Bone morphogenetic protein-13 (BMP-13) plays an important role in skeletal development. In the light of a recent report that mutations in the BMP-13 gene are associated with spine vertebral fusion in Klippel-Feil syndrome, we hypothesized that BMP-13 signaling is crucial for regulating embryonic endochondral ossification. In this study, we found that BMP-13 inhibited the osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro. The endogenous BMP-13 gene expression in MSCs was examined under expansion conditions. The MSCs were then induced to differentiate into osteoblasts in osteo-inductive medium containing exogenous BMP-13. Gene expression was analysed by real-time PCR. Alkaline phosphatase (ALP) expression and activity, proteoglycan (PG) synthesis and matrix mineralization were assessed by cytological staining or ALP assay. Results showed that endogenous BMP-13 mRNA expression was higher than BMP-2 or -7 during MSC growth. BMP-13 supplementation strongly inhibited matrix mineralization and ALP activity of osteogenic differentiated MSCs, yet increased PG synthesis under the same conditions. In conclusion, BMP-13 inhibited osteogenic differentiation of MSCs, implying that functional mutations or deficiency of BMP-13 may allow excess bone formation. Our finding provides an insight into the molecular mechanisms and the therapeutic potential of BMP-13 in restricting pathological bone formation.
BMP-13; GDF6; CDMP-2; osteogenic differentiation; mesenchymal stromal cells
Bone morphogenetic proteins (BMPs) induce bone formation but are difficult to localize, and subsequent diffusion from the site of interest and short half-life reduce the efficacy of the protein. Currently, spine fusion requires stripping, decortications of the transverse processes, and an autograft harvest procedure. Even in combination with BMPs, clinical spinal fusion has a high failure rate, presumably because of difficulties in localizing sufficient levels of BMP.
The goal was to achieve reliable spine fusion through a single injection of a cell-based gene therapy system without the need for any surgical intervention.
Eighty-seven immunodeficient (n=44) and immune-competent (n=43) mice were injected along the paraspinous musculature to achieve rapid induction of heterotopic ossification (HO) and ultimately spinal arthrodesis.
Immunodeficient and immune-competent mice were injected with fibroblasts, transduced with an adenoviral vector to express BMP2, along the paraspinous musculature. Bone formation was evaluated via radiographs, microcomputed tomography, and biomechanical analysis.
ew bridging bone between the vertebrae and the fusion to adjacent skeletal bone was obtained as early as 2 weeks. Reduction in spine flexion-extension also occurred as early as 2 weeks after injection of the gene therapy system, with greater than 90% fusion by 4 weeks in all animals regardless of their genetic background.
Injection of our cell-based system into the paraspinous musculature induces spinal fusion that is dependent neither on the cell type nor on the immune status. These studies are the first to harness HO in an immune-competent model as a noninvasive injectable system for clinically relevant spinal fusion and may one day impact human spinal arthrodesis.
Gene therapy; Spine fusion; Heterotopic ossification; BMP2; Spinal arthrodesis
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 PPARγ2. 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 PPARγ2 not only promoted adipogenic differentiation, but also enhanced osteogenic differentiation upon BMP-2, BMP-6, and BMP-9 stimulation. Conversely, MSCs with PPARγ2 knockdown or mouse embryonic fibroblasts derived from PPARγ2−/− mice exhibited a marked decrease in adipogenic differentiation, coupled with reduced osteogenic differentiation and diminished mineralization upon BMP-9 stimulation, suggesting that PPARγ2 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.
The combined delivery of mesenchymal stem cells (MSCs), vascular endothelial growth factor (VEGF), and bone morphogenetic protein (BMP) to sites of bone injury results in enhanced repair compared to the administration of a single factor or a combination of two factors. Based on these findings, we hypothesized that coexpression of VEGF and BMP-6 genes would enhance the osteoblastic differentiation of rat bone-marrow-derived stem cells (rMSCs) and osteogenesis by comparison to rMSCs that do not express VEGF and BMP-6. We prepared a GFP tagged adenovirus vector (Ad-VEGF+BMP-6) that contained DNA encoding the hVEGF and hBMP-6 genes. rMSCs were transduced with the virus, and the successful transduction was confirmed by green fluorescence and by production of VEGF and BMP-6 proteins. The cells were cultured to assess osteoblastic differentiation or administered in the Fischer 344 rats to assess bone formation. Mineralization of rMSCs transduced with Ad-VEGF+BMP-6 was significantly enhanced over the nontransduced rMSCs. Only transduced rMSCs could induce osteogenesis in vivo, whereas Ad-VEGF+BMP-6 or nontransduced rMSCs alone did not induce osteogenesis. The data suggests that the combined delivery of MSCs, VEGF, and BMP-6 is an attractive option for bone repair therapy.
