Influence Of Bone Morphogenetic Proteins Research Articles

Bone morphogenetic protein 2/7 promotes repair of bone defect via induction of endochondral ossification

Purpose: To study the influence of bone morphogenetic protein 2/7 (BMP2/7) on repair of bone defect, and the mechanism involved.Methods: Bone marrow stromal cells (BMSCs) were randomly assigned to 2 groups: control and transfection groups. The cells were transfected with rBMP2/7 recombinant adenovirus. Cell growth and alkaline phosphatase (ALP) activity were determined in both groups. Rabbit model of femoral bone defect was prepared using standard methods. Male New Zealand white rabbits were randomly assigned to 3 groups, each of which had 10 rabbits: control, gelatin and BMP2/7 groups. Histopathological and xray examinations, and three-point bending flexural test were used to compare the potential of gelatin and BMP2/7 to repair bone defects.Results: Transfection of BMSCs with rBMP2/7 recombinant adenovirus significantly enhanced their growth (p < 0.05). Alkaline phosphatase (ALP) level was also markedly and time-dependently higher in transfection group than in control group (p < 0.05). Rabbits with grade 4 bone healing or above were more in BMP2/7-treated category than in control and gelatin groups. New bone hyperplasia with typical lamellar bone structure, irregular medullary cavity, as well as transition from osteoblast to osteocyte were observed in BMP2/7 group. Moreover, maximum flexural strength and repair were significantly higher in BMP2/7-transfected group than in control.Conclusion: These findings indicate that BMP2/7 promotes the repair of bone defect via induction of endochondral ossification in rabbits. Thus, this protein may be useful for the repair of bone defects in humans.
 Keywords: Alkaline phosphatase, Bone lesions, BMP2/7, Endochondral ossification

ИССЛЕДОВАНИЕ МЕТАБОЛИЧЕСКОЙ АКТИВНОСТИ И СИНТЕЗА ПРОТЕОГЛИКАНОВ КЛЕТКАМИ МЕЖПОЗВОНКОВОГО ДИСКА ПОД ВОЗДЕЙСТВИЕМ КОСТНОГО МОРФОГЕНЕТИЧЕСКОГО БЕЛКА2 В ЭКСПЕРИМЕНТЕ

Modern therapeutic strategies for intervertebral disc repair mainly focus on targeting molecular pathways of extracel-lular matrix degeneration. Anabolic strategies for regeneration are aimed to increase production of major extracellular molecules. Members of TGF-b superfamily proteins, particularly the bone morphogenetic proteins (BMP) have a high regenerative potential regarding the mesenchymal cells. The goal of this study is to study production of proteoglycans by the intervertebral disc cells under the influence of bone morphogenetic protein 2.Material and methods. The experiment was carried out on the cell cultures derived from the annulus fibrosis cells and nucleus pulposus cells of the human intervertebral disc. We studied cell livability, metabolic activity and proteoglycan expression. Cell livability was assessed using the trypan blue staining. Alamar blue test was used for the estimation of metabolic activity. Amount of sulfated glycosaminoglycans was assessed using the assay based on the reaction with 1,9-Dimethylmethylene Blue.Results. Cultivation with bone morphogenetic protein 2 in different concentrations did not decrease livability of the cells. Study cell cultures with application of bone morphogenetic protein 2 in different concentrations showed significant increase in metabolic activity and proteoglycan synthesis by the annulus fibrosis cells. Despite the relative increase in the number of the nucleus pulposus cells treated with the bone morphogenetic protein 2, the differences in metabolic and synthetic activity compared with control group was not significant. Conclusion. The bone morphogenetic protein 2 has an anabolic effect towards the intervertebral disc cells, particularly in the production of proteoglycans by the annulus fibrosis cells.

Recombinant Human Bone Morphogenetic Protein-2 in Development and Progression of Oral Squamous Cell Carcinoma.

Bone morphogenetic proteins (BMPs), belonging to the transforming growth factor-β superfamily, regulate many cellular activities including cell migration, differentiation, adhesion, proliferation and apoptosis. Use of recombinant human bone morphogenic protein?2 (rhBMP?2) in oral and maxillofacial surgery has seen a tremendous increase. Due to its role in many cellular pathways, the influence of this protein on carcinogenesis in different organs has been intensively studied over the past decade. BMPs also have been detected to have a role in the development and progression of many tumors, particularly disease-specific bone metastasis. In oral squamous cell carcinoma - the tumor type accounting for more than 90% of head and neck malignancies- aberrations of both BMP expression and associated signaling pathways have a certain relation with the development and progression of the disease by regulating a range of biological functions in the altered cells. In the current review, we discuss the influence of BMPs -especially rhBMP-2- in the development and progression of oral squamous cell carcinoma.

