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Tissue Engineering and Regenerative Medicine

, Volume 15, Issue 6, pp 793–801 | Cite as

Combined Treatment with Low-Level Laser and rhBMP-2 Promotes Differentiation and Mineralization of Osteoblastic Cells under Hypoxic Stress

  • Jin-Ho Heo
  • Jeong-Hun Choi
  • In-Ryoung Kim
  • Bong-Soo Park
  • Yong-Deok Kim
Original Article
  • 28 Downloads

Abstract

Background:

The aim of this study was to evaluate the combined effect of low-level laser treatment (LLLT) and recombinant human bone morphological protein-2 (rhBMP-2) applied to hypoxic-cultured MC3T3-E1 osteoblastic cells and to determine possible signaling pathways underlying differentiation and mineralization of osteoblasts under hypoxia.

Methods:

MC3T3-E1 cells were cultured under 1% oxygen tension for 72 h. Cell cultures were divided into four groups: normoxia control, low-level laser (LLL) alone, rhBMP-2 combined with LLLT, and rhBMP-2 under hypoxia. Laser irradiation was applied at 0, 24, and 48 h. Cells were treated with rhBMP-2 at 50 ng/mL. Alkaline phosphatase activity was measured at 3, 7, and 14 days to evaluate osteoblastic differentiation. Cell mineralization was determined with Alizarin red S staining at 7 and 14 days. Western blot assays were performed to evaluate whether p38/protein kinase D (PKD) signaling was involved.

Results:

The results indicate that LLLT and rhBMP-2 synergistically increased alkaline phosphatase (ALP) activity and mineralization. Western blot analyses showed that expression of type I collagen, runt-related transcription factor 2 (RUNX2), and Osterix (Osx), increased and expression of hypoxia-inducible factor 1-alpha (HIF-1α), decreased more in the LLLT and rhBMP-2 combined group than in the rhBMP-2 or LLL alone groups. Moreover, LLLT and rhBMP-2 stimulated p38 phosphorylation and rhBMP-2 and LLLT increased Prkd1 phosphorylation.

Conclusion:

Combined treatment with rhBMP-2 and LLL induced differentiation and mineralization of hypoxic-cultured MC3T3-E1 osteoblasts by activating p38/PKD signaling in vitro.

Keywords

Hypoxia Osteoblast Laser p38 PKD 

Notes

Acknowledgements

This study was supported by National Research Foundation of Korea (NRF-2018R1A2B2006546) and a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI17C0708).

Authors contribution

JHH carried out the molecular genetic studies and prepared the manuscript. JHC participated in the design of the study and performed the statistical analysis. IRK carried out the molecular studies. BSP conceived of the study, and participated in its design and coordination and helped to draft the manuscript. YDK designed the study, drafted the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

There are no animal experiments carried out for this article.

