Modulator of bone morphogenetic protein activity in the progression of kidney diseases

Tubular damage and interstitial fibrosis is a final common pathway leading to end-stage renal disease, and once tubular damage is established, it cannot be reversed by currently available treatment. The administration of bone morphogenetic protein-7 (BMP-7) in pharmacological doses repairs established tubular damages and improves renal function in several kidney disease models; however, pathophysiological role of endogenous BMP-7 and regulatory mechanism of its activities remain elusive. The activity of BMP is precisely regulated by certain classes of molecules termed BMP agonist/antagonist. In this review, roles of BMP agonist/antagonists possibly modulating the activity of BMP in kidney diseases are discussed. Our group demonstrated that uterine sensitization-associated gene-1 (USAG-1), a novel BMP antagonist abundantly expressed in the kidney, is the central negative regulator of BMP-7 in the kidney, and that mice lacking USAG-1 (USAG-1(-/-) mice) are resistant to kidney injuries. USAG-1(-/-) mice exhibited markedly prolonged survival and preserved renal function in acute and chronic renal injuries. Renal BMP signaling, assessed by phosphorylation of Smad proteins, is significantly enhanced in USAG-1(-/-) mice during renal injury, indicating that the preservation of renal function is attributed to enhancement of endogenous BMP-7 signaling. Furthermore, the administration of neutralizing antibody against BMP-7 abolished renoprotection in USAG-1(-/-) mice, indicating that USAG-1 plays a critical role in the modulation of renoprotective action of BMP, and that inhibition of USAG-1 will be promising means of development of novel treatment for kidney diseases.     

骨形态蛋白在脊柱外科应用的最新进展

骨形态发生蛋白(bone morphogenetic proteins,BMPs)不仅能够诱导异位骨化,而且能够作用于成骨细胞和骨祖细胞,提高其成骨分化的能力.在骨的形成及骨折愈合过程中发挥作用,尽管BMPs在矫形外科中的应用仍处于初期阶段,但是其发展前景极其广阔,了解植骨融合的确切机制具有重要的意义.     

The use of bone morphogenetic protein in spine fusion

Background contextBecause pseudarthrosis remains a clinically significant complication after spinal arthrodesis, the role of recombinant bone morphogenetic proteins (BMPs) is continually evaluated in spine surgery.PurposeThis article reviews the important literature in clinical research involving the use of BMPs in the augmentation of spinal fusion.Study design/settingReview article.MethodsA literature search was performed via MEDLINE through PubMed with the dates January 1960 to July 2007 using the keywords “bone morphogenetic protein, BMP, spinal arthrodesis, and/or bone healing.” Pertinent preclinical and clinical publications were chosen based on relevance and quality for inclusion in this study.ResultsPublications focused on the historical context and potential clinical applications using BMP were selected to delineate the risks, benefits, and current indications for the augmentation of spinal arthrodesis.ConclusionsAlthough multiple commercially available recombinant BMPs have demonstrated clinical success in interbody and posterolateral fusions, the associated costs preclude its routine use in spinal arthrodesis. The spine surgeon must assess each patient individually based on age, bone quality, diagnosis, comorbidities, and risks of nonunion to determine the cost effectiveness of the use of BMP to augment spinal fusion.     

Preservation of bone morphogenetic protein in heat-treated bone.

In operations of bone tumors, reimplantation of resected bone after boiling or autoclaving is a simple means of obtaining both tumor necrosis and skeletal reconstruction. However, such reimplants lose their osteogenesity. We investigated whether bone inductive ability could be maintained in heat-treated bone. Bone morphogenetic protein (BMP) extracted from rabbit bone after heating for various periods at different temperatures was implanted into the muscles of mice to evaluate osteogenetic activity. The maximum new bone formation was observed in specimens treated at 70 degrees C for 10 minutes, followed by those treated at 70 degrees C for 15 minutes. We then measured the temperature in the center of a cortical bone heated in 0.15 N NaCl solution at 50 degrees, 60 degrees, 70 degrees, 80 degrees, and 90 degrees C. Cortical bone center temperature reached that of the surrounding solution within 2.5 minutes. These results indicated that heating at 70 degrees for 10 to 15 minutes was suitable for heat treated-bone to maintain bone inductive ability.     

