Expression of bone sialoprotein and osteopontin in breast cancer bone metastases

Bone sialoprotein (BSP) and osteopontin (OPN) are prominent, mineral-associated proteins in the extracellular matrix of bone that have been implicated in the metastatic activity of cancer cells. The expression of BSP, which is normally restricted to mineralizing tissues, has been observed in cancers with a high propensity for forming bone metastases. To investigate the relationship between BSP expression and the formation of bone metastases we have conducted an initial study of the expression of BSP in 10 intraductal breast carcinoma bone metastases using immunostaining and in situ hybridization, and compared the expression with OPN. The metastases were characterized by the infiltration of tumour cells into bone with extensive bone resorption evident. Moderate to strong staining for BSP was observed in all (100%) carcinomas, which also expressed BSP mRNA as determined by in situ hybridization. Variable staining for BSP was also observed in the mineralized bone and expression of BSP mRNA could be observed in osteoblastic cells on the bone surface and in some osteocytes at sites of bone remodelling. Contrary to a previous report, BSP expression could be demonstrated by PCR in three breast cancer cell lines, MCF-7, T47-D and MDA-MB-231. Moreover, in sub-cutaneous tumours formed by MDA-MB-231 breast cancer cells injected into athymic mice, higher immunostaining for BSP was seen in large ulcerating tumours in which mineral deposits were formed. In contrast to BSP, staining for OPN in bone metastases was generally restricted to the interface between tumor cells and bone surface of the carcinomas. While OPN staining was also observed in the cytoplasm of osteoclasts, which showed strong hybridization to a digoxygenin-labelled OPN cRNA probe, expression of OPN was not clearly detectable in the tumour cells. These studies provide the first demonstration of BSP expression by tumour cells in bone metastases and support the concept that BSP may have a role in targeting metastatic cells to bone. Expression of OPN in bone metastases appears to be related to increased bone resorptive activity by osteoclasts. This revised version was published online in July 2006 with corrections to the Cover Date.     

Dissolution of synthetic hydroxyapatite in the presence of acidic polypeptides.

This article deals with the effect of two acidic polypeptides [polyaspartic acid (PA) and polyglutamic acid (PG)] onto hydroxyapatite (HAP) dissolution by separately considering their influence when they are present only at the HAP interface and when they are both adsorbed and present in the bulk solution. We first determined the amount of adsorbed PA and PG at pH 7.0 and 5.0 onto 10 mg of HAP. Dissolution experiments were performed at pH 5.0 under pH stat conditions by continuously following the consumed protons and released calcium versus time with the aid of specific electrodes. The released phosphate ions were determined by spectrophotometric analysis. The data show that, because of their calcium chelating properties, the polypeptides act as a driving force for HAP dissolution when PA and PG remain present in solution and the interfacial beneficial effect of the adsorbed peptides is erased by the chelating properties of PA and PG present in the solution. When the polypeptides are only adsorbed at the interface, even if a partial PA or PG desorption occurs, HAP dissolution inhibition is still observed.     

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.     

Correlation between calcification and bone sialoprotein and osteopontin in papillary thyroid carcinoma

The correlation between calcification and papillary thyroid carcinoma has received increasing attention. We investigated the ability of bone sialoprotein (BSP) and osteopontin (OPN) protein levels to diagnose papillary thyroid carcinoma (PTC), and explored the correlation between BSP and OPN protein levels and calcification in PTC. Archival PTC specimens from patients with PTC with calcification and lateral cervical lymph node metastasis (LNM) were included in this retrospective immunohistochemical study. The protein levels of BSP and OPN were analysed immunohistochemically using routinely prepared tissue sections. PTC specimens from 66 patients with PTC were reviewed retrospectively (25 patients with histological calcification seen in paraffin sections, 41 patients without calcification; 35 patients with lateral cervical LNM, 31 patients without LNM). The percentage of samples that had cells that demonstrated positive protein staining differed significantly between PTC specimens, benign thyroid nodules, and adjacent normal follicular epithelium (BSP: 87.88%, 55.00%, and 42.50%, respectively; OPN: 83.33%, 70.00% and 50.00%, respectively). There was a significant difference in the immunohistochemical score (IHS) for BSP and OPN protein staining between PTC specimens with and without calcification (P < 0.05). The level of BSP protein staining was found to be significantly correlated with the level of OPN protein staining in PTC specimens. We conclude that the strong correlation between BSP and OPN and PTC suggests a role for BSP and OPN in calcification and tumor progression of PTC. BSP and OPN might be useful tumour markers for the diagnosis of PTC with limited value, because both of them had low specificity.     

