Bone Morphogenetic Protein Group Research Articles

Erythropoietin modulates the structure of bone morphogenetic protein 2-engineered cranial bone.

The ideally engineered bone should have similar structural and functional properties to the native tissue. Although structural integrity is critical for functional bone regeneration, we know less about modulating the structural properties of the engineered bone elicited by bone morphogenetic protein (BMP) than efficacy and safety. Erythropoietin (Epo), a primary erythropoietic hormone, has been used to augment blood transfusion in orthopedic surgery. However, the effects of Epo on bone regeneration are not well known. Here, we determined the role of Epo in BMP2-induced bone regeneration using a cranial defect model. Epo administration improved the quality of BMP2-induced bone and more closely resembled natural cranial bone with a higher bone volume (BV) fraction and lower marrow fraction when compared with BMP2 treatment alone. Epo increased red blood cells (RBCs) in peripheral blood and also increased hematopoietic and mesenchymal stem cell (MSC) populations in bone marrow. Consistent with our previous work, Epo increased osteoclastogenesis both in vitro and in vivo. Results from a metatarsal organ culture assay suggested that Epo-promoted osteoclastogenesis contributed to angiogenesis because angiogenesis was blunted when osteoclastogenesis was blocked by alendronate (ALN) or osteoprotegerin (OPG). Earlier calcification of BMP2-induced temporary chondroid tissue was observed in the Epo+BMP group compared to BMP2 alone. We conclude that Epo significantly enhanced the outcomes of BMP2-induced cranial bone regeneration in part through its actions on osteoclastogenesis and angiogenesis.

Effect of grafting BMP2-derived peptide to nanoparticles on osteogenic and vasculogenic expression of stromal cells

Bone morphogenetic protein-2 (BMP2) plays a major role in initiating the cascade of osteogenesis. However, high doses of exogenous BMP2 coupled with diffusion away from the intended site cause adverse side-effects. An alternative is to use biodegradable polymeric nanoparticles (NPs) grafted with peptides of the active domains of BMP2. NPs present a multivalent form of the peptide for stronger interaction with cell surface receptors, leading to a stronger activation of osteogenic signalling pathways. The objective of this work was to compare osteogenic activity of the BMP2 peptide (BMP2Pe), corresponding to residues 73-92 of BMP2 protein (BMP2Pr), grafted to biodegradable NPs with that of BMP2 protein (BMP2Pr). BMP2Pe was functionalized with a cysteine residue and grafted to poly(lactide fumarate) and poly(lactide-co-ethylene oxide fumarate) (PLAF/PLEOF) NPs via a thioether link. The calcium content of bone marrow stromal (BMS) cells cultured in osteogenic medium supplemented with BMP2 peptide/protein-grafted NPs (BMP2Pe-gNP and BMP2Pr-gNP) was slightly higher than other BMP2-treated groups, but all osteogenic groups showed similar levels of mineralization after 21 days. The expression pattern of master transcription factors Dlx5 and Runx2 indicated that BMP2 protein induced faster osteogenic signalling than the BMP peptide. The expression level of Osteopontin (OP), Osteocalcin (OC) and PECAM-1 in the NP-grafted BMP2 groups was significantly higher than those of ungrafted BMP2Pr and BMP2Pe groups, which may be due to a more effective presentation of the peptide/protein to cell surface receptors, thus leading to a stronger interaction of the peptide/protein with clustered cell surface receptors.

Regenerative repair of bone defects with osteoinductive hydroxyapatite fabricated to match the defect and implanted with combined use of computer-aided design, computer-aided manufacturing, and computer-assisted surgery systems: a feasibility study in a canine model

