Bone Morphogenetic Protein Group Research Articles

Effect of bone morphogenetic protein 2 and dexamethason on proliferation and differentiation of human dental pulp cells in vitro

To investigate the effect of bone morphogenetic protein 2 (BMP-2) and dexamethason (DXM) on proliferation and differentiation of human dental pulp cells in vitro. Primary human dental pulp cells were cultured in vitro by tissue culture method. The 3rd generation cells were used to identify cell phenotype for vimentin and cytokeratin by immunocytochemistry staining. The 3-5 generations of human dental pulp cells were randomly divided into 4 groups: 100 ng/mL BMP-2 (group A), 1×10 -8 mol/L DXM (group B), and both 100 ng/mL BMP-2 and 1×10 -8 mol/L DXM (group C) were added; neither BMP-2 nor DXM was added in group D as control group. The cell growth curve was drawn at 1, 3, 5, and 7 days after culture. The expressions of osteo/dentanogenic genes including alkaline phosphatase (ALP), dentin sialophoshoprotein (DSPP), and dentin matrix protein 1 (DMP-1) were detected by RT-PCR analysis at 5 and 7 days after culture, the ratio between the positive staining area and the total area by ALP staining at 14 days, and absorbance ( A) value at 562 nm by alizarin red staining at 21 days after culture. Human dental pulp cells were successfully isolated and cultured, which were long fusiform and showed a positive reaction for vimentin and a negative reaction for cytokeratin. The growth curve indicated that cells increased with the extending of incubation time, reached a peak at 5 days, then reduced at 7 days to the level at 3 days. At 5 days after culture, the cells were significantly more in groups A, B, and C than group D ( P<0.05), in group C than group A ( P<0.05), and in group A than group B ( P<0.05). RT-PCR analysis showed that the mRNA expressions of ALP, DSPP, and DMP-1 at 5 days were significantly higher in groups A, B, and C than group D ( P<0.05), and in group C than groups A and B ( P<0.05), but no significant difference was found between groups A and B ( P>0.05); the mRNA expression of DSPP in groups A, B, and C was significantly higher than that in group D ( P<0.05), but there was no significant difference in mRNA expressions between other groups at 7 days ( P>0.05). At 14 days, positive staining in varying degrees was observed in each group, especially in group C; the ratio between the positive staining area and the total area was significantly higher in group C than groups A, B, and D ( P<0.05), and in groups A and B than group D ( P<0.05), but there was no significant difference between groups A and B ( P>0.05). At 21 days, there were a variety of mineralized nodules in groups A, B, and C in nonuniformly scattered or clustered distribution, but no mineralized nodules were observed in group D. The A values of mineralized nodules showed significant difference between groups ( P<0.05). BMP-2 may be more effective in promoting proliferation of human dental pulp cells than DXM. Combined application of BMP-2 and DXM can remarkably promote the proliferation and differentiation of human dental pulp cells.

Relationship between in vitro growth of bovine oocytes and steroidogenesis of granulosa cells cultured in medium supplemented with bone morphogenetic protein-4 and follicle stimulating hormone

Bone morphogenetic protein-4 (BMP-4) and FSH play important regulatory roles in follicular growth and steroidogenesis in vivo. The purpose of this study was to investigate the effects of BMP-4 and FSH on in vitro growth (IVG) and steroidogenesis of bovine oocyte-cumulus-granulosa complexes (OCGCs). We cultured OCGCs collected from early antral follicles (0.5–1 mm) in medium without BMP-4 and FSH for 4 days and investigated the appearance of OCGCs and their steroidogenesis. During the first 4 days of IVG, morphologically normal OCGCs produced more estradiol-17β (E2), but less progesterone (P4). Morphologically normal OCGCs were subjected to an additional culture in medium supplemented with BMP-4 (0, 10, and 50 ng/mL) and FSH (0 and 0.5 ng/mL) until day 12. We examined the viability and steroidogenesis of OCGCs after 8 and 12 days of culture. Oocyte growth, characteristics of granulosa cells, and the maturational competence of oocytes were also investigated. On day 8, the viability of OCGCs cultured without FSH was higher in the 10 ng/mL BMP-4 group than in the 50 ng/mL BMP-4 group (P < 0.05). No significant difference was observed in the viability of groups cultured with FSH, regardless of the addition of BMP-4, and FSH improved the viability of 50 ng/mL BMP-4 group similar to 10 ng/mL BMP-4 group. The total number of granulosa cells was larger in 10 ng/mL BMP-4 group cultured with FSH than in 50 ng/mL BMP-4 group cultured with FSH on day 8 (P < 0.05). E2 production decreased from days 8–12, and P4 production increased throughout IVG culture, regardless of the addition of BMP-4 and FSH (P < 0.05). No significant differences in E2 production were observed between groups from days 4–8, regardless of whether BMP-4 was added without FSH; however, E2 production in the group cultured with 50 ng/mL BMP-4 was suppressed by FSH. BMP-4 suppressed E2 production from days 8–12, regardless of whether FSH was added. The group cultured with 10 ng/mL BMP-4 without FSH showed the lowest P4 production among all groups for all culture periods. OCGCs that produced mature oocytes tended to secrete more E2 and less P4 than OCGCs that produced immature oocytes. In conclusion, until day 8 of the IVG culture, P4 production by OCGCs was suppressed by the addition of 10 ng/mL BMP-4 in the absence of FSH, without inhibiting E2 production. These conditions appear to mimic growing follicles until day 8 and mimic degenerating follicles from days 8–12 of culture.

