Acid solutions enhance bone-inducing activity of a murine osteosarcoma

Heterotopic osteogenesis induced by lyophilized powder of a murine osteosarcoma was greatly enhanced by treating the powder with the following acids: 0.01 N HCl (pH 2.0), 0.1 N HCl (pH 1.7), 1 N HCl (pH 1.2), 0.5 M acetic acid (pH 2.8), 0.5 M lactic acid (pH 2.0), and 0.5 M citric acid (pH 1.8). The acid-treated preparations were neutralized before implantation into back muscles of ddY mice. Acid treatment resulted in almost a fivefold increase in the amount of new bone formation, as measured by uptake of strontium-85 into the implants. In addition bone-inducing activity was increased by longer exposure to 0.01 N HCl up to 24 h. These findings suggest that the bone-inducing activity of the osteosarcoma is enhanced by various acids below pH 2.8. A possible role for hydrogen ion concentration in the regulation of osteogenesis is discussed.     

Nature of bone morphogenetic protein (BMP) from decalcified rabbit bone matrix

Rabbit bone morphogenetic protein (BMP) from demineralized and defatted rabbit bone matrix was partially purified. BMP activity was examined by the implantation of fractionated materials into the thigh muscle pouch of the mouse. Rabbit BMP was solubilized by both 4M guanidine hydrochloride (GuHCl) and 6M urea solutions. Crude BMP had isoelectric point precipitation at pH 3 in 6M urea and showed bone morphogenesis. Fractions eluted with 0 and 0.2 N NaCl in DEAE CL-6B ion exchange chromatography showed bone morphogenesis in each individual pH of pH 4 to pH 7 but the fraction eluted with 1.0 N NaCl did not show any activity. Sephadex G-75 filtration separated the crude material into three peaks and the peak of about 23,000 showed bone morphogenesis. In sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and isoelectric focusing, rabbit BMP was thought to be an acidic protein having a molecular weight of 24,000 with an isoelectric point around 4.85.     

Solubilized bone morphogenetic protein (BMP) from mouse osteosarcoma and rat demineralized bone matrix

A selection of proteins including bone morphogenetic protein (BMP) was extracted in a disaggregated form from Dunn osteosarcoma or rat demineralized bone matrix by 4M guanidine hydrochloride (GuHCl) solution without losing its biological activity. The GuHCl extracts of Dunn osteosarcoma were divied into 4 different fractions by cesium chloride (CsCl) density gradients. Under a dissociative condition, the highest new bone yield was obtained in the low dense top one-third fraction, and BMP acitivity declined with increase in the density of each fraction. No BMP potential was observed in the surface-gel fraction under dissociative conditions. Under an associative condition (low GuHCl concentrations), BMP activity appears in the surface-gel fraction, while under a dissociative condition (high concentrations of GuHCl) BMP appears in the fraction below the surface gel. These facts suggest that under associative conditions, BMP aggregates with other low dense proteins in the surface-gel fraction and that this may be the state of aggregation of BMP in cells and matrix in nature. Present observations support the assumption that BMP is a relatively low density protein and excludes the idea of BMP activity in the collagen molecule, per se. A specific protein, with an apparent molecular weight of 63,000 daltons, is present in all fractions that exhibit BMP activity, and absent in fractions that do not exhibit this activity. BMP is not species-specific; rat BMP induces bone formation in mice. CsCl density-gradient centrifugation is an efficient tool for further purification and isolation of BMP.     

Roles of bone morphogenetic protein signaling in osteosarcoma

Purpose

Since the original extraction of bone morphogenetic proteins (BMPs) from bovine bone, research interest and clinical use has increased exponentially. With this, a concomitant analysis of BMP expression in bone tumours has been performed. BMP ligands, receptors, and signaling activity have been observed in diverse benign and malignant bone tumours. However, the reported expression, function, and importance of BMPs in bone tumours, and specifically osteosarcomas, have been far from uniform. This review highlights recent advances in understanding the role of BMP signaling in osteosarcoma biology, focusing on the sometimes divergent findings by various researchers and the challenges inherent in the study of osteosarcoma.

Methods

We performed a literature review of all studies examining BMP signaling in osteosarcoma.