BackgroundCurrently, regenerative repair of large bone defects that result from bone tumor resection or severe trauma is a challenging issue because of the limited regenerative potential of bone and treatment modalities. The aim of this study was to achieve repair of large bone defects to the original three-dimensional (3D) anatomical state by combining computer-aided technologies and local delivery of bone morphogenetic protein (BMP) in a canine model. MethodsComputed tomography (CT) images of the pelvic bone of each dog were obtained, and an imaginary spherical malignant bone tumor of 15-mm diameter was placed in the left ilium of a canine on the 3D CT image. Resection of the whole tumor with a 10-mm margin of healthy bone was planned preoperatively by using computer-aided design (CAD) software. In addition, an image of the implant to be used to fill the resulting bone defect was constructed on the computer image. A porous hydroxyapatite (HA) implant identical to the imaged bone defect was made by shaving a tetragonal porous apatite block (40 × 20 × 10mm) with a computer-aided manufacturing system operated by using the CT-image data of the bone defect obtained from the CAD system. To resect the iliac bone as planned preoperatively on the 3D CT image, computer-aided surgery was performed using the CT data. The defect was filled with the HA implant fabricated as described and coated with a putty carrier either with BMP-2 (BMP group, n = 6) or without BMP-2 (control group, n = 6). ResultsIn the BMP group, new bone formation was noted around each implant on CT images at 3weeks after surgery and was remodeled to restore the original anatomy of the ilium on serial CT images. At 12weeks, the implant was enclosed within new bone, and histological analysis revealed bone formation on and within the implant. Little bone formation was noted in the control group. ConclusionsThis new method may enable efficacious and precise regenerative repair of large bone defects without bone grafting.

Periodontal regeneration using an injectable bone cement combined with BMP-2 or FGF-2

Periodontitis is a frequently diagnosed oral disease characterized by bone resorption and soft tissue loss around teeth. Unfortunately, currently available therapies only slow or arrest progress of the disease. Ideally, treatment of periodontal defects should be focused on complete regeneration of the lost tissues [(bone and periodontal ligament (PDL)]. As a result, this study used intrabony defects to evaluate the regenerative potential of an injectable macroporous calcium phosphate cement (CaP) in combination with bone morphogenetic protein-2 (BMP-2) or fibroblast growth factor-2 (FGF-2). After creating 30 periodontal defects in 15 Wistar rats, three treatment strategies were conducted: application of CaP only, CaP + BMP-2 and CaP + FGF-2. Animals were euthanized after 12 weeks and processed for histology and histomorphometry. Using CaP alone resulted in limited effects on PDL and bone healing. CaP + BMP-2 showed a good response for bone healing; a significant 2.4 fold increase in bone healing score was observed compared to CaP. However, for PDL healing, CaP + BMP-2 treatment showed no difference compared to the CaP group. The best results were observed with the combined treatment of CaP + FGF-2, which showed a significant 3.3 fold increase in PDL healing score compared to CaP + BMP-2 and a significant 2.6 fold increase compared to CaP. For bone healing, CaP + FGF-2 showed a significant 1.9 fold increase compared to CaP but no significant difference was noted compared to the CaP + BMP-2 group. The combination of a topical application of FGF-2 and an injectable CaP seems to be a promising treatment modality for periodontal regeneration.

An experimental setup to evaluate innovative therapy options for the enhancement of bone healing using BMP as a benchmark – a pilot study

Critical or delayed bone healing in rat osteotomy (OT) models is mostly achieved through large defects or instability. We aimed to design a rat OT model for impaired bone healing based on age, gender and parity. The outcome should be controllable through variations of the haematoma in the OT including a bone morphogenetic protein (BMP) 2 guided positive control. Using external fixation to stabilise femoral a 2 mm double OT in 12 month old, female Sprague Dawley rats after a minimum of 3 litters healing was characterised following in situ haematoma formation (ISH-group)), transplantation of a BMP charged autologous blood clot (BMP-group) and the artificial blood clot only (ABC-group) into the OT-gap. In vivo micro-computer tomography (µCT) scans were performed after 2, 4 and 6 weeks. After 6 weeks specimens underwent histological analyses. In µCT examinations and histological analyses no bony bridging was observed in all but one animal in the ISH-group. In the BMP group complete bridging was achieved in all animals. The ABC-group showed less mineralised tissue formation and smaller bridging scores during the course of healing than the ISH-group. In this pilot study we introduce a model for impaired bone healing taking the major biological risk factors into account. We could show that the in situ fracture haematoma is essential for bone regeneration. Using BMP as a positive control the presented experimental setup can serve to evaluate innovative therapeutical concepts in long bone application.

