Axial mechanical loading to ex vivo mouse long bone regulates endochondral ossification and endosteal mineralization through activation of the BMP-Smad pathway during postnatal growth

Article

Author: Yoshikawa, Hideki ; Nakata, Ken ; Miyamoto, Satoshi

Mechanical loading contributes to bone development, growth, and metabolism. However, the mechanisms underlying long bone mineralization via changes in loading during the growth period are unclear. The aim of the present study was to investigate the regulatory mechanisms underlying endochondral ossification and endosteal mineralization by developing an ex vivo organ culture model with cyclic axial mechanical loads. The metacarpal bones of 3-week-old C57BL/6 mice were exposed to mechanical loading (0, 7.8, and 78 mN) for 1 h/day for 4 days. Histomorphometry revealed that axial mechanical loading regulated the thickness of the calcified zone in the growth plate and endosteal mineralization in the diaphysis in a load-dependent manner. Mechanical loading also resulted in load-dependent upregulation of endochondral ossification and bone mineralization-related genes, including bone morphogenetic protein 2 (Bmp2). Recombinant human BMP-2 administration caused similar changes in tissue structures. Conversely, inhibition of the BMP-Smad pathway diminished the stimulatory effects of mechanical loading and BMP-2 administration, suggesting that the effects of mechanical loading may be exerted through activation of the BMP-Smad pathway with the results of gene ontology and pathway analyses. Mechanical loading increased alkaline phosphatase activity and decreased carbonic anhydrase IX (Car9) mRNA expression, resulting in a significant pH increase in the culture supernatant. We hypothesize that, through activation of the BMP-Smad pathway, mechanical loading downregulates Car9, which may alkalize the local milieu, thereby inducing bone formation and long bone mineralization. Our results showed that cyclic axial mechanical loading increased endochondral ossification and endosteal mineralization in developing mouse long bones, which may have resulted from changes in the pH, ALP activity, and Pi/PPi of the extracellular environment. These findings advance our understanding of the regulation of mineralization mechanisms by mechanical loading mediated through activation of the BMP-Smad pathway.

01 Jun 2021·Bone Reports

Low magnetic field promotes recombinant human BMP-2-induced bone formation and influences orientation of trabeculae and bone marrow-derived stromal cells

Article

Author: Tateiwa, Daisuke ; Kushioka, Junichi ; Nakano, Takayoshi ; Kodama, Joe ; Zeynep, Bal ; Yoshikawa, Hideki ; Kawakami, Minoru ; Ukon, Yuichiro ; Ishimoto, Takuya ; Yamato, Kai ; Okada, Rintaro ; Kaito, Takashi

Effects of high magnetic fields [MFs, ≥ 1 T (T)] on osteoblastic differentiation and the orientation of cells or matrix proteins have been reported. However, the effect of low MFs (< 1 T) on the orientation of bone formation is not well known. This study was performed to verify the effects of low MFs on osteoblastic differentiation, bone formation, and orientation of both cells and newly formed bone. An apparatus was prepared with two magnets (190 mT) aligned in parallel to generate a parallel MF. In vitro, bone marrow-derived stromal cells of rats were used to assess the effects of low MFs on cell orientation, osteoblastic differentiation, and mineralization. A bone morphogenetic protein (BMP)-2-induced ectopic bone model was used to elucidate the effect of low MFs on microstructural indices, trabecula orientation, and the apatite c-axis orientation of newly formed bone. Low MFs resulted in an increased ratio of cells oriented perpendicular to the direction of the MF and promoted osteoblastic differentiation in vitro. Moreover, in vivo analysis demonstrated that low MFs promoted bone formation and changed the orientation of trabeculae and apatite crystal in a direction perpendicular to the MF. These changes led to an increase in the mechanical strength of rhBMP-2-induced bone. These results suggest that the application of low MFs has potential to facilitate the regeneration of bone with sufficient mechanical strength by controlling the orientation of newly formed bone.

01 Feb 2020·Regenerative BiomaterialsQ2 · ENGINEERING & TECHNOLOGY

Quantification of rhBMP2 in bioactive bone materials

Q2 · ENGINEERING & TECHNOLOGY

ArticleOA

Author: Han, Qianqian ; Wang, Chunren ; Lian, Huan ; Wang, Han

AbstractBone morphogenetic protein (BMP), belongs to transforming growth factor-β (TGF-β) superfamily except BMP-1. Implanting BMP into muscular tissues induces ectopic bone formation at the site of implantation, which provides opportunity for the treatment of bone defects. Recombinant human BMP-2 (rhBMP-2) has been used clinically, but the lack of standard methods for quantifying rhBMP-2 biological activity greatly hindered the progress of commercialization. In this article, we describe an in vitro rhBMP-2 quantification method, as well as the data analyzation pipeline through logistic regression in RStudio. Previous studies indicated that alkaline phosphatase (ALP) activity of C2C12 cells was significantly increased when exposed to rhBMP-2, and showed dose-dependent effects in a certain concentration range of rhBMP-2. Thus, we chose to quantify ALP activity as an indicator of rhBMP-2 bioactivity in vitro. A sigmoid relationship between the ALP activity and concentration of rhBMP-2 was discovered. However, there are tons of regression models for such a non-linear relationship. It has always been a major concern for researchers to choose a proper model that not only fit data accurately, but also have parameters representing practical meanings. Therefore, to fit our rhBMP-2 quantification data, we applied two logistic regression models, three-parameter log-logistic model and four-parameter log-logistic model. The four-parameter log-logistic model (adj-R2 > 0.98) fits better than three-parameter log-logistic model (adj-R2 > 0.75) for the sigmoid curves. Overall, our results indicate rhBMP-2 quantification in vitro can be accomplished by detecting ALP activity and fitting four-parameter log-logistic model. Furthermore, we also provide a highly adaptable R script for any additional logistic models.

100 Deals associated with Recombinant human interleukin-2(Changchun Institute of Biological Products)

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R&D Status

10 top approved records.

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Indication	Country/Location	Organization	Date
Autoimmune Diseases	China	Changchun Institute of Biological Products Co., Ltd.	01 Jan 1997
Bladder Cancer	China	Changchun Institute of Biological Products Co., Ltd.	01 Jan 1997
Breast Cancer	China	Changchun Institute of Biological Products Co., Ltd.	01 Jan 1997
Candidiasis	China	Changchun Institute of Biological Products Co., Ltd.	01 Jan 1997
Hepatitis B	China	Changchun Institute of Biological Products Co., Ltd.	01 Jan 1997
Leprosy	China	Changchun Institute of Biological Products Co., Ltd.	01 Jan 1997
Liver Cancer	China	Changchun Institute of Biological Products Co., Ltd.	01 Jan 1997
Lung Cancer	China	Changchun Institute of Biological Products Co., Ltd.	01 Jan 1997
Lymphoma	China	Changchun Institute of Biological Products Co., Ltd.	01 Jan 1997
Malignant ascites	China	Changchun Institute of Biological Products Co., Ltd.	01 Jan 1997
Malignant Pleural Effusion	China	Changchun Institute of Biological Products Co., Ltd.	01 Jan 1997
Melanoma	China	Changchun Institute of Biological Products Co., Ltd.	01 Jan 1997
Pulmonary Tuberculosis	China	Changchun Institute of Biological Products Co., Ltd.	01 Jan 1997
Rectal Cancer	China	Changchun Institute of Biological Products Co., Ltd.	01 Jan 1997
Renal Cell Carcinoma	China	Changchun Institute of Biological Products Co., Ltd.	01 Jan 1997
Rheumatoid Arthritis	China	Changchun Institute of Biological Products Co., Ltd.	01 Jan 1997
Sjogren's Syndrome	China	Changchun Institute of Biological Products Co., Ltd.	01 Jan 1997
Systemic Lupus Erythematosus	China	Changchun Institute of Biological Products Co., Ltd.	01 Jan 1997
Thrombocytopenia	China	Changchun Institute of Biological Products Co., Ltd.	01 Jan 1997

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