Results

Overall, multiple BMP ligands and receptors are expressed in most osteosarcoma cell lines and subtypes, although BMP signaling may be reduced in comparison with benign bone-forming tumours. Studies suggest that osteosarcomas with different lineages of differentiation may have differential expression of BMP ligands. Although significant disagreement in the literature exists, the presence of BMP signaling in osteosarcoma may impart a worse prognosis. On the cellular level, BMP signaling appears to mediate promigratory effects in osteosarcoma and chondrosarcoma cell types, possibly via interaction and activation of Integrin β1.

Conclusions

BMP signaling has clear biologic importance in osteosarcoma, although it is not yet fully understood. Future questions for study include assessing the utility of BMP signaling in prognostication of osteosarcoma and the potential modulation of BMP signaling for inhibition of osteosarcomagenesis, growth and invasion.     

Hydrolysis of pyrophosphate and ester phosphates by bone extracts

A study was conducted of the enzymatic hydrolysis of pyrophosphate and ester phosphates by extracts of bones of young rabbits. The proximal ends of humeri and distal ends of femurs were homogenized in barbital buffer (7.5 mM) at pH 7.4. The extracted pyrophosphatase activity had acid and alkaline pH optima. The acid pyrophosphatase activity at pH 5.0 was independent of magnesium up to a concentration of 3 mM and was inhibited by an excess of this ion. Pyrophosphatase activities at both pH 5.0 and 7.4 were inhibited by fluoride. The Michaelis constant of pyrophosphatase with pH 5.0 optimum activity was 1.4×10^–3 M. The bone extract, when loaded on a column of Sephadex G-200 and eluted with barbital buffer (7.5 mM) at pH 7.4 containing 1% sodium chloride, allowed a separation of the two pyrophosphatase activities; that at pH 5.0 optimum activity was associated with acid phosphatase activity. Results of experiments of the two pyrophosphatase activities and alkaline phosphatase in bones of rabbits as they aged from 5 to 60 days are presented.This investigation was supported by Grant DE-1850 from the National Institute of Dental Research, National Institute of Health, Bethesda, Maryland.The authors wish to acknowledge the help of Miss Doris Bauder in the preparation of the Sephadex columns.     

The relationship between bone morphogenetic protein and neoplastic bone diseases

The monoclonal antibody against bovine bone morphogenetic protein was used for demonstration of bone morphogenetic protein (BMP) in neoplastic bone diseases. The avidin-biotin-peroxidase complex method demonstrated that BMP mainly exists in the cytoplasm of tumor cells of osteosarcoma and chondrosarcoma. Immunostaining showed that a majority of osteosarcomas and all of the chondrosarcoma cells contained a large quantity of BMP. Conversely, none of the fibrosarcomas showed positive staining. Thus, it was possible to differentiate osteosarcomas from fibrosarcomas by immunostaining. In fibrous dysplasia of bone, BMP was abundant in the fibrocellular tissue that had osteogenic activity. In contrast, fibrous tissue of ossifying fibroma showed weak positive staining; only the osteoblasts rimming the bone showed a positive reaction. Immunostaining showed that BMP was also detected in other neoplastic bone diseases such as osteoma, chondroma, and other tumors.     

一种新型生物活性人工骨的制备及成骨活性的研究

目的：研制CPC/BMP复合人工骨,检测其成骨活性。方法：制备CPC/BMP及CPC骨块,扫描电子显微镜观察表面结构。用小鼠肌袋植入实验观察材料的成骨活性。结果：BMP在CPC中呈微球状均匀分布。CPC植入小鼠肌袋内不能诱导,CPC/BMP植入后1周有软骨细胞出现,2周有编织骨,4周以后小梁骨生成,16周出现成熟的板层骨。同时材料出现降解迹象。有机质含量、碱性磷酸酶浓度在CPC/BMP组出现升高,扫描电镜结果同样证实有新骨形成。结论：CPC/BMP生物活性人工骨可异位诱导成骨,可望成为新型的骨缺损修复材料。     