Our aim was to further elucidate the cardiac lineage development of bone marrow-derived mesenchymal stem cells (MSC) and to identify cells which had the potential for cardiac myogenic differentiation when compared to skeletal muscle satellite (Sk-sat) myogenesis. Unlike Sk-sat, MSC expressed the early cardiac markers Nkx2.5 and GATA4. Their expression was significantly increased by culturing MSC with Bone Morphogenetic Protein 4 (BMP4). Enhanced cardiac myogenic lineage differentiation and loss of stem cell characteristics induced by BMP4 were further confirmed by flow cytometry of cells stained for Nkx2.5 and Sca-1 expression. MSC also expressed skeletal genes (MyoG, ssTnI, Sk-Act) early in culture but their expression was suppressed when BMP4 was added from day 0–6 (p < 0.05). BMP4 treated MSC also exhibited a 6-fold increase in cTnI expression by day 12 in culture. The average MSC action potential time duration at 90% (APD90) was 32.3 ± 4 ms, with some cells exhibiting action potentials closer to Sk-sat APD90 of 13.7 ± 0.9 ms. After treatment with BMP4, MSC significantly increased their APD90 to 54.4 ± 7.6 ms, shifting from the shorter skeletal-like signature, towards a longer action potential duration more characteristic of a cardiomyocyte signature. Our results show that MSC and Sk-sat exhibit similarities in myogenic lineage development early in culture but that BMP4 clearly enhances cardiac myogenic development, suppresses skeletal myogenesis, and leads to loss of “stemness” in MSC. These findings provide novel information regarding the use of BMP4 to accelerate cardiac myogenic development in harvested MSC and further support the use of MSC in cardiac regenerative therapy.
Bone morphogenetic proteins (BMPs) are critical growth factors in the osteogenic differentiation of progenitor cells during development in embryos and fracture repair in adults. Although recombinant BMPs are in use clinically, their clinical efficiency needs to be improved. The biological activities of BMPs are naturally regulated by extracellular binding proteins. The specific hypotheses tested in this study were as follows: the BMP inhibitor chordin is produced endogenously during the osteogenic differentiation of human mesenchymal stem cells (MSCs); and blockade of the activity of the BMP inhibitor increases the rate of osteogenic differentiation of human MSCs in vitro.
Human MSCs were derived from bone marrow from an iliac crest aspirate and from patients undergoing hip hemiarthroplasty. The MSCs were induced down the osteogenic pathway using standard osteogenic differentiation media, and expressions of BMP-2 and chordin were determined by gene expression analysis. During osteogenic differentiation, chordin knockdown was induced using RNA interference. Osteogenic differentiation was assessed by measuring the expression of alkaline phosphatase and calcium deposition. The differences in expression of osteogenic makers between groups were compared by analysis of variance, followed by Gabriel post hoc test.
We demonstrate the expression of BMP-2 and chordin in human MSCs during osteogenic differentiation. Knockdown of chordin by RNA interference in vitro resulted in a significant increase in the expression of the osteogenic marker alkaline phosphatase and the deposition of extracellular mineral, in response to osteogenic stimulation.
We conclude that endogenously produced chordin constrains the osteogenic differentiation of human MSCs. The targeting of BMP inhibitors, such as chordin, may provide a novel strategy for enhancing bone regeneration.
The expression patterns of (bone morphogenetic proteins) BMPs during fracture repair and pre-natal bone development suggests that these processes are regulated through the coordinated actions of multiple BMPs. Murine bone marrow stromal cells (MSCs) in culture provide a well recognized ex vivo system of mesenchymal stem cell differentiation in which the effects of BMPs can be examined. Studies were performed to determine if MSC differentiation is dependent on the endogenous expression of multiple BMPs and to characterize their interactions. MSCs were harvested from the bone marrow of tibiae and femora of 8 to 10 week old male C57/B6 mice and prepared by standard methods. Osteogenic differentiation was assessed by histological assays, alkaline phosphatase enzyme activity and assays for the expression of multiple mRNAs for BMPs and osteogenic development. The role of autogenously expressed BMPs in controlling the osteogenic differentiation of marrow stromal cells in vitro was assessed in both gain-of-function and loss-of-function experiments. Gain of function experiments were carried out in the presence of exogenously added BMP-2 or 7 and loss of function experiments were carried out by BMP antagonism with noggin and BMP-2 antibody blockade. Osteogenic differentiation was concurrent with and proportional to increases in the expression of BMPs 2, 3, 4, 5, 6, and 8A. BMP antagonism with either noggin or BMP-2 antibody blockade inhibited osteogenic differentiation by 50% to 80% respectively and reduced the expression of endogenous levels of BMPs 2, 3, 5, and 8A. In contrast, antagonism induced the expression of BMP-4 and 6. The addition of rhBMP-2 or 7 enhanced osteogenic differentiation and produced a reciprocal expression profile in the endogenous BMPs expression as compared to BMP antagonism. BMP antagonism could be rescued through the competitive addition of rhBMP-2. These studies demonstrated that osteogenic differentiation was regulated by a complex network of multiple BMPs that showed selective increased and decreased expression during differentiation. They further demonstrated that BMP-2 was a central regulator in this network.