Differential circRNA expression profiles during the BMP2-induced osteogenic differentiation of MC3T3-E1 cells

ObjectiveRecent studies have indicated that circular RNAs (circRNAs) might play important roles in various diseases. However, little is known about the functions of circRNAs in the skeletal system, and the role of circRNAs in the mechanism by which bone morphogenetic protein 2 (BMP2) promotes bone differentiation remains unknown. Here, we performed RNA-seq to analyze differential expression of circRNA during different osteoblast differentiation stages and investigated the relevant mechanisms. Materials and methodsAlkaline phosphatase (ALP) staining and activity were performed to assess osteogenic differentiation in MC3T3-E1 cells. The expression of osteogenic markers in MC3T3-E1 cells and the differential expression levels of circRNAs were measured and validated by qRT-PCR. Osteogenic marker proteins were measured by western blot. RNA-seq was performed to detect differential expression of circRNAs during the osteogenic differentiation of MC3T3-E1 cells induced by BMP2. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and PANTHER pathway analyses were performed to predict the functions of differentially expressed circRNAs and potentially co-expressed target genes. The microRNA (miRNA) targets of the circRNAs and circRNA–miRNA interactions were predicted by miRanda. The circRNA–miRNA co-expression network was constructed based on the correlation analysis between the differentially expressed circRNAs and miRNAs. A graph of the circRNA–miRNA network was created using Cytoscape 3.01. ResultsThe Cell Counting Kit 8 (CCK-8) assay showed that BMP2 promoted the proliferation of osteoblasts in vitro. Both the intracellular ALP content and activity were increased in BMP2-treated MC3T3-E1 cells. In addition, the mRNA and protein levels of the osteoblastic markers ALP, Sp7 transcription factor (SP7) and runt-related transcription factor 2 (RUNX2) were substantially up-regulated. In the present study, 158 circRNAs were differentially expressed by a fold-change ≥2.0, P<0.05 and false discovery rate <0.05. Among these, 74 circRNAs were up-regulated, while 84 circRNAs were down-regulated. In addition, the expression levels of circRNA.5846, circRNA.19142 and circRNA.10042 were significantly increased in the BMP2 group. Furthermore, by analyzing the target mRNAs of miR-7067-5p using GO and PANTHER pathway analyses, circ19142 and circ5846 were found to be not only strongly associated with the biological process of the positive regulation of developmental processes but also related to the fibroblast growth factor, epidermal growth factor, platelet-derived growth factor and Wnt signaling pathways, which are involved in cell growth and differentiation. ConclusionThe present study identified circ19142 and circ5846 as being associated with osteoblast differentiation and BMP2 may induce osteogenic differentiation through a circ19142/circ5846-targeted miRNA–mRNA axis.

The Efficacy of Cyclic Injection of Bone Morphogenetic Protein-2 in Large-Scale Calvarial Bone Defects.

Bone morphogenetic protein-2 (BMP-2) appears to be one of the most potent growth factors thus far studied. However, recent publications on the clinical application of BMP-2 revealed that its correct control is the paramount issue in clinical practice. For improving BMP-2 delivery, the cyclic administration might be an alternative. Accordingly, the authors cyclically injected BMP-2 in a cyclic injection model of large cranial defects to maintain the proper dosage during the bone healing process. A 10-mm diameter calvarial bone defect was produced using a round drill in 8-week-old Sprague-Dawley rats. Silk-hydroxyapatite scaffolds soaked in the appropriate concentration of BMP-2 were implanted into the defect. The animals were split into 4 single-injection groups and 3 multiple-injection groups; the latter groups received weekly subcutaneous injections of BMP-2 solution (1, 5, and 10 μg/mL) for 4 weeks, whereas the former groups received a single injection of BMP-2 at these concentrations. Each rat underwent computed tomography at 8 weeks. In terms of total volumes of the new bone, the 5 μg/mL multiple-injection BMP-2 group had significantly greater increases in bone volume than the single-injection groups. In terms of bone thickness, the multiple-injection groups had better outcomes than the single-injection groups. Thus, the cyclic injection protocol restored the original thickness without overgrowth. Cyclic injection of BMP-2 permits more accurate dosage control than single injection and improves thickness and dense bone regeneration. Therefore, it may represent a promising approach for future clinical trials. Further investigation using a greater number of animals is required.

Epimedin C Promotes Vascularization during BMP2-Induced Osteogenesis and Tumor-Associated Angiogenesis.

