Generation and Use of Recombinant Human Bone Sialoprotein and Osteopontin for Hydroxyapatite Studies

Bone sialoprotein (BSP) and osteopontin (OPN) are two extracellular bone matrix proteins that have the ability to modulate the growth of hydroxyapatite in vitro. Studies of BSP/OPN hydroxyapatite interactions in the past have been directed toward the identification of essential structural elements that allow these two proteins to modulate hydroxyapatite growth. However, these studies are limited by the finite quantities of purified extracellular matrix proteins. I have utilized a recombinant E. coli expression system to obtain milligram quantities of human bone sialoprotein and human osteopontin that may be used to study extracellular matrix protein interactions with hydroxyapatite.     

Binding of Bone Sialoprotein,Osteopontin and Synthetic Polypeptides to Hydroxyapatite

The phosphorylated acidic glycoproteins bone sialoprotein (BSP) and osteopontin (OPN) bind to hydroxyapatite (HA) crystals and may be involved in the regulation of bone mineralization. The HA-binding properties of these proteins have been attributed to glutamic acid-rich sequences in BSP and aspartic acid-rich sequences in OPN. The present study examines the roles of these polycarboxylate sequences in the binding of BSP and OPN to HA.

Porcine BSP, OPN and the synthetic polypeptides poly-L-glutamic acid [Poly(Glu)] and poly-L-aspartic acid [Poly(Asp)] were labeled with fluorescein isothiocyanate and their binding to HA determined by fluorimetry. From the binding isotherms, dissociation constants (K_Ds) for all the reagents tested were determined to be in the micromolar range. The saturation binding capacities of HA for Poly(Glu), Poly(Asp), BSP and OPN were similar (500–600 μg/m²). To investigate the role of glutamic acid-rich and aspartic acid-rich sequences in the binding to HA of BSP and OPN, respectively, competitive binding studies with Poly(Glu) and Poly(Asp) were performed. Poly(Glu) was able to displace a maximum of 100% of Poly(GIu), 81% of OPN, 68% of BSP and 65% of Poly(Asp). Poly(Asp) was able to displace a maximum of 100% of Poly(Glu), 99% of Poly(Asp), 95% of OPN and 89% of BSP. These results are consistent with the view that BSP and OPN bind to HA via their polycarboxylate sequences, but suggest a complex mode of interaction between polyelectrolytes and ionic crystals.     

Role of Two Mineral-Associated Adhesion Molecules,Osteopontin and Bone Sialoprotein,during Cementogenesis

Adhesion molecules and their cell membrane receptors are known to play important regulatory roles in cell differentiation. Consequently, the following experiments were conducted to determine the role of two adhesion molecules, bone sialoprotein (BSP) and osteopontin (OPN) in tooth root formation. Developing murine molar tooth germs at sequential stages of development (developmental days 21–42) were analyzed using immunohistochemical and in situ hybridization techniques. While BSP was localized to alveolar bone and odontoblasts early in development, BSP was distinctly localized to the cemental root surface at latter periods coincident with the initiation of root formation and cementogenesis. Conversely, OPN was distributed in a nonspecific fashion throughout the PDL and the eruption pathway of the forming tooth. In situ hybridization confirmed that cells lining the root surface express BSP. The fact that BSP is specifically localized to the cemental surface suggests that this protein is involved in cementoblast differentiation and/or early mineralization of the cementum matrix. Localization of OPN to non-mineralized tissues further suggests that OPN functions as an inhibitor of mineralization during periodontal ligament formation. These findings collectively suggest that BSP and OPN are intimately involved in the sequence of cellular and molecular events accompanying cementogenesis.     