Influence of bone morphogenetic protein and proportion of hydroxyapatite on new bone formation in biphasic calcium phosphate graft: Two pilot studies in animal bony defect model

PurposeThe purpose of these two pilot studies using animal bony defect models was to evaluate the influence of bone morphogenetic protein (BMP) and proportion of hydroxyapatite (HA)/beta-tricalcium phosphate (β-TCP) in biphasic calcium phosphate (BCP) graft on new bone formation. MethodsIn this study, four kinds of synthetic osteoconductive bone materials known for bone growth scaffold, OSTEON™II(HA:β-TCP 30:70), OSTEON™III (HA:β-TCP 20:80), OSTEON™II Collagen, and OSTEON™III Collagen, were prepared as BCP graft materials. In pilot study 1, three BCP materials (OSTEON™II, OSTEON™III, and OSTEON™II Collagen) were grafted in rabbit calvarial defects after impregnating in rhBMP-2. OSTEON™II without the rhBMP-2 impregnation was included in the study as the control. The amount of new bone was examined and measured histologically at 2, 4, and 8 weeks. In pilot study 2, four BCP materials (OSTEON™II, OSTEON™III, OSTEON™II Collagen, and OSTEON™III Collagen) were grafted in beagle dog mandibular defects after soaking in the rhBMP-2. The amount of total bone and new bone were measured three-dimensionally using microCT and healing process was examined histologically at 2, 4, and 8 weeks. ResultsIn pilot study 1, rhBMP-2 impregnated groups showed more new bone formation than the rhBMP-2 free group. In pilot study 2, increased new bone formation was observed in time-dependent manner after graft of BCP and BCP-collagen (OSTEON™II, OSTEON™III, OSTEON™II Collagen, and OSTEON™III Collagen) impregnated with rhBMP-2. Also, BCP with a higher proportion of HA (30% HA) showed more favorable result in new bone formation and space maintenance, especially at the 8 weeks. ConclusionFrom the results of the pilot studies, rhBMP-2 played positive roles in new bone formation and BCP could become a scaffold candidate for rhBMP-2 impregnation to induce new bone formation. Moreover, BCP with a higher proportion of HA (30% HA) could be considered more appropriate for rhBMP-2 carrier.

Role of BMPs in the regulation of sclerostin as revealed by an epigenetic modifier of human bone cells

Sclerostin, encoded by the SOST gene, is specifically expressed by osteocytes. However osteoblasts bear a heavily methylated SOST promoter and therefore do not express SOST. Thus, studying the regulation of human SOST is challenged by the absence of human osteocytic cell lines. Herein, we explore the feasibility of using the induction of SOST expression in osteoblasts by a demethylating agent to study the mechanisms underlying SOST transcription, and specifically, the influence of bone morphogenetic proteins (BMPs).Microarray analysis and quantitative PCR showed that AzadC up-regulated the expression of several BMPs, including BMP-2, BMP-4 and BMP-6, as well as several BMP downstream targets. Recombinant BMP-2 increased the transcriptional activity of the SOST promoter cloned into a reporter vector. Likewise, exposing cells transfected with the vector to AzadC also resulted in increased transcription. On the other hand, inhibition of the canonical BMP signaling blunted the effect of AzadC on SOST.These results show that the AzadC-induced demethylation of the SOST promoter in human osteoblastic cells may be a valuable tool to study the regulation of SOST expression. As a proof of concept, it allowed us to demonstrate that BMPs stimulate SOST expression by a mechanism involving BMPR1A receptors and downstream Smad-dependent pathways.

Influence of Bone Morphogenetic Protein in Composition of Biocomposite Material upon Osteogenesis and Bone Mineralization

Influence of bone morphogenetic protein (BMP-2) in its local application in combination with demineralized lyophilized bone implant (DLBI) upon the process of osteogenesis and bone mineral density in the zone of surgical intervention and the whole segment (tibia) was studied in experiment (40 rats). The animals were divided into 2 groups. In animals from the study group the defect zone was filled with both DLBI and BMP-2 while in control group only with DLBI. In evaluation of morphologic changes by points at terms 7 and 12 weeks no reliable differences between the groups were noted. However at 12 weeks in study group cases of marked osteogenesis with full remodeling of bone implant were observed. It was shown that use of BMP-2 in combination with DLBI reliably increased bone mineral density both in the zone of surgical intervention and in the segment as a whole. That fact was regarded as the evidence of BMP-2 positive effect upon the mechanical strength of the forming regenerate.