Reconstruction of radial bone defects using the reinforced tissue-engineered periosteum

The objective of this study was to compare the osteogenic potential of reinforced and conventional tissue-engineered periosteum.Adipose-derived stromal cells of rabbits were induced into osteoblasts. Osteoinduced cells were seeded onto chitosan-tricalcium-phosphate-gelatin (Cs-TCP-Gel) and chitosan (Cs) scaffold, thus constructing the reinforced and conventional tissue-engineered periostea, respectively. Alkaline phosphatase (ALP) and von Kossa staining protocols were used to assess osteoblast phenotype.We surgically created a 15-mm-long bone defect in the right radii of New Zealand rabbits. The defects were treated with reinforced biomimetic periosteum in group A (n = 30) and treated with conventional tissue-engineered periosteum in group B (n = 30).Group C (n = 30) received CS-TCP-Gel scaffold alone, and group D (n = 30) served as untreated side (sham group). Radiologic,histologic, immunohistochemical, and histomorphometric studies were used to analyze healing pattern.ALP was remarkably expressed in the osteoinduced cells, indicating that osteoblastic differentiation was stable. Extracellular matrix calcification with dark nodule was detected by von Kossa staining. Compared with groups B and C, histologic results demonstrated that de novo osteogenesis proliferated in group A at 4 weeks. This was further confirmed by radiographic findings, which displayed the segmental gap completely healed by mature bone at 12 weeks. Robust expression of bone morphogenetic protein-2 in group A was also evident, whereas group D displayed poor osteogenic performance. Furthermore, histomorphometric and biomechanical results in group A demonstrated statistical significance over those in other groups (p 0.05).Our findings show that the reinforced tissue-engineered periosteum is superior to conventional one as a better biomimetic tissue,further indicating that it can repair the weight-bearing defects.

Enhancement of Human Peripheral Blood Mononuclear Cell Transplantation-Mediated Bone Formation

Recent studies have demonstrated the existence of osteoblast progenitor cells in circulating blood. Here we show that local delivery of bone morphogenetic protein-2 (BMP-2) to cell transplantation sites induces in situ osteogenic differentiation of transplanted human peripheral blood mononuclear cells (PBMNCs) and enhances in vivo bone formation mediated by PBMNC transplantation. Human PBMNCs were seeded on scaffolds with or without BMP-2 and implanted subcutaneously into athymic mice. Nonseeded scaffolds with BMP-2 were also implanted. Eight weeks later, radiographic and histological analyses showed that the PBMNC + BMP-2 group had undergone much more extensive bone formation than either the PBMNC group or BMP-2 group. Only the PBMNC + BMP-2 group expressed human Cbfa1, osteonectin, and osteocalcin, suggesting in situ osteogenic differentiation of and bone formation by transplanted human PBMNCs, while the other groups did not express these genes. This study provides a method to enhance human PBMNC transplantation-mediated bone formation.

Intramarrow Bone Morphogenetic Protein 4 Gene Delivery Enhances Early Implant Stability in Femurs of Ovariectomized Rabbits

Sufficient early implant stability is critical to prevent excessive micromovement of the implant during peri-implant healing and to ensure the success of osseointegration. Implants placed in osteoporotic bones are often associated with low early implant stability. The purpose of this study is to determine the effects of intramarrow bone morphogenetic protein 4 (BMP4) gene delivery on early implant stability and peri-implant healing. Adenoviruses encoding human BMP4 or LacZ were introduced into the femoral osteotomy sites immediately before implant placement in ovariectomized rabbits. The implant stability was determined by resonance frequency analysis at weeks 0, 4, and 8. Changes in cortical bone thickness and intrascrew bone formation at weeks 4 and 8 were evaluated by microcomputed tomography analysis and undecalcified histologic examination, respectively. Intramarrow BMP4 gene delivery resulted in more improvement in implant stability at both weeks 4 and 8. Increased increment in peri-implant cortical bone thickness and better intrascrew bone formation were found in the BMP4 group compared to the LacZ group. The results of this study suggest that intramarrow adenoviral gene delivery of BMP4 enhances peri-implant bone healing and improves early implant stability in osteoporotic rabbit femurs.