人牙髓细胞骨基质蛋白表达的研究进展

骨基质蛋白在骨骼和牙齿形成，以及损伤修复过程中起着重要作用。本对骨涎蛋白(BSP)、骨桥蛋白(OPN)、牙本质涎蛋白(DSP)、牙本质磷蛋白(DPP)、牙本质涎磷蛋白(DSPP)的表达，以及在牙齿形成、损伤修复，对组织工程中成骨组织作用等研究进展进行了概述。     

纳米羟基磷灰石人工骨修复骨缺损的再血管化研究

目的通过动物实验探讨多孔纳米羟基磷灰石人工骨（Nano-HA）的骨缺损修复作用并观察骨重建过程中再血管化过程。方法采用新西兰大白兔24只在单侧桡骨制备骨缺损动物模型，然后用纳米羟基磷灰石人工骨材料进行植入骨缺损处进行修复作为实验组，以普通羟基磷灰石人工骨材料作为对照组；术后2，4，8和12周分别行钟^99mTc-亚甲基二膦酸盐（MDP）放射性核素骨显像监测在骨缺损修复中再血管化的过程，并以x线、组织学检查作比较。结果通过ROI方法定量对比两种材料植入区和正常区放射性浓集比值，Nano-HA人工骨组成骨代谢明显优于HA人工骨组，骨缺损修复能力明显优于后者，差异有统计学意义（P〈0．05）。结论纳米羟基磷灰石人工骨具有优异的成骨能力，可望成为新型的骨缺损修复材料，放射性核素骨显像是监测骨修复再血管化过程敏感、直观、可靠的方法。     

Protein Kinases of Cultured Chicken Osteoblasts That Phosphorylate Extracellular Bone Proteins

Cytosolic and microsomal protein kinase preparations from cultured chicken osteoblasts were found to phosphorylate up to six major proteins with M_rS 66, 58, 50, 36, 32, and 22 kDa in chicken bone extract. Use of heparin led to the conclusion that these proteins were predominantly phosphorylated by factor-independent protein kinase (FIPK) present both in microsomal and cytosolic preparations. It was confirmed that microsomal preparation contained predominantly FIPK, whereas cytosolic preparation contained additional kinases, that can phosphorylate the bone proteins. Use of purified chicken bone osteopontin (OPN) (58 kDa) and recombinant OPN led to the same conclusions. The identify of the protein kinases was clearly established by using a series of synthetic peptide substrates. Quantitative analysis utilizing pure protein kinases and purified chicken bone OPN, recombinant mouse OPN, and bovine bone OPN and BSP led to introduction of -9 moles of phosphate/mole of OPN and 6.6 moles phosphate/mole bovine bone sialo-protein (BSP) by casein kinase II. cGMP-dependent protein kinase and protein kinase C both introduced 0.5–1.2 moles phosphate/mole of OPN and BSP, whereas cAMP-dependent protein kinase led to no significant phosphorylation of OPN or BSP. Consistent with the above results, sites of phosphorylation identified for OPN (metabol-ically labeled) and BSP (labeled by casein kinase II) revealed that predominant phosphorylated sites have recognition sequences for FIPK.     

Differential expression of osteopontin and bone sialoprotein in bone metastasis of breast and prostate carcinoma

Breast and prostate cancer often metastasise to the skeleton. Interestingly, the histopathological characteristics of the bone lesions that arise from these two cancer types differ. Breast tumours give rise to metastases in the skeleton with a mixed lytic/sclerotic pattern, whereas a predominantly sclerotic pattern is seen in metastases from prostate tumours. Osteopontin (OPN) and bone sialoprotein (BSP) are bone matrix proteins that have been implicated in the selective affinity of cancer cells for bone. In the present study, 21 patient cases with skeletal metastasis and their respective primary tumours (12 with breast cancer, 9 with prostate cancer) were investigated by immunohistochemistry in order to assess the level of OPN and BSP. Moderate to strong OPN expression was found in 42% of all breast tumours and in 56% of all prostate tumours. Significantly more breast cancer bone metastases exhibited high OPN expression, 83%, as compared with prostate tumour bone metastases, 11% (P=0.0019). In contrast, moderate to strong BSP expression was found in 33% of breast tumours and in 89% of prostate tumours. In the bone lesions, only 33% of breast tumour metastases showed moderate/strong BSP expression compared to 100% of prostate tumour metastases (P=0.0046). This divergent pattern of OPN/BSP expression could be an important determinant for the different characteristics of these two types of bone metastasis, i.e., lytic vs. sclerotic, consistent with the proposed role of OPN in differentiation and activation of osteoclasts and of BSP as a stimulator of bone mineralisation. This revised version was published online in July 2006 with corrections to the Cover Date.     