Smads在骨形态发生蛋白诱导成骨中的作用

Smads蛋白是骨形态发生蛋白细胞内信号传导中起重要作用的转录因子,在骨形态发生蛋白诱导骨形成中发挥着关键作用.骨形态发生蛋白可以激活多种Smads蛋白并使其转移至细胞核,通过直接与特定DNA序列相结合、与核调节因子形成复合体、募集转录共活化或共抑制因子等3种方式调节目的基因的转录,最终诱导骨形成.该文就Smads蛋白的定义与分类、Smads信号转导通路及其在骨形态发生蛋白诱导成骨中的作用机制、BMP/Smads信号调节通路与其他通路之间的关系等相关研究作一综述.     

Separation and purification of porcine bone morphogenetic protein

Porcine bone morphogenetic protein (pBMP) was separated and purified from porcine bone by repeated solubilization and precipitation of the protein with different concentrations of urea and GuHCl. The molecular weight of pBMP was determined to be 19.7 k. The property of inductive osteogenesis of pBMP caused new bone formation in muscular and subcutaneous tissues of strain No. 615 mice. Osteoid tissues were observed as early as ten days after implantation of pBMP, and lamellated new bones with marrow cavities were first found on the 15th day of implantation. A gradual transformation of mesenchymal-type cells into active chondroblasts and osteoblasts and finally into heterotopic new bone was observed and studied histologically and ultrastructurally.     

Bone morphogenetic protein signaling in bone homeostasis

Bone morphogenetic proteins (BMPs) are cytokines belonging to the transforming growth factor-β (TGF-β) superfamily. They play multiple functions during development and tissue homeostasis, including regulation of the bone homeostasis. The BMP signaling pathway consists in a well-orchestrated manner of ligands, membrane receptors, co-receptors and intracellular mediators, that regulate the expression of genes controlling the normal functioning of the bone tissues. Interestingly, BMP signaling perturbation is associated to a variety of low and high bone mass diseases, including osteoporosis, bone fracture disorders and heterotopic ossification. Consistent with these findings, in vitro and in vivo studies have shown that BMPs have potent effects on the activity of cells regulating bone function, suggesting that manipulation of the BMP signaling pathway may be employed as a therapeutic approach to treat bone diseases. Here we review the recent advances on BMP signaling and bone homeostasis, and how this knowledge may be used towards improved diagnosis and development of novel treatment modalities. This article is part of a Special Issue entitled “Muscle Bone Interactions”.     

骨形态发生蛋白缓释载体的研究进展

骨形态发生蛋白（bonemorphogeneticprotein,BMP）是研究较早的骨生长因子,其诱导成骨的作用已被多次实验所证实。它能诱导未分化的间充质细胞分化,表达骨与软骨细胞表型,还能刺激软骨细胞和成骨细胞分化,并提高细胞ALP的活性犤１犦。但是BMP在骨内的含量极微,约１mg／kg湿骨,而外源性BMP来源有限、成本较高,且在体内迅速降解或随体液扩散犤２犦,不能均匀持久地分布在大的骨缺损部位,难以发挥出成骨效应。在以往实验中单纯应用BMP治疗骨缺损的效果并不理想,必须有合适的载体材料与其复合,起到令BMP缓释及提供骨细胞生长…     

Effect of sterilization on bone morphogenetic protein

Demineralized bone matrix and bone morphogenetic protein have been used clinically to accelerate bone regeneration. However, the best method of sterilization has been the subject of controversy. Some investigators have used ethylene oxide, but others have reported that doses adequate for sterilization destroyed the osteoinductivity of demineralized bone matrix and that gamma irradiation was less harmful in this respect. We used partially purified bone morphogenetic protein and type-I collagen to investigate the effects of sterilization by ethylene oxide and gamma irradiation on the activity of bone morphogenetic protein. Osteoinductivity was reduced considerably after sterilization by gamma irradiation at 2.5 Mrad and by ethylene oxide at 37°C for 4 hours and at 55°C for 1 hour; however, the reduction induced by ethylene oxide at 29°C for 5 hours was about half of the control values. This study showed that ethylene oxide at 29°C for 5 hours can be used clinically for sterilization of bone morphogenetic protein. We also investigated the effect of gamma irradiation on bone morphogenetic protein and the collagen carrier separately and found that collagen was far more labile than bone morphogenetic protein.     