Epimedin C is one of the chemical markers and major flavonoids in Herba Epimedii (Yinyanghuo), which is traditionally used to treat bone diseases and gonadal dysfunction in China. Our previous study indicated that epimedin C could induce endothelial-like, but not osteogenic differentiation of C3H/10T1/2 cells in vitro. As vasculogenesis plays a pivotal role in bone formation, this study used the bone morphogenetic protein 2 (BMP2) induced ectopic bone formation model and mice 4T1 breast cancer cells co-implanted with luciferase labeled C3H/10T1/2 cells (4T1 [Formula: see text] C3H/10T1/2-Luc) model to examine the in vivo effects of Epimedin C on vasculogenesis. As a result, Epimedin C significantly increased the bone weight and blood perfusion of mice in the BMP2 induced ectopic osteogenesis model, and the bone in Epimedin C [Formula: see text] BMP2 group was more mature than that in BMP2 group. In addition, the tumor weight, blood perfusion and tumor-associated angiogenesis were also significantly increased in the Epimedin C treated 4T1 tumor bearing mice. The mRNA levels of endothelial markers, such as the platelet endothelial adhesive factor-1(CD31), the endothelial cell specific molecule-1(ESM-1), and the vascular von Willebrand factor (vWF) in mouse 4T1 mammary tumor tissue, were commonly found to occur alongside the luciferase (labeled in C3H/10T1/2 cells) expression and significantly increased after Epimedin C treatment. Taken together, Epimedin C can effectively promote vascularization both in the BMP2-depended bone formation model and in the 4T1 mammary tumor-bearing model by inducing an endothelial-like differentiation of C3H/10T1/2 in BALB/c nude mice.

Anterior Cruciate Ligament–Derived Stem Cells Transduced With BMP2 Accelerate Graft-Bone Integration After ACL Reconstruction

Background: Strong graft-bone integration is a prerequisite for successful graft remodeling after reconstruction of the anterior cruciate ligament (ACL) using soft tissue grafts. Novel strategies to accelerate soft tissue graft-bone integration are needed to reduce the need for bone-tendon-bone graft harvest, reduce patient convalescence, facilitate rehabilitation, and reduce total recovery time after ACL reconstruction. Hypothesis: The application of ACL-derived stem cells with enhanced expression of bone morphogenetic protein 2 (BMP2) onto soft tissue grafts in the form of cell sheets will both accelerate and improve the quality of graft-bone integration after ACL reconstruction in a rat model. Study Design: Controlled laboratory study. Methods: ACL-derived CD34+ cells were isolated from remnant human ACL tissues, virally transduced to express BMP2, and embedded within cell sheets. In a rat model of ACL injury, bilateral single-bundle ACL reconstructions were performed, in which cell sheets were wrapped around tendon autografts before reconstruction. Four groups containing a total of 48 rats (96 knees) were established (n = 12 rats; 24 knees per group): CD34+BMP2 (100%), CD34+BMP2 (25%), CD34+ (untransduced), and a control group containing no cells. Six rats from each group were euthanized 2 and 4 weeks after surgery, and each graft was harvested for immunohistochemical and histological analyses. The remaining 6 rats in each group were euthanized at 4 and 8 weeks to evaluate in situ tensile load to failure in each femur-graft-tibia complex. Results: In vitro, BMP2 transduction promoted the osteogenic differentiation of ACL-derived CD34+ cells while retaining their intrinsic multipotent capabilities. Osteoblast densities were greatest in the BMP2 (100%) and BMP2 (25%) groups. Bone tunnels in the CD34+BMP2 (100%) and CD34+BMP2 (25%) groups had the smallest cross-sectional areas according to micro–computed tomography analyses. Graft-bone integration occurred most rapidly in the CD34+BMP2 (25%) group. Tensile load to failure was significantly greater in the groups containing stem cells at 4 and 8 weeks after surgery. Tensile strength was greatest in the CD34+BMP2 (100%) group at 4 weeks, and in the CD34+BMP2 (25%) group at 8 weeks. Conclusion: ACL-derived CD34+ cells transduced with BMP2 accelerated graft-bone integration after ACL reconstruction using soft tissue autografts in a rat model, as evidenced by improved histological appearance and graft-bone interface biology along with tensile load to failure at each time point up to 8 weeks after surgery. Clinical Relevance: A primary disadvantage of using soft tissue grafts for ACL reconstruction is the prolonged time required for bony ingrowth, which delays the initiation of midsubstance graft remodeling. The lack of consistent correlation between the appearance of a “healed” ACL on postoperative magnetic resonance imaging and readiness to return to sport results in athletes being released to sport before the graft is ready to handle high-intensity loading. Therefore, it is desirable to identify strategies that accelerate graft-bone integration, which would reduce the time to biologic fixation, improve the reliability of biologic fixation, allow for accelerated rehabilitation, and potentially reduce the incidence of early graft pullout and late midsubstance failure.

The treatment of nonunions with application of BMP-7 increases the expression pattern for angiogenic and inflammable cytokines: a matched pair analysis.