磷酸钙骨水泥作为骨形成蛋白载体修复节段性骨缺损及相关研究

目的 制备CPC/BMP复合人工骨，通过动物实验研究其对骨缺损的修复作用及相关问题，探讨临床应用的可能性。方法 参考有关文献方法合成CPC，并将其作为BMP的载体制成CPC/BMP复合物，植入兔桡骨15mm骨缺损处，术后不同时间处死动物。通过生物力学测定，组织学染色分析，电镜扫描及X射线电子能谱分析。X线摄片，无机质含量测定以及骨密度测定等手段观察新骨形成和材料降解情况。同时以单纯的CPC及空白组作为对照研究。综合评价CPC/BMP对骨缺损的修复能力及对机体的影响。结果 术后CPC/BMP和CPC两组动物均无毒性反应。随着时间的延长，血清中碱性磷酸酶浓度逐渐升高，尤以CPC/BMP组显著，提示CPC/BMP复合物和单纯的CPC均可以促进新骨形成，前者新骨形成量大，骨修复能力明显好于后者。CPC/BMP植入2周时可见大量间充质细胞分化，在材料与骨端之间出现一层软骨细胞。4周时软骨细胞向编织骨分化，16周时板骨层骨长人材料并与之相互分割包裹，24周时骨缺损初步修复，新骨密度明显高于CPC组，说明BMP的加入不仅有效地促进了新骨的形成，同时也加速了新骨的钙化。24周组标本生物力学测定结果表明，新骨形成的同时伴随材料的降解，CPC组材料降解速度缓慢，CPC/BMP组降解速度优于CPC组，但24周时仍有部分材料残存。在新骨形成和材料降解过程中可出现血清钙浓度的一过性升高。结论 CPC是BMP的理想载体。CPC/BMP生物活性人工骨对骨缺损有较强的修复能力，可望成为新型的骨缺损修复材料。     

聚乳酸作为骨形态发生蛋白载体修复骨缺损的实验研究

目的 探讨聚乳酸（ｐｏｌｙｌｄａｃ ａｃｉｄ,ＰＬＡ）作为骨形态发生蛋白（ｂｏｎ ｅ ｍｏｒｐｈｏｇｅｎｅｔｉｃ ｐｒｏ－ｔｅｉｎ,ＢＭＰ）载体的可行性及观察其诱导成骨能力。方法 手术造成日本大耳白兔左尺骨中上段１２ｍｍ骨缺损实验模型。随机分为实验。对照及空白组,实验组植入以ＰＬＡ为载体的ＢＭＰ１０ｍｇ、对照组植入以牛松质骨基质为载体的ＢＭＰ１０ｍｇ、空白组不做任何处理,术后摄Ｘ线片观察各组不同时相骨缺损修复情况,并于术后第４、８、１２周观察各组缺损内组织学变化。图像分析骨小梁的生成量。结果 实验修复情况优于对照组,无论是骨连接发生时间还是骨成熟时间,实验组均较对照组提前２周左右,同期骨生成量也明显多于对照组,而空白组缺损内主要形成纤维组织。结论 ＰＬＡ可以作为ＢＭＰ的载体修复骨缺损,它比异种松质骨基质载体的成骨效     

Bone morphogenesis in implants of residues of radioisotope labelled bone matrix

Bone matrix demineralized in 0.6 N HCl at 2° for 24 h and implanted in muscle in allogeneic rats possesses consistently reproducible bone morphogenetic activity. Experiments on implants of matrix, obtained from donors injected with³H-tyrosine or³H-tryptophan, or Na³⁵SO₄, suggest that bone morphogenetic property is a protein or apart of a protein that is (1) insoluble in buffer solutions, pH 3.6 and 5.0; (2) degraded in buffer solutions at pH 7.4 by an endogenous sulfhydryl-group neutral proteinase; (3) digested by trypsin at 15° within 8 h without solubilization of the helical regions, possibly even without degradation of the nonhelical ends of the bone collagen molecule, and without any loss of the periodic ultrastructure of the collagen fibrils; (4) degraded or removed by 0.1 N NaOH at 2° within 24 h without solubilization of collagen; (5) biologically active even after nitration of tyrosyl groups with tetranitromethane. The release of only one-third of the radioactivity with loss of nearly all yield of new bone by limited tryptic digestion of³H-borohydride-reduced matrix indicates that the bone morphogenetic response is the function of a non-collagenous component. Autoradiographs of implants of matrix with non-collagenous proteins labelled with³H-tryptophan,³H-tyrosine, or both³H-tyrosine and³H-phenyl-alanine demonstrate random dissemination of the radioactive constituents and no evidence of local transfer of labelled proteins or soluble protein derivatives. Hypothetically, the bone morphogenetic response is controlled by an insoluble acidic bone morphogenetic protein or polypeptide (BMP) and a soluble neutral proteinase (BMP-ase) resembling trypsin in activity except functionally more specific for BMP. Firmly bound but separable from bone collagen, BMP is one of many short-lived morphogenetic substances appearing and disappearing throughout embryonic development and persisting in postfetal life. Where the BMP receptor resides and how it activates cell mechanisms of differential repression and derepression of such genes as code for osteogenesis is unknown.     

