Use of recombinant human bone morphogenetic protein-2 in spine surgery

Bone morphogenetic proteins are osteoinductive factors which have gained popularity in orthopaedicsurgery and especially in spine surgery. The use of recombinant human bone morphogenetic protein-2 has been officially approved by the United States Food and Drug Administration only for single level anterior lumbar interbody fusion, nevertheless it is widely used by many surgeons with off-label indications. Despite advantages in bone formation, its use still remains a controversial issue and several complications have been described by authors who oppose their wide use.

Implantation of xenogeneic bone combined with recombinant human bone morphogenetic protein-2 into bone defect—An scanning electron microscopic study

To determine the ability of a new type of composite xenogeneic bone grafting to repair bone defect. Methods: The new type of composite xenogeneic bone was obtained by combining the chemically treated cance1lous bone with recombinant human bone morphogenetic protein-2 (rhBMP-2). It was implanted on the bone defect of rabbit. Results: There was a large amount of new bone formation within the combined material and the amount was increasing as the time elapsed. In contrast, there was a lot of fibrous tissue with a little new bone formed on the area of the bone defect when the treated cancellous bone was implanted alone. Conclusion: The results imply that the rhBMP-2 plays a very important role in new bone formation and the composite xenogeneic bone appear to be an ideal material for repair of bone defect.

Expression of mature peptide of human bone morphogenetic protein-2 in Escherichia coli

To express die mature peptide of human bone morphogenetic protein-2 in Escherichia coil. Methods: TheDNA fragment encoding the mature peptide of human bone morphogenetic protein-2 (hBMP-2m) was inserted into expression vectorpDH in which foreign gene was controlled by PRPL promoters. E. coli DH5a transformed with recombinant plasmid pDHB2m wasinduced at 42℃to express the target protein. The expressed product was partially purified and refolded, and then implanted intorat thigh muscles to assay its bone inductive activity. Results: After induction, a protein band on SDS-PAGE gel with an apparentmol. wt. of 13kD was observed to anticipate in the strain carrying pDHB2m, but not in the control. The expressed hBMP-2m accounted for 45%-60% of the total bacterial protein. The expressed product existed in a form of inclusion body. After partially purified and refolded, rhBMP-2m could induce the formation of cartilage and bone tissue heterotopically. Conclusion: The maturepeptide of human bone morphogenetic protein-2 has ben successfully expressed in E. coli and the product has ectopic bone inductive activity.

Bone morphogenetic protein-6 suppresses TGF-β_(2)-induced epithelial-mesenchymal transition in retinal pigment epithelium

AIM:To evaluate the effect of bone morphogenetic protein-6(BMP-6)on transforming growth factor(TGF)-β_(2)-induced epithelial-mesenchymal transition(EMT)in retinal pigment epithelium(RPE).METHODS:Adult retinal pigment epithelial cell line(ARPE-19)were randomly divided into control,TGF-β_(2)(5μg/L),and BMP-6 small interfering RNA(siRNA)group.The cell morphology was observed by microscopy,and the cell migration ability were detected by Transwell chamber.The EMT-related indexes and BMP-6 protein levels were detected by Western blotting.Furthermore,a BMP-6 overexpression plasmid was constructed and RPE cells were divided into the control group,TGF-β_(2)+empty plasmid group,BMP-6 overexpression group,and TGF-β_(2)+BMP-6 overexpression group.The EMT-related indexes and extracellular regulated protein kinases(ERK)protein levels were detected.RESULTS:Compared with the control group,the migration of RPE cells in the TGF-β_(2) group was significantly enhanced.TGF-β_(2) increased the protein expression levels ofα-smooth muscle actin(α-SMA),fibronectin and vimentin but significantly decreased the protein levels of E-cadherin and BMP-6(P<0.05)in RPE.Similarly,the migration of RPE cells in the BMP-6 siRNA group was also significantly enhanced.BMP-6 siRNA increased the protein expression levels ofα-SMA,fibronectin and vimentin but significantly decreased the protein expression levels of E-cadherin(P<0.05).Overexpression of BMP-6 inhibited the migration of RPE cells induced by TGF-β_(2) and prevented TGF-β_(2) from affecting EMT-related biomarkers(P<0.05).CONCLUSION:BMP-6 prevents the EMT in RPE cells induced by TGF-β_(2),which may provide a theoretical basis for the prevention and treatment of proliferative vitreoretinopathy.

