VEGF and BMP-6 enhance bone formation mediated by cloned mouse osteoprogenitor cells

New strategies such as combined utilization of growth factors may provide a better treatment for difficult fractures. We have demonstrated enhanced angiogenesis and osteogenesis through the actions of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-6 (BMP-6) on the osteogenic differentiation of a cloned mouse osteoprogenitor cell in vitro and ectopic bone formation in vivo. Human VEGF and BMP-6 genes expressed together produced a significant increase in alkaline phosphatase activity, expression of the RunX2 and osteocalcin genes and mineralization. Microcomputed tomographic analysis of subcutaneous implants consisting of cells transfected with VEGF and BMP-6 cDNA and delivered on a 3D poly (lactic-co-glycolic acid) scaffold confirmed the additive effects between VEGF and BMP-6. Ectopic bone formation in the VEGF plus BMP-6 group was greatest compared to that in either VEGF or BMP-6 alone. This is the first study that demonstrates osteogenesis in vitro and in vivo through the additive effects of VEGF and BMP-6.     

Exogenously added BMP-6, BMP-7 and VEGF may not enhance the osteogenic differentiation of human adipose stem cells

Abstract

In the present study bone morphogenetic protein (BMP)-6 alone or in synergy with BMP-7 and vascular endothelial growth factor (VEGF) were tested with human adipose stem cells (hASCs) seeded on cell culture plastic or 3D bioactive glass. Osteogenic medium (OM) was used as a positive control for osteogenic differentiation. The same growth factor groups were also tested combined with OM. None of the growth factor treatments could enhance the osteogenic differentiation of hASCs in 3D- or 2D-culture compared to control or OM. In 3D-culture OM promoted significantly total collagen production, whereas in 2D-culture OM induced high total ALP activity and mineralization compared to control and growth factors groups, but also high cell proliferation. In this study, hASCs did not respond to exogenously added growth although various parameters of the study set-up may have affected these findings contradictory to the previous literature.     

The role of BMP-6, IL-6, and BMP-4 in mesenchymal stem cell-dependent bone development: effects on osteoblastic differentiation induced by parathyroid hormone and vitamin D(3)

Human bone marrow-derived mesenchymal stem cells (MSCs) represent an ideal source for cell therapy for inherited and degenerative diseases, bone and cartilage repair, and as target for gene therapy. The role of the combination of human parathyroid hormone (PTH) and vitamin D(3) in bone formation and mineralization has been established in several osteoblast cell culture studies. The aim of the present study was to evaluate the role of this hormonal combination alone and in the presence of bone morphogenetic protein-4 (BMP-4) or-6 (BMP-6) in inducing osteogenic differentiation of human MSC. Human MSC derived from adult normal bone marrow that are positive for CD29, CD44, CD105, and CD166 and negative for CD14, CD34, and CD45, were treated with the PTH and 1,25-dihydroxyvitamin D(3) in the presence and absence of recombinant human BMP-4 or BMP6. PTH and vitamin D(3) induced high levels of expression of two key markers of bone formation: osteocalcin and alkaline phosphatase by MSCs. BMP-6 but not BMP-4 increased osteocalcin expression induced by PTH and vitamin D(3). Both BMPs enhanced calcium formation in MSC cultures and this response was potentiated by PTH and vitamin D(3). The present results revealed a novel potent effect of PTH and vitamin D(3) plus BMPs in inducing bone development by human MSCs. These results may facilitate therapeutic utility of MSCs for bone disease and help clarify mechanisms involved in stem cell-mediated bone development.     

BMP-4 increases activin A gene expression during osteogenic differentiation of mouse embryonic stem cells

Abstract

Activin A is a growth factor released by mature osteoblasts that has a critical effect on bone formation. We investigated the effect of bone morphogenetic protein (BMP)-4 on activin A gene expression during in vitro osteogenic differentiation of mouse embryonic stem (ES) cells. Embryoid bodies were cultured in retinoic acid (RA) for three days and then without RA for two days. Seeded cells received osteogenic medium with β-glycerophosphate, L-ascorbic acid 2-phosphate and dexamethasone during 19 days, with or without BMP-4. Six independent experiments were carried out. Real-time PCR was used to detect gene expression of activin A, Oct-4, Nanog, osteocalcin, RUNX2 and bone alkaline phosphatase. Immunofluorescence was used to co-localize activin A with the undifferentiation marker stage-specific embryonic antigen 1. Cells treated with BMP-4 had an increased gene expression of activin A, osteocalcin and bone alkaline phosphatase (p?<?0.05). In conclusion, BMP-4 increases activin A gene expression during mouse ES cell differentiation into bone precursors.     