Novel complexities regarding BMPs and fracture healing

Novel complexities regarding BMPs and fracture healing

Modulation of sclerostin expression by mechanical loading and bone morphogenetic proteins in osteogenic cells

The anabolic effect of dynamic mechanical loading on skeletal architecture has been repeatedly demonstrated, but the cellular and molecular events occurring between load and ultimate bone formation remain obscure. The discovery of sclerostin, an antagonist of Wnt/Lrp5 signaling, and the sclerosing bone dysplasias that result from its mutation suggest its pivotal role in modulating bone formation. We examined expression of Sost mRNA across a variety of clonal cell lines spanning the osteogenic phenotype from immature osteoblast to mature osteocyte. No sclerostin expression was detected in immature MC3T3-E1 osteoblasts and, surprisingly, mature MLO-Y4 osteocytes, whereas immature MLO-A5 osteocytic cells expressed very low levels of Sost. Highest expression was observed in mature UMR 106.01 osteoblasts. We examined the influence of bone morphogenetic proteins on Sost expression. Treatment with BMP-2, -4 or -6 was without effect on Sost in mature MLO-Y4 osteocytes but elicited a robust increase in Sost expression in immature MLO-A5 osteocytes. Oscillatory fluid flow applied to mature UMR 106.01 osteoblasts transiently decreased expression of sclerostin at both the mRNA and protein level. Overall, our results indicate that BMP treatment and in vitro mechanical loading demonstrate opposite effects upon sclerostin expression.

Sphingosine‐1‐phosphate acts as a developmental stage specific inhibitor of platelet‐derived growth factor‐induced chemotaxis of osteoblasts

The development and maintenance of a healthy skeleton depends on the migration of cells to areas of new bone formation. Osteoblasts, the bone forming cells of the body, mature from mesenchymal stem cells under the influence of bone morphogenetic protein. It is unclear at what developmental stage the osteoblasts start to migrate to their functional location. We have studied migration of immature pre-osteoblasts and of mature osteoblasts in response to Platelet-derived growth factor (PDGF) and sphingosine-1-phosphate (S1P). PDGF is a growth factor involved in bone remodeling and fracture healing whereas S1P is a circulating sphingolipid known to control cell trafficking. Our data indicate that PDGF acts as a chemotactic cue for pre-osteoblasts. In contrast, S1P is a chemorepellent to these cells. Upon Bone Morphogenetic Protein 2-induced conversion to the osteoblast phenotype, the chemotaxis response to PDGF is retained whereas the sensitivity to S1P is lost. By RNA interference and overexpression experiments we showed that the expression level of the S1P2 receptor is the sole determinant controlling responsiveness to S1P. The combined data indicate that migration of osteoblasts is controlled by the balance between PDGF, S1P and the differentiation state of the cells. We propose that this mechanism preserves the osteoprogenitor pool in the bone marrow, only allowing the more differentiated cell to travel to sites of bone formation.

BMPs: Conserved Morphogens in Neural Patterning

BMPs: Conserved Morphogens in Neural Patterning

Id Genes Are Direct Targets of Bone Morphogenetic Protein Induction in Embryonic Stem Cells

Bone morphogenetic proteins (BMPs) are morphogenetic signaling molecules essential for embryonic patterning. To obtain molecular insight into the influence of BMPs on morphogenesis, we searched for new genes directly activated by BMP signaling. In vitro cultured mouse embryonic stem (ES) cells were used, cultivated in chemically defined growth medium (CDM). CDM-cultured ES cells responded very selectively to stimulation by various mesoderm inducers (BMP2/4, activin A, and basic fibroblast growth factor). BMP2/4 rapidly induced transcript levels of the homeobox genes Msx-1 and Msx-2 and the proto-oncogene JunB, whereas c-jun transcripts displayed delayed albeit prolonged increase. Using differential display cDNA cloning, six direct BMP target genes were identified. These include Id3, which showed strong mRNA induction, and the moderately induced Cyr61, DEK, and eIF4AII genes, as well as a gene encoding a GC-binding protein. Besides Id3, also the Id1 and Id2 genes were activated by BMP4 in both ES cells and a range of different cell lines. Id genes encode negative regulators of basic helix-loop-helix transcription factors. In vivo we observed local ectopic expression of Id3 and Msx-2 mRNAs in Ft/+ embryos at overlapping regions of ectopic Bmp4 misexpression. We therefore propose that the Msx and Id genes are direct target genes of embryonic BMP4 signaling in vivo.

