Article

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

ABSTRACT

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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    • "However local application of BMP-7 lead to an increase in TGF-β after the second week that lasted longer than in every other group. It has been shown that the concentrations of bone morphogenic proteins were decreased in patients suffering from non-union, thereby indicating that a down-regulation in expression of osteogenic BMPs might be responsible for failed fracture heal- ing [32, 33]. In our study patients with failed fracture healing were treated with adjunct application of BMP-7 serving as a substitution of known lower BMP concentrations in patients with failed fracture healing, thereby modulating the microenviroment in the fracture gap and increasing local concentration of BMP-7. "
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    ABSTRACT: Background: In this study we sought to determine if application of bone morphogenic protein 7 (BMP-7) promotes physiological bone healing of non-unions and to investigate if serum cytokine analysis may serve as a promising tool in the analysis of adjunct non-union therapy. Therefore we analyzed the influence of BMP-7 application on the serum cytokine expression patterns on patients with impaired bone healing compared to patients that showed proper bone healing. Methods: Our study involved analyzing blood samples from 208 patients with long bone fractures together with patients that subsequently developed non-unions. From this large pool, 15 patients with atrophic non-union were matched to 15 patients with atrophic non-union treated with local application of BMP-7 as well as normal bone healing. Changes in the cytokine expression patterns were monitored during the 1st, 2nd, 4th, 8th, 12th and 52nd week. The patients were followed both clinically and radiologically for the entire duration of the study. Serum cytokine expression levels of transforming growth factor beta (TGF-β), platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) were analyzed and compared. Results: Serum expression of TGF-β were nearly parallel in all three groups, however serum concentrations were significantly higher in patients with proper bone healing and those treated with BMP-7 than in patients with non-unions (p < 0.05). bFGF serum concentrations increased initially in patients with proper bone healing and in those treated with BMP-7. Afterwards, values decreased; bFGF serum concentrations in the BMP-7 group were significantly higher than in the other groups (p < 0.05). PDGF serum concentration levels were nearly parallel in all groups, serum concentrations were significantly higher in patients with proper bone healing and those treated with BMP-7 than in patients with non-unions (p < 0.05). Conclusion: Treatment with BMP-7 in patients with former non-unions led to similar cytokine expression patterns after treatment as those found in patients with proper bone healing. Our results suggest that treatment with BMP-7 promote healing of non-unions. Furthermore, quantitative measurement of serum cytokine expression is a promising tool for evaluating the effectiveness of additional non-union therapies such as adjunct application of growth factors.
    Full-text · Article · Dec 2016 · Journal of Inflammation
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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.
    Full-text · Article · Jul 2014 · PLoS ONE
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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.
    Full-text · Article · Jul 2013 · Arthritis & Rheumatology
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