Histidinoalanine-containing phosphoprotein in bone

Summary Histidinoalanine is a naturally occurring cross-linking amino acid found in mammalian bone, dentin, cartilage and aorta, and also in the extracellular fluid of bivalve clams. The exact components from which this amino acid derives have not been characterized in the case of bone. In this study, the origin of histidinoalanine in bone was investigated in order to clarify the possible role of the matrix component containing this amino acid in bone mineralization. An extract of bovine bone powder with 0.5 M EDTA/1M NaCl was desalted and separated by calcium-induced precipitation. The supernatant fraction was chromatographed successively on Sepharose CL-6B, hydroxyapatite, DEAE cellulose, and reverse-phase high-performance liquid chromatography columns. The fraction with the highest affinity for hydroxyapatite was found to contain the highest concentration of histidinoalanine. A phosphoprotein with a molecular weight of about 24 K that contained 1.2 mol of histidinoalanine per molecule was isolated. The amino acid composition of this fraction showed a high content of acidic amino acids and serine, at least 42% of which was phosphorylated. These results suggested a possible formation mechanism of histidinoalanine that includes the beta-elimination of phosphoserine, producing a dehydroalanine residue and an addition of histidine on it. It was concluded that this low molecular weight phosphoprotein is one of the major sources of histidinoalanine in bovine bone.     

Selective extractability of noncollagenous proteins from chicken bone

Quantitative analyses of a wide variety of different solvents used for the extraction of several of the noncollagenous proteins of fully mineralized chicken bone powder were carried out to compare both the effectiveness of various procedures and the distribution of specific proteins which were solubilized. Extraction procedures included solutions of 6 M guanidine-HCl, pH 7.0, 0.5 M EDTA, pH 7.4, 0.3 N citric acid, 0.3 N HCl, 0.3 N formic acid, and 0.3 N acetic acid. Chelation of calcium ions by EDTA and dissolution of the mineral phase by acid extraction released 95% or more of the total calcium content of the bone powder by 48 hours, guanidine-HCl released less than 20% or less of the total calcium content even when extraction was carried out by 168 hours. Moreover, although guanidine-HCl solubilized a significant amount of collagen as gelatin, essentially none of the phosphoproteins, osteocalcin, or the proteoglycan decorin were solubilized, as detected by immunological techniques. In contrast, extraction of the mineralized bone powder by HCl and formic acid was very efficient in selectively solubilizing osteocalcin and osteopontin, while bone sialoprotein was selectively released by EDTA, and solubilized to a lesser extent by formic acid. Similarly, EDTA selectively removed decorin compared with HCl, formic, acetic, or citric acids. Only small amounts of osteopontin and osteocalcin were detected in the acetic acid extracts. These results provide methods for the selective solubilization of several different major, noncollagenous proteins from mineralized bone which should significantly aid in maximizing the amount of the specific protein recovered, and the ease with which the various proteins can be purified. The data also provide some insight into the intrinsic solubility characteristics of collaten, the specific noncollagenous proteins, and their potential association with each other and the mineral phase.     

A microscopic method for determining bone crystal solubility

Anin vitro system was used to induce growth and dissolution of bone crystals by manipulating the composition of their fluid environment. The induced changes could be detected with the polarizing microscope because of the extrinsic birefringence of the bone tissue. Supporting observations were made with the electron microscope and by determining⁴⁵Ca uptake. The bone crystals were found to be heterogeneous with regard to their solubility. When induced crystal changes were minimal by polarization microscopy the overall solubility product of the bone mineral expressed asp [Ca]+p[P] was 6.51±0.07 (S.D.). This result corroborated the findings of other investigators who determined bone powder solubility by conventional methods. Solubility of the crystals in thein vitro system varied inversely with hydroxyl ion and fluoride ion concentration, but directly with citrate ion concentration.This work was supported by PHS Grant AM-11460 from the National Institute of Arthritis and Metabolic Diseases.     