Adhesion of MC3T3-E1 cells to bone sialoprotein and bone osteopontin specifically bound to collagen I

Bone sialoprotein (BSP) and bone osteopontin (OPN) are members of the SIBLING (small integrin-binding ligand, N-linked glycoproteins) family of proteins commonly found in mineralized tissues. Previously, OPN was shown to exhibit a preferential orientation for MC3T3-E1 cell adhesion when it was specifically bound to collagen. In this work, the orientation of BSP under similar circumstances is examined and compared with OPN. Radiolabeled adsorption isotherms were obtained for BSP bound to both tissue culture polystyrene (TCPS) and collagen-coated TCPS. The results show that collagen has the capacity to bind almost twice as much OPN under identical conditions. An in vitro MC3T3-E1 cell adhesion assay was then performed to compare the cell binding ability of BSP on either TCPS or collagen-coated TCPS with identical amounts of adsorbed protein. It was found that there is no significant difference in the cell binding ability of BSP on either of the substrates. For cell binding studies on collagen-coated TCPS, it was shown that there are a greater number of cells bound to substrates with adsorbed OPN as compared with BSP. The preferable orientation of OPN for cell binding coupled with the higher binding capability of collagen for OPN indicates that OPN is more important than BSP for osteoblast adhesion to the collagen matrix. In addition, a cell inhibition assay was performed to show that all of the cell binding that occurred throughout these studies was dependent upon integrin interactions with the RGD cell binding moiety.     

Nanotexturing of titanium-based surfaces upregulates expression of bone sialoprotein and osteopontin by cultured osteogenic cells

Bone formation around implants is influenced by surface geometry. Since cell/matrix/substrate interactions associated with cell signaling occur in the nanoscale dimension, we have evaluated the influence of nanotexturing of titanium-based surfaces on the expression of matrix proteins by cultured osteogenic cells at initial time points. Cells were obtained by enzymatic digestion of newborn rat calvaria and grown on titanium and titanium alloy discs with nanotextured or machined surfaces, and on glass coverslips for periods of 6 h, 1 day, and 3 days, under standard culture conditions. Cultures were processed for single or dual immunolabeling with monoclonal and/or polyclonal antibodies against bone sialoprotein (BSP), fibronectin (FN), osteopontin (OPN), type-I pro-collagen, or tubulin, followed by corresponding fluorophore-conjugated secondary antibodies. Some samples were processed for scanning electron microscope analysis of morphology and immunogold labeling. After 6 h, nanotextured surfaces exhibited up to a nine-fold increase in the proportion of cells with peripheral OPN labeling. At day 3, the proportion of OPN and BSP labeled cells was higher, and the intensity of immunoreactivity dramatically increased. No significant differences were observed in the expression pattern and the proportion of cells immunoreactive for FN or type-I pro-collagen. Our results demonstrate that nanotexturing of titanium-based surfaces upregulates the early expression of BSP and OPN in osteogenic cell cultures.     

Setting properties of bone cement with added synthetic hydroxyapatite

Synthetic hydroxyapatite powder was added to commercial bone cement to improve its physico-mechanical properties. The study of the setting properties showed the considerable effect of hydroxyapatite (HA) on the polymerization process; in particular statistical treatment of the experimental results revealed a significant relative minimum of the exothermic peak value at HA 5% weight percentage--an effect attributed to the HA 'crumbling' action on the microporosity features. The relationship between the interface (air and monomer bubbles/acrylic resin) versus dough composition was evaluated: the comparison between this last one and the corresponding internal energy variation supports the above hypothesis.     