In Vivo and In Vitro Phosphorylation Regions of Bone Sialoprotein

The present study for the first time evaluated both the in vitro and in vivo phosphorylation regions of bone sialoprotein (BSP) by utilizing multiple approaches and techniques. The in vitro phosphorylation sites were determined by 32 P-labeling of native BSP using purified casein kinase II (CKII), followed by peptide mapping and solid-phase N-terminal sequence analyses. The in vivo phosphorylation sites were determined by (i) derivatization with 1-S-[ 14 C]carboxymethyl-dithiothreitol ([ 14 C] CM-DTT) of the proteolytic digests of BSP, followed by isolation and N-terminal peptide sequence analysis; and (ii) analyzing the proteolytic peptides of native BSP using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Native BSP incorporated ~2.5 mol of phosphate/mol of BSP by CKII, which were distributed over four major peptide peaks and three shoulder peaks within the peptide map with varying degrees of phosphorylation. Further studies using the [ 14 C] CM-DTT thiol reagent indicated that native and deglycosylated BSP incorporated 5.84 and 5.80 mol of 14 C/mol of BSP, respectively. This confirmed that there were ~5.8 mol P-Ser/mol of BSP naturally (in vivo) occurring phosphorylation sites and that there was no overlap between the phosphorylation and glycosylation sites. The 5.8 mol P-Ser/mol BSP reflects the total number of mols of naturally occurring phosphorylation, phosphorylated in vivo by CKII (4.1 mol), protein kinase C (0.9 mol), and cGMP-dependent kinase (0.8 mol). Peptide N-terminal sequence analyses of both in vitro ( 32 P) and in vivo ( 14 C) phosphorylated peptides indicated that the phosphorylated residues were predominantly on the N-terminal half of the protein that included recognition sequences for CKII, e.g., LESDEENGVFK (residues 12-22).     

聚-DL-乳酸膜引导兔桡骨缺损再生的实验研究

本实验旨在观察可降解聚 - DL-乳酸膜引导兔桡骨缺损再生的现象 ,并探讨其作用和机制。取 4 0只成年新西兰大白兔建立双侧桡骨缺损模型。左侧为实验侧 ,以聚 - DL-乳酸膜卷成管状桥接骨缺损 ;右侧为对照侧 ,桡骨缺损不做处理。术后 2、4、8、12周分别处死动物 ,行大体观察、X线摄片、组织学观察和骨生物力学检测。实验侧术后 2周 ,可见隔膜管两端已为软组织覆盖 ,膜管外两端可见多量新生骨痂形成 ,膜管内骨断端间充满与膜管形状相适应的血肿和纤维骨痂。术后 12周膜管颜色明显变白 ,仍然保持原有外形无塌陷 ,膜管外骨痂已基本消失 ,膜管内骨缺损均已骨性愈合。对照侧术后 2周骨缺损区被大量的结缔组织充填 ,12周时无 1例愈合 ,骨断端均已封闭而形成典型的骨不连。对照侧均不能进行力学测试 ,实验侧 12周组新生骨的生物力学指标明显优于 8周组 (P<0 .0 5 )。因此 ,聚 - DL-乳酸膜能够成功引导骨再生 ,可以用于临床治疗骨缺损和骨不连。     

豌豆骨移位代月骨的应用解剖

在15只成年上肢标本和57套腕骨标本上观察了豌豆骨的形态和血供。豌豆骨的骨内、外血管网发达,吻合丰富,有3～5支营养血管。以尺侧腕屈肌腱为蒂进行移位代替月骨,血供有保证。豌豆骨的纵径刚好与月骨的横径相近,故将纵置的豌豆骨改为横置方位:适当保留豌豆骨周围部分软组织,可以填补月骨空间,又能保存骨周围血管网。     

羟基磷灰石基人工骨的研究进展

目的 研究羟基磷灰石基人工骨作为最有发展前途的生物硬组织替代材料之一，在生物医用材料和医学研究领域的应用。方法制备具有良好生物相容性、力学性能、骨诱导性与治疗功能的羟基磷灰石基人工骨，用于人体骨组织的缺失的修复，及相关疾病的治疗。结果通过制备性能优良的羟基磷灰石粉体，结合纳米技术、组织工程技术和生物技术将不同材料通过合适的方法组合成羟摹磷灰石人工骨，从而获得良好的临床应用性能。结论从临床角度，综合了国内外近十年相关文献的基础上，评述了羟基磷灰石人工骨的研究进展。     