The local application of bone morphogenetic protein-7 (BMP-7) in combination with the transplantation of autologous bone graft improves the outcome in nonunion treatment; however, the specific reasons remain unclear. In this study, we sought to determine if the local application of BMP-7 contributes to improved bone regeneration in nonunion therapy by modulation of the angiogenic and inflammable cytokine expression patterns of the early inflammation response. Therefore, we utilized the analysis of serological cytokine expression patterns. As a matched pair analysis, best-fitting patients who were treated with transplantation of autologous bone graft (G1, n=10) were compared with patients who were treated with additional application of BMP-7 (G2, n=10). The changes in the cytokine expression patterns were monitored and correlated to clinical data of bone healing. Significant differences in angiogenesis potential (vascular endothelial growth factor [VEGF] serum levels) could be found in the first days after surgery (P<0.05). Furthermore, the increase and absolute amount of VEGF levels in the BMP-7 group were considerably higher than in the control group during the first 2 weeks after surgery. The expression pattern of inflammable cytokines showed noticeable differences in the time point of significant elevated levels, in particular, inflammable cytokines showed an earlier peak in G2. Furthermore, interleukin-6 was significantly elevated within the first week only, comparing G2 to G1 (P<0.05). Our findings indicate that BMP-7 induces an early and more intense expression of VEGF via a direct and postulated indirect pathway, thereby providing a favorable environment for bone healing. Moreover, application of BMP-7 leads to an earlier expression of known proinflammatory cytokines. The results of this study show that application of BMP-7 leads to costimulatory effect on both angiogenic and inflammable cytokine expression patterns that may serve as a possible stimulus for bone regeneration.

Reoperation rates for symptomatic nonunions in posterior cervicothoracic fusions with and without bone morphogenetic protein in a cohort of 450 patients.

OBJECTIVE Fusions across the cervicothoracic junction have been challenging because of the large biomechanical forces exerted resulting in frequent reoperations for nonunions. The objective of this study was to investigate a retrospective cohort using chart review of posterior cervicothoracic spine fusions with and without bone morphogenetic protein (BMP) and to determine the reoperation rates for symptomatic nonunions in both groups. METHODS Between January 2009 and September 2013, posterior cervicothoracic spine fusion cases were identified from a large spine registry (Kaiser Permanente). Demographics, diagnoses, operative times, lengths of stay, and reoperations were extracted from the registry. Reoperations for symptomatic nonunions were adjudicated via chart review. Logistic regression was used to estimate odds ratios and 95% confidence intervals. Kaplan-Meier curves for the non-BMP and BMP groups were generated and compared using the log-rank test. RESULTS In this cohort there were 450 patients (32.7% with BMP) with a median follow-up of 1.4 years (interquartile range [IQR] 0.5-2.7 years). Kaplan-Meier curves showed no significant difference in reoperation rates for nonunions using the log-rank test (p = 0.088). In a subset of patients with more than 1 year of follow-up, 260 patients were identified (43.1% with BMP) with a median follow-up duration of 2.4 years (IQR 1.6-3.3 years). There was no statistically significant difference in the symptomatic operative nonunion rates for posterior cervicothoracic fusions with and without BMP (0.0% vs 2.7%, respectively; p = 0.137) for more than 1 year of follow-up. CONCLUSIONS This study presents the largest series of patients using BMP in posterior cervicothoracic spine fusions. Reoperation rates for symptomatic nonunions with more than 1 year of follow-up were 0% with BMP and 2.7% without BMP. No statistically significant difference in the reoperation rates for symptomatic nonunions with or without BMP was found.

Enhancement of posterolateral lumbar spine fusion using recombinant human bone morphogenetic protein-2 and mesenchymal stem cells delivered in fibrin glue.

Mesenchymal stem cells have shown great potential for accelerating bone healing. In the present study, we evaluate the efficacy of fibrin glue/mesenchymal stem cells/recombinant human bone morphogenetic protein-2 composite for posterolateral spinal fusion in a rabbit model. Forty adult rabbits underwent posterolateral intertransverse fusion at the L5-L6 level. The animals were randomly divided into four groups based on the implant material: fibrin glue, fibrin glue/mesenchymal stem cells composite, fibrin glue-recombinant human bone morphogenetic protein-2 (fibrin glue/recombinant human bone morphogenetic protein-2) composite, and fibrin glue/mesenchymal stem cells/recombinant human bone morphogenetic protein-2 composite. After six weeks, the rabbits were euthanized for manual palpation, radiographic examination, biomechanical testing, and histology. Manual palpation results showed that the fusion rate for fibrin glue, fibrin glue/mesenchymal stem cells, fibrin glue/recombinant human bone morphogenetic protein-2, and fibrin glue/mesenchymal stem cells/recombinant human bone morphogenetic protein-2 was 0, 0, 40%, and 70%, respectively. Moreover, fusion rate determined by radiographic examination for fibrin glue, fibrin glue/mesenchymal stem cells, fibrin glue/recombinant human bone morphogenetic protein-2, and fibrin glue/mesenchymal stem cells/recombinant human bone morphogenetic protein-2 was 0, 0, 40%, and 80%, respectively. Gray analysis showed that fibrin glue/recombinant human bone morphogenetic protein-2 group had higher ossification area and density than fibrin glue group; and fibrin glue/mesenchymal stem cells/recombinant human bone morphogenetic protein-2 group had higher ossification area and density than fibrin glue/recombinant human bone morphogenetic protein-2 group. Formation of continuous bone masses between L5 and L6 level in mesenchymal stem cells/recombinant human bone morphogenetic protein-2/fibrin glue group was further confirmed by computed tomography scanning and three-dimensional reconstruction. Biomechanical testing demonstrated that the fusion strength (flexion, extension, lateral bending, and axial rotation) in fibrin glue/mesenchymal stem cells/recombinant human bone morphogenetic protein-2 group is significantly higher than that in fibrin glue/recombinant human bone morphogenetic protein-2 group. The formation of mature bone tissues between transverse processes of the fused specimens from both fibrin glue/recombinant human bone morphogenetic protein-2, and fibrin glue/mesenchymal stem cells/recombinant human bone morphogenetic protein-2 groups was confirmed by HE staining, and quantitative real-time polymerase chain reaction results showed the upregulation of CD31, type I collagen, osteocalcin, and osteonectin in the fibrin glue/mesenchymal stem cells/recombinant human bone morphogenetic protein-2 group. In conclusion, our findings show that mesenchymal stem cells delivered with recombinant human bone morphogenetic protein-2 using fibrin glue as carrier are more effective in enhancing spine fusion than recombinant human bone morphogenetic protein-2 without mesenchymal stem cells in the rabbit model.

