strength and grinding properties compared to those obtainable with glass slides. Sample cracking and distortion is minimized and the tissue-implant interface can be kept intact The expense of start-up equipment for this technique is minimal.     

Surface Staining of Sawed Sections of Undecalcified Bone Containing Alloplastic Implants

A simple new method is described for the histological evaluation of bones containing alloplastic implants of ceramic and/or metallic materials. the undecalcified bone is embedded in acrylic resin and section at 50-200 μm using a sawing microtome. One surface of the preparation is stained up to 10 μm deep by floating the preparation on Giemsa stain. Other staining procedures are possible. Microscopic detail is Satisfactory for histological and morphometric evaluation.     

骨涎蛋白在大鼠磨牙第三期牙本质形成过程中的表达

目的:观察大鼠牙髓修复第三期牙本质形成过程中骨涎蛋白(bone sialoprotein,BSP)的表达变化及意义。方法:选取6周龄雄性Wistar大鼠10只,建立实验大鼠模型,利用免疫组化法检测大鼠第三期牙本质中骨涎蛋白的表达情况。结果:盖髓2周后,在盖髓处下方有第三期牙本质形成。与原发性牙本质(PD)相比,第三期牙本质小管数目少且形态不规则。BSP在原发性牙本质中没有表达,但在盖髓下方和髓角下第三期牙本质中都有表达。结论:BSP可能通过参与羟基磷灰石(HA)的形成以及调节新分化的成牙本质细胞向新形成的牙本质基质的粘附来参与早期的第三期牙本质的生成。     

Radiologic Evaluation of Bone Loss at Implants with Biocide Coated Titanium Abutments: A Study in the Dog

The objective of the present study is to evaluate bone loss at implant abutments coated with a soda-lime glass containing silver nanoparticles subjected to experimental peri-implantitis. Five beagle dogs were used in the experiments, 3 implants were installed in each quadrant of the mandibles. Glass/n-Ag coted abutments were connected to implant platform. Cotton floss ligatures were placed in a submarginal position around the abutment necks and the animals were subject to a diet which allowed plaque accumulation, and after 15 weeks the dogs were sacrificed. Radiographs of all implant sites were obtained at the beginning and at the end of the experimentally induced peri-implantitis. The radiographic examination indicated that significant amounts of additional bone loss occurred in implants without biocide coating, considering both absolute and relative values of bone loss. Percentages of additional bone loss observed in implants dressed with a biocide coated abutment were about 3 times lower (p<0.006 distal aspect; and p<0.031 at mesial aspect) than the control ones. Within the limits of the present study it seems promising the use of soda-lime glass/nAg coatings on abutments to prevent peri-implant diseases.     

Collagen Self-Assembly on Orthopedic Magnesium Biomaterials Surface and Subsequent Bone Cell Attachment

Magnesium (Mg) biomaterials are a new generation of biodegradable materials and have promising potential for orthopedic applications. After implantation in bone tissues, these materials will directly interact with extracellular matrix (ECM) biomolecules and bone cells. Type I collagen, the major component of bone ECM, forms the architecture scaffold that provides physical support for bone cell attachment. However, it is still unknown how Mg substrate affects collagen assembly on top of it as well as subsequent cell attachment and growth. Here, we studied the effects of collagen monomer concentration, pH, assembly time, and surface roughness of two Mg materials (pure Mg and AZ31) on collagen fibril formation. Results showed that formation of fibrils would not initiate until the monomer concentration reached a certain level depending on the type of Mg material. The thickness of collagen fibril increased with the increase of assembly time. The structures of collagen fibrils formed on semi-rough surfaces of Mg materials have a high similarity to that of native bone collagen. Next, cell attachment and growth after collagen assembly were examined. Materials with rough surface showed higher collagen adsorption but compromised bone cell attachment. Interestingly, surface roughness and collagen structure did not affect cell growth on AZ31 for up to a week. Findings from this work provide some insightful information on Mg-tissue interaction at the interface and guidance for future surface modifications of Mg biomaterials.     

Isoform-specific O-Glycosylation of Osteopontin and Bone Sialoprotein by Polypeptide N-Acetylgalactosaminyltransferase-1

Mucin-type O-glycan biosynthesis is regulated by the family of UDP-GalNAc polypeptide:N-acetylgalactosaminlytransfersases (ppGalNAcTs) that catalyzes the first step in the pathway by transferring GalNAc to Ser or Thr residues in a protein from the sugar donor UDP-GalNAc. Because not all Ser/Thr residues are glycosylated, rules must exist that signal which hydroyxamino acids acquire sugar. To date, no universal consensus signal has emerged. Therefore, strategies to deduce the subset of proteins that will be glycosylated by distinct ppGalNAcTs must be developed. Mucin-type O-glycoproteins are present abundantly in bone, where we found multiple ppGalNAcT isoforms, including ppGalNAcT-1, to be highly expressed. Thus, we compared glycoproteins expressed in wild-type and Galnt1-null mice to identify bone-associated proteins that were glycosylated in a ppGalNAcT-1-dependent manner. A reduction in the apparent molecular masses of two SIBLINGs (small integrin binding ligand N-linked glycoproteins), osteopontin (OPN) and bone sialoprotein (BSP) in the Galnt1-null mice relative to those of the wild-type was observed. Several synthetic peptides derived from OPN and BSP sequences were designed to include either known or predicted (in silico) glycosylation sites. In vitro glycosylation assays of these peptides with recombinant ppGalNAcT-1, ppGalNAcT-2, or ppGalNAcT-3 demonstrated that both SIBLINGs contained Thr/Ser residues that were preferentially glycosylated by ppGalNAcT-1. In addition, lysates prepared from wild-type, but not those from Galnt1-null derived osteoblasts, could glycosylate these peptides efficiently, suggesting that OPN and BSP contain sites that are specific for ppGalNAcT-1. Our study presents a novel and systematic approach for identification of isoform-specific substrates of the ppGalNAcT family and suggests ppGalNAcT-1 to be indispensable for O-glycosylation at specific sites of the bone glycoproteins OPN and BSP.     

A Polymethyl Methacrylate Method for Large Specimens of Mineralized Bone with Implants

A simple modified polymethyl methacrylate method is described for large mineralized bone specimens with implants and bioactive materials which produces consistently good histological preservation of the interface between bone and implant. Human femoral heads, whole rabbit condyles and canine tibias and femurs containing implants consisting of hydroxyapatite, smooth polyethylene, porous polyethylene and carbon were dehydrated in ascending grades of ethanol and cleared with xylene on an automated tissue processor which alternated vacuum and pressure for 22 hr. Infiltration was done with washed polymethyl methacrylate at 4 C under vacuum for 13 days. Polymerization was carried out in wide-mouth glass jars at 38 C for 36 hr so that the total processing time was less than 20 days. The only important modification was in the polymethyl methacrylate, which had less plasticizer than usual in order to give a harder block. This enabled production of 4 μm sections with good preservation of mineralized and cellular areas for the study of metabolic bone diseases, morphometry, fluorochrome labelling and interface analysis with the implant in situ.     

Functionalization of Titanium with Chitosan via Silanation: Evaluation of Biological and Mechanical Performances

Complications in dentistry and orthopaedic surgery are mainly induced by peri-implant bacterial infections and current implant devices do not prevent such infections. The coating of antibacterial molecules such as chitosan on its surface would give the implant bioactive properties. The major challenge of this type of coating is the attachment of chitosan to a metal substrate. In this study, we propose to investigate the functionalization of titanium with chitosan via a silanation. Firstly, the surface chemistry and mechanical properties of such coating were evaluated. We also verified if the coated chitosan retained its biocompatibility with the peri-implant cells, as well as its antibacterial properties. FTIR and Tof-SIMS analyses confirmed the presence of chitosan on the titanium surface. This coating showed great scratch resistance and was strongly adhesive to the substrate. These mechanical properties were consistent with an implantology application. The Chitosan-coated surfaces showed strong inhibition of Actinomyces naeslundii growth; they nonetheless showed a non significant inhibition against Porphyromonas gingivalis after 32 hours in liquid media. The chitosan-coating also demonstrated good biocompatibility to NIH3T3 fibroblasts. Thus this method of covalent coating provides a biocompatible material with improved bioactive properties. These results proved that covalent coating of chitosan has significant potential in biomedical device implantation.     

Skeletal Development of Mice Lacking Bone Sialoprotein (BSP) - Impairment of Long Bone Growth and Progressive Establishment of High Trabecular Bone Mass