Systemically available bone morphogenetic protein two and seven affect bone metabolism

Purpose

Bone morphogenetic protein (BMP)-2 and -7 are used in patients with long-bone fractures, nonunions and spinal fusions. It is unknown whether their potential systemic bioavailability following local bone administration might affect skeletal metabolism. To answer this question, we examined effects of systemically administered BMP-2 and -7 on bone in a newly developed rat model with a low level of calciotropic hormones.

Methods

Removal of thyroid and parathyroid glands (TPTx) in rats resulted in a decreased level of calciotropic hormones and subsequent bone loss assessed by micro computed tomography (micro-CT) and measurement of serum bone formation and resorption markers, including osteocalcin, C-telopeptide, osteoprotegerin and receptor activator of nuclear factor kappa-B ligand. Results were complemented with in vitro studies on osteoblast and osteoclast activity by both BMP-2 and -7. The doses used were calculated from published pharmacodynamic studies and bioavailability results from preclinical BMP-2 and -7 studies.

Results

TPTx resulted in bone loss, which was restored by systemic administration of 10–70 μg/kg of BMP-2 and 10–250 μg/kg of BMP-7. BMP-2 showed a higher capacity for enhancing trabecular microarchitecture, whereas BMP-7 augmented trabecular thickness. In vitro experiments revealed that BMP-2 and -7 when uncoupled increased the number and activity of both osteoblasts and osteoclasts.

Conclusions

Surprisingly, both BMP-2 and -7 showed an increased bone volume in an in vivo environment of low calciotropic hormones. Locally administered BMP-2 and -7 from bone devices might become partially available in circulation but will not mediate systemic bone loss.     

细胞外囊泡与衰老的关系及其在骨代谢疾病中的研究进展

随着老龄化社会的到来,骨质疏松等骨代谢性疾病越来越受到人们的重视。细胞衰老已被证明是导致骨代谢过程中骨量丢失的重要原因之一,其中细胞外囊泡是由细胞分泌的在细胞间传递信息的重要媒介,囊泡内含有不同种类的miRNAs、蛋白质、脂质、mRNAs、tRNAs等生物活性物质,在细胞信息交流中起着重要作用,目前被认为具有与衰老相关分泌表型,即SASP因子相似的功能。本文将总结近年来机体或细胞在衰老时分泌的细胞外囊泡与SASP因子之间关系的研究进展,并展望细胞外囊泡在未来衰老引起的骨代谢性疾病治疗研究中存在的潜在价值。     

Demineralized bone matrix and native bone morphogenetic protein in orthopaedic surgery

The recognition that demineralized bone matrix could induce bone formation when placed in mammalian skeletal muscle led to preclinical studies of crude native insoluble bone morphogenetic protein and noncollagenous protein, followed by the clinical application of demineralized bone matrix, chemosterilized autolyzed antigen-extracted allogenic bone, and autolyzed antigen-extracted allogenic bone matrix gelatin. Cultural norms and regulatory agencies influence the availability of different demineralized bone matrix preparations in different parts of the world, but there is continued interest in the biologic structure of native insoluble bone morphogenetic protein and noncollagenous protein aggregates and the applied science of osteoinduction and osteoconduction in reconstructive orthopaedic surgery. Demineralized bone matrix is not widely available in Asia, but tissue processing facilities in the United States distribute demineralized bone matrix materials with different carriers, handling properties, and possibly osteoinductive potential. The purpose of the current study was to review the development and use of various preparations of demineralized bone matrix materials.     