Intramarrow Bone Morphogenetic Protein 4 Gene Delivery Improves Local Bone Quality in Femurs of Ovariectomized Rabbits

Poor bone quality at implant recipient site is a major risk factor for implant failure. The purpose of this study is to examine the potential of intramarrow bone morphogenetic protein 4 (BMP4) gene delivery for local bone quality improvement. Adenoviral vector encoding human BMP4 (Ad-BMP4) was constructed. Adenovirus encoding β-galactosidase (Ad-LacZ) was used as a control virus. Ad-BMP4 and Ad-LacZ were injected into femurs of ovariectomized rabbits. The temporal changes in bone mineral density at injected areas were determined by repeated measurements by dual-energy x-ray absorptiometry at 0, 1, 2, 4, and 8 weeks after injection. The effects of gene delivery on cortical bone and cancellous bone were evaluated by microcomputed tomography analysis and histologic examination at 8 weeks. The bone mineral density of the BMP4 group was significantly higher than the LacZ group at 4 and 8 weeks by 61% and 35%, respectively. Results from microcomputed tomography analysis and histologic examination at 8 weeks indicated thicker cortical bone and denser cancellous bone in the BMP4 group compared to the LacZ group. Intramarrow gene delivery of BMP4 effectively improved local bone quality for at least 8 weeks. The sustained delivery of osteogenic factors via local gene therapy approach may reduce implant failures associated with poor local bone quality.

Local Administration of Ibandronate and Bone Morphogenetic Protein-2 After Ischemic Osteonecrosis of the Immature Femoral Head

Bisphosphonate therapy has been shown to preserve the osteonecrotic femoral head in experimental and short-term clinical studies. However, a lack of new bone formation within the preserved femoral head due to the inhibition of bone remodeling is a concern. The purpose of this investigation was to determine if combined therapy consisting of ibandronate and bone morphogenetic protein-2 (BMP-2) can preserve the shape of the femoral head and stimulate new bone formation in an immature animal model of ischemic osteonecrosis. Ischemic osteonecrosis was surgically induced in immature pigs. Four groups were studied: normal, treated with saline solution, treated with ibandronate, and treated with both ibandronate and BMP-2 (the ibandronate + BMP-2 group). The animals were killed eight weeks after surgery. Radiographic, histological, and histomorphometric assessments were performed. Radiographic assessment showed better preservation of the femoral head shape-i.e., a 54% (CI [95% confidence interval]: 22%, 86%) higher mean epiphyseal quotient-in the ibandronate + BMP-2 group than in the saline group. Histological assessment showed increased trabecular bone in the ibandronate + BMP-2 group as compared with that in the saline group. The mean values for trabecular bone volume, thickness, and number and for osteoblast surface were an average of 400% (CI: 242%, 558%), 212% (CI: 166%, 259%), 71% (CI: 6%, 137%), and 2402% (CI: 2113%, 2693%) higher, respectively, in the ibandronate + BMP-2 group than in the saline group. The osteoclast number was significantly reduced in the ibandronate + BMP-2 group compared with that in the saline group (-59% [CI: -75%, -42%]). The mean osteoblast surface value in the ibandronate + BMP-2 group was significantly higher (2567% [CI: 2258%, 2877%]) than that in the ibandronate group. Heterotopic ossifications were present in the capsule of the hip joint in the ibandronate + BMP-2 group. A combination of ibandronate and BMP-2 decreased femoral head deformity while stimulating bone formation in an immature animal model of ischemic osteonecrosis.

Effect of transfected bone morphogenetic protein-2 gene and insulin-like growth factor gene on gene expression of Wistar rat mesenchymal stem cells