Marrow Stromal Stem Cells; Bone Morphogenetic Proteins; BMP; Osteoinduction; Noggin
Introduction: While several studies report that bone morphogenetic proteins (BMPs) and vascular endothelial growth factor (VEGF) can act synergistically to improve bone tissue engineering, others suggest that VEGF inhibits osteogenesis. The purpose of these experiments was therefore to evaluate the effect of dual transfection of these growth factors and potential mechanisms of interaction on gene expression and osteogenesis in vitro and in vivo. Methods: Marrow-derived mesenchymal stem cells (MSCs) were exposed to recombinant VEGF protein or transfected with adenoviruses encoding BMP2, VEGF, or LacZ in a variety of ratios. Alterations in gene and protein expression in vitro as well as bone formation in vivo were assessed. Results: MSC exposure to AdV-VEGF or recombinant VEGF inhibited BMP2 mRNA expression, protein production, and MSC differentiation. Coculture experiments revealed that BMP2 suppression occurs through both an autocrine and a paracrine mechanism, occurring at the transcriptional level. Compared to controls, cotransfection of VEGF and BMP2 transgenes prevented ectopic bone formation in vivo. Conclusion: VEGF is a potent inhibitor of BMP2 expression in MSCs, and supplementation or overexpression of VEGF inhibits osteogenesis in vitro and ectopic bone formation in vivo. Strategies to utilize MSCs in bone tissue engineering therefore require careful optimization and precise delivery of growth factors for maximal bone formation.
Parathyroid hormone (PTH) stimulates bone remodeling and induces differentiation of bone marrow mesenchymal stromal/stem cells (MSCs) by orchestrating activities of local factors such as bone morphogenetic proteins (BMPs). The activity and specificity of different BMP ligands are controlled by various extracellular antagonists that prevent binding of BMPs to their receptors. Low-density lipoprotein receptor-related protein 6 (LRP6) has been shown to interact with both the PTH and BMP extracellular signaling pathways by forming a complex with PTH1R and sharing common antagonists with BMPs. We hypothesized that PTH-enhanced differentiation of MSCs into the osteoblast lineage through enhancement of BMP signaling occurs by modifying the extracellular antagonist network via LRP6. In vitro studies using multiple cell lines, including Sca-1+CD45−CD11b− MSCs showed that a single injection of PTH enhanced phosphorylation of Smad1 and could also antagonize the inhibitory effect of noggin. PTH treatment induced endocytosis of a PTH1R/LRP6 complex and resulted in enhancement of phosphorylation of Smad1 that was abrogated by deletion of PTH1R, β-arrestin, or chlorpromazine. Deletion of LRP6 alone lead to enhancement of pSmad1 levels that could not be further increased with PTH treatment. Finally, knockdown of LRP6 increased the exposure of endogenous cell-surface BMPRII significantly in C2C12 cells and PTH treatment significantly enhanced cell surface binding of 125I-BMP2 in a dose- and time-dependent manner, implying that LRP6 organizes an extracellular network of BMP antagonists that prevent access of BMPs to BMP receptors. In vivo studies in C57BL/6J mice and of transplanted GFP-labeled Sca-1+CD45−CD11b− MSCs into bone marrow cavity of Rag2−/− immunodeficient mice showed PTH-enhanced phosphorylation of Smad1 and increased commitment of MSCs to osteoblast lineage, respectively. These data demonstrate that PTH-enhancement of MSCs differentiation to the osteoblast lineage occurs through a PTH and LRP6 dependent pathway by endocytosis of LRP6/PTH1R complex, allowing enhancement of BMP signaling.
PARATHYROID HORMONE; BONE MORPHOGENETIC PROTEINS; DIFFERENTIATION; MESENCHYMAL STEM CELLS; LOW-DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN 6
Spinal fusion is a common orthopaedic procedure that has been previously modeled using canine, lapine, and rodent subjects. Despite the increasing availability of genetically modified mouse strains, murine models have only been infrequently described.
To present an efficient and minimally traumatic procedure for achieving spinal fusion in a mouse model and determine the optimal rhBMP-2 dose to achieve sufficient fusion mass.
MicroCT reconstructions of the unfused mouse spine and human spine were compared to design a surgical approach. In phase 1, posterolateral lumbar spine fusion in the mouse was evaluated using 18 animals allocated to three experimental groups. Group 1 received decortication only (n = 3), Group 2 received 10 μg rhBMP-2 in a collagen sponge bilaterally (n = 6), and Group 3 received 10 μg rhBMP-2 + decortication (n = 9). The surgical technique was assessed for intra-operative safety, efficacy, access and reproducibility. Spines were harvested for analysis at 3 weeks (Groups 1, 2) and 1, 2, and 3 weeks (Group 3). In phase 2, a dose response study was carried out in an additional 18 animals with C57BL6 mice receiving sponges containing 0, 0.5, 1, 2.5, 5 μg of rhBMP-2 per sponge bilaterally.