Osteogenesis by human mesenchymal stem cells cultured on silk biomaterials: comparison of adenovirus mediated gene transfer and protein delivery of BMP-2

Bone tissue engineering, gene therapy based on human mesenchymal stem cells (MSCs) and silk fibroin biomaterials were combined to study the impact of viral transfection on MSC osteogenic performance in vitro. MSCs were transduced with adenovirus containing a human BMP-2 (Ad-BMP-2) gene at clinically reasonable viral concentrations and cultured for 4 weeks. Controls with nontransfected MSCs, but exposed to exogenous BMP-2 concentrations on an analogous time profile as that secreted by the Ad-BMP-2 group, were compared. Both the Ad-BMP-2 MSC group and the exogenous protein BMP-2 group strongly expressed osteopontin and bone sialoprotein. Cells secreted a matrix that underwent mineralization on the silk fibroin scaffolds, forming clusters of osseous material, as determined by micro-computed tomography. The expression of osteogenic marker proteins and alkaline phosphatase was significantly higher in the Ad-BMP-2 MSC group than in the exogenous protein BMP-2 group, and no significant differences in mineralization were observed in two of the three MSC sources tested. The results demonstrate that transfection resulted in higher levels of expression of osteogenic marker genes, no change in proliferation rate and did not impact the capacity of the cells to calcify tissues on these protein scaffolds. These findings suggest additional options to control differentiation where exogenous additions of growth factors or morphogens can be replaced with transfected MSCs.     

Small spheroids of adipose‐derived stem cells with time‐dependent enhancement of IL‐8 and VEGF‐A secretion

Wound‐healing factors secreted from mesenchymal stem cells (MSCs) modulate the immune response and facilitate proliferation of neighboring cells. Although in vitro three‐dimensional (3D) culture techniques have improved the therapeutic potential of MSCs, no studies have focused on the effects of cell aggregation alone. In this study, the effect of cell aggregation on the up‐regulation of wound‐healing proteins secretions was investigated by constructing small spheroids of human adipose‐derived stem cells (hADSCs) on a micropatterned surface. These spheroids were mostly unaffected by the secondary effects of cell aggregation, such as hypoxia, low‐nutrient supply, and metabolic waste accumulation. Small spheroids of hADSCs, which were of 100 μm in diameter, were successfully constructed using micropatterned surface. Expression of the wound‐healing‐related factors, VEGF‐A and IL‐8, was markedly enhanced at the gene and protein levels, whereas the enhancement of VEGF‐A expression was transient and IL‐8 enhancement was maintained for a long time.     

Effect of local sequential VEGF and BMP-2 delivery on ectopic and orthotopic bone regeneration

Bone regeneration is a coordinated cascade of events regulated by several cytokines and growth factors. Angiogenic growth factors are predominantly expressed during the early phases for re-establishment of the vascularity, whereas osteogenic growth factors are continuously expressed during bone formation and remodeling. Since vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs) are key regulators of angiogenesis and osteogenesis during bone regeneration, the aim of this study was to investigate if their sequential release could enhance BMP-2-induced bone formation. A composite consisting of poly(lactic-co-glycolic acid) microspheres loaded with BMP-2 embedded in a poly(propylene) scaffold surrounded by a gelatin hydrogel loaded with VEGF was used for the sequential release of the growth factors. Empty composites or composites loaded with VEGF and/or BMP-2 were implanted ectopically and orthotopically in Sprague–Dawley rats (n = 9). Following implantation, the local release profiles were determined by measuring the activity of ¹²⁵I-labeled growth factors using scintillation probes. After 8 weeks blood vessel and bone formation were analyzed using microangiography, μCT and histology. The scaffolds exhibited a large initial burst release of VEGF within the first 3 days and a sustained release of BMP-2 over the full 56-day implantation period. Although VEGF did not induce bone formation, it did increase the formation of the supportive vascular network (p = 0.03) in ectopic implants. In combination with local sustained BMP-2 release, VEGF significantly enhanced ectopic bone formation compared to BMP-2 alone (p = 0.008). In the orthotopic defects, no effect of VEGF on vascularisation was found, nor was bone formation higher by the combination of growth factors, compared to BMP-2 alone. This study demonstrates that a sequential angiogenic and osteogenic growth factor release may be beneficial for the enhancement of bone regeneration.     