Objective To investigate whether bone morphogenetic protein-2 (BMP-2) and insulin-like growth factor-1 ( IGF-1 ) in high glucose can be transfected into Wistar rat mesenchymal stem cells,and the gene expression after transfection. Methods Adenovirus-mediated BMP-2 and IGF-1 genes were transfected into the Wistar rat mesenchymal stem cells. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA) were used to detect the gene replication and expression. Results After the transfection of adenovirus-mediated BMP-2 and(or) IGF-1 gene into the cells in high glucose, all of bone marrow stromal cells survived, bone marrow stromal cells could express the target gene product with the peak of BMP-2 gene product expression at in 72nd h after transfection. The grey degree and gene product concentration of BMP-2 in BMP-2 group were ( 1.86 ± 0. 06) and (58.60 ±1.61) μg/L, and those in BMP-2 ± IGF-1 group (2.37 ±0.34) and (67.61 ±1.91) μg/L,respectively (all P ＜ 0. 05 ). The peak of IGF-1 gene expression occurred at 48th h after transfection. The grey degree and gene product concentration of IGF-1 in IGF-1 group were ( 1.48 ± 0. 03 ) and ( 65.17 ±3.64) μg/L, and those in BMP-2 + IGF-1 group were (1.93 ±0. 17) and (73. 94 ±4. 30) μg/L, respectively ( all P ＜0. 05). Conclusion Adenovirus-mediated BMP-2 and IGF-1 gene could be transfected the Wistar rat bone marrow stromal cells in high glucose. The gene products after transfection were increased significantly. There was a synergistic effect on gene expression between BMP-2 and IGF-1. Key words: BMP-2; IGF-1; High glucose; Bone marrow mesenchymal stem cells

Reconstruction of the alveolar cleft: can growth factor-aided tissue engineering replace autologous bone grafting? A literature review and systematic review of results obtained with bone morphogenetic protein-2

The alveolar cleft in patients with clefts of lip, alveolus and palate (CLAP) is usually reconstructed with an autologous bone graft. Harvesting of autologous bone grafts is associated with more or less donor site morbidity. Donor site morbidity could be eliminated if bone is fabricated by growth factor-aided tissue engineering. The objective of this review was to provide an oversight on the current state of the art in growth factor-aided tissue engineering with regard to reconstruction of the alveolar cleft in CLAP. Medline, Embase and Central databases were searched for articles on bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 7, transforming growth factor beta, platelet-derived growth factor, insulin-like growth factor, fibroblast growth factor, vascular endothelial growth factor and platelet-rich plasma for the reconstruction of the alveolar cleft in CLAP. Two-hundred ninety-one unique search results were found. Three articles met our selection criteria. These three selected articles compared BMP-2-aided bone tissue engineering with iliac crest bone grafting by clinical and radiographic examinations. Bone quantity appeared comparable between the two methods in patients treated during the stage of mixed dentition, whereas bone quantity appeared superior in the BMP-2 group in skeletally mature patients. Favourable results with BMP-2-aided bone tissue engineering have been reported for the reconstruction of the alveolar cleft in CLAP. More studies are necessary to assess the quality of bone. Advantages are shortening of the operation time, absence of donor site morbidity, shorter hospital stay and reduction of overall cost.

Enhanced bone regeneration around dental implant with bone morphogenetic protein 2 gene and vascular endothelial growth factor protein delivery

To evaluate the synergistic effect of bone morphogenetic protein 2 (BMP-2) and vascular endothelial growth factor (VEGF) on the repair of bone defects around dental implants. Five groups of scaffold were fabricated by a freeze-drying method, including pure chitosan/collagen scaffold; scaffold loaded with adenoviruses expressing BMP-2, adenoviruses expressing VEGF, both adenoviruses expressing BMP-2 and VEGF, VEGF protein and adenovirus expressing BMP-2. In vitro studies examined whether bone marrow stromal cells were responsive to these scaffolds over time. Bone formation capacity, bone-to-implant contact, as well as removal torque values were investigated in vivo. Differences between the various groups were statistically analyzed using the one-way analysis of variance test. The in vitro study revealed a burst and rapid release of VEGF with a sustained high-level expression of BMP-2 in scaffold combined with VEGF protein and adenoviruses expressing BMP-2. Histomorphometry demonstrated that scaffolds expressing BMP-2 enhanced more bone formation compared with other groups; VEGF alone is insufficient to promote bone formation. New bone formation in the bone defects around dental implants, bone-to-implant contact and mean peak removal torque showed statistically significant difference for the adenoviral vector encoding human bone morphogenetic protein 2 (Ad-BMP-2) and VEGF protein and adenovirus expressing BMP-2 groups. Furthermore, scaffold combined with VEGF protein and Ad-BMP-2 represented the best outcomes in this model. A combination of BMP-2 gene and VEGF protein could have a synergistic effect in promoting bone healing.