References

  1. 1.
    Dimitriou R, Jones E, McGonagle D, Giannoudis PV. Bone regeneration: current concepts and future directions. BMC Med. 2011;9:66.CrossRefGoogle Scholar
  2. 2.
    Giannoudis PV, Einhorn TA, Marsh D. Fracture healing: the diamond concept. Injury. 2007;38 Suppl 4:S3-6.CrossRefGoogle Scholar
  3. 3.
    Pyo SJ, Song WW, Kim IR, Park BS, Kim CH, Shin SH, et al. Low-level laser therapy induces the expressions of BMP-2, osteocalcin, and TGF-β1 in hypoxic-cultured human osteoblasts. Lasers Med Sci. 2013;28:543–50.CrossRefGoogle Scholar
  4. 4.
    Harrison JS, Rameshwar P, Chang V, Bandari P. Oxygen saturation in the bone marrow of healthy volunteers. Blood. 2002;99:394.CrossRefGoogle Scholar
  5. 5.
    Lewis JS, Lee JA, Underwood JC, Harris AL, Lewis CE. Macrophage responses to hypoxia: relevance to disease mechanisms. J Leukoc Biol. 1999;66:889–900.CrossRefGoogle Scholar
  6. 6.
    Susperregui AR, Viñals F, Ho PW, Gillespie MT, Martin TJ, Ventura F. BMP-2 regulation of PTHrP and osteoclastogenic factors during osteoblast differentiation of C2C12 cells. J Cell Physiol. 2008;216:144–52.CrossRefGoogle Scholar
  7. 7.
    Prolo DJ, Rodrigo JJ. Contemporary bone graft physiology and surgery. Clin Orthop Relat Res. 1985;200:322–42.Google Scholar
  8. 8.
    Szpalski M, Gunzburg R. Recombinant human bone morphogenetic protein-2: a novel osteoinductive alternative to autogenous bone graft. Acta Orthop Belg. 2005;71:133–48.PubMedGoogle Scholar
  9. 9.
    Merli LA, Santos MT, Genovese WJ, Faloppa F. Effect of low-intensity laser irradiation on the process of bone repair. Photomed Laser Surg. 2005;23:212–5.CrossRefGoogle Scholar
  10. 10.
    Renno AC, McDonnell PA, Parizotto NA, Laakso EL. The effects of laser irradiation on osteoblast and osteosarcoma cell proliferation and differentiation in vitro. Photomed Laser Surg. 2007;25:275–80.CrossRefGoogle Scholar
  11. 11.
    AlGhamdi KM, Kumar A, Moussa NA. Low-level laser therapy: a useful technique for enhancing the proliferation of various cultured cells. Lasers Med Sci. 2012;27:237–49.CrossRefGoogle Scholar
  12. 12.
    Kiyosaki T, Mitsui N, Suzuki N, Shimizu N. Low-level laser therapy stimulates mineralization via increased Runx2 expression and ERK phosphorylation in osteoblasts. Photomed Laser Surg. 2010;28 Suppl 1:S167–72.CrossRefGoogle Scholar
  13. 13.
    Ozawa Y, Shimizu N, Kariya G, Abiko Y. Low-energy laser irradiation stimulates bone nodule formation at early stages of cell culture in rat calvarial cells. Bone. 1998;22:347–54.CrossRefGoogle Scholar
  14. 14.
    Marshall CJ. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell. 1995;80:179–85.CrossRefGoogle Scholar
  15. 15.
    Pearson G, Robinson F, Beers Gibson T, Xu BE, Karandikar M, Berman K, et al. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev. 2001;22:153–83.Google Scholar
  16. 16.
    Matsuda N, Morita N, Matsuda K, Watanabe M. Proliferation and differentiation of human osteoblastic cells associated with differential activation of MAP kinases in response to epidermal growth factor, hypoxia, and mechanical stress in vitro. Biochem Biophys Res Commun. 1998;249:350–4.CrossRefGoogle Scholar
  17. 17.
    Suzuki A, Guicheux J, Palmer G, Miura Y, Oiso Y, Bonjour JP, et al. Evidence for a role of p38 MAP kinase in expression of alkaline phosphatase during osteoblastic cell differentiation. Bone. 2002;30:91–8.CrossRefGoogle Scholar
  18. 18.
    Lin FH, Chang JB, Brigman BE. Role of mitogen-activated protein kinase in osteoblast differentiation. J Orthop Res. 2011;29:204–10.CrossRefGoogle Scholar
  19. 19.
    Hug H, Sarre TF. Protein kinase C isoenzymes: divergence in signal transduction? Biochem J. 1993;291:329–43.CrossRefGoogle Scholar
  20. 20.
    Lemonnier J, Ghayor C, Guicheux J, Caverzasio J. Protein kinase C-independent activation of protein kinase D is involved in BMP-2-induced activation of stress mitogen-activated protein kinases JNK and p38 and osteoblastic cell differentiation. J Biol Chem. 2004;279:259–64.CrossRefGoogle Scholar
  21. 21.
    Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science. 1992;258:607–14.CrossRefGoogle Scholar
  22. 22.
    Toker A. Signaling through protein kinase C. Front Biosci. 1998;3:D1134–47.CrossRefGoogle Scholar
  23. 23.
    Celil AB, Campbell PG. BMP-2 and insulin-like growth factor-I mediate Osterix (Osx) expression in human mesenchymal stem cells via the MAPK and protein kinase D signaling pathways. J Biol Chem. 2005;280:31353–9.CrossRefGoogle Scholar
  24. 24.
    Yamada T, Ezura Y, Hayata T, Moriya S, Shirakawa J, Notomi T, et al. β2 adrenergic receptor activation suppresses bone morphogenetic protein (BMP)-induced alkaline phosphatase expression in osteoblast-like MC3T3E1 cells.. J Cell Biochem. 2015;116:1144–52.CrossRefGoogle Scholar
  25. 25.
    D’Ippolito G, Diabira S, Howard GA, Roos BA, Schiller PC. Low oxygen tension inhibits osteogenic differentiation and enhances stemness of human MIAMI cells. Bone. 2006;39:513–22.CrossRefGoogle Scholar
  26. 26.
    Grayson WL, Zhao F, Izadpanah R, Bunnell B, Ma T. Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs. J Cell Physiol. 2006;207:331–9.CrossRefGoogle Scholar
  27. 27.
    Hirao M, Hashimoto J, Yamasaki N, Ando W, Tsuboi H, Myoui A, et al. Oxygen tension is an important mediator of the transformation of osteoblasts to osteocytes. J Bone Miner Metab. 2007;25:266–76.CrossRefGoogle Scholar
  28. 28.
    Steinbrech DS, Mehrara BJ, Saadeh PB, Chin G, Dudziak ME, Gerrets RP, et al. Hypoxia regulates VEGF expression and cellular proliferation by osteoblasts in vitro. Plast Reconstr Surg. 1999;104:738–47.CrossRefGoogle Scholar
  29. 29.
    Zahm AM, Bucaro MA, Srinivas V, Shapiro IM, Adams CS. Oxygen tension regulates preosteocyte maturation and mineralization. Bone. 2008;43:25–31.CrossRefGoogle Scholar
  30. 30.
    Stein A, Benayahu D, Maltz L, Oron U. Low-level laser irradiation promotes proliferation and differentiation of human osteoblasts in vitro. Photomed Laser Surg. 2005;23:161–6.CrossRefGoogle Scholar
  31. 31.
    Karu T. Photobiology of low-power laser effects. Health Phys. 1989;56:691–704.CrossRefGoogle Scholar
  32. 32.
    Karu T. The science of low-power laser therapy. London: Gordon and Breach Science Publishers; 1998.Google Scholar
  33. 33.
    Jawad MM, Husein A, Azlina A, Alam MK, Hassan R, Shaari R. Effect of 940 nm low-level laser therapy on osteogenesis in vitro. J Biomed Opt. 2013;18:128001.CrossRefGoogle Scholar
  34. 34.
    Tim CR, Pinto KN, Rossi BR, Fernandes K, Matsumoto MA, Parizotto NA, et al. Low-level laser therapy enhances the expression of osteogenic factors during bone repair in rats. Lasers Med Sci. 2014;29:147–56.CrossRefGoogle Scholar
  35. 35.
    Gallea S, Lallemand F, Atfi A, Rawadi G, Ramez V, Spinella-Jaegle S, et al. Activation of mitogen-activated protein kinase cascades is involved in regulation of bone morphogenetic protein-2-induced osteoblast differentiation in pluripotent C2C12 cells. Bone. 2001;28:491–8.CrossRefGoogle Scholar
  36. 36.
    Guicheux J, Lemonnier J, Ghayor C, Suzuki A, Palmer G, Caverzasio J. Activation of p38 mitogen-activated protein kinase and c-Jun-NH2-terminal kinase by BMP-2 and their implication in the stimulation of osteoblastic cell differentiation. J Bone Miner Res. 2003;18:2060–8.CrossRefGoogle Scholar
  37. 37.
    Park JH, Park BH, Kim HK, Park TS, Baek HS. Hypoxia decreases Runx2/Cbfa1 expression in human osteoblast-like cells. Mol Cell Endocrinol. 2002;192:197–203.CrossRefGoogle Scholar
  38. 38.
    Utting JC, Robins SP, Brandao-Burch A, Orriss IR, Behar J, Arnett TR. Hypoxia inhibits the growth, differentiation and bone-forming capacity of rat osteoblasts. Exp Cell Res. 2006;312:1693–702.CrossRefGoogle Scholar
  39. 39.
    Hirata S, Kitamura C, Fukushima H, Nakamichi I, Abiko Y, Terashita M, et al. Low-level laser irradiation enhances BMP-induced osteoblast differentiation by stimulating the BMP/Smad signaling pathway. J Cell Biochem. 2010;111:1445–52.CrossRefGoogle Scholar

Copyright information

© The Korean Tissue Engineering and Regenerative Medicine Society and Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • Jin-Ho Heo
    • 1
  • Jeong-Hun Choi
    • 1
  • In-Ryoung Kim
    • 2
  • Bong-Soo Park
    • 2
  • Yong-Deok Kim
    • 1
    • 3
  1. 1.Department of Oral and Maxillofacial SurgeryPusan National UniversityYangsan-siRepublic of Korea
  2. 2.Department of Oral AnatomyPusan National UniversityYangsan-siKorea
  3. 3.Dental Research Institute and Institute of Translational Dental SciencesPusan National UniversityYangsan-siKorea

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