Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Sep 25;13(9):e0204306.
doi: 10.1371/journal.pone.0204306. eCollection 2018.

Extracellular matrix with defective collagen cross-linking affects the differentiation of bone cells

Takako Ida  1 , Masaru Kaku  1 , Megumi Kitami  1 , Masahiko Terajima  2 , Juan Marcelo Rosales Rocabado  1 , Yosuke Akiba  1 , Masako Nagasawa  1 , Mitsuo Yamauchi  2 , Katsumi Uoshima  1
Affiliations

Extracellular matrix with defective collagen cross-linking affects the differentiation of bone cells

Takako Ida et al. PLoS One. .

Abstract

Fibrillar type I collagen, the predominant organic component in bone, is stabilized by lysyl oxidase (LOX)-initiated covalent intermolecular cross-linking, an important determinant of bone quality. However, the impact of collagen cross-linking on the activity of bone cells and subsequent tissue remodeling is not well understood. In this study, we investigated the effect of collagen cross-linking on bone cellular activities employing a loss-of-function approach, using a potent LOX inhibitor, β-aminopropionitrile (BAPN). Osteoblastic cells (MC3T3-E1) were cultured for 2 weeks in the presence of 0-2 mM BAPN to obtain low cross-linked collagen matrices. The addition of BAPN to the cultures diminished collagen cross-links in a dose-dependent manner and, at 1 mM level, none of the major cross-links were detected without affecting collagen production. After the removal of cellular components from these cultures, MC3T3-E1, osteoclasts (RAW264.7), or mouse primary bone marrow-derived stromal cells (BMSCs) were seeded. MC3T3-E1 cells grown on low cross-link matrices showed increased alkaline phosphatase (ALP) activity. The number of multinucleate tartrate-resistant acid phosphatase (TRAP)-positive cells increased in RAW264.7 cells. Initial adhesion, proliferation, and ALP activity of BMSCs also increased. In the animal experiments, 4-week-old C57BL/6 mice were fed with BAPN-containing diet for 8 weeks. At this point, biochemical analysis of bone demonstrated that collagen cross-links decreased without affecting collagen content. Then, the diet was changed to a control diet to minimize the direct effect of BAPN. At 2 and 4 weeks after the change, histological samples were prepared. Histological examination of femur samples at 4 weeks showed a significant increase in the number of bone surface osteoblasts, while the bone volume and surface osteoclast numbers were not significantly affected. These results clearly demonstrated that the extent of collagen cross-linking of bone matrix affected the differentiation of bone cells, underscoring the importance of collagen cross-linking in the regulation of cell behaviors and tissue remodeling in bone. Characterization of collagen cross-linking in bone may be beneficial to obtain insight into not only bone mechanical property, but also bone cellular activities.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Effect of β-aminopropionitrile (BAPN) on matrix formation and collagen components produced by MC3T3-E1 cells.
(a) BAPN treatment did not affect the proliferation of MC cells, as demonstrated by MTS assay. (b) Expression of core-binding factor alpha 1/runt-related transcription factor 2 (Runx2/Cbfa1) and type I collagen α2 chain (Col1a2) did not change, while the levels of lysyl oxidase (Lox) were significantly elevated after BAPN treatment in a dose-dependent manner. (c) Collagen components were analyzed by electrophoresis. In the control, all chains (α, β, and γ) were clearly observable. However, BAPN treatment led to the inhibition of β- and γ-chain formation. Picrosirius Red staining was visualized under bright-field (d) and polarized light (e). Collagen quantity was not affected by BAPN, while the alignment of collagenous fibers and matrix maturation were impaired. Bar: 100 μm. (f) Collagen content was slightly increased following 0.5 mM BAPN treatment, but it decreased after the administration of 2 mM BAPN. Hyl/Hyp × 300 decreased after the treatment with 2 mM BAPN. Divalent DHLNL and HLNL levels decreased with 0.5 mM BAPN treatment and were not detectable following the treatment with 1.0 and 2.0 mM BAPN. Trivalent pyridinoline levels significantly decreased after the application of 0.5 mM BAPN, while they were undetectable following the treatment with 1.0 and 2.0 mM BAPN. *p < 0.05, compared to the control.
Fig 2
Fig 2. Effect of low cross-linked matrices on osteoblast and osteoclast.