Adult Ibsp-knockout mice (BSP−/−) display shorter stature, lower bone turnover and higher trabecular bone mass than wild type, the latter resulting from impaired bone resorption. Unexpectedly, BSP knockout also affects reproductive behavior, as female mice do not construct a proper "nest" for their offsprings. Multiple crossing experiments nonetheless indicated that the shorter stature and lower weight of BSP−/− mice, since birth and throughout life, as well as their shorter femur and tibia bones are independent of the genotype of the mothers, and thus reflect genetic inheritance. In BSP−/− newborns, µCT analysis revealed a delay in membranous primary ossification, with wider cranial sutures, as well as thinner femoral cortical bone and lower tissue mineral density, reflected in lower expression of bone formation markers. However, trabecular bone volume and osteoclast parameters of long bones do not differ between genotypes. Three weeks after birth, osteoclast number and surface drop in the mutants, concomitant with trabecular bone accumulation. The growth plates present a thinner hypertrophic zone in newborns with lower whole bone expression of IGF-1 and higher IHH in 6 days old BSP−/− mice. At 3 weeks the proliferating zone is thinner and the hypertrophic zone thicker in BSP−/− than in BSP+/+ mice of either sex, maybe reflecting a combination of lower chondrocyte proliferation and impaired cartilage resorption. Six days old BSP−/− mice display lower osteoblast marker expression but higher MEPE and higher osteopontin(Opn)/Runx2 ratio. Serum Opn is higher in mutants at day 6 and in adults. Thus, lack of BSP alters long bone growth and membranous/cortical primary bone formation and mineralization. Endochondral development is however normal in mutant mice and the accumulation of trabecular bone observed in adults develops progressively in the weeks following birth. Compensatory high Opn may allow normal endochondral development in BSP−/− mice, while impairing primary mineralization.     

Versatile Utilization of Massive Bone Allografts in Orthopedic Surgery

The clinical use of massive bone allografts in orthopaedic surgery has become common practice in tumour operations and primary and revision total joint replacement. In certain special clinical situations associated with large bone loss, such as trauma, limb-length discrepancy repair or even infection, massive bone allografts can be successfully used. We present our treatment results of 47 patients who suffered from major bone loss due either to trauma, limb-length discrepancy repair, or infection. Our results (>2 years minimum follow-up to allow full-bone allograft incorporation) indicate that the use of massive bone allografts in these special and delicate medical conditions is feasible, and have good functional results.     

Animal Modelling of Lumbar Corpectomy and Fusion and in vivo Growth of Spine Supporting Bone by Titanium Cage Implants: An Experimental Study

##正## In this study a lumbar spinal fusion animal model is established to assess the effect of spinal fusion cage,and explore theminimum area ratio of titanium cage section to vertebral section that ensures bone healing and biomechanical property.Lumbarcorpectomy was conducted by posterolateral approach with titanium cage implantation combined with plate fixation.Titaniumcages with the same length but different diameters were used.After implantation of titanium cages,the progress of bone healingwas observed and the bone biomechanical properties were measured,including deformation and displacement in axial compression,flexion,extension,and lateral bending motion.The factors affecting the in vivo growth of spine supporting body wereanalyzed.The results show that the area ratio of titanium cage section to vertebral section should reach 1/2 to ensure the bonehealing,sufficient bone intensity and biomechanical properties.Some bone healing indicators,such as BMP,suggest that there isa relationship between the peak time and the peak value of bone formation and metabolism markers and the bone healing strength.     

In Vitro Electrochemical Corrosion and Cell Viability Studies on Nickel-Free Stainless Steel Orthopedic Implants

The corrosion and cell viability behaviors of nanostructured, nickel-free stainless steel implants were studied and compared with AISI 316L. The electrochemical studies were conducted by potentiodynamic polarization and electrochemical impedance spectroscopic measurements in a simulated body fluid. Cytocompatibility was also evaluated by the adhesion behavior of adult human stem cells on the surface of the samples. According to the results, the electrochemical behavior is affected by a compromise among the specimen''s structural characteristics, comprising composition, density, and grain size. The cell viability is interpreted by considering the results of the electrochemical impedance spectroscopic experiments.     

High Bone Sialoprotein (BSP) Expression Correlates with Increased Tumor Grade and Predicts a Poorer Prognosis of High-Grade Glioma Patients

Objectives

To investigate the expression and prognostic value of bone sialoprotein (BSP) in glioma patients.

Methods

We determined the expression of BSP using real-time RT-PCR and immunohistochemistry in tissue microarrays containing 15 normal brain and 270 glioma samples. Cumulative survival was calculated by the Kaplan-Meier method and analyzed by the log-rank test. Univariate and multivariate analyses were performed by the stepwise forward Cox regression model.