Domestic porous tantalum loaded with bone morphogenetic 7 in repairing osteochondral defect in rabbits

?To investigate the ability to repair osteochondral defect and the biocompatibility of porous tantalum loaded with bone morphogenetic protein 7 (BMP-7) by observing the effect of porous tantalum loaded with BMP-7 in repairing articular cartilage and subchondral bone defect. ?The cartilage defect models of medial femoral condyle were established in 48 New Zealand white rabbits, which were randomly divided into 3 groups (n=16): porous tantalum material+BMP-7 (group A) and porous tantalum material (group B) were implanted into the right side of the medial femoral condyle; and no material was implanted as control (group C). The general condition of animals was observed after operation, then the specimens were harvested for gross observation, histological observation, and scanning electron microscope (SEM) observation at 4, 8, and 16 weeks after implantation, micro-CT was used to observe the cartilage and bone ingrowth and bone formation around porous tantalum at 16 weeks after implantation. ?No animal died after operation and wound healed well. Gross observation showed that defects of groups A and B were covered with new cartilage with time, but earlier new cartilage formation and better repair were observed in group A than group B, no repair occurred at the site of bone defects, and defect surface was filled with fibrous tissue in group C. Cartilage repair gross score of group A was significantly higher than that of group B at 8 and 16 weeks (P<0.05) but no significant difference was found between groups A and B at 4 weeks (P>0.05). SEM observation showed that the number of new cartilage and osteoblasts increased gradually with time, and the implanted material was gradually covered with the extracellular matrix, and the new bone tissue grew into the pores of the material; the neonatal bone tissue and extracellular matrix secretion of group A were significantly more than those of group B. The toluidine blue staining results showed that new cartilage and bone tissue gradually increased in the porous tantalum interface, and new bone trabecula formed and grew in the pores, the bone and the porous tantalum contact tended to close, and cartilage defect was gradually covered with cartilage like tissue, cartilage tissue and porous tantalum combined more closely in groups A and B at 4, 8 and 16 weeks. New cartilage and bone tissue of group A was more than that of group B. Micro-CT analysis indicated that the bone mineral density, trabecular thickness, trabecular number, and bone volume fraction of group A were significantly higher than those of group B at 16 weeks (P<0.05), but the trabecular bone space was significantly lower than that of group B (P<0.05). ?The domestic porous tantalum has good biocompatibility, domestic porous tantalum loaded with BMP-7 can promote the formation of a stable connection with the host and has a good effect on cartilage and subchondral bone defect repair.

The effect of SDF-1α on low dose BMP-2 mediated bone regeneration by release from heparinized mineralized collagen type I matrix scaffolds in a murine critical size bone defect model.

The treatment of critical size bone defects represents a challenge. The growth factor bone morphogenetic protein 2 (BMP-2) is clinically established but has potentially adverse effects when used at high doses. The aim of this study was to evaluate if stromal derived factor-1 alpha (SDF-1α) and BMP-2 released from heparinized mineralized collagen type I matrix (MCM) scaffolds have a cumulative effect on bone regeneration. MCM scaffolds were functionalized with heparin, loaded with BMP-2 and/or SDF-1α and implanted into a murine critical size femoral bone defect (control group, low dose BMP-2 group, low dose BMP-2 + SDF-1α group, and high dose BMP-2 group). After 6 weeks, both the low dose BMP-2 + SDF-1α group (5.8 ± 0.6 mm³, p = 0.0479) and the high dose BMP-2 group (6.5 ± 0.7 mm³, p = 0.008) had a significantly increased regenerated bone volume compared to the control group (4.2 ± 0.5 mm³). There was a higher healing score in the low dose BMP-2 + SDF-1α group (median grade 8; Q1-Q3 7-9; p = 0.0357) than in the low dose BMP-2 group (7; Q1-Q3 5-9) histologically. This study showed that release of BMP-2 and SDF-1α from heparinized MCM scaffolds allows for the reduction of the applied BMP-2 concentration since SDF-1α seems to enhance the osteoinductive potential of BMP-2. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2126-2134, 2016.