Endogenous and Ectopic Expression ofnogginSuggests a Conserved Mechanism for Regulation of BMP Function during Limb and Somite Patterning

The genenoggin,originally cloned inXenopus,encodes a secreted factor expressed in the Spemann organizer, where it functions to oppose the ventralizing influence of bone morphogenetic proteins (BMPs). Noggin protein acts by binding directly to BMPs, thereby preventing them from interacting with their receptors. Here we describe the pattern of expression of the chickennoggingene during somite and limb development, two tissues in which BMPs have been postulated to play essential patterning roles. We find thatnogginis expressed in dynamic restricted patterns consistent with an important role in the modulation of BMP signaling. Using a replication competent retrovirus we have ectopically expressednogginin developing somitic and limb bud mesoderm and observed phenotypes consistent with complete block of BMP activity. This includes suppression of lateral somite differentiation and, in the limb, complete inhibition of chondrogenesis and local suppression of programmed cell death. In addition, we find that ectopicnogginexpression in the limb has no effect on anteroposterior limb pattern, suggesting that BMPs are unlikely to play a significant role in this process. Taken together, our results indicate thatnogginis a key regulator of vertebrate limb and somite patterning and suggest that the antagonistic Noggin–BMP interaction is a widely used mechanism to modulate BMP signaling during multiple inductive events in vertebrate embryogenesis.

Effects of osteogenic protein-1 (OP-1, BMP-7) on bone matrix protein expression by fetal rat calvarial cells are differentiation stage specific.

Bone morphogenetic proteins (BMPs) are a group of cytokines that are characterized by their ability to stimulate osteoblast differentiation and bone formation. However, the influence of BMPs on osteoblastic cells at different stages of differentiation is not known. Since bone matrix proteins are differentially regulated during bone formation we have studied the effects of recombinant human osteogenic protein-1 (rhOP-1; BMP-7) on the expression of these proteins by fetal rat calvarial cells (FRCCs) at discrete stages of osteoblast differentiation. Continuous administration of rhOP-1 to FRCCs, beginning at confluence (day 7), produced a dose-dependent increase in the number, size and mineralization of bone-like nodules formed in the presence of vitamin C and beta-glycerophosphate. Within 9 h of administration, rhOP-1 stimulated a 3-fold increase in OPN mRNA which was reflected in a comparable increase in the low phosphorylated, 55 kDa form of osteopontin. In contrast, changes in type 1 collagen, alkaline phosphatase and bone sialoprotein mRNAs followed the differentiation of preosteoblastic cells, and were increased 2-, 4- and 5-fold, respectively, after 8 days (day 15). When administered at intermediate stages of osteoblast differentiation (days 12, 15 and 18) BSP remained refractory to rhOP-1 whereas the ALP was increased almost 2-fold, independent of the constitutive levels of mRNA expression. To determine the effects on osteoblasts, FRCCs were first grown to the bone nodule-forming stage (day 21) before rhOP-1 was administered. Only modest, transient increases in the expression of ALP and OPN mRNAs were evident whereas OC expression was increased more than 3-fold. In contrast, collagen type 1 and BSP mRNA levels were not changed significantly. These results suggest that rhOP-1 increases bone formation by promoting osteoblastic differentiation, as indicated by the increased number of bone forming colonies and by increasing the number of osteoblastic cells in the colonies, but not by increasing matrix production by individual osteoblasts. It is also evident that the regulation of bone matrix proteins by rhOP-1 is dependent upon the differentiated state of the cell.

Bone morphogenetic protein inhibits differentiation and affects expression of helix‐loop‐helix regulatory molecules in myoblastic cells

Bone morphogenetic protein (BMP) reproducibly induces chondrogenesis and osteogenesis when implanted into skeletal muscle. The exact identity of the cell that responds to BMP is not known. Furthermore, controversy exists regarding the possibility that myoblastic cells may transdifferentiate to chondrocytes and osteoblasts under the influence of BMP. We have therefore, undertaken studies on the effects of BMP on differentiation in L6 and C2C12 cells, two rodent myoblastic cell lines. To gain insights into the mechanisms of action of BMP, we have studied the effects of BMP on the levels of expression of the four known myogenic determination genes: myogenin, Myo D, herculin, and myf-5. BMP inhibited myogenesis in myoblastic cells. Convincing evidence of transdifferentiation of myoblasts to chondrocytes or osteoblasts was not seen. BMP inhibited the expression of all four myogenic determination genes.