Local treatment of cancellous bone grafts with BMP-7 and zoledronate increases both the bone formation rate and bone density

Background and purpose The remodeling of morselized bone grafts in revision surgery can be enhanced by an anabolic substance such as a bone morphogenetic protein (BMP). On the other hand, BMPs boost catabolism and might cause a premature resorption, both of the graft and of the new-formed bone. Bisphosphonates inactivate osteoclasts and can be used to control the resorption. We studied a combination of both drugs as a local admix to a cancellous allograft.Methods Cancellous bone allografts were harvested and freeze-dried. Either saline, BMP-7, the bisphosphonate zoledronate, or a combination of BMP-7 and zoledronate were added in solution. The grafts were placed in bone conduction chambers and implanted in the proximal tibia of 34 rats. The grafts were harvested after 6 weeks and evaluated by histomorphometry.Results Bone volume/total volume (BV/TV) was 50% in the grafts treated with the combination of BMP-7 and zoledronate and 16% in the saline controls (p < 0.001). In the zoledronate group BV/TV was 56%, and in the BMP group it was 14%. The ingrowth distance of new bone into the graft was 3.5 mm for the combination of BMP-7 and zoledronate and 2.6 mm in the saline control (p = 0.002). The net amount of retained remodeled bone was more than 4 times higher when BMP-7 and zoledronate were combined than in the controls.Interpretation An anabolic drug like BMP-7 can be combined with an anti-catabolic bisphosphonate as local bone graft adjunct, and the combination increases the amount of remaining bone after remodeling is complete.

Mandibular Alveolar Bony Defect Repair Using Bone Morphogenetic Protein 2-Expressing Autologous Mesenchymal Stem Cells

Mandibular bone regeneration is stepped up by human recombinant bone morphogenetic protein 2 (BMP-2) whose application is also related to limited cementum and periodontal ligament regeneration, local root resorption, and ankylosis. The alveolar bone grafting without traditional autologous bone grafts remains a challenge for plastic surgeons. Bilateral mandibular alveolar and periodontal defects were created over the premolar areas in 9 mature male beagles. The defects were randomly assigned for either the adenovirus BMP-2 (advBMP-2) group with BMP-2-expressing mesenchymal stem cells (MSCs) or the control with MSCs alone. The regenerated periodontal attachment apparatus was evaluated histologically, and the whole regenerated bone volume was scrutinized from three-dimensional computed tomography analysis. Periodontal apparatus regeneration was significantly better in the advBMP-2 group. New cementum and Sharpey fibers were observed on the denuded root surfaces in the advBMP-2 group, whereas incomplete healing with localized root surface resorption was noted in the control group. Eight weeks after implantation, the advBMP-2 group showed significant increase in bone regeneration than the control one. Thus, the use of ex vivo BMP-2-engineered autologous MSCs boosted bone and periodontal apparatus regeneration in mandibular periodontal defects. This de novo approach might be suitable for clinical mandibular bone repair and periodontal apparatus repair.

Comparative in vivo study of injectable biomaterials combined with BMP for enhancing tendon graft osteointegration for anterior cruciate ligament reconstruction

This study was to compare effect of osteointegration of grafted tendon in bone tunnels between injected calcium phosphate cement (ICPC) and injected fibrin sealant (IFS) combined with bone morphogenetic protein (BMP) after anterior cruciate ligament (ACL) reconstruction. ACL reconstruction was performed bilaterally in 51 rabbits. ICPC-BMP composite was injected into one knee, with the contralateral knee IFS-BMP composite. The rabbits were killed at postoperative weeks 2, 6, and 12 for testing. Histological observations showed the ICPC composite gradually increased the new bone formation during the whole healing process, while the IFS composite had a burst effect on enhancing the healing of tendon-to-bone at 2 and 6 weeks. By 12 weeks, there was more new cartilage and new bone in the interface in the ICPC-bBMP group. Micro-CT showed that the values of BMD in the ICPC-bBMP group were lower than those in the IFS-bBMP group at 6 weeks, while the values in the ICPC-bBMP group were higher than those in the IFS-bBMP group at 12 weeks (p > 0.05). Fluorescent labels showed that the rate of new bone formation of IFS-BMP composite was significantly higher than that of ICPC composite at 6 weeks (3.45 ± 0.62 µm/day vs. 2.93 ± 0.51 µm/day), but the rate was decreased compared with ICPC composite at 12 weeks (2.58 ± 0.72 µm/day vs. 3.05 ± 0.68 µm/day; p < 0.05). Biomechanically, the ultimate failure load in the ICPC-BMP group was always higher than that in the IFS-BMP group. It is evident that the ICPC composite achieved a more prolonged osteogenic effect than that by IFS composite.