Reaction of bone to HA, carbonate-HA, hydroxyapatite + calcium orthophosphate and to hydroxyapatite + calcium ortho- and pyrophosphate.

Slip cast conical implants of HA, carbonate-HA, calcium orthophosphate/hydroxyapatite and hydroxyapatite/calcium pyrophosphate/calcium orthophosphate with weight ratios 75/25 and 50/30/20 were implanted in rabbit tibia. The bone-implant interfaces were evaluated histologically, by means of SEM/EDX analysis and by push-out test. Thirty-six implanted samples were investigated after 2, 8, and 24 weeks. The triphasic calcium phosphate showed a moderate disintegration. This material showed 2 weeks after implantation a bonding between new bone and implant could be seen in parts of the operation site by SEM. Eight weeks after implantation an intimate relationship between the bone tissue and all specimens of each material was found. After 24 weeks the Ca/P ratio in the bone had reached the Ca/P ratio of mature bone determined by SEM/EDX analysis.     

Compressive properties of polymer coated synthetic hydroxyapatite for bone grafting

A porous hydroxyapatite material hydrothermally converted from the calcium carbonate exoskeleton of the coral, genus Goniopora (CHAG) was either microcoated using polymethylmethacrylate (PMMA) or polylactic acid (PLA) to cover all internal surfaces, or externally coated to produce a shell, with the objective of reducing the brittleness of the material. Compressive testing showed that while CHAG, externally coated with PMMA, showed the largest increases in strength, stiffness, and energy absorption, the uncoated hydroxyapatite core cracked at low loads although the shell remained intact. CHAG internally microcoated with PMMA showed a 3.84 increase in compressive strength while specimens with internal PLA coatings had 1.81 times the compressive strength of uncoated CHAG specimens. Compared to the mechanical properties of cancellous graft material, specimens internally microcoated with either polymer could be produced having properties equivalent to or greater than those of cancellous graft.     

Osteoclast adhesion and activity on synthetic hydroxyapatite, carbonated hydroxyapatite, and natural calcium carbonate: relationship to surface energies.

This study investigates the adhesion, cytoskeletal changes, and resorptive activity of disaggregated rat osteoclasts cultured on polished slices of three biomaterials: crystalline synthetic hydroxyapatite (HA), carbonated hydroxyapatite (C-HA), and natural calcium carbonate (C). The surface chemistry of each substrate was defined by X-ray diffraction and IR spectroscopy, surface wettability by the dispersive, and the polar components of the surface energies. Osteoclast adhesion was modulated by the polar component of the surface energy: fewer (p < 0.01) osteoclasts adhered to C-HA (97 +/- 20/slice, surface energy 9 +/- 5 mJ/m2) than to HA (234 +/- 16/slice, surface energy 44 +/- 2 mJ/m2) or to C (268 +/- 37/slice, surface energy 58 +/- 0.5 mJ/m2). Actin rings, which are the cytoskeletal structure essential for resorption, developed on all three materials. The area of the actin ring, which is resorbed by local acidification, and the osteoclast area, which reflects osteoclast spreading, were both greater in osteoclasts cultured on HA and C-HA than in those cultured on C. C was resorbed, but HA and C-HA were not. Thus, the surface energy plays an essential role in osteoclast adhesion, whereas osteoclast spreading may depend on the surface chemistry, especially on protein adsorption and/or on newly formed apatite layers. Resorption may be limited to the solubility of the biomaterial.     

New polymer syntheses, 3. Binding of nucleosides to basic polypeptides via isocyanato-isothiocyanates

New aliphatic α-isocyanato-ω-isothiocyanates of various chain lengths were synthesized from ω-isothiocyanatocarboxylic acid chlorides and trimethylsilyl azide. The 5′-OH group of 2′-O,3′-O-isopropylideneuridine and of N⁶-benzoyl-2′-O,3′-O-isopropylideneadenosine was added selectively onto the isocyanate group of aromatic and aliphatic α-isocyanato-ω-isothiocyanates. The isothiocyanate group of the resulting nucleoside derivatives was then reacted with the free amino groups of polylysine, isopolylysine, polyornithine and isopolyornithine. The resulting polypeptides with pending nucleosides were characterized by elemental analyses and 360 MHz ¹H NMR spectra. A new “one-pot-procedure” was developed to protect the primary amino group (N⁶) of 2′-O,3′-O-isopropylideneadenosine.     