Results

Both BSP mRNA and protein levels were significantly elevated in high-grade glioma tissues compared with those of normal brain and low-grade glioma tissues, and BSP expression positively correlated with tumor grade (P<0.001). Univariate and multivariate analysis showed high BSP expression was an independent prognostic factor for a shorter progression-free survival (PFS) and overall survival (OS) in both grade III and grade IV glioma patients [hazard ratio (HR) = 2.549 and 3.154 for grade III glioma, and HR = 1.637 and 1.574 for grade IV glioma, respectively]. Patients with low BSP expression had a significantly longer median OS and PFS than those with high BSP expression. Small extent of resection and lineage of astrocyte served as independent risk factors of both shorter PFS and OS in grade III glioma patients; GBM patients without O⁶-methylguanine (O⁶-meG) DNA methyltransferase (MGMT) methylation and Karnofsky performance score (KPS) less than 70 points were related to poor prognosis. Lack of radiotherapy related to shorter OS but not affect PFS in both grade III and grade IV glioma patients.

Conclusion

High BSP expression occurs in a significant subset of high-grade glioma patients and predicts a poorer outcome. The study identifies a potentially useful molecular marker for the categorization and targeted therapy of gliomas.     

高压蒸汽灭菌对钛合金内植入物的疲劳的影响

分析了制约骨折手术成功的因素,一是钛合金内植人物经受长期反复高压蒸汽灭菌造成材料疲劳引起断裂;二是AO手术的引起应力集中和疲劳断裂。提出了发展骨内固定植入物的建议。     

The Impairment of Osteogenesis in Bone Sialoprotein (BSP) Knockout Calvaria Cell Cultures Is Cell Density Dependent

Bone sialoprotein (BSP) belongs to the "small integrin-binding ligand N-linked glycoprotein" (SIBLING) family, whose members interact with bone cells and bone mineral. BSP is strongly expressed in bone and we previously showed that BSP knockout (BSP-/-) mice have a higher bone mass than wild type (BSP+/+) littermates, with lower bone remodelling. Because baseline bone formation activity is constitutively lower in BSP-/- mice, we studied the impact of the absence of BSP on in vitro osteogenesis in mouse calvaria cell (MCC) cultures. MCC BSP-/- cultures exhibit fewer fibroblast (CFU-F), preosteoblast (CFU-ALP) and osteoblast colonies (bone nodules) than wild type, indicative of a lower number of osteoprogenitors. No mineralized colonies were observed in BSP-/- cultures, along with little/no expression of either osteogenic markers or SIBLING proteins MEPE or DMP1. Osteopontin (OPN) is the only SIBLING expressed in standard density BSP-/- culture, at higher levels than in wild type in early culture times. At higher plating density, the effects of the absence of BSP were partly rescued, with resumed expression of osteoblast markers and cognate SIBLING proteins, and mineralization of the mutant cultures. OPN expression and amount are further increased in high density BSP-/- cultures, while PHEX and CatB expression are differentiatlly regulated in a manner that may favor mineralization. Altogether, we found that BSP regulates mouse calvaria osteoblast cell clonogenicity, differentiation and activity in vitro in a cell density dependent manner, consistent with the effective skeletogenesis but the low levels of bone formation observed in vivo. The BSP knockout bone microenvironment may alter the proliferation/cell fate of early osteoprogenitors.     

多孔钛合金不同孔径大小对新骨长入的影响

目的:评价不同孔径多孔钛合金植入物在骨缺损区对新骨长入的影响。方法:采用电子束熔融(EBM)技术制备三种不同孔径(孔径分别为1.0 mm,2.0 mm,3.0 mm)的多孔钛合金材料,其孔隙率依次为73%,79%,86%。将18只家犬随机分为1.0 mm孔径材料组,2.0 mm孔径材料组,3.0 mm孔径材料组,每组6只。制备家犬双侧股骨外侧髁缺损模型,然后植入各孔径组材料,于术后4周,8周,12周分别行大体标本观察,X线片观察,组织形态学观察三组不同孔径材料与周围骨的整合情况及孔隙中的新骨长入情况。结果:通过大体标本观察和X线片观察显示,12周后三组材料均与周围紧密骨连接。其中1.0 mm孔径组材料中心明显成骨,2.0 mm孔径组和3.0 mm孔径组中心仍为较多白色组织填充。组织学观察显示,12周时2.0 mm孔径组和3.0 mm孔径组材料周围有骨质包绕,但中心空洞,基本无骨质形成。1.0 mm孔径组材料周围骨质包绕紧密,孔中新生骨形成较多,且有大量纤维母细胞和软骨细胞形成。各时间点1.0 mm孔径组新生骨面积百分比明显高于2.0 mm孔径组和3.0 mm孔径组,P<0.01,差异具有统计学意义。2.0 mm孔径组和3.0 mm孔径组相比,P>0.05,无显著差异。结论:孔径大小影响多孔钛合金材料的骨长入,适当孔径的设计将更有利于材料的传导成骨。