High-dose bone morphogenetic protein–induced ectopic abdomen bone growth

Background contextInfuse [bone morphogenetic protein (BMP)] is increasingly used in spinal fusion surgery.PurposeThe authors report a rare complication of BMP use.Study designThis is a case report.Patient sampleA 55-year-old male underwent a thoracic T8 to the pelvis fusion for degenerative lumbar disc disease and pseudarthrosis at another institution. The procedure involved an anterior and posterior approach with the use of multiple units of BMP.MethodsThe patient presented to our institution with complaints of weight loss, pain, tenderness, and increasing solid growth in the left lower quadrant several months after his surgery. A computed tomography revealed ectopic bone growth in the retroperitoneal area and pelvis contiguous to the anterior lumbar exposure.ResultsThe anterior wound was reexplored, and a large sheet of ectopic bone was removed from the retroperitoneal space.ConclusionsWe report a rare case of extraspinal ectopic bone growth because of the use of multiple packages of BMP.     

Periosteal bone formation elicited by partially purified bone morphogenetic protein

A small amount of partially purified, water-soluble murine osteosarcoma-derived bone morphogenetic protein (BMP) was implanted into the dorsal muscles of mice in combination with calf skin gelatin or collagen as carriers. Changes in the ribs adjacent to the implants were then chronologically observed. On implantation of BMP with gelatin, the gelatin was rapidly absorbed and no ectopic bone formation was observed, but periosteal cellular proliferation with subsequent formation of periosteal cartilage and bone was seen in ribs adjacent to the implant. Implantation of the same amount of BMP fraction or gelatin alone as controls did not result in either any ectopic bone formation in situ or any periosteal bone formation in adjacent ribs. Implantation of BMP with collagen resulted consistently in both ectopic bone formation in situ and periosteal bone formation in adjacent ribs. These results suggest that BMP is diffusible in vivo and is capable of eliciting a response from periosteum to stimulate periosteal bone formation.     

Purification and chemical modification of porcine bone morphogenetic protein

Implantation of porcine bone morphogenetic protein (pBMP) in the muscle induces differentiation of mesenchymal-type cells and results in endochondral bone formation. pBMP was isolated from porcine demineralized bone matrix and purified by hydroxyapatite chromatography, Sephadex G75 gel filtration, preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), preparative isoelectric focusing (IEF), and chromatofocusing fast protein liquid chromatography (FPLC). Porcine BMP has an MW of 26 K and a range of pI from 4.65 to 4.73 determined by SDS-PAGE and IEF, respectively. Reconstitution with the citrate buffer supernatant fraction enables as little as 50 micrograms of the soluble pBMP fractions to induce osteogenesis in an in vivo assay. Chemical modification studies indicate that the osteoinductive potential of the pBMP molecule depends on tyrosine, carboxyl groups, and disulfide bonds and can be increased by modification of sulfhydryl groups. Modification of arginine and tryptophan has no effect on bioactivity. By pepsin-limited proteolysis, fragments of pBMP with an MW of 6-14 K show definite, although reduced, BMP activity.     

Circulating bone morphogenetic protein levels and delayed fracture healing

Objective

Despite adequate treatment 5–30 % of bone fracture patients experience delayed union. During normal fracture union, bone morphogenetic proteins (BMPs) induce healing through a sequential cascade of events. Improved fracture healing after BMP-2 or -7 supplementation in patients with impaired fracture union suggests a deficiency of one or more of these factors. We postulated that low levels of circulating BMPs may result in delayed bone healing. The aim of this study was to quantify differences in levels of circulating BMP-2, -4, -6, -7, and −9 in patients that have demonstrated normal or delayed fracture healing.

Patients and methods

Blood samples were collected from an unselected cohort of 65 patients that had been treated for a diaphyseal tibia or femur fracture. Patients were divided into a group with fracture healing within nine months after injury and a group with delayed fracture union. BMP plasma concentrations were quantified using ELISAs and compared between these two groups.

Results

Circulating plasma levels of BMP-2, -4, -6, and -7 did not differ between 34 patients with normal fracture healing and 31 patients with delayed fracture healing. Also the median BMP-9 plasma levels were not statistically different between the two groups of patients. However, the distribution in the patients with normal union showed a wider range (72–2496 pg/ml) compared with the delayed union group (120–816 pg/ml).