A comprehensive analysis of the dual roles of BMPs in regulating adipogenic and osteogenic differentiation of mesenchymal progenitor cells

Pluripotent mesenchymal stem cells (MSCs) are bone marrow stromal progenitor cells that can differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. Several signaling pathways have been shown to regulate the lineage commitment and terminal differentiation of MSCs. Here, we conducted a comprehensive analysis of the 14 types of bone morphogenetic protein (BMPs) for their abilities to regulate multilineage specific differentiation of MSCs. We found that most BMPs exhibited distinct abilities to regulate the expression of Runx2, Sox9, MyoD, and PPARgamma2. Further analysis indicated that BMP-2, BMP-4, BMP-6, BMP-7, and BMP-9 effectively induced both adipogenic and osteogenic differentiation in vitro and in vivo. BMP-induced commitment to osteogenic or adipogenic lineage was shown to be mutually exclusive. Overexpression of Runx2 enhanced BMP-induced osteogenic differentiation, whereas knockdown of Runx2 expression diminished BMP-induced bone formation with a decrease in adipocyte accumulation in vivo. Interestingly, overexpression of PPARgamma2 not only promoted adipogenic differentiation, but also enhanced osteogenic differentiation upon BMP-2, BMP-6, and BMP-9 stimulation. Conversely, MSCs with PPARgamma2 knockdown or mouse embryonic fibroblasts derived from PPARgamma2(-/-) mice exhibited a marked decrease in adipogenic differentiation, coupled with reduced osteogenic differentiation and diminished mineralization upon BMP-9 stimulation, suggesting that PPARgamma2 may play a role in BMP-induced osteogenic and adipogenic differentiation. Thus, it is important to understand the molecular mechanism behind BMP-regulated lineage divergence during MSC differentiation, as this knowledge could help us to understand the pathogenesis of skeletal diseases and may lead to the development of strategies for regenerative medicine.     

#br# rhTGF-β1对SD大鼠MSCs骨向分化的影响及机制研究

 目的：通过观察重组人转化生长因子 β1(rhTGF-β1)对大鼠骨髓间充质干细胞(MSCs)增殖和骨向分化能力的影响,以及对骨形态发生蛋白2(BMP-2)、Smad4及核心结合因子α1(Cbfa1)的作用,阐释其对MSCs骨向分化影响以及可能的作用机制。方法：用全骨髓贴壁法分离、纯化SD大鼠MSCs;用MTT法检测0、5、10、20、40、80和100 μg/L rhTGF-β1对MSCs增殖活性的影响;以碱性磷酸酶(ALP)活性及ALP染色阳性率确定rhTGF-β1的最佳促MSCs骨向分化浓度,并以该浓度对MSCs骨向分化进行干预。按是否添加经典成骨诱导液将实验分为：正常组、经典组、rhTGF-β1组和rhTGF-β1+经典组。通过检测ALP、I型胶原、骨钙素表达和钙化结节的数目,评价各组骨向分化能力;通过检测BMP-2、Smad4和Cbfa1 mRNA的表达,评价各组促MSCs骨向分化的可能作用机制。结果：rhTGF-β1最佳促MSCs增殖浓度为10 μg/L,最佳促MSCs骨向分化浓度为5 μg/L。经典组、rhTGF-β1组和rhTGF-β1+经典组均能促进MSCs骨向分化,刺激BMP-2分泌,并上调Smad4和Cbfa1 mRNA的表达,且rhTGF-β1对MSCs成骨分化的早期、中期效果好,而rhTGF-β1+经典组对MSCs成骨分化的晚期效果更为明显。结论:经典组、rhTGF-β1组和rhTGF-β1+经典组均有促MSCs骨向分化的作用,其机制可能是促进BMP-2的分泌,通过TGF-β超家族/Smads信号通路调控骨向分化。     

BMP-9 induced osteogenic differentiation of mesenchymal stem cells: molecular mechanism and therapeutic potential

Promoting osteogenic differentiation and efficacious bone regeneration have the potential to revolutionize the treatment of orthopaedic and musculoskeletal disorders. Mesenchymal Stem Cells (MSCs) are bone marrow progenitor cells that have the capacity to differentiate along osteogenic, chondrogenic, myogenic, and adipogenic lineages. Differentiation along these lineages is a tightly controlled process that is in part regulated by the Bone Morphogenetic Proteins (BMPs). BMPs 2 and 7 have been approved for clinical use because their osteoinductive properties act as an adjunctive treatment to surgeries where bone healing is compromised. BMP-9 is one of the least studied BMPs, and recent in vitro and in vivo studies have identified BMP-9 as a potent inducer of osteogenic differentiation in MSCs. BMP-9 exhibits significant molecular cross-talk with the Wnt/ β-catenin and other signaling pathways, and adenoviral expression of BMP-9 in MSCs increases the expression of osteogenic markers and induces trabecular bone and osteiod matrix formation. Furthermore, BMP-9 has been shown to act synergistically in bone formation with other signaling pathways, including Wnt/ β-catenin, IGF, and retinoid signaling pathways. These results suggest that BMP-9 should be explored as an effective bone regeneration agent, especially in combination with adjuvant therapies, for clinical applications such as large segmental bony defects, non-union fractures, and/or spinal fusions.     

Short-term BMP-2 expression is sufficient for in vivo osteochondral differentiation of mesenchymal stem cells

Currently available murine models to evaluate mesenchymal stem cell (MSC) differentiation are based on cell injection at ectopic sites such as muscle or skin. Due to the importance of environmental factors on the differentiation capacities of stem cells in vivo, we investigated whether the peculiar synovial/cartilaginous environment may influence the lineage specificity of bone morphogenetic protein (BMP)-2-engineered MSCs. To this aim, we used the C3H10T1/2-derived C9 MSCs that express BMP-2 under control of the doxycycline (Dox)-repressible promoter, Tet-Off, and showed in vitro, using the micropellet culture system that C9 MSCs kept their potential to differentiate toward chondrocytes. Implantation of C9 cells, either into the tibialis anterior muscles or into the joints of CB17-severe combined immunodeficient bg mice led to the formation of cartilage and bone filled with bone marrow as soon as day 10. However, no differentiation was observed after injection of na?ve MSCs or C9 cells that were repressed to secrete BMP-2 by Dox addition. The BMP-2-induced differentiation of adult MSCs is thus independent of soluble factors present in the local environment of the synovial/cartilaginous tissues. Importantly, we demonstrated that a short-term expression of the BMP-2 growth factor is necessary and sufficient to irreversibly induce bone formation, suggesting that a stable genetic modification of MSCs is not required for stem cell-based bone/cartilage engineering.     

Characterization of human ethmoid sinus mucosa derived mesenchymal stem cells (hESMSCs) and the application of hESMSCs cell sheets in bone regeneration

Mesenchymal stem cells (MSCs) have been extensively applied in the field of tissue regeneration. MSCs derived from various tissues exhibit different characteristics. In this study, a cluster of cells were isolated from human ethmoid sinus mucosa membrane and termed as hESMSCs. hESMSCs was demonstrated to have MSC-specific characteristics of self-renewal and tri-lineage differentiation. In particular, hESMSCs displayed strong osteogenic differentiation potential, and also remarkably promoted the proliferation and osteogenesis of rat bone marrow mesenchymal stem cells (rBMSCs) in vitro. Next, hESMSCs were prepared into a cell sheet and combined with a PSeD scaffold seeded with rBMSCs to repair critical-sized calvarial defects in rats, which showed excellent reparative effects. Additionally, ELISA assays revealed that secreted cytokines, such as BMP-2, BMP-4 and bFGF, were higher in the hESMSCs conditioned medium, and immunohistochemistry validated that hESMSCs cell sheet promoted the expression of BMP signaling downstream genes in newly formed bone. In conclusion, hESMSCs were demonstrated to be a class of mesenchymal stem cells that possessed high self-renewal capacity along with strong osteogenic potential, and the cell sheet of hESMSCs could remarkably promote new bone regeneration, indicating that hESMSCs cell sheet could serve as a novel and promising alternative strategy in the management of bone regeneration.     

Comparative study of osteogenic differentiation potential of mesenchymal stem cells derived from bone marrow and adipose tissue of osteoporotic female rats

Osteoporosis causes reduction of osteogenic differentiation of mesenchymal stem cells (MSCs) from bone marrow and adipose tissue. This study was designed to compare the osteogenic potential of bone marrow mesenchymal stem cells (BMMSCs) and adipose-derived stem cells (ADSCs) of ovariectomized (OVX) rats. MSC were harvested from bone marrow and inguinal fat pads of six OVX rats. The limitations of this report are that cells from different animals were pooled for the purpose of the experiments that were carried out in this study. At 7, 14 and 21?d of osteogenic differentiation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion, alkaline phosphatase activity and gene expression for collagen I, osteocalcin, bone sialoprotein, osteopontin and bone morphogenetic protein-2 bone morphogenetic protein-2 (BMP-2) were analyzed. At 21?d, percentage of cells per field and percentage of mineralized nodule were analyzed. The data were subjected to analysis of variance, and the means were compared by Student–Newman–Keuls test. The cells, regardless of group, showed phenotypic characteristics consistent with stem cells. MTT conversion, alkaline phosphatase activity, percentage of mineralized nodule and expression of collagen I, osteocalcin and BMP-2 of ADSCs from OVX rats were higher when compared to BMMSCs from OVX rats in at least one of the evaluated periods (p?p?相似文献   

Perlecan domain I-conjugated, hyaluronic acid-based hydrogel particles for enhanced chondrogenic differentiation via BMP-2 release

We have developed a biomimetic growth factor delivery system that effectively stimulates the chondrogenic differentiation of the cultured mesenchymal stem cells via the controlled presentation of bone morphogenetic protein-2 (BMP-2). Hyaluronic acid (HA)-based, microscopic hydrogel particles (HGPs) with inherent nanopores and defined functional groups were synthesized by an inverse emulsion polymerization technique. Recombinantly produced, heparan sulfate (HS)-bearing perlecan domain I (PlnDI) was covalently immobilized to HA HGPs (HGP–P₁) via a flexible poly(ethylene glycol) (PEG) linker through the lysine amines in the core protein of PlnDI employing reductive amination. Compared to HGP without PlnDI, HGP–P₁ exhibited significantly (p < 0.05) higher BMP-2 binding capacity and distinctly different BMP-2 release kinetics. Heparitinase treatment increased the amount of BMP-2 released from HGP–P₁, confirming the HS-dependent BMP-2 binding. While BMP-2 was released from HGPs with a distinct burst release followed by a minimal cumulative release, its release from HGP–P₁ exhibited a minimal burst release followed by linear release kinetics over 15 days. The bioactivity of the hydrogel particles was evaluated using micromass culture of multipotent mesenchymal stem cells (MSCs), and the chondrogenic differentiation was assessed by the production of glycosaminoglycan, aggrecan and collagen type II. Our results revealed that BMP-2 loaded HGP–P₁ stimulates more robust cartilage specific ECM production as compared to BMP-2 loaded HGP, due to the ability of HGP–P₁ to potentiate BMP-2 and modulate its release with a near zero-order release kinetics. The PlnDI-conjugated, HA HGPs provide an improved BMP-2 delivery system for stimulating chondrogenic differentiation in vitro, with potential therapeutic application for cartilage repair and regeneration.     

Osteogenic differentiation of rabbit mesenchymal stem cells in thermo-reversible hydrogel constructs containing hydroxyapatite and bone morphogenic protein-2 (BMP-2)

The aim of this study was to assess the efficacy of ectopic bone formation in a three-dimensional hybrid scaffold in combination with hydroxyapatite (HA) and poly(NiPAAm-co-AAc) as an injectable vehicle in the form of a supporting matrix for the osteogenic differentiation of rabbit mesenchymal stem cells (MSCs). Osteogenic differentiation of MSCs in the hybrid scaffold was greatly influenced by the addition of growth factors. When the osteoinduction activity of hybrid scaffold was studied following implantation into the back subcutis of nude mouse in terms of histological and biochemical examinations, significantly homogeneous bone formation was histologically observed throughout the hybrid scaffolds containing growth factor (BMP-2: bone morphogenic protein-2). The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of hybrid scaffolds were significantly high for the perfusion group compared with those in static culture group. We conclude that combination of MSC-seeded hybrid scaffold containing BMP-2 was a promising method by which to enhance in vitro osteogenic differentiation of MSC and in vivo ectopic bone formation.     

Combined VEGF and LMP-1 delivery enhances osteoprogenitor cell differentiation and ectopic bone formation

A novel strategy to enhance bone repair is to combine angiogenic factors and osteogenic factors. We combined vascular endothelial growth factor (VEGF) and LIM mineralization protein-1 (LMP-1) by using an internal ribosome entry site to link the genes within a single plasmid. We then evaluated the effects on osteoblastic differentiation in vitro and ectopic bone formation in vivo with a subcutaneously placed PLAGA scaffold loaded with a cloned mouse osteoprogenitor cell line, D1, transfected with plasmids containing VEGF and LMP-1 genes. The cells expressing both genes elevated mRNA expression of RunX2 and β-catenin and alkaline phosphatase activity compared to cells from other groups. In vivo, X-ray and micro-CT analysis of the retrieved implants revealed more ectopic bone formation at 2 and 3 weeks but not at 4 weeks compared to other groups. The results indicate that the combination of the therapeutic growth factors potentiates cell differentiation and may promote osteogenesis.