Reconstruction of orbital defect in rabbits with composite of calcium phosphate cement and recombinant human bone morphogenetic protein-2

Background Calcium phosphate cement （CPC） is a biocompatible and osteoconductive bone substitute, and recombinant human bone morphogenetic protein-2 （rhBMP-2） has strong osteoinductibility, therefore we developed a composite bone substitute with CPC and rhBMP-2 and evaluate its reconstruction effect in rabbit orbital defect.Methods Thirty-six rabbits were randomly divided into two groups and a 5 mmx5 mmx2 mm bone defect in the infraorbital rim was induced by surgery in each orbit （72 orbits in all）. The orbital defects were treated with pure CPC or composite of CPC and rhBMP-2. The osteogenesis ability of different bone substitute was evaluated by gross observation, histological examination, histomorphometrical evaluation, compressive load-to-failure testing, and scanning electron microscope （SEM）.Results Gross observation showed that both bone substitutes were safe and effective for reconstruction of orbital defect. However, histological examination, histomorphometrical evaluation and SEM showed that CPC/rhBMP-2 group had faster speed in new bone formation and degradation of substitute material than CPC group. Compressive load-to-failure testing showed that CPC/rhBMP-2 group had stronger compressive strength than CPC group at every stage with significant difference （P ＜0.05）.Conclusion Composite of CPC/rhBMP-2 is an ideal bioactive material for repairing orbital defect, with good osteoconductibility and osteoinductibility.

A single nucleotide polymorphism in the human bone morphogenetic protein-2 gene （109T〉G） affects the Smad signaling pathway and the predisposition to ossification of the posterior longitudinal ligament of the spine

Background Although various systemic and local factors such as abnormal carbohydrate or calcium metabolism, aging, and hormonal disturbances have been suggested as causes of ossification of the posterior longitudinal ligament （OPLL）, the etiology of OPLL is not fully understood. The purpose of this study was to investigate whether bone morphogenetic protein （BMP）-2 is a candidate gene to modify the susceptibility of OPLL and the mechanism of signal transduction in ossification. Methods A total of 420 OPLL patients and 506 age- and sex-matched controls were studied. The complete coding sequence of the human BMP-2 gene was analyzed using polymerase chain reaction （PCR） and direct sequencing. All single nucleotide polymorphisms （SNPs） were detected and genotyped. BMP-2 expression vectors containing positive polymorphisms were constructed and transfected into the C3H10T1/2 cells. The expression of BMP-2 and the Smad signal pathway in positive cell clones were detected by Western blotting. The alkaline phosphatase （ALP） activity was determined using quantitative detection kits. Results The frequencies for the 109T〉G and 570A〉T polymorphisms were different between the case and control groups. The ＂TG＂ genotype in 109T〉G polymorphism is associated with the occurrence of OPLL, the frequency of the ＂G＂ allele is significantly higher in patients with OPLL than in control subjects （P 〈0.001）. The ＂AT＂ genotype in 570A〉T polymorphism is associated with the occurrence of OPLL, the frequency of the ＂T＂ allele is significantly higher in patients with OPLL than in control subjects （P=0.005）. Western blotting analysis revealed that the expression of P-Smadl/5/8 protein transfected by wild-type or mutant expression vectors were significantly higher than control groups （P 〈0.05）, but there was no statistical difference in each experimental group （P 〉0.05）. The expression of Smad4 protein transfected by wild-type or mutant expression vectors was significantly higher than control groups （P 〈0.05）. The expression of Smad4 protein transfected by pcDNA3.1-BMP2 （109G） and pcDNA3.1-BMP2 （109G, 570T） was significantly higher than the other experimental groups （P 〈0.05）. The increase in ALP activity has been detected in pcDNA3.1-BMP2 （109G） and pcDNA3.1-BMP2 （109G, 570T） transfected cells up to 4 weeks after stable transfection. Activity of ALP was （30.56±0.46） nmol.min^-1.mg^-1 protein and （29.62±0.68） nmol.min^-1.mg^-1 protein, respectively. This was statistically different compared with the other experimental groups （P 〈0.05）. Conclusions BMP-2 is the predisposing gene of OPLL. The ＂TG＂ genotype in the 109T〉G and the ＂AT＂ genotype in the 570A〉T polymorphisms are associated with the occurrence of OPLL. The 109T〉G polymorphism in exon-2 of the BMP-2 gene is positively associated with the level of Smad4 protein expression and the activity of ALP. The Smad mediated sicjnaling pathway plays an important role durincl the Datholoqical process of OPLL induced by SNPs of BMP-2 aene.

CORAL AS A CARRIER FOR RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN-2

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Drilling Combined with Adipose-derived Stem Cells and Bone Morphogenetic Protein-2 to Treat Femoral Head Epiphyseal Necrosis in Juvenile Rabbits

This study was designed to evaluate the effects of drilling through the growth plate and using adipose-derived stem cells （ADSCs） and bone morphogenetic protein-2 （BMP-2） to treat femoral head epiphyseal ischemic necrosis, which can be done in juvenile rabbits. Passagefour bromodeoxyuridine （BrdU）-labeled ADSCs were cultured, assayed with MTT to determine their viability and stained with alizarin red dye to determine their osteogenic ability. Twomonth-old, healthy male rabbits （1.2 to 1.4 kg, n=45） underwent ischemic induction and were randomly divided into five groups （group A： animal model control; group B： drilling; group C： drilling ＆ ADSCs; group D： drilling ＆ BMP-2; and group E： drilling ＆ ADSCs ＆ BMP-2）. Magnetic resonance imaging （MRI）, X-ray imaging, hematoxylin and eosin staining and BrdU immunofluorescence detection were applied 4, 6 and 10 weeks after treatment. Approximately 90% of the ADSCs were labeled with BrdU and showed good viability and osteogenic ability. Similar results were observed in the rabbits in groups C and E at weeks 6 and 10. The animals of groups C and E demonstrated normal hip structure and improved femoral epiphyseal quotients and trabecular areas compared with those of the groups A and B （P〈0.01）. Group D demonstrated improved femoral epiphyseal quotients and trabecular areas compared with those of groups A and B （P〈0.05）. In summary, drilling through the growth plate combined with ADSC and BMP-2 treatments induced new bone formation and protected the femoral head epiphysis from collapsing in a juvenile rabbit model of femoral head epiphyseal ischemic necrosis.

The Effect of Simvastatin on mRNA Expression of Transforming Growth Factor-β1,Bone Morphogenetic Protein-2 and Vascular Endothelial Growth Factor in Tooth Extraction Socket

Aim To determine the effect of local simvastatin application on the mRNA expression level of transforming growth factor-β1 （TGF-β1）, bone morphogenetic protein-2 （BMP-2） and vascular endothelial growth factor （VEGF） in the tooth sockets of rat. Methodology Forty-eight male Wistar rats were randomly divided into experimental and control groups （n=24）. Polylactic acid/polyglycolic acid copolymer carriers, with or without simvastatin, were implanted into extraction sockets of right mandibular incisors. The expression of TGF-β1, BMP-2 and VEGF mRNA was determined by in situ hybridization in the tooth extraction socket at five days, one week, two weeks and four weeks after implantation. Results The fusiform stroma cells in the tooth extraction socket began to express TGF-β1, BMP-2 and VEGF mRNA in both experimental and control groups from one week after tooth extraction until the end of experiment. The expression of TGF-131 and BMP-2 mRNA in the experimental group was significantly up-regulated after one, two and four weeks, and expression of VEGF mRNA was significantly increased after one and two weeks compared with that in the control group. Conclusion The findings indicate that local administration of simvastatin can influence alveolar bone remodeling by regulating the expression of a school of growth factors which are crucial to osteogenesis in the tooth extraction socket.

Heterotopic ossification after the use of recombinant human bone morphogenetic protein-7

AIM To present the incidence of heterotopic ossification after the use of recombinant human bone morphogenetic protein-7(rhB MP-7) for the treatment of nonunions.METHODS Bone morphogenetic proteins(BMPs) promote bone formation by auto-induction. Recombinant human BMP-7 in combination with bone grafts was used in 84 patients for the treatment of long bone nonunions. All patients were evaluated radiographicaly for the development of heterotopic ossification during the standard assessment for the nonunion healing. In all patients(80.9%) with radiographic signs of heterotopic ossification, a CT scan was performed. Nonunion site palpation and ROM evaluation of the adjacent jointswere also carried out. Factors related to the patient(age, gender), the nonunion(location, size, chronicity, number of previous procedures, infection, surrounding tissues condition) and the surgical procedure(graft and fixation type, amount of rhB MP-7) were correlated with the development of heterotopic ossification and statistical analysis with Pearsons χ~2 test was performed.RESULTS Eighty point nine percent of the nonunions treated with rh BMP-7, healed with no need for further procedures. Heterotopic bone formation occurred in 15 of 84 patients(17.8%) and it was apparent in the routine radiologi-cal evaluation of the nonunion site, in a mean time of 5.5 mo after the rh BMP-7 application(range 3-12). The heterotopic ossification was located at the femur in 8 cases, at the tibia in 6, and at the humerus in οne patient. In 4 patients a palpable mass was present and only in one patient, with a para-articular knee nonunion treated with rhB MP-7, the size of heterotopic ossification affected the knee range of motion. All the patients with heterotopic ossification were male. Statistical analysis proved that patient's gender was the only important factor for the development of heterotopic ossification(P = 0.007). CONCLUSION Heterotopic ossification after the use of rh BMP-7 in nonunions was common but it did not compromise the final clinical outcome in most cases, and affected only male patients.