羟基磷灰石涂层与骨组织界面生物相容性与毒理学研究

依据卫生部新近制定的《生物材料和制品的生物学评价标准》(简称《标准》),确定五项评价项目,按照标准的生物学试验方法,对目前临床应用的羟基磷灰石制品进行系统研究。结果表明：国产羟基磷灰石制品除有轻微细胞毒性外,其致敏性、溶血性、致突变性和组织相容性均符合《标准》要求,临床应用安全。     

骨移植体及骨移植替代物在体内的血管化

探讨了骨移植体及骨移植替代物在体内血管化的机理、意义及过程,以更好地促进骨缺损移植修复的效果.通过对近期有关文献所作相关研究的综述,总结出各类骨移植体及骨移植替代物体内血管化的特点及影响因素.骨移植体及骨移植替代物体内血管化过程是移植手段能够奏效的重要保障.组织工程化人工骨作为一种日益重要的骨移植替代物,进一步提高其在体内的血管化进程,是更好发挥其生命活力的关键.     

In situ hybridization and immunohistochemistry of bone sialoprotein and secreted phosphoprotein 1 (osteopontin) in the developing mouse mandibular condylar cartilage compared with limb bud cartilage

Mandibular condylar cartilage is often classified as a secondary cartilage, differing from the primary cartilaginous skeleton in its rapid progress from progenitor cells to hypertrophic chondrocytes. In this study we used in situ hybridization and immunohistochemistry to investigate whether the formation of primary (tibial) and secondary (condylar) cartilage also differs with respect to the expression of two major non‐collagenous glycoproteins of bone matrix, bone sialoprotein (BSP) and secreted phosphoprotein 1 (Spp1, osteopontin). The mRNAs for both molecules were never expressed until hypertrophic chondrocytes appeared. In the tibial cartilage, hypertrophic chondrocytes first appeared at E14 and the expression of BSP and Spp1 mRNAs was detected in the lower hypertrophic cell zone, but the expression of BSP mRNA was very weak. In the condylar cartilage, hypertrophic chondrocytes appeared at E15 as soon as cartilage tissue appeared. The mRNAs for both molecules were expressed in the newly formed condylar cartilage, although the proteins were not detected by immunostaining; BSP mRNA in the condylar cartilage was more extensively expressed than that in the tibial cartilage at the corresponding stage (first appearance of hypertrophic cell zone). Endochondral bone formation started at E15 in the tibial cartilage and at E16 in the condylar cartilage. At this stage (first appearance of endochondral bone formation), BSP mRNA was also more extensively expressed in the condylar cartilage than in the tibial cartilage. The hypertrophic cell zone in the condylar cartilage rapidly extended during E15–16. These results indicate that the formation process of the mandibular condylar cartilage differs from that of limb bud cartilage with respect to the extensive expression of BSP mRNA and the rapid extension of the hypertrophic cell zone at early stages of cartilage formation. Furthermore, these results support the hypothesis that, in vivo, BSP promotes the initiation of mineralization.     

The Linkage Region in the Polypeptide of Pig Costal Cartilage Proteoglycan

Proteoglycan from pig costal cartilage and fragments obtained by proteolytic digestion were characterized by equilibrium ultracentrifugation and amino acid analysis.

The proteoglycan extractable in 4 M guanidinium chloride yielded, after proteolytic digestion with trypsin and chymotrypsin, a chondroitin sulfate peptide containing four chains of polysaccharide. The unextractable residue yielded chondroitin sulfate peptide containing only two chains. The amino acid composition indicated a fairly uniform spacing between all four chains with an average of eight amino acid residues between the serine residues involved in linkage.

Following the alkaline sulfite elimination-addition reaction, free peptide was isolated and found to contain one unsubstituted serine residue for every two linked glycosidically. Glycine and glutamic acid were the only two amino acids sufficiently abundant to be part of an invariant sequence near to serine residues destined to be glycosy-lated. The linkage region of the polypeptide also contains some substituted serine residues which do not carry a full chondroitin sulfate chain.