(a) The ablation of cellular components by DOC was confirmed by DAPI staining. Bar: 50 μm. (b)The Lox activity of prepared matrices before and after the DOC treatment was analyzed. The Lox activity was significantly decreased in the matrices produced by BAPN-treated cells. DOC treatment did not significantly affect the Lox activity of prepared matrices, regardless of the BAPN concentration. (c) The proliferation of MC cells was significantly increased in the low cross-link density matrices. (d) Alkaline phosphatase (ALP) activity increased in cells seeded on low cross-linked matrices. (e) Gene expression of Cbfa1/Runx2, type I collagen α2 chain (Col1a2), alkaline phosphatase (Alpl), and osteocalcin (Spp1) significantly increased in these matrices at 3 and 7 days of culture. (f) Osteoclasts were cultured on differentially cross-linked matrices for 6 days under the differentiation condition. The number of multi-nuclear tartrate-resistant acid phosphatase (TRAP)-positive cells increased in the low cross-link density matrices. (g) Gene expression of cathepsin K (Ctsk), nuclear factor of activated T-cells cytoplasmic 1 (Nfatc1), and dendritic cell-specific transmembrane protein (DCstamp) increased in low cross-link density matrices. *p < 0.05, compared to the control.
Fig 3
Fig 3. Effect of low cross-linked matrices on osteoblastic differentiation of BMSCs.
(a) BMSCs to the prepared matrices was shown. Cell nuclei were stained using DAPI (blue), and the cytoskeleton was stained with phalloidin (red). Initial adhesion of cells cultured on low cross-link density matrix increased. (b) Proliferation of BMSCs was significantly elevated in low cross-linked matrices. (c) ALP activity of BMSCs increased in low cross-link density matrices. (d) Gene expression of Col1a2, Alpl, and Spp1 was significantly increased, while that of Cbfa1/Runx2 did not change in low cross-link density matrices after 1 and 2 weeks. *p < 0.05, compared to the control. Bars: 20 μm.
Fig 4
Fig 4. Effects of β-aminopropionitrile (BAPN)-containing diet on body weight, bone volume, and collagen cross-linking in mice.
(a) The body weight of mice increased steadily throughout the experimental period, and no differences were observed between groups. (b) Histology of Picrosirius Red-stained samples at 12 weeks of age (after 8-week of the BAPN consumption) is shown. Under polarized light, positive pixel ratio of immature matrix (green) increased with BAPN administration, while that of mature matrix (red) did not change Bar: 200 μm. (c) Collagen Cross-links were analyzed at 12 weeks of age (after 8-week of the BAPN consumption). Collagen content and hydroxylysine (Hyl)/hydroxyproline (Hyp) × 300 were not affected by BAPN, but dehydrodihydroxylysinonorleucine (DHLNL) and dehydrohydroxylysinonorleucine (HLNL) levels decreased in a dose-dependent manner. Pyridinoline, deoxypyridinoline, and total aldehyde contents were significantly reduced in mice fed with BAPN-containing diet. *p < 0.05, compared to the control.
Fig 5
Fig 5. Histological and histomorphometric analysis.
(a) Hematoxylin and eosin, and tartrate-resistant acid phosphatase (TRAP)-stained histological sections of the distal femur epiphysis at 0 week (after 8-week of the BAPN consumption). (b) No differences of osteoblast and osteoclasts activities were observed at 0 and 2 weeks after replacing to the control diet. After 4 weeks, number of osteoblasts per bone surface (N.Ob/BS) and osteoblast surface per bone surface (Ob.S/BS), representing osteoblast activity, significantly increased, while number of osteoclasts per bone surface (N.Oc/BS) and osteoclast surface per bone surface (Oc.S/BS), representing osteoclast activity did not change. *p < 0.05, compared to the control. Bar: 50 μm. (c) Picrosirius red-stained samples at 16 weeks of age (after 8-week of the BAPN consumption followed by 4-week of control diet) are analyzed under polarized light. After 4-week of control diet, immature/irregular collagen matrix still detected both in cortical and cancellous bone. Bar: 50 μm. Quantitative data also confirmed that the immature collagens, detected in green, retained high value after 4-week of control diet in cancellous bone.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Canty EG, Kadler KE. Procollagen trafficking, processing and fibrillogenesis. Journal of cell science. 2005;118(7):1341–53. 10.1242/jcs.01731 - DOI - PubMed
    1. Yamauchi M, Sricholpech M. Lysine post-translational modifications of collagen. Essays Biochem. 2012;52:113–33. Epub 2012/06/20. 10.1042/bse0520113 ; PubMed Central PMCID: PMC3499978. - DOI - PMC - PubMed
    1. Knott L, Bailey AJ. Collagen cross-links in mineralizing tissues: a review of their chemistry, function, and clinical relevance. Bone. 1998;22(3):181–7. . - PubMed
    1. Shiiba M, Arnaud SB, Tanzawa H, Kitamura E, Yamauchi M. Regional alterations of type I collagen in rat tibia induced by skeletal unloading. Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research. 2002;17(9):1639–45. 10.1359/jbmr.2002.17.9.1639 . - DOI - PubMed
    1. Shiiba M, Arnaud SB, Tanzawa H, Uzawa K, Yamauchi M. Alterations of collagen matrix in weight-bearing bones during skeletal unloading. Connective tissue research. 2001;42(4):303–11. . - PubMed

Publication types

Grants and funding

This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS, JP15J03831 to TI, JP26293407 JP15KK0337, and JP15K15704 to MKa).