The operative procedure via midline access was rapid and reproducible, and fusion of the murine articular processes was found to be analogous to the human procedure. Unlike reports from other species, decortication alone (Group 1) yielded no new bone formation. Addition of rhBMP-2 (Groups 2 and 3) yielded a significant bone mass that bridged the L4-L6 vertebrae. The subsequent dose response experiment revealed that 0.5 μg rhBMP-2 per sponge was sufficient to create a fusion mass.
We describe a new approach for mouse lumbar spine fusion that is safe, efficient, and highly reproducible. The technique we employed is analogous to the human midline procedure and may be highly suitable for genetically modified mouse models.
Spine; Fusion; Arthrodesis; Mouse; BMP
Efficient osteogenic differentiation and bone formation from mesenchymal stem cells (MSCs) should have clinical applications in treating nonunion fracture healing. MSCs are adherent bone marrow stromal cells that can self-renew and differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. We have identified bone morphogenetic protein 9 (BMP-9) as one of the most osteogenic BMPs. Here we investigate the effect of insulin-like growth factor 2 (IGF-2) on BMP-9-induced bone formation. We have found that endogenous IGF-2 expression is low in MSCs. Expression of IGF-2 can potentiate BMP-9-induced early osteogenic marker alkaline phosphatase (ALP) activity and the expression of later markers. IGF-2 has been shown to augment BMP-9-induced ectopic bone formation in the stem cell implantation assay. In perinatal limb explant culture assay, IGF-2 enhances BMP-9-induced endochondral ossification, whereas IGF-2 itself can promote the expansion of the hypertropic chondrocyte zone of the cultured limb explants. Expression of the IGF antagonists IGFBP3 and IGFBP4 leads to inhibition of the IGF-2 effect on BMP-9-induced ALP activity and matrix mineralization. Mechanistically, IGF-2 is further shown to enhance the BMP-9-induced BMPR-Smad reporter activity and Smad1/5/8 nuclear translocation. PI3-kinase (PI3K) inhibitor LY294002 abolishes the IGF-2 potentiation effect on BMP-9-mediated osteogenic signaling and can directly inhibit BMP-9 activity. These results demonstrate that BMP-9 crosstalks with IGF-2 through PI3K/AKT signaling pathway during osteogenic differentiation of MSCs. Taken together, our findings suggest that a combination of BMP-9 and IGF-2 may be explored as an effective bone-regeneration agent to treat large segmental bony defects, nonunion fracture, and/or osteoporotic fracture. © 2010 American Society for Bone and Mineral Research.
BMP-9; bone formation; fracture healing; IGF-2; osteoblastic differentiation
Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily and play a critical role in skeletal development, bone formation and stem cell differentiation. Disruptions in BMP signaling result in a variety of skeletal and extraskeletal anomalies. BMP9 is a poorly characterized member of the BMP family and is among the most osteogenic BMPs, promoting osteoblastic differentiation of mesenchymal stem cells (MSCs) both in vitro and in vivo. Recent findings from various in vivo and molecular studies strongly suggest that the mechanisms governing BMP9-mediated osteoinduction differ from other osteogenic BMPs. Many signaling pathways with diverse functions have been found to play a role in BMP9-mediated osteogenesis. Several of these pathways are also critical in the differentiation of other cell lineages, including adipocytes and chondrocytes. While BMP9 is known to be a potent osteogenic factor, it also influences several other pathways including cancer development, angiogenesis and myogenesis. Although BMP9 has been demonstrated as one of the most osteogenic BMPs, relatively little is known about the specific mechanisms responsible for these effects. BMP9 has demonstrated efficacy in promoting spinal fusion and bony non-union repair in animal models, demonstrating great translational promise. This review aims to summarize our current knowledge of BMP9-mediated osteogenesis by presenting recently completed work which may help us to further elucidate these pathways.
BMP; BMP9; bone regeneration; IGF; osteogenesis; TGF-β; Wnt; signal transduction; mesenchymal stem cells
Hyperbaric oxygen (HBO) therapy has been proved in improving bone healing, but its effects on mesenchymal stem cells (MSCs) in vivo is not clear. The aims of this study are to clarify whether the HBO therapy has the same enhancing effect on MSCs with regard to bone formation and maturation and to ascertain whether the transplanted MSCs survive in the grafted area and contribute to new bone formation.
Twenty-three adult rabbits underwent posterolateral fusion at L4-L5 level. The animals were divided into three groups according to the material implanted and subsequent treatment: (1) Alginate carrier (n = 6); (2) Alginate-MSCs composite (n = 11); and (3) Alginate-MSCs composite with HBO therapy (n = 6). After 12 weeks, spine fusion was examined using radiographic examination, manual testing, and histological examination. Using a PKH fluorescence labeling system, whether the transplanted MSCs survived and contributed to new bone formation in the grafted area after HBO therapy was also examined.