Effect of bone morphogenetic protein-2 on proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells in vitro

Objective To study the effects of the bone morphogenetic protein-2 (BMP-2) on proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs) in vitro. Methods hUCMSCs were cultured in vitro, and BMP-2 (20 mg/L) was added into the culture medium. The proliferation effect of BMP-2 on hUCMSCs was examined by MTT assay. The proportion of antigen-positive cells for STRO-1 was observed by flow cytometry (FCM). The osteopontin ( OPN), alkaline phosphatase (ALP) and type 1 collagen ( COL1 ) mRNA levels was detected by reverse transcription-polymerase chain reaction ( RT-PCR). The ALP activity was tested by ALP staining array. Von Kossa staining method was used to study the calcification effects. Results The proliferation rate was increased 1-, 3-,5-, 7-day BMP-2 (20 mg/L) groups and no BMP-2 group, but there was no statistically significant difference among groups (P ＞ 0. 05). When the concentration of serum in the culture medium decreased from 10% to 2%, the proliferation was just increasing 10% and no different between BMP-2 group and no BMP-2 group (P＞0.05). The percentages of STRO-1 positive cells was increasing from 25.1 ±4.0 to 51.1 ±6.4 after culture with BMP-2( P ＜ 0.01 ). The BMP-2 increased the ALP and COL1 gene expression, induced the OPN gene expression. A lot of hUCMSCs was positive in ALP staining array in BMP-2 medium. Many mineralized nodes was detected in the Von kossa staining method after culture 28 days in the BMP-2 medium.Conclusion BMP-2 can promote the osteogenic differentiation,and have a little effect on the prolferation of hUCMSCs. Key words: Bone morphogenetic protein-2; Umbilical cord mesenchymal stem cell; Osteogenic differntiation

The role of BMP7 in periosteal cells in vitro

Objective To study the role of bone morphogenetic protein-7 in the osteogenic differentiation of periosteal cellsin vitro. Methods Periosteal cells, obtained from adult tibial periosteum, were cultured by routine method in vitro, and divided into two groups. One group cultured with BMP7 and the supplements of 100 nmol dexametasone, 10 mmol b-glycerophosphate and 50 mg/mL L-ascorbic acid (BMP7 group), the other cultured with the supplements alone as the control (control group). Ultrastructure and morphological changes of periosteal cells were observed by contrast phase microscope and electron microscope. In order to test the expression of markers of osteoblastic differantiation in periosteal cells, involved mineralized node and alkaline phosphatase. Each group was tested at the time of 5 d, 10 d, 15 d, 20 d, respectively, using ALP kit stain and Von Kossa stain with 3 samples at each time. Results The periosteal cells cultured by routine method and induced into osteoblast differentiation with BMP7 were both growing well, in vitro. Microscope observations showed that the periosteal cells were spindle-shaped, well-stacked, transparent and three-dimensional in the early stage, and cube-shaped or puncheon shaped in the mitotic phase, gradually became wide shuttle and irregular shape with a lot secretion in telophase. The positive cells were visible by the ALP kit staining and Von Kossa staining of calcium nodules at 5 d, 10 d, 15 d and 20 d in both groups.A difference of positive rate at each time point was found between BMP7 group and control group at 5 d, 10 d, 15 d, 20 d, and the difference was statistically significant (P ＜ 0.01). Conclusion It displayed well regeneration and osteogenesis ability in the periosteal cell. BMP7 has definite osteo-inductive activity, which can obviously enhance the proliferation and ossifyng differentiation of periosteal cells. Key words: Bone morphogenetic protein-7 (BMP7); Periosteal cell; Osteoblast; Cell culture

Tumor necrosis factor-α inhibits osteoblast differentiation by interfering with the bone morphogentic protein-2 signaling pathway