EDTA-insoluble,calcium-binding proteoglycan in bovine bone

A calcium ion precipitable, trypsin-generated proteoglycan fragment has been isolated from the demineralized, EDTA-insoluble matrices of bone. The demineralized matrix was completely digested with trypsin, increasing concentrations of CaCl₂ were added to the supernatant, and the resulting precipitates were analyzed. The amount of precipitate gradually increased with higher concentrations of calcium and was reversibly solubilized by EDTA. After molecular sieve and anion exchange chromatography, a proteoglycan-containing peak was obtained. Immunochemical analysis showed that this peak contained chondroitin 4-sulfate and possibly keratan sulfate. Amino acid analysis showed that this proteoglycan contained high amounts of aspartic acid/asparagine (Asx), serine (Ser), glutamic acid/glutamine (Glx), proline (Pro), and glycine (Gly); however, it contained little leucine (Leu) which suggests that it is not a member of the leucine-rich small proteoglycan family. In addition, significant amounts of phosphoserine (P-Ser) and hydroxyproline (Hyp) were identified in hydrolysates of this fraction. A single band (Mr 59 kDa) was obtained on SDS-PAGE that stained with Stains-all but not with Coomassie Brilliant Blue R-250. If bone powder was trypsinized prior to demineralization, this proteoglycan-containing fraction was not liberated. Collectively, these results indicate that a proteoglycan occurs in the demineralized matrix that is precipitated with CaCl₂ and is closely associated with both mineral and collagen matrices. Such a molecule might facilitate the structural network for the induction of mineralization in bone.     

Macrophage-mediated bone resorption occurs in an acidic environment

Summary Resorption is a continuous skeletal process involving the degratation of both the organic and inorganic matrices of bone. This process has received much attention, but little is known about the physical mechanism by which resorptive cells degrade the skeleton. In this study, we examined the pH at the resorptive cell-bone interface using a fluorescent dye, fluorescein isothiocyanate, conjugated to the organic matrix of devitalized rat bone. Because the visible spectrum of fluorescein differs in acidic and neutral environments, the relative magnitudes of fluorescence at two different wave lengths indicates whether the pH at the fluorescing site is above or below the pKa of the dye. Our studies indicate that macrophage-mediated bone resorption occurs at pH below 6.0. The presence of parathyroid hormone or calcitonin, however, has no effect on the cell-matrix interface pH after 6 hours of incubation. In contrast to resorptive macrophages, fibroblasts, which bind to bone without resorbing it, do not generate an acidic environment at the matrix attachment site. This work was supported in part by NIH Grant No. AM32788 and by a Nenezian donation.     

人骨形态发生蛋白4成熟肽在大肠杆菌中的表达及其活性测定

目的 利用基因工程技术表达人骨形态发生蛋白4（hBMP4),为深入研究基结构、诱骨机理及临床应用将起积极作用。方法 应用逆转录－聚合酶链反应（RT－PCR）技术,扩增出hBMP4成熟区基因,将其克隆于表达载体pBV220的PRPL的启动子下游。分别经随机挑选、限制性内切酶酶切鉴定及十二烷基硫酸钠－聚丙烯酰 胺凝胶电泳（SDS－PAGE）筛选证实得到5株重组粒pBV/BMP4的转化菌株,以非融合蛋白形式表达hBMP4。结果 DS－PAGE显示,此蛋白相对分子量为14,表达量占菌体蛋白15.6%;细胞培养碱性磷酸酶（ALP）活性测定证明,细胞分泌ALP的活性与rhBMP4浓度呈剂量依赖关系。结论 hBMP4在大肠杆菌中的表达产物复性后具有生物学活性,确证为hBMP4蛋白。     

BMP促进去神经后废用性骨质疏松骨折愈合的实验研究

为了观察ＢＭＰ对去神经后废用性骨质疏松性骨折愈合的作用，作考选用６月龄健康标准大鼠，严格配对分组，切除双侧坐骨神经后３周证实有明显骨质疏松。然后在腔骨人工骨折骨缺损处分别植入酸解胶状鼠尾胶原（对照）及ＢＭＰ与胶原混和物（实验组）。通过血清钙浓度、碱性磷酸酶活性及骨组织形态计量学多项参数的观察，显示ＢＭＰ对去神经后废用性骨质疏松骨折愈合有明显促进作用（Ｐ＜０．０５～０．０１）     

Studies on a factor responsible for new bone formation from osteosarcoma in mice

The bone inducing factor derived from BF osteosarcoma was purified in the following manner. Step 1. The sarcoma, grown in CBA mice, was excised and lyophilized. Step 2. The powder was washed with chilled acetone. Step 3. The acetone-treated powder was then homogenized with chilled distilled water. Step 4. Washing with 0.15M KCl. Step 5. The precipitate was incubated in 0.2 N NH₂OH, pH 7.0, for 48 h at 25°. After Step 5, the bone-forming activity showed a slight increase; however, the factor remained insoluble. The properties of the factor were as follows. The factor is relatively heat stable; the osteogenic activity survived the treatment at 75° for 15 min or at 55° for 19 h. The activity was easily lost by mechanical shaking. Incubation with DNase, RNase, neuraminidase, chondroitinase ABC and β-galactosidase left the osteogenic activity intact, but treatment with either pronase or collagenase destroyed this activity. The results suggest that the factor may be a protein. The activity was seen with the lyopholized BF osteosarcoma cells (without matrix), and it is probable that the factor was exclusively synthesized in the cells. The bone formation, observed across a millipore filter when living BF osteosarcoma enclosed in a millipore chamber was implanted in mice, suggests the synthesis and secretion of the factor from the cells.     

The clinical use of bone morphogenetic proteins revisited: a novel biocompatible carrier device OSTEOGROW for bone healing

Purpose

The purpose of this study was to revise the clinical use of commercial BMP2 (Infuse) and BMP7 (Osigraft) based bone devices and explore the mechanism of action and efficacy of low BMP6 doses in a novel whole blood biocompatible device OSTEOGROW.

Methods

Complications from the clinical use of BMP2 and BMP7 have been systemically reviewed in light of their role in bone remodeling. BMP6 function has been assessed in Bmp6-/- mice by μCT and skeletal histology, and has also been examined in mesenchymal stem cells (MSC), hematopoietic stem cells (HSC) and osteoclasts. Safety and efficacy of OSTEOGROW have been assessed in rats and rabbits.

Results

Clinical use issues of BMP2 and BMP7 have been ascribed to the limited understanding of their role in bone remodeling at the time of device development for clinical trials. BMP2 and BMP7 in bone devices significantly promote bone resorption leading to osteolysis at the endosteal surfaces, while in parallel stimulating exuberant bone formation in surrounding tissues. Unbound BMP2 and BMP7 in bone devices precipitate on the bovine collagen and cause inflammation and swelling. OSTEOGROW required small amounts of BMP6, applied in a biocompatible blood coagulum carrier, for stimulating differentiation of MSCs and accelerated healing of critical size bone defects in animals, without bone resorption and inflammation. BMP6 decreased the number of osteoclasts derived from HSC, while BMP2 and BMP7 increased their number.

Conclusions

Current issues and challenges with commercial bone devices may be resolved by using novel BMP6 biocompatible device OSTEOGROW, which will be clinically tested in metaphyseal bone fractures, compartments where BMP2 and BMP7 have not been effective.     

Establishment of a cell line producing bone morphogenetic protein from a human osteosarcoma

A human osteosarcoma cell line was established from a biopsy specimen from a 13-year-old girl. The osteosarcoma tissue was maintained in athymic nude mice (Balb C nu/nu) by serial transplantation for three years. The tumor was excised from a host mouse and digested with collagenase. The isolated cells were cultured by 98 passages in 14 months, and clones of osteosarcoma cells were obtained by limiting dilution. A clone named human osteosarcoma cell 6 (H-OS-6) that showed the osteoblastic phenotypes of productions of bone morphogenetic protein (BMP) and alkaline phosphatase and a response to human parathyroid hormone (h-PTH 1-34) was selected. The morphology of its chromosomes indicated its human origin. This human osteosarcoma cell line is unique in producing BMP under in vitro conditions.