Niikura, T. et al. Global gene profiling reveals a downregulation of BMP gene expression in experimental atrophic nonunions compared to standard healing fractures. J. Orthop. Res. 24, 1463-1471

Center for Tissue Regeneration and Repair, Department of Orthopaedic Surgery, University of California, Davis, 4860 Y Street, Suite 3800, Sacramento, California 95817, USA.
Journal of Orthopaedic Research (Impact Factor: 2.99). 07/2006; 24(7):1463-71. DOI: 10.1002/jor.20182
Source: PubMed


Nonunion is a challenging problem that may occur following certain bone fractures. However, there has been little investigation of the molecular basis of nonunions. Bone morphogenetic proteins (BMPs) play a significant role in osteogenesis. However, little is known about the expression patterns of BMPs in abnormal bone healing that results in nonunion formation. These facts prompted us to investigate and compare the gene expression patterns of BMPs and their antagonists in standard healing fractures and nonunions using rat experimental models. Standard closed healing fractures and experimental atrophic nonunions produced by periosteal cauterization at the fracture site were created in rat femurs. At postfracture days 3, 7, 10, 14, 21, and 28, total RNA was extracted from the callus of standard healing fracture and fibrous tissue of nonunion (n=4 per each time point and each group). Gene expression of BMPs, BMP antagonists, and other regulatory molecules were studied by methods including Genechip microarray and real-time quantitative RT-PCR. Gene expression of BMP-2, 3, 3B, 4, 6, 7, GDF-5, 7, and BMP antagonists noggin, drm, screlostin, and BAMBI were significantly lower in nonunions compared to standard healing fractures at several time points. Downregulation in expression of osteogenic BMPs may account for the nonunions of fracture. The balance between BMPs and their endogenous antagonists is critical for optimal fracture healing.