Objective To investigate the influence of tumor necrosis factor-α(TNF-α) activation on osteoblast differentiation through interfering with the bone morphogenetic protein-2(BMP-2) signaling pathway. Methods Mouse C2C12 myoblast cells stimulated with BMP-2 were used as the in vitro osteoblast differentiation model. The stimulated cells were cultured with growth factors TNF-α and BMP-2 of different concentrations. We had one blank control group and 3 experimental groups. In the BMP-2 group, 100 ng/mL of BMP-2 was added. In the TNF-α group, 5 ng/mL of TNF-α was added. In the BMP-2 + TNF-α group, 100 ng/mL of BMP-2 and 5 ng/mL of TNF-α were added. After culture for 7 days, ALP staining was used to determine the level of osteoblast differentiation. The P-Nitrophenyl phosphate (PNPP) method was used to measure the activity of alkaline phosphatase (ALP). Results The ALP activity was 0. 260 ± 0. 245 in the BMP-2 group, 7. 311 ±0. 772 in the TNF-α group, and 1. 344 ±0. 133 in the BMP-2 +TNF-α group. The differences between any 2 groups were significant ( P ＜ 0. 05). At a constant concentration of 100 ng/mL of BMP-2, the ALP activities were 10. 207 ±0. 459, 6. 183 ±0. 374, 1. 873 ±0. 388, 0. 096 ±0. 023 respectively when TNF-α of 0, 2, 5, 10 ng/mL were added. The differences between any 2 groups were significant ( P ＜ 0. 05) . ALP activities in C2C12 cells were significantly decreased by TNF-α treatment, but significantly increased by BMP-2. Conclusions TNF-α may inhibit osteoblast differentiation induced by BMP-2 in a dose-dependent manner. However, the inhibition of osteoblast differentiation by TNF-α can be counter balanced if the concentration of BMP-2 treatment is high enough. Key words: Osteoblasts; Bone morphogentic protein; Tumor necrosis factor-α; Cell differentiation

Phase 1 safety and tolerability study of BMP-7 in symptomatic knee osteoarthritis

BackgroundThere are no proven therapies that modify the structural changes associated with osteoarthritis (OA). Preclinical data suggests that intra-articular recombinant human BMP-7 (bone morphogenetic protein-7) has reparative effects on cartilage, as well as on symptoms of joint pain. The objective of this study was to determine the safety and tolerability as well as dose-limiting toxicity and maximal tolerated dose of intra-articular BMP-7. The secondary objectives were to determine the effect on symptomatic responses through 24 weeks.MethodsThis was a Phase 1, double-blind, randomized, multi-center, placebo-controlled, single-dose escalation safety study consisting of 4 dosing cohorts in participants with knee OA. Each cohort was to consist of 8 treated participants, with treatment allocation in a 3:1 active (intra-articular BMP-7) to placebo ratio. Eligible participants were persons with symptomatic radiographic knee OA over the age of 40. The primary objective of this study was to determine the safety and tolerability of BMP-7 including laboratory assessments, immunogenicity data and radiographic assessments. Secondary objectives were to determine the proportion of participants with a 20%, 50%, and 70% improvement in the WOMAC pain and function subscales at 4, 8, 12, and 24 weeks. Other secondary outcomes included the change from baseline to 4, 8, 12, and 24 weeks for the OARSI responder criteria.ResultsThe mean age of participants was 60 years and 73% were female. All 33 participants who were enrolled completed the study and most adverse events were mild or moderate and were similar in placebo and BMP-7 groups. The 1 mg BMP-7 group showed a higher frequency of injection site pain and there was no ectopic bone formation seen on plain x-rays. By week 12, most participants in both the BMP-7 and placebo groups experienced a 20% improvement in pain and overall the BMP-7 group was similar to placebo with regard to this measurement. In the participants who received 0.1 mg and 0.3 mg BMP-7, there was a trend toward greater symptomatic improvement than placebo. The other secondary endpoints showed similar trends including the OARSI responder criteria for which the BMP-7 groups had more responders than placebo.ConclusionsThere was no dose limiting toxicity identified in this study. The suggestion of a symptom response, together with the lack of dose limiting toxicity provide further support for the continued development of this product for the treatment of osteoarthritis.