Regulation of scleral fibroblast differentiation by bone morphogenetic protein-2

Bone morphogenesis proteins(BMPs) are multi-functional growth factors. They are expressed in retina,retinal pigment epithelium(RPE) and sclera and serve as a regulator in the growth and development of the eye. This article reviewed the chondrogenic potency of the sclera,biochemical and pathological changes of myopic scleral tissue and the differentiation of chondrogenesis by BMP-2. We proposed the hypothesis that BMP-2 can regulate differentiate of scleral fibroblasts and affect the development of myopia.

Bone morphogenetic protein-4 affects both trophoblast and non-trophoblast lineage-associated gene expression in human embryonic stem cells

Human embryonic stem cells (hESC) can be induced to differentiate to trophoblast by bone morphogenetic proteins (BMPs) and by aggregation to form embryoid bodies (EB), but there are many differences and controversies regarding the nature of the differentiated cells. Our goals herein were to determine if BG02 cells form trophoblast-like cells (a) in the presence of BMP4-plus-basic fibroblast growth factor (FGF-2) and (b) upon EB formation, and (c) whether the BMP4 antagonist noggin elicits direct effects on gene expression and hormone production in the cells. Transcriptome profiling of hESC incubated with BMP4/FGF-2 showed a down-regulation of pluripotency-associated genes, an up-regulation of trophoblast-associated genes, and either a down-regulation or no change in gene expression for many markers of the three embryonic germ layers. Yet, there was up-regulation of several genes associated with mesoderm, ectoderm, and endoderm, strongly suggesting that differentiation to trophoblast-like cells under the conditions used does not yield a homogeneous cell type. Several genes, heretofore unreported, were identified that are altered in hESC in response to BMP4-mediated differentiation. The production of human chorionic gonadotropin (hCG), progesterone, and estradiol in the differentiated cells confirmed that trophoblast-like cells were obtained. Gene expression by EB was characterized by an up-regulation of a number of genes associated with trophoblast, ectoderm, endoderm, and mesoderm, and the production of hCG and progesterone confirmed that trophoblast-like cells were formed. These results suggest that, in the presence of FGF-2, BG02 cells respond to BMP4 to yield trophoblast-like cells, which are also obtained upon EB formation. Thus, BMP4-mediated differentiation of hESC represents a viable cell system for studying early developmental events post-implantation;however, up-regulation of non-trophoblast genes suggests a somewhat diverse response to BMP4/FGF-2. Noggin altered the transcription of a limited number of genes but, not surprisingly, did not lead to secretion of hormones.

EFFECTS OF TRANSFORMING GROWTH FACTOR β AND RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN 2 ON HUMAN PERIODONTAL LIGAMENT FIBROBLASTS

Objective To evaluate the effects of transforming growth factor β(TGF-β) and recombinant human bone morphogenetic protein 2 (rhBMP2) on human periodontal ligament fibroblasts (HPDLFs). Methods HPDLFs were done primary culture to detect the distinct concentrations of TGF-P and rhBMF2 on its proliferation, alkaline phosphatase (ALP) activity, osteocalcin (OC) synthesis and formation of the minerali-zed nodules, respectively. Results TGF-β (5～100ng/ml) significantly stimulated the proliferation of HPDLFs. The ALP activity of HPDLFs was evaluated evidently by 5ng/ml TGF-β. TGF-β( 0. 5 ～ 100ng/ml) had no effects on OC synthesis and formation of the mineralized nodules of HPDLFs. rhBMP2 (0. 25～2mg/ ml) had no remarkable effect on the proliferation of HPDLFs. The ALP activity, OC synthesis and forma-tion of the mineralized nodules of HPDLFs were significantly stimulated by 0. 5～ 2mg /ml rhBMP2. Conclusion The effects of TGF-β and rhBMP2 on HPDLFs are dose-dependent. TGF-P can stimulate HPDLFs to express the early marker of osteoblastic phenotype, and it lacks the ability to promote maturation of the osteogenic phenotype. rhBMP2 can not only stimulate the expression but also promote the maturation of osteoblas-tic phenotype of HPDLFs.

Construction of bicistronic green fluorescent protein labeled pSELECT GFPzeo human bone morphogenetic protein 2 eukaryotic expression vector

Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector.Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by using polymerase

Influence of bone morphogenetic proteins-2 and strontium chloride on the human umbilical cord mesenchymal stem cells proliferation and osteogenic differentiation

Objective To study the effects of combination of bone morphogenetic protein-2 (BMP-2) and strontium chloride on proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells(hUCM-SCs)in vitro culture.

Effect of dopamine on bone morphogenesis protein-2 expression in human retinal pigment epithelium

AIM：To investigate the effect of dopamine on bone morphogenesis protein-2（BMP-2）expression in retinal pigment epithelium（RPE）cells in vitro.METHODS：ARPE-19 cells as a human RPE cell line were cultured with dopamine for different times（2,4,6,8,12,16and 24h）or with different concentrations（0.1,1,2,5,10,20,and 100μg/m L）in vitro.BMP-2 m RNA expression level in ARPE-19 cells was analyzed with real-time polymerase chain reaction（PCR）analysis and BMP-2 protein level was measured with Western blot analysis.The active form of BMP-2 in the culture medium was measured with enzymelinked immunosorbent assay（ELISA）.RESULTS：The expression level of BMP-2 increased significantly cultured with 20μg/m L dopamine,at different time points（P〈0.05）.BMP-2 m RNA level peaked 2h and the protein level peaked at 6 and 8h after treatment.The concentrations of secreted BMP-2 elevated at 12h and peaked at 24h（P〈0.05）in a time-dependent manner.Treated with 100μg/m L dopamine for 6h,the expression levels of BMP-2 m RNA and protein in ARPE-19 cells were enhanced significantly compared to that in the untreated cells（P〈0.05）.And secreted BMP-2 protein in the cell culture supernatant was also increased（P〈0.05）.CONCLUSION：Dopamine up-regulate BMP-2 expression in RPE cells,and this may be associated with its inhibitive effect on myopia development.

Bone morphogenetic protein-7 induced bone marrow stromal cells differentiate into neuron-like cells

Bone morphogenetic protein-7 is widely accepted as an inducer for bone marrow stem cells differentiating into osteoblasts and chondrocytes. Whether bone marrow stromal cells differentiate into neuron-like cells remains unclear. The current study examined the presence of positive cells for intermediate filament protein and microtubule associated protein-2 in the cytoplasm of bone marrow stromal cells induced by bone morphogenetic protein-7 under an inverted microscope, while no expression of glial fibrillary acidic protein was found. Reverse transcription PCR electrophoresis also revealed a positive target band for intermediate filament protein and microtubule-associated protein 2 mRNA. These results confirmed that bone morphogenetic protein-7 induces rat bone marrow stromal cells differentiating into neuron-like cells.

Three-dimensional kagome structures in a PCL/HA-based hydrogel scaffold to lead slow BMP-2 release for effective bone regeneration

Osteoconductive function is remarkably low in bone disease in the absence of bone tissue surrounding the grafting site,or if the bone tissue is in poor condition.Thus,an effective bone graft in terms of both osteoconductivity and osteoinductivity is required for clinical therapy.Recently,the three-dimensional(3D)kagome structure has been shown to be advantageous for bone tissue regeneration due to its mechanical properties.In this study,a polycaprolactone(PCL)kagome-structure scaffold containing a hyaluronic acid(HA)-based hydrogel was fabricated using a 3D printing technique.The retention capacity of the hydrogel in the scaffold was assessed in vivo with a rat calvaria subcutaneous model for 3 weeks,and the results were compared with those obtained with conventional 3D-printed PCL grid-structure scaffolds containing HA-based hydrogel and bulk-type HA-based hydrogel.The retained hydrogel in the kagome-structure scaffold was further evaluated by in vivo imaging system analysis.To further reinforce the osteoinductivity of the kagome-structure scaffold,a PCL kagome-structure scaffold with bone morphogenetic protein-2(BMP-2)containing HA hydrogel was fabricated and implanted in a calvarial defect model of rabbits for 16 weeks.The bone regeneration characteristics were evaluated with hematoxylin and eosin(H&E),Masson’s trichrome staining,and micro-CT image analysis.