Theoretical study of bone sialoprotein in bone biomineralization

Bone sialoprotein (BSP) is an acidic, non-collagenous protein specific to bone proposed previously to promote hydroxyapatite (HAP) nucleation and modulate HAP nanocrystal growth. Specifically, two phosphorylated acidic amino acid sequences in BSP, highly conserved across several vertebrates, are the proposed active sites. We selected one of these sites, i.e. (Sp)(2)E(8), where Sp represents a phosphoserine as a model peptide to study the role of BSP. We used molecular dynamics simulations to determine whether an α-helix or a random coil peptide conformation promotes templated HAP nucleation. A bioinformatics method helps infer preferential crystal growth directions by predicting the likely peptide conformations adsorbed on the (001), (100), and (110) crystal faces of HAP. Results suggest that, independent of conformation, no stable nucleating template is formed and, thus, the ion distributions in the vicinity of the peptide that eventually lead to a stable nucleus start out with disordered arrangements of ions. When adsorbed on all three faces, the Sp residues bind strongly regardless of the peptide conformation, and the Glu residues show different propensities to form helical conformations. The lack of geometrical templating between the peptide residues and all HAP surface sites indicates that adsorption and subsequent crystal growth modulation may be structurally nonspecific.     

Compressive properties of cancellous bone defects in a rabbit model treated with particles of natural bone mineral and synthetic hydroxyapatite.

A rabbit model was developed to evaluate the compressive mechanical properties of cancellous bone defects treated with particles of selected bone graft substitute materials. A novel feature of the model was the precise retrieval of the site of implantation. A notable finding was a 9-fold increase in the modulus of elasticity of the defect implanted with a synthetic hydroxyapatite material after 26 weeks when compared to the modulus of the trabecular bone normally at the site. The compressive modulus of lesions treated with particles of a natural bovine bone mineral (anorganic bovine bone) was closer to the normal modulus of the cancellous bone at the site. While the compressive strength of the anorganic bone particles was less than that of normal bone, the site implanted with the bone mineral particles achieved compressive strength greater than normal after 6 weeks. Moreover, the anorganic bone particles accelerated the increase in strength of the lesion, at 6 weeks exceeding the strength achieved by the untreated defect after 26 weeks. The potential problem associated with the disparity in the compressive modulus between sites implanted with the synthetic HA particles and surrounding bone is discussed.     

Antigenicity and tolerance of some synthetic polypeptides related to collagen

Guinea-pigs immunized with poly-L-proline, poly-L-proline—glycine or poly-L-proline—glycine—acetyl-hydroxy-L-proline develop antibodies which appear to be exclusively directed against determinants composed of sequences of proline.

(2) All three antigens also induce delayed hypersensitivity but the determinants involved include both glycine- and acetyl-hydroxyproline when the antigen is poly-proline—glycine—acetyl-hydroxyproline and glycine when the antigen is poly-proline—glycine.

(3) The administration of either polyproline or poly-proline—glycine in incomplete adjuvant produces a state of tolerance as revealed by subsequent injection of the peptides with Freund's complete adjuvant.

(4) Tolerance induced with poly-proline cannot be broken by immunization with poly-proline—glycine with regard to circulating antibody but is broken with regard to delayed hypersensitivity.