The bilateral fusion areas in each animal were evaluated independently. By radiographic examination and manual palpation, union for the Alginate, Alginate-MSCs, and Alginate-MSCs-HBO groups was 0 of 12, 10 of 22, and 6 of 12 respectively. The difference between the Alginate-MSCs and Alginate-MSCs-HBO groups was not significant (P = 0.7997). The fluorescence microscopy histological analysis indicated that the transplanted PKH67-labeled MSCs survived and partly contributed to new bone formation in the grafted area.
This study demonstrated that the preconditioned MSCs could survive and yield bone formation in the grafted area. HBO therapy did not enhance the osteogenic ability of MSCs and improve the success of spine fusion in the rabbit model. Although there was no significant effect of HBO therapy on MSCs for spine fusion, the study encourages us to research a more basic approach for determining the optimal oxygen tension and pressure that are required to maintain and enhance the osteogenic ability of preconditioned MSCs. Further controlled in vivo and in vitro studies are required for achieving a better understanding of the effect of HBO treatment on MSCs.
At present, due to the growing attention focused on the issue of tendon–bone healing, we carried out an animal study of the use of genetic intervention combined with cell transplantation for the promotion of this process. Here, the efficacy of bone marrow stromal cells infected with bone morphogenetic protein-2 (BMP-2) on tendon–bone healing was determined. A eukaryotic expression vector containing the BMP-2 gene was constructed and bone marrow-derived mesenchymal stem cells (bMSCs) were infected with a lentivirus. Next, we examined the viability of the infected cells and the mRNA and protein levels of BMP-2-infected bMSCs. Gastrocnemius tendons, gastrocnemius tendons wrapped by bMSCs infected with the control virus (bMSCs+Lv-Control), and gastrocnemius tendons wrapped by bMSCs infected with the recombinant BMP-2 virus (bMSCs+Lv-BMP-2) were used to reconstruct the anterior cruciate ligament (ACL) in New Zealand white rabbits. Specimens from each group were harvested four and eight weeks postoperatively and evaluated using biomechanical and histological methods. The bMSCs were infected with the lentivirus at an efficiency close to 100%. The BMP-2 mRNA and protein levels in bMSCs were significantly increased after lentiviral infection. The bMSCs and BMP-2-infected bMSCs on the gastrocnemius tendon improved the biomechanical properties of the graft in the bone tunnel; specifically, bMSCs infected with BMP-2 had a positive effect on tendon–bone healing. In the four-week and eight-week groups, bMSCs+Lv-BMP-2 group exhibited significantly higher maximum loads of 29.3 ± 7.4 N and 45.5 ± 11.9 N, respectively, compared with the control group (19.9 ± 6.4 N and 21.9 ± 4.9 N) (P = 0.041 and P = 0.001, respectively). In the eight-week groups, the stiffness of the bMSCs+Lv-BMP-2 group (32.5 ± 7.3) was significantly higher than that of the bMSCs+Lv-Control group (22.8 ± 7.4) or control groups (12.4 ± 6.0) (p = 0.036 and 0.001, respectively). Based on the histological findings, there was an increased amount of perpendicular collagen fibers formed between the tendon and bone in the bMSCs+Lv-Control and bMSCs+Lv-BMP-2 group, compared with the gastrocnemius tendons. The proliferation of cartilage-like cells and the formation of fibrocartilage-like tissue were highest within the bone tunnels in the bMSCs+Lv-BMP-2 group. These results suggest that this lentivirus can be used to efficiently infect bMSCs with BMP-2. Furthermore, tendons wrapped by bMSCs+Lv-BMP-2 improved tendon–bone healing.
tendon-bone healing; anterior cruciate ligament (ACL); reconstruction; bone marrow-derived mesenchymal stem cells
Current clinical therapies for critical-sized bone defects (CSBDs) remain far from ideal. Previous studies have demonstrated that engineering bone tissue using mesenchymal stem cells (MSCs) is feasible. However, this approach is not effective for CSBDs due to inadequate vascularization. In our previous study, we have developed an injectable and porous nano calcium sulfate/alginate (nCS/A) scaffold and demonstrated that nCS/A composition is biocompatible and has proper biodegradability for bone regeneration. Here, we hypothesized that the combination of an injectable and porous nCS/A with bone morphogenetic protein 2 (BMP2) gene-modified MSCs and endothelial progenitor cells (EPCs) could significantly enhance vascularized bone regeneration. Our results demonstrated that delivery of MSCs and EPCs with the injectable nCS/A scaffold did not affect cell viability. Moreover, co-culture of BMP2 gene-modified MSCs and EPCs dramatically increased osteoblast differentiation of MSCs and endothelial differentiation of EPCs in vitro. We further tested the multifunctional bone reconstruction system consisting of an injectable and porous nCS/A scaffold (mimicking the nano-calcium matrix of bone) and BMP2 genetically-engineered MSCs and EPCs in a rat critical-sized (8 mm) caviarial bone defect model. Our in vivo results showed that, compared to the groups of nCS/A, nCS/A+MSCs, nCS/A+MSCs+EPCs and nCS/A+BMP2 gene-modified MSCs, the combination of BMP2 gene -modified MSCs and EPCs in nCS/A dramatically increased the new bone and vascular formation. These results demonstrated that EPCs increase new vascular growth, and that BMP2 gene modification for MSCs and EPCs dramatically promotes bone regeneration. This system could ultimately enable clinicians to better reconstruct the craniofacial bone and avoid donor site morbidity for CSBDs.