Conclusion

In general, circulating BMP concentrations are not statistically different between patients who demonstrated normal or delayed fracture healing. High circulating BMP-9 levels seem to be associated with faster fracture healing, but are apparently not decisive.     

The effect of heat-treated human bone morphogenetic protein on clinical implantation

For the clinical usage of human-derived bones, it is necessary to treat bones to reduce the risk of contamination by microorganisms. Bone morphogenetic protein is vulnerable to chemicals, but shows resistance to thermal heat to 70 degrees C in a short time. In this experiment, crude human bone morphogenetic protein was extracted from heat-treated bones at 60 degrees C for 10 hours and from nonheated bones. Sodium dodecyl sulfate polyacrylamide gel electrophoresis for these specimens was done. Gelatin capsules containing 5 mg of crude human bone morphogenetic protein extracted from heated and nonheated bones were implanted into thigh muscle pouches of five mice. At 20 days after implantation, the heterotopic bone formation was compared by evaluating the radiographic and histologic analyses. The sodium dodecyl sulfate polyacrylamide gel electrophoresis pattern of the human bone morphogenetic proteins showed five main bands (16, 22, 28, 35, and 67 kDa) that were almost identical. Heterotopic bone formation observed on the radiograph was induced by crude human bone morphogenetic protein from heated bones in a manner similar to that used for nonheated bones. The results from this study show that heat-treated bone preserves osteoinduction.     

Measurement of total and local bone morphogenetic protein concentration in bone tumours

Summary. Bone morphogenetic protein (BMP) has been shown to be one of the significant factors in the prognosis of bone tumours. In normal development BMP induces new bone formation and later takes part in fracture healing, but its function in malignant tumours is not known. In this study the concentration of bone morphogenetic protein was measured in primary bone tumours by two methods. Local staining intensity was detected immunohistologically by the avidin-biotin-peroxidase method determining the highest dilution of anti-serum against bovine bone morphogenetic protein. The total amount of BMP in a tumour sample was measured by an enzyme-linked immunosorbent assay technique after digesting the tissue with collagenase to remove proteins from the connective tissue. Immunohistochemical staining showed that bone morphogenetic protein was present in the cytoplasm and in reactive bone formed by malignant cells. The local concentration was highest in the tissue of giant cell tumours compared to chondrosarcoma, osteosarcoma and benign bone tumours. The total amount in malignant bone tumours was 2.4 times higher compared to benign bone tumours.

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Résumé. Il a été démontré que la protéine morphogénétique osseuse (BMP) est un facteur important dans le pronostic des affections osseuses. Dans un développement osseux normal, cette protéine induit la formation de nouveau tissu osseux et contribue, par exemple, dans les fractures, à la consolidation. Dans les affections malignes, la fonction précise de la BMP reste inconnue. Dans la présente étude, la concentration de protéine morphogénétique osseuse a été mesurée dans des tumeurs osseuses primaires à l’aide de deux méthodes. L’intensité de coloration locale de la BMP a été détectée immunohistochimiquement par la méthode avidine-biotine-péroxydase, déterminant le titre le plus élevé de dilution d’antisérum par rapport à la BMP bovine (bBMP); le résultat de la coloration a été positif. Le total de protéine morphogénétique osseuse présent dans un échantillon de tumeur a été mesuréà l’aide d’un dosage enzymatique immuno-absorbant (ELISA) aprés digestion du tissu avec de la collagénase pour retirer les protéines du tissu conjonctif. La coloration immunohistochimique a montré que la BMP se localisait dans les cytoplasmes et dans le tissu osseux réactif formé par les cellules malignes. La plus forte concentration locale de BMP a été trouvée dans le tissu des cellules géantes de la tumeur dans chondrosarcome, l’ostéosarcome et les tumeurs osseuses bénignes. Le total de BMP présent dans les tumeurs osseuses malignes était 2.4 fois supérier à celui décelé dans les tumeurs osseuses bénignes.

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Accepted: 2 May 1996