Cartilaginous Transdifferentiation of Rat Tenosynovial Cells Under the Influence of Bone Morphogenetic Protein in Tissue Culture

In an in vitro system cartilage tissue was formed from the patellar ligament of young adult rats in the crevices of bone matrix gelatin (BMG) under the influence of bone morphogenetic protein (BMP). The rate of cartilage induction was 75% (six of eight) for the explants using patellar ligaments of rat knee joints. Almost the same rate was observed, 67% (four of six), for explants of ligament with fatty tissue. Tenosynovial cells and synovial cells from the surface of ligaments are potent responders to BMP, and this property may be applied to accelerate the biologic attachment of excised tendons and ligaments in clinical practice.

Differentiation of Cartilage on Three Substrata Under the Influence of an Aggregate of Morphogenetic Protein and Other Bone Tissue Noncollagenous Proteins (BMP/iNCP)

A cellulose acetate membrane was fashioned into a cone to serve as a substratum for an outgrowth of connective tissue from normal neonatal muscle, and a container for diffusion of bone morphogenetic protein (BMP) of an aggregate of BMP and cold water insoluble noncollagenous protein (BMP/iNCP). The BMP/iNCP was prepared by dissociative extraction and differential precipitation with other bone matrix proteins that slowly become soluble and diffusible in culture media at 37 degrees. The BMP/iNCP was applied either on, within, or beneath the surface of the explants or suspended in the culture medium. Under the influence of BMP in tissue cultures, without any bone matrix or bone collagen in the system, connective tissue outgrowths of muscle differentiate into cartilage on three substrata: (1) cellulose acetate membranes with pore size of 0.45-5.0 micron; (2) remnants of undissolved BMP/iNCP; and (3) degenerating myofibers. The cartilage developed in the interior of muscle, possibly by phenotypic cell transformation, when the pore size of the membrane was 0.1-0.22 micron too small to sustain anchorage of the explant. Cartilage developed on particle surfaces when the muscle tissue and BMP/iNCP particle were minced and mixed before explantation. The cartilage preferentially grew out directly onto the cellulose acetate membrane when the pore size was optimal for anchorage and the BMP/iNCP was suspended on the surface of the explant to either simultaneously percolate through the explant or diffuse through the culture medium. The biosynthetic activity of cells proliferating before and associated with cell differentiation was measured by 35S uptake in total glycosaminoglycan (GAG) per microgram of DNA. When the pore size was 8.0 micron, large enough to permit cells to migrate across the membrane, a thick plate of fibrous connective tissue developed on the undersurface of the membrane without any evidence of cartilage cell differentiation in any location. Repeated doses of BMP/iNCP with each change of culture medium produced a greater incidence and quantity of cartilage than a single dose, but the 35S incorporation into GAG always reached peak levels, in the interval between four and ten days, irrespective of the schedule of administration or dosage. These observations suggested that the exogenous or endogenous noncollagenous proteins are a carrier for BMP and can substitute for whole bone matrix or bone collagen.

The reaction of the dura to bone morphogenetic protein (BMP) in repair of skull defects.

Trephine defects in the adult rat skull 0.8 cm in diameter, which do not spontaneously heal, were filled with a bovine bone morphogenetic protein (BMP) fraction. The defects healed not only by bony ingrowth from the trephine rim, but also by proliferation of pervascular mesenchymal-type cells (pericytes) of the dura mater. Under the influence of BMP, dural pericytes differentiated into chondroid and woven bone. Between three and four weeks postimplantation, sinusoids formed and the woven bone remodelled into lamellar bone. Concurrently, blood-borne bone marrow cells colonized the bone deposits, and the diploe were restored. Demonstrating that it is soluble in interstitial fluid, and diffusible across a nucleopore membrane (which isolated the bony margins of the skull), BMP induced new bone formation in the underlying dura and complete repair of the defect. The response of the dura to the BMP fraction produced more new bone than the response to allogeneic bone matrix. The BMP-induced repair was dose dependent; the quantity of new bone was proportional to the dose of the implanted BMP.