We characterized the differentiation of rat bone marrow-derived mesenchymal stem cells (BM-MSCs) into tenocyte-like cells in response to bone morphogenetic protein-12 (BMP-12). BM-MSCs were prepared from Sprague-Dawley rats and cultured as monolayers. Recombinant BMP-12 treatment (10 ng/ml) of BM-MSCs for 12 hours in vitro markedly increased expression of the tenocyte lineage markers scleraxis (Scx) and tenomodulin (Tnmd) over 14 days. Treatment with BMP-12 for a further 12-hour period had no additional effect. Colony formation assays revealed that ∼80% of treated cells and their progeny were Scx- and Tnmd-positive. BM-MSCs seeded in collagen scaffolds and similarly treated with a single dose of BMP-12 also expressed high levels of Scx and Tnmd, as well as type I collagen and tenascin-c. Furthermore, when the treated BM-MSC-seeded scaffolds were implanted into surgically created tendon defects in vivo, robust formation of tendon-like tissue was observed after 21 days as evidenced by increased cell number, elongation and alignment along the tensile axis, greater matrix deposition and the elevated expression of tendon markers. These results indicate that brief stimulation with BMP-12 in vitro is sufficient to induce BM-MSC differentiation into tenocytes, and that this phenotype is sustained in vivo. This strategy of pretreating BM-MSCs with BMP-12 prior to in vivo transplantation may be useful in MSC-based tendon reconstruction or tissue engineering.
The study is a retrospective case series. The objective is to review the results after off-label recombinant human BMP-2 (rhBMP-2) use in the pediatric spine after previously failed spinal fusion. Non-union in the pediatric spine is a challenging condition associated with increased morbidity due to instability, neurological impairment or multiple revision surgeries. BMP has been used with good results in the adult spine; however, information on its use in the pediatric population is still lacking. rhBMP-2 was used at our institution at revision posterior spinal surgery in three patients. Solid spinal fusion was achieved in all three cases despite underlying bone dysplasia (Hurler’s disease), instability or bony substance loss. No adverse reactions due to rhBMP-2 use were observed. rhBMP-2 should be considered as potential option to achieve spinal fusion in children with compromised bone healing due to congenital, local or systemic conditions.
rhBMP-2; Immature spine; Bone dysplasia
We recently demonstrated a novel effective therapeutic regimen for treating hamster heart failure based on injection of bone marrow mesenchymal stem cells (MSCs) or MSC-conditioned medium into the skeletal muscle. The work highlights an important cardiac repair mechanism mediated by the myriad of trophic factors derived from the injected MSCs and local musculature that can be explored for non-invasive stem cell therapy. While this therapeutic regimen provides the ultimate proof that MSC-based cardiac repair is mediated by the trophic actions independent of MSC differentiation or stemness, the trophic factors responsible for cardiac regeneration after MSC therapy remain largely undefined. Toward this aim, we took advantage of the finding that human and porcine MSCs exhibit species-related differences in expression of trophic factors. We demonstrate that human MSCs when compared to porcine MSCs express and secrete 5-fold less vascular endothelial growth factor (VEGF) in conditioned medium (40 ± 5 and 225 ± 17 pg/ml VEGF, respectively). This deficit in VEGF output was associated with compromised cardiac therapeutic efficacy of human MSC-conditioned medium. Overexpression of VEGF in human MSCs however completely restored the therapeutic potency of the conditioned medium. This finding indicates VEGF as a key therapeutic trophic factor in MSC-mediated myocardial regeneration, and demonstrates the feasibility of human MSC therapy using trophic factor-based cell-free strategies, which can eliminate the concern of potential stem cell transformation.
VEGF; mesenchymal stem cell; heart failure; cell therapy
To determine the effectiveness of bone marrow mesenchymal stem cell (BM-MSC) transplantation in isolation or within a synthetic extracellular matrix (sECM) for tissue regeneration of the scarred vocal fold lamina propria.
In vitro stability and compatibility of mouse BM-MSC embedded in sECM was assessed by flow cytometry detection of BM-MSC marker expression and proliferation. Eighteen rats were subjected to vocal fold injury bilaterally, followed by one month post-treatment with unilateral injections of saline or sECM hydrogel (Extracel), GFP-mouse BM-MSC or BM-MSC suspended in sECM. Outcomes measured one month after treatment included procollagen-III, fibronectin, hyaluronan synthase-III (HAS3), hyaluronidase (HYAL3), smooth muscle actin (SMA) and transforming growth factor-beta 1(TGF-β1) mRNA expression. The persistence of GFP BM-MSC, proliferation, apoptosis and myofibroblast differentiation was assessed by immunofluorescence.