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    • "It involves a variety of cell types and signaling molecules. Deficiencies in mesenchymal stem cells (MSCs) [1]–[2], angiogenesis induced by vascular endothelial growth factor (VEGF) [3]–[4] and bone morphogenetic proteins (BMPs) signaling [5]–[7] are associated with fractures that do not heal. It is estimated that of the 7.9 million fractures sustained each year in the United States, 5% to 20% result in delayed or impaired healing [8]. "
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    ABSTRACT: Clinical trials on fracture repair have challenged the effectiveness of bone morphogenetic proteins (BMPs) but suggest that delivery of mesenchymal stem cells (MSCs) might be beneficial. It has also been reported that BMPs could not increase mineralization in several MSCs populations, which adds ambiguity to the use of BMPs. However, an exogenous supply of MSCs combined with vascular endothelial growth factor (VEGF) and BMPs is reported to synergistically enhance fracture repair in animal models. To elucidate the mechanism of this synergy, we investigated the osteoblastic differentiation of cloned mouse bone marrow derived MSCs (D1 cells) in vitro in response to human recombinant proteins of VEGF, BMPs (-2, -4, -6, -9) and the combination of VEGF with BMP-6 (most potent BMP). We further investigated ectopic bone formation induced by MSCs pre-conditioned with VEGF, BMP-6 or both. No significant increase in mineralization, phosphorylation of Smads 1/5/8 and expression of the ALP, COL1A1 and osterix genes was observed upon addition of VEGF or BMPs alone to the cells in culture. The lack of CD105, Alk1 and Alk6 expression in D1 cells correlated with poor response to BMPs indicating that a greater care in the selection of MSCs is necessary. Interestingly, the combination of VEGF and BMP-6 significantly increased the expression of ALP, COL1A1 and osterix genes and D1 cells pre-conditioned with VEGF and BMP-6 induced greater bone formation in vivo than the non-conditioned control cells or the cells pre-conditioned with either VEGF or BMP-6 alone. This enhanced bone formation by MSCs correlated with higher CADM1 expression and OPG/RANKL ratio in the implants. Thus, combined action of VEGF and BMP on MSCs enhances osteoblastic differentiation of MSCs and increases their bone forming ability, which cannot be achieved through use of BMPs alone. This strategy can be effectively used for bone repair.
    PLoS ONE 07/2014; 9(7):e103060. DOI:10.1371/journal.pone.0103060 · 3.23 Impact Factor
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    • "β-actin was used as an internal control. Relative messenger RNA expression levels were calculated as described by Niikura et al (26). PCR primers were as follows: for aggrecan, 5′-GCAGGGATAACGGACTGAAG-3′ (forward) and 5′-GAGTAAAGTGGTCATAGTTCAGCTTG-3′ (reverse); for COL1A1, 5′-TCCTGGCAAGAACGGAGAT-3′ (forward) and 5′-CAGGAGGTCCACGCTCAC-3′ (reverse); for COL2A1, 5′-CCAGGTCCTGCTGGAAAA-3′ (forward) and 5′-CCTCTTTCTCCGGCCTTT-3′ (reverse). "
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    ABSTRACT: We examined whether BMP-7 could induce ectopic cartilage formation in the tendon, and whether transplantation of tendon treated with BMP-7 promoted meniscal regeneration. Additionally, we analyzed the relative contributions of host and donor cells on the healing process after tendon transplantation in a rat model. BMP-7 was injected in situ into the Achilles tendon of rats, and the histological findings and the gene profile were evaluated. Achilles tendon injected with 1 μg BMP-7 was transplanted into a rat meniscal defect. The regenerated meniscus and articular cartilage were evaluated at 4, 8, and 12 weeks. The Achilles tendon of LacZ rats was transplanted into the meniscal defect of wild type rats, and vice versa. Injection of BMP-7 into the Achilles tendon induced the fibrochondrocyte differentiation of tendon cells and changed the collagen gene profile of tendon tissue to more closely approximate meniscus tissue. Transplantation of the Achilles tendon into a meniscal defect increased meniscal size. The rats who received the tendon treated with BMP-7 had meniscus matrix that exhibited increased safranin-o and type II collagen staining, and showed a delay in articular cartilage degradation. Using LacZ transgenic rats, we were able to determine that the regeneration of the meniscus was the resultant function of both donor and host cells. BMP-7 induced ectopic cartilage formation in tendons. Transplantation of Achilles tendon treated with BMP-7 promoted meniscus regeneration and prevented cartilage degeneration in a rat massive meniscus defect model. Native cells in the Achilles tendon contributed to meniscal regeneration.
    Arthritis & Rheumatology 07/2013; 65(11). DOI:10.1002/art.38099 · 7.76 Impact Factor
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    • "After an initial denaturation step (95°C for 10 minutes), amplification was performed for 40 cycles (95°C for 15 seconds, 60°C for 60 seconds). Relative amounts of mRNA were calculated and standardized as previously described [13,14]. "
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    ABSTRACT: Transplantation of mesenchymal stem cells (MSCs) derived from synovium is a promising therapy for cartilage regeneration. For clinical application, improvement of handling operation, enhancement of chondrogenic potential, and increase of MSCs adhesion efficiency are needed to achieve a more successful cartilage regeneration with a limited number of MSCs without scaffold. The use of aggregated MSCs may be one of the solutions. Here, we investigated the handling, properties and effectiveness of aggregated MSCs for cartilage regeneration. Human and rabbit synovial MSCs were aggregated using the hanging drop technique. The gene expression changes after aggregation of synovial MSCs were analyzed by microarray and real time RT-PCR analyses. In vitro and in vivo chondrogenic potential of aggregates of synovial MSCs was examined. Aggregates of MSCs cultured for three days became visible, approximately 1 mm in diameter and solid and durable by manipulation; most of the cells were viable. Microarray analysis revealed up-regulation of chondrogenesis-related, anti-inflammatory and anti-apoptotic genes in aggregates of MSCs. In vitro studies showed higher amounts of cartilage matrix synthesis in pellets derived from aggregates of MSCs compared to pellets derived from MSCs cultured in a monolayer. In in vivo studies in rabbits, aggregates of MSCs could adhere promptly on the osteochondral defects by surface tension, and stay without any loss. Transplantation of aggregates of MSCs at relatively low density achieved successful cartilage regeneration. Contrary to our expectation, transplantation of aggregates of MSCs at high density failed to regenerate cartilage due to cell death and nutrient deprivation of aggregates of MSCs. Aggregated synovial MSCs were a useful source for cartilage regeneration considering such factors as easy preparation, higher chondrogenic potential and efficient attachment.
    Arthritis research & therapy 06/2012; 14(3):R136. DOI:10.1186/ar3869 · 3.75 Impact Factor
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