Study on transfection of dog bone mesenchymal stem cells with BMP-2/VEGF165-based lentivirus vector

Objective To transfect the dog bone mesenchymal stem cells (BMSCs) with lentivirus-mediated vascular endothelial growth factor 165 (VEGF165) and bone morphogenetic protein 2 (BMP-2) and observe the expressions of VEGF165 and BMP-2 in BMSCs. Methods Lentivirus vectors carrying VEGF165 and BMP-2 genes were constructed and used to transfect dog third-generation BMSCs, including untransfected group, VEGF165 group, BMP-2 group and double-gene (VEGF165+ BMP-2) group. Expressions of VEGF165 and BMP-2 mRNA were detected by RT-PCR 7 d after the transfection.Expressions of VEGF165 and BMP-2 protein were detected by Western blotting 5 d and 28 d after the transfection. Cell growth was detected by MTT assay. Results RT-PCR showed high expression of VEGF165 mRNA in VEGF165 and double-gene groups, and BMP-2 mRNA in BMP-2 and double-gene groups, but there was no significant difference in group comparison(P>0.05). Western blotting showed specific performance of VEGF165 protein in VEGF165 and double-gene groups, and BMP-2 protein in BMP-2 and double-gene groups. In MTT assay cells were found to have 2-3 d relatively stationary phase in the early period, proliferated rapidly at 3 d, reaching the logarithmic growth, and entered the plateau phase with slow propagation. There was no significant difference in absorbance value before and after the transfection (P>0.05). Conclusion BMSCs with highly expressed VEGF165 and BMP-2 can be successfully obtained by lentiviral transduction. Key words: Tissue engineering; Mesenchymal stem cells; Lentivirus; Transfection

Influence of biomechanical and biochemical stimulation on the proliferation and differentiation of bone marrow stromal cells seeded on polyurethane scaffolds.

The aim of the present investigation was to compare the effects of cyclic compression, perfusion, dexamethasone (DEX) and bone morphogenetic protein-7 (BMP-7) on the proliferation and differentiation of human bone marrow stromal cells (hBMSCs) in polyurethane scaffolds in a perfusion bioreactor. Polyurethane scaffolds seeded with hBMSCs were cultured under six different conditions, as follows: 10% Cyclic compression at 0.5 and 5 Hz; 10 ml/min perfusion; 100 nM DEX; 100 ng/ml BMP-7; and 1 ml/min perfusion without mechanical and biochemical stimulation (control). On days 7 and 14, samples were tested for the following data: Cell proliferation; mRNA expression of Runx2, COL1A1 and osteocalcin; osteocalcin content; calcium deposition; and the equilibrium modulus of the tissue specimen. The results indicated that BMP-7 and 10 ml/min perfusion promoted cell proliferation, which was inhibited by 5 Hz cyclic compression and DEX. On day 7, the 5 Hz cyclic compression inhibited Runx2 expression, whereas the 0.5 Hz cyclic compression and BMP-7 upregulated the COL1A1 mRNA levels on day 7 and enhanced the osteocalcin expression on day 14. The DEX-treated hBMSCs exhibited downregulated osteocalcin expression. After 14 days, the BMP-7 group exhibited the highest calcium deposition, followed by the 0.5 Hz cyclic compression and the DEX groups. The equilibrium modulus of the engineered constructs significantly increased in the BMP-7, 0.5 Hz cyclic compression and DEX groups. In conclusion, the present results suggest that BMP-7 and perfusion enhance cell proliferation, whereas high frequency cyclic compression inhibits the proliferation and osteogenic differentiation of hBMSCs. Low frequency cyclic compression is more effective than DEX, but less effective compared with BMP-7 on the osteogenic differentiation of hBMSCs seeded on polyurethane scaffolds.

The impact of bone morphogenetic protein 4 (BMP4) on breast cancer metastasis in a mouse xenograft model

Bone morphogenetic protein 4 (BMP4) is a key regulator of cell proliferation and differentiation. In breast cancer cells, BMP4 has been shown to reduce proliferation in vitro and interestingly, in some cases, also to induce migration and invasion. Here we investigated whether BMP4 influences breast cancer metastasis formation by using a xenograft mouse model. MDA-MB-231 breast cancer cells were injected intracardially into mice and metastasis formation was monitored using bioluminescence imaging. Mice treated with BMP4 developed metastases slightly earlier as compared to control animals but the overall number of metastases was similar in both groups (13 in the BMP4 group vs. 12 in controls). In BMP4-treated mice, bone metastases were more common (10 vs. 7) but adrenal gland metastases were less frequent (1 vs. 5) than in controls. Immunostaining revealed no differences in signaling activation, proliferation rate, blood vessel formation, EMT markers or the number of cancer-associated fibroblasts between the treatment groups. In conclusion, BMP4 caused a trend towards accelerated metastasis formation, especially in bone. More work is needed to uncover the long-term effects of BMP4 and the clinical relevance of these findings.

Reoperation rates for symptomatic nonunions in posterior cervical (subaxial) fusions with and without bone morphogenetic protein in a cohort of 1158 patients.

Bone morphogenetic protein (BMP) was first approved in 2002 for use in single-level anterior lumbar fusions as an alternative to iliac crest grafts. Subsequent studies have concluded that BMP provides superior fusions rates and therefore reduces reoperations for nonunions. The purpose of this study was to determine the reoperation rates for symptomatic nonunions in posterior cervical (subaxial) spinal fusions with and without the use of BMP and to determine if the nonunion rates are statistically significantly different between the two groups. Between January 2009 and September 2013, the authors identified 1158 posterior cervical spinal fusion cases in the subaxial spine (C2-7) from a large spine registry (Kaiser Permanente). Patient characteristics, diagnoses, operative times, lengths of stay, and reoperations were extracted from the registry. Reoperations for symptomatic nonunions were adjudicated via chart review. Logistic regression was conducted to produce estimates of odds ratios (OR) and 95% confidence intervals (CIs). Kaplan-Meier curves for the non-BMP and BMP groups were generated and compared using the log-rank test. In this cohort there were 1158 patients (19.3% with BMP) with a median follow up of 1.7 years (interquartile range [IQR] 0.7-2.9 years) and median duration to operative nonunion of 0.63 years (IQR 0.44-1.57 years). Kaplan-Meier curves showed no significant difference in reoperation rates for nonunions using the log-rank test (p = 0.179). In a subset of patients with more than 1 year of follow-up, 788 patients were identified (22.5% with BMP) with a median follow-up duration of 2.5 years (IQR 1.7-3.4 years) and a median time to operative nonunion of 0.73 years (IQR 0.44-1.57 years). There was no statistically significant difference in the symptomatic operative nonunion rates for posterior cervical (subaxial) fusions with BMP compared with non-BMP (1.1% vs. 0.7%; crude OR 1.73, 95% CI 0.32-9.55, p = 0.527) for more than 1 year of follow-up. This study presents the largest series of patients using BMP in posterior cervical (subaxial) spinal fusions. Reoperation rates for symptomatic nonunions with more than 1 year of follow-up were found to be 1.1% with BMP and 0.7% without BMP. There was no significant difference in the reoperation rates for symptomatic nonunions with or without BMP.

In VivoEvaluation of Adipose-Derived Stromal Cells Delivered with a Nanofiber Scaffold for Tendon-to-Bone Repair

Rotator cuff tears are common and cause a great deal of lost productivity, pain, and disability. Tears are typically repaired by suturing the tendon back to its bony attachment. Unfortunately, the structural (e.g., aligned collagen) and compositional (e.g., a gradient in mineral) elements that produce a robust attachment in the healthy tissue are not regenerated during healing, and the repair is prone to failure. Two features of the failed healing response are deposition of poorly aligned scar tissue and loss of bone at the repair site. Therefore, the objective of the current study was to improve tendon-to-bone healing by promoting aligned collagen deposition and increased bone formation using a biomimetic scaffold seeded with pluripotent cells. An aligned nanofibrous poly(lactic-co-glycolic acid) scaffold with a gradient in mineral content was seeded with adipose-derived stromal cells (ASCs) and implanted at the repair site of a rat rotator cuff model. In one group, cells were transduced with the osteogenic factor bone morphogenetic protein 2 (BMP2). The healing response was examined in four groups (suture only, acellular scaffold, cellular scaffold, and cellular BMP2 scaffold) using histologic, bone morphology, and biomechanical outcomes at 14, 28, and 56 days. Histologically, the healing interface was dominated by a fibrovascular scar response in all groups. The acellular scaffold group showed a delayed healing response compared to the other groups. When examining bone morphology parameters, bone loss was evident in the cellular BMP2 group compared to other groups at 28 days. When examining repair-site mechanical properties, strength and modulus were decreased in the cellular BMP2 groups compared to other groups at 28 and 56 days. These results indicated that tendon-to-bone healing in this animal model was dominated by scar formation, preventing any positive effects of the implanted biomimetic scaffold. Furthermore, cells transduced with the osteogenic factor BMP2 led to impaired healing, suggesting that this growth factor should not be used in the tendon-to-bone repair setting.

A population-based review of bone morphogenetic protein: associated complication and reoperation rates after lumbar spinal fusion.

OBJECT The authors compared the rates of postoperative adverse events and reoperation of patients who underwent lumbar spinal fusion with bone morphogenetic protein (BMP) to those of patients who underwent lumbar spinal fusion without BMP. METHODS The authors retrospectively analyzed the PearlDiver Technologies, Inc., database, which contains the Medicare Standard Analytical Files, the Medicare Carrier Files, the PearlDiver Private Payer Database (UnitedHealthcare), and select state all-payer data sets, from 2005 to 2010. They identified patients who underwent lumbar spinal fusion with and without BMP. The ICD-9-CM code 84.52 was used to identify patients who underwent spinal fusion with BMP. ICD-9-CM diagnosis codes identified complications that occurred during the initial hospital stay. ICD-9-CM procedural codes were used to identify reoperations within 90 days of the index procedure. The relative risks (and 95% CIs) of BMP use compared with no BMP use (control) were calculated for the association of any complication with BMP use compared with the control. RESULTS Between 2005 and 2010, 460,773 patients who underwent lumbar spinal fusion were identified. BMP was used in 30.7% of these patients. The overall complication rate in the BMP group was 18.2% compared with 18.7% in the control group. The relative risk of BMP use compared with no BMP use was 0.976 (95% CI 0.963-0.989), which indicates a significantly lower overall complication rate in the BMP group (p < 0.001). In both treatment groups, patients older than 65 years had a statistically significant higher rate of postoperative complications than younger patients (p < 0.001). CONCLUSIONS In this large-scale institutionalized database study, BMP use did not seem to increase the overall risk of developing a postoperative complication after lumbar spinal fusion surgery.