BM-MSC grown in vitro within sECM express Sca-1, are positive for hyaluronan receptor CD44 and continue to proliferate. In the in vivo study, groups injected with BM-MSC had detectable GFP-labeled BM-MSC remaining, showed proliferation and low apoptotic or myofibroblast markers compared to the contralateral side. Embedded BM-MSC in sECM group exhibited increased levels of procollagen III, fibronectin and TGF-β1. BM-MSC within sECM downregulated the expression of SMA compared to BM-MSC alone, exhibited upregulation of HYAL3 and no change in HAS3 compared to saline.
Treatment of vocal fold scarring with BM-MSC injected in a sECM displayed the most favorable outcomes in ECM production, hyaluronan metabolism, myofibroblast differentiation and production of TGF-β1. Furthermore, the combined treatment had no detectable cytotoxicity and preserved local cell proliferation.
Mesenchymal stem cells; tissue engineering; hydrogel; extracellular matrix; myofibroblast differentiation
Adult stem cells have therapeutic potential because of their intrinsic capacity for self-renewal, especially for bone regeneration. The present study demonstrates the utility of ex vivo modified mesenchymal stem cells (MSC) to enhance bone density in an immunocompetent mouse model of osteopenia. MSC were transduced ex vivo with a recombinant adeno-associated virus 2 (rAAV) expressing BMP-2 under the transcriptional control of collagen type-1α promoter. To enrich bone homing in vivo, the cells were further modified to transiently express the mouse α-4 integrin. The modified MSC were systemically administered to ovariectomized, female C57BL/6 mice. Effects of the therapy were determined by dual energy x-ray absorptiometry, 3D micro-CT, histology, and immunohistochemistry for up to six months. Results indicated that mice transplanted with MSC expressing BMP-2 showed significant increase in bone mineral density and bone mineral content(p<0.001) with relatively better proliferative capabilities of bone marrow stromal cells and higher osteocompetent pool of cells compared to control animals. Micro-CT analysis of femora and other bone histomorphometric analyses indicated more trabecular bone following MSC-BMP-2 therapy. Results obtained by transplanting genetically modified MSC from GFP transgenic mouse suggested that production of BMP2 from transplanted MSC also influenced the mobilization of endogenous progenitors for new bone formation.
Stromal derived factor-1 (SDF-1) is a chemokine signaling molecule that binds to its transmembrane receptor CXC chemokine receptor-4 (CXCR4). While we previously detected that SDF-1 was co-required with bone morphogenetic protein 2 (BMP2) for differentiating mesenchymal C2C12 cells into osteoblastic cells, it is unknown whether SDF-1 is similarly involved in the osteogenic differentiation of mesenchymal stem cells (MSCs). Therefore, here we examined the role of SDF-1 signaling during BMP2-induced osteogenic differentiation of primary MSCs that were derived from human and mouse bone marrow. Our data showed that blocking of the SDF-1/CXCR4 signal axis or adding SDF-1 protein to MSCs significantly affected BMP2-induced alkaline phosphatase (ALP) activity and osteocalcin (OCN) synthesis, markers of preosteoblasts and mature osteoblasts, respectively. Moreover, disrupting the SDF-1 signaling impaired bone nodule mineralization during terminal differentiation of MSCs. Furthermore, we detected that blocking of the SDF-1 signaling inhibited the BMP2-induced early expression of Runt-related factor-2 (Runx2) and osterix (Osx), two “master” regulators of osteogenesis, and the SDF-1 effect was mediated via intracellular Smad and Erk activation. In conclusion, our results demonstrated a regulatory role of SDF-1 in BMP2-induced osteogenic differentiation of MSCs, as perturbing the SDF-1 signaling affected the differentiation of MSCs towards osteoblastic cells in response to BMP2 stimulation. These data provide novel insights into molecular mechanisms underlying MSC osteogenesis, and will contribute to the development of MSC therapies for enhancing bone formation and regeneration in broad orthopaedic situations.
Bone morphogenetic protein 2; CXC chemokine receptor-4; Mesenchymal stem cell; Osteogenic differentiation; Stromal derived factor-1
Bone marrow-derived human mesenchymal stem cells (hMSCs) have become valuable candidates for cell-based therapeutical applications including neuroregenerative and anti-tumor strategies. Yet, the molecular mechanisms that control hMSC trans-differentiation to neural cells and hMSC tropism toward glioma remain unclear. Here, we demonstrate that hMSCs incubated with 50 ng/ml tumor necrosis factor alpha (TNF-α) acquired astroglial cell morphology without affecting proliferation, which was increased at 5 ng/ml. TNF-α (50 ng/ml) upregulated expression of numerous genes important for neural cell growth and function including LIF (leukemia inhibitory factor), BMP2 (bone morphogenetic protein 2), SOX2 (SRY box 2), and GFAP (glial fibrillary acidic protein), whereas NES (human nestin) transcription ceased suggesting a premature neural phenotype in TNF-α-differentiated hMSCs. Studies on intracellular mitogen-activated protein kinase (MAPK) signaling revealed that inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) activity abolished the TNF-α-mediated regulation of neural genes in hMSCs. In addition, TNF-α significantly enhanced expression of the chemokine receptor CXCR4 (CXC motive chemokine receptor 4), which facilitated the chemotactic invasiveness of hMSCs toward stromal cell-derived factor 1 (SDF-1) alpha. TNF-α-pretreated hMSCs not only exhibited an increased ability to infiltrate glioma cell spheroids dependent on matrix metalloproteinase activity in vitro, but they also showed a potentiated tropism toward intracranial malignant gliomas in an in vivo mouse model. Taken together, our results provide evidence that culture-expansion of hMSCs in the presence of TNF-α triggers neural gene expression and functional capacities, which could improve the use of hMSCs in the treatment of neurological disorders including malignant gliomas.
mesenchymal stem cells; TNF-α; ERK1/2 MAPK; CXCR4/SDF-1 axis; glioma mouse model; in vivo MPLSM
Nanomaterials have unique advantages in controlling stem cell function due to their biomimetic characteristics and special biological and mechanical properties. Controlling adhesion and differentiation of stem cells is critical for tissue regeneration.
This in vitro study investigated the effects of nano-hydroxyapatite, nano-hydroxyapatite-polylactide- co-glycolide (PLGA) composites, and a bone morphogenetic protein (BMP-7)- derived short peptide (DIF-7c) on osteogenic differentiation of human mesenchymal stem cells (MSC). The peptide was chemically functionalized onto nano-hydroxyapatite, incorporated into a nanophase hydroxyapatite-PLGA composite or PLGA control, or directly injected into culture media.
Unlike the PLGA control, the nano-hydroxyapatite-PLGA composites promoted adhesion of human MSC. Importantly, nano-hydroxyapatite and nano-hydroxyapatite-PLGA composites promoted osteogenic differentiation of human MSCs, comparable with direct injection of the DIF-7c peptide into culture media.
Nano-hydroxyapatite and nano-hydroxyapatite-PLGA composites provide a promising alternative in directing the adhesion and differentiation of human MSC. These nanocomposites should be studied further to clarify their effects on MSC functions and bone remodeling in vivo, eventually translating to clinical applications.
human mesenchymal stem cells; osteogenesis; stem cell differentiation; bone morphogenetic protein; peptide delivery; nanocomposites
Osteogenic differentiation of human mesenchymal stem cells (hMSCs) is classically thought to be mediated by different cytokines such as the bone morphogenetic proteins (BMPs). Here, we report that cell adhesion to extracellular matrix (ECM), and its effects on cell shape and cytoskeletal mechanics, regulates BMP-induced signaling and osteogenic differentiation of hMSCs. Using micropatterned substrates to progressively restrict cell spreading and flattening against ECM, we demonstrated that BMP-induced osteogenesis is progressively antagonized with decreased cell spreading. BMP triggered rapid and sustained RhoA/Rho-associated protein kinase (ROCK) activity and contractile tension only in spread cells, and this signaling was required for BMP-induced osteogenesis. Exploring the molecular basis for this effect, we found that restricting cell spreading, reducing ROCK signaling, or inhibiting cytoskeletal tension prevented BMP-induced SMA/mothers against decapentaplegic (SMAD)1 c-terminal phosphorylation, SMAD1 dimerization with SMAD4, and SMAD1 translocation into the nucleus. Together, these findings demonstrate the direct involvement of cell spreading and RhoA/ROCK-mediated cytoskeletal tension generation in BMP-induced signaling and early stages of in vitro osteogenesis, and highlight the essential interplay between biochemical and mechanical cues in stem cell differentiation.
The purpose of this study was to investigate the feasibility and advantages of constructing a novel tissue engineering bone, using β-tricalcium phosphate (β-TCP) and rat bone marrow mesenchymal stem cells (MSCs), modified with human bone morphogenetic protein 2 gene (hBMP2) and human vascular endothelial growth factor 165 gene (hVEGF165), through lentiviral transfection. Both genes were successfully co-expressed in the co-transfection group for up to eight weeks confirmed by enzyme-linked immunosorbent assay (ELISA). After seeding MSCs onto the scaffolds, scanning electron microscopy (SEM) observation showed that MSCs grew and proliferated well in co-transfection group at 7 and 14 days. There was no significant difference among all the groups in hoechst DNA assay for cell proliferation for 14 days after cell seeding (P > 0.05), but the highest alkaline phosphatase (ALP) activity was observed in the co-transfection group at 14 days after cell seeding (p < 0.01). These results demonstrated that it was advantageous to construct tissue engineering bone using β-TCP combined with MSCs lentivirally co-transfected with BMP2 and VEGF165, providing an innovative way for treating bone defects.
bone morphogenetic protein 2; co-transfection; lentiviral vector; tissue engineering bone; vascular endothelial growth factor; β-tricalcium phosphate (β-TCP)