     

Tolerance to thymus-independent antigens. Characteristics of induction of tolerance to thymus-independent synthetic polypeptides

The conditions have been studied for induction of tolerance in mice to two thymus-independent multichain synthetic polypeptides, poly(DTyr,DGlu)-polyDPro--polyDLys, and poly(DTyr,DGlu)-polyLPro--polyLLys. The antigens are of very similar structure, differing only in the optical configuration of proline and lysine residues.The tolerance thresholds were established by giving a single injection of antigen in solution in saline, followed later by challenge of antigen in adjuvant. For both antigens it was possible to induce high dose tolerance, which was preceded by a zone of priming at lower doses. In neither case was low zone tolerance observed. The induction of high zone tolerance was accompanied by a passing phase of antibody production in the case of poly(DTyr,DGlu)-polyDPro--polyDLys, but not in the case of poly(DTyr,DGlu)-polyLPro--polyLLys. Furthermore, the threshold doses for high zone tolerance for the two antigens differed by two orders of magnitude, being at about 100 μg per mouse for the former and at 1 μg per mouse for the latter. It appears that thymus-independent antigens, even if very similar structurally, can differ very markedly in the characteristics of their interaction with B cells.     

Immune responses to some proteins and synthetic polypeptides in inbred strains of rats.

The antibody responses to DNP-bovine gamma globulin, bovine gamma globulin, human serum albumin, ovalbumin, pepsin and five synthetic polypeptides were examined in strains of inbred rats representative of eight common major histocompatibility complex (RTI) haplotypes. With each antigen the antibody response varied considerably among strains, and the data provide many potential strain and antigen combinations with which to study the genetic control of the immune response.     

Binding affinity of surface functionalized gold nanoparticles to hydroxyapatite

Gold nanoparticles (Au NPs) have been investigated for a number of biomedical applications, including drug and gene delivery vehicles, thermal ablation therapy, diagnostic sensors, and imaging contrast agents. Surface functionalization with molecular groups exhibiting calcium affinity can enable targeted delivery of Au NPs to calcified tissue, including damaged bone tissue. Therefore, the objective of this study was to investigate the binding affinity of functionalized Au NPs for targeted delivery to bone mineral, using hydroxyapatite (HA) crystals as a synthetic analog in vitro. Au NPs were synthesized to a mean particle size of 10-15 nm and surface functionalized with either L-glutamic acid, 2-aminoethylphosphonic acid, or alendronate, which exhibit a primary amine for binding gold opposite carboxylate, phosphonate, or bisphosphonate groups, respectively, for targeting calcium. Bisphosphonate functionalized Au NPs exhibited the most rapid binding kinetics and greatest binding affinity to HA, followed by glutamic acid and phosphonic acid. All functional groups reached complete binding after 24 h. Equilibrium binding constants in de-ionized water, determined by nonlinear regression of Langmuir isotherms, were 3.40, 0.69, and 0.25 mg/L for bisphosphonate, carboxylate, and phosphonate functionalized Au NPs, respectively. Functionalized Au NPs exhibited lower overall binding in fetal bovine serum compared to de-ionized water, but relative differences between functional groups were similar.     

利用人源化噬菌体抗体库筛选骨唾液酸蛋白的单链抗体

目的:从人源化噬菌体抗体库中筛选高亲和、特异结合人骨唾液酸蛋白(BsP)的人源可变区单链抗体(scFv).方法:采用噬菌体表面展示系统,以重组的BSP蛋白为包被抗原,从噬菌体可变区scFv中筛选特异性结合BSP的小分子scFv,经过3轮亲和富集筛选后,再用ELISA方法进一步鉴定与抗原BSP有特异结合活性的scFv阳性克隆,PCR扩增鉴定阳性克隆的插入轻、重链基因片段,并对阳性scFv分子测序和序列分析.结果:筛选得到的scFv片段可以特异地结合BSP蛋白,PCR扩增都得到了长为368 bp、527 bp和935bp的轻链、重链和轻链-连接片段-重链的基因片段,测序结果分析发现上述scFv片段在轻链有11处的氨基酸组成不同,在重链区域氨基酸组成则有3处不同.基因序列分析结果表明符合人源单链可变区抗体基因序列的结构特征.结论:利用噬菌体抗体库技术成功获得BSP蛋白的特异性人源单链可变区抗体.