Effects of low dose FGF-2 and BMP-2 on healing of calvarial defects in old mice

There is an age-associated reduction in the bone healing activity of bone morphogenetic protein-2 (BMP-2) that is currently addressed by administering higher doses of BMP-2 in elderly patients. The unwanted medical complications from high dose BMP-2 motivated this investigation to determine whether the addition of a low dose of fibroblast growth factor 2 (FGF-2) could enhance the ability of a lower dose of BMP-2 to heal calvarial bone defects in old mice (18–20months old). FGF-2 (5ng) and BMP-2 (2μg) were administered by a controlled release two-phase biomaterial scaffold placed into the bone defect. FGF-2 released more rapidly and completely in vitro than BMP-2 (40% vs 2%). In vivo, both BMP-2 and FGF-2+BMP-2 groups formed more new bone in calvarial defects than scaffold alone (p<0.001) or FGF-2 only groups (p<0.01). The overall total volume of new bone was not statistically increased by the addition of FGF-2 to BMP-2 as measured by microCT, but the pattern of bone deposition was different. In old mice, but not young, there was enhanced bony fill in the central bone defect area when the BMP-2 was supplemented with FGF-2. Histological analysis of the center of the defect revealed an increased bone volume (%BV/TV (p=0.004)) from the addition of FGF-2. These studies suggest that combining a low dose of FGF-2 with a low dose of BMP-2 has the potential to increase bone healing in old mice relative to BMP-2 alone.

Transforming growth factor beta 1 augments calvarial defect healing and promotes suture regeneration.

Repair of complex cranial defects is hindered by a paucity of appropriate donor tissue. Bone morphogenetic protein 2 (BMP2) and transforming growth factor beta 1 (TGFβ1) have been shown separately to induce bone formation through physiologically distinct mechanisms and potentially improve surgical outcome for cranial defect repair by obviating the need for donor tissue. We hypothesize that a combination of BMP2 and TGFβ1 would improve calvarial defect healing by augmenting physiologic osteogenic mechanisms. Coronal suturectomies (3×15 mm) were performed in 10-day-old New Zealand White rabbits. DermaMatrix™ (3×15mm) patterned with four treatments (vehicle, 350 ng BMP2, 200 ng TGFβ1, or 350 ng BMP2+200 ng TGFβ1) was placed in suturectomy sites and rabbits were euthanized at 6 weeks of age. Two-dimensional (2D) defect healing, bone volume, and bone density were quantified by computed tomography. Regenerated bone was qualitatively assessed histologically. One-way analysis of variance revealed significant group main effects for all bone quantity measures. Analysis revealed significant differences in 2D defect healing, bone volume, and bone density between the control group and all treatment groups, but no significant differences were detected among the three growth factor treatment groups. Qualitatively, TGFβ1 treatment produced bone with morphology most similar to native bone. TGFβ1-regenerated bone contained a suture-like tissue, growing from the lateral edge of the defect margin toward the midline. Unique to the BMP2 treatment group, regenerated bone contained lacunae with chondrocytes, demonstrating the presence of endochondral ossification. Total healing in BMP2 and TGFβ1 treatment groups is not significantly different. The combination of BMP2+TGFβ1 did not significantly increase bone healing compared with treatment with BMP2 or TGFβ1 alone postoperatively at 4 weeks. We highlight the potential use of TGFβ1 to regenerate calvarial bone and cranial sutures. TGFβ1 therapy significantly augmented bony defect healing at an earlier time point when compared with control, regenerated bone along the native intramembranous ossification pathway, and (unlike BMP2 alone or in combination with TGFβ1) permitted normal suture reformation. We propose a novel method of craniofacial bone regeneration using low-dose, spatially controlled growth factor therapies to minimize potentially harmful effects while maximizing local bioavailability and regenerating native tissues.

Combination of Root Surface Modification with BMP-2 and Collagen Hydrogel Scaffold Implantation for Periodontal Healing in Beagle Dogs.

Objective : Biomodification of the root surface plays a major role in periodontal wound healing. Root surface modification with bone morphogenetic protein (BMP) stimulates bone and cementum-like tissue formation; however, severe ankylosis is simultaneously observed. Bio-safe collagen hydrogel scaffolds may therefore be useful for supplying periodontal ligament cells and preventing ankylosis. We examined the effects of BMP modification in conjunction with collagen hydrogel scaffold implantation on periodontal wound healing in dogs. Material and Methods: The collagen hydrogel scaffold was composed of type I collagen sponge and collagen hydrogel. One-wall infrabony defects (5 mm in depth, 3 mm in width) were surgically created in six beagle dogs. In the BMP/Col group, BMP-2 was applied to the root surface (loading dose; 1 µg/µl), and the defects were filled with collagen hydrogel scaffold. In the BMP or Col group, BMP-2 coating or scaffold implantation was performed. Histometric parameters were evaluated at 4 weeks after surgery. Results: Single use of BMP stimulated formation of alveolar bone and ankylosis. In contrast, the BMP/Col group frequently enhanced reconstruction of periodontal attachment including cementum-like tissue, periodontal ligament and alveolar bone. The amount of new periodontal ligament in the BMP/Col group was significantly greater when compared to all other groups. In addition, ankylosis was rarely observed in the BMP/Col group. Conclusion: The combination method using root surface modification with BMP and collagen hydrogel scaffold implantation facilitated the reestablishment of periodontal attachment. BMP-related ankylosis was suppressed by implantation of collagen hydrogel.