Lasers in Medical Science

, Volume 32, Issue 7, pp 1489–1496 | Cite as

Laser irradiation promotes the proliferation of mouse pre-osteoblast cell line MC3T3-E1 through hedgehog signaling pathway

  • Qiushi Li
  • Yingxin Chen
  • Shujun Dong
  • Shujie Liu
  • Xiaodan Zhang
  • Xi Si
  • Yanmin ZhouEmail author
Original Article


Low-level laser could promote osteoblast proliferation, and it has been applied in clinical practice to promote wound healing and tissue regeneration. However, the mechanism related to laser irradiation remains unclear. This study aimed to investigate the effects of low-level laser irradiation on the cell proliferation and the expressions of hedgehog signaling molecules Indian hedgehog (Ihh), Ptch, and Gli in vitro. In our present study, the MTT method was used to evaluate the effect on cell proliferation of laser irradiation on MC3T3-E1 cells. And cell cycle was examined by flow cytometry. Gene and protein expressions of hedgehog signaling molecules, including Ihh, Ptch, Smoothened (Smo), and Gli, were examined by qRT-PCR and western blot analysis. The results showed that laser irradiation at dosage of 3.75 J/cm2 enhances the proliferation of MC3T3-E1 cells compared with control groups (p = 0.00). Moreover, laser irradiation (3.75 J/cm2) increased the cell amount at S phase (p = 0.00). In addition, the expressions of Ihh, Ptch, Smo, and Gli were significantly increased compared to the control during laser irradiation (3.75 J/cm2)-induced MC3T3-E1 osteoblast proliferation. After adding the hedgehog signaling inhibitor CY (cyclopamine), cell proliferation and Ihh, Ptch, Smo, and Gli expressions were inhibited (p = 0.00), and the cell amount at S phase was reduced compared with combination groups (p = 0.00). These results indicated that laser irradiation promotes proliferation of MC3T3-E1 cells through hedgehog signaling pathway. Our findings provide insights into the mechanistic link between laser irradiation-induced osteogenesis and hedgehog signaling pathway.


Low-level laser Osteoblast proliferation Hedgehog signaling pathway MC3T3-E1Cs 



The authors acknowledge the help from Dr. Yanmin Zhou.

Compliance with ethical standards

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.


There is no funding to support this study.

Conflict of interest

The authors declare that they have no conflict of interest.

Informed consent

Informed consent was obtained from all individual participants included in the study.


  1. 1.
    Mester E, Szende B, Gartner P (1968) The effect of laser beams on the growth of hair in mice. Radiobiol Radiother (Berl) 9(5):621–626Google Scholar
  2. 2.
    Mester E, Mester AF, Mester A (1985) The biomedical effects of laser application. Lasers Surg Med 5(1):31–39CrossRefPubMedGoogle Scholar
  3. 3.
    Pejcic A, Kojovic D, Kesic L, Obradovic R (2010) The effects of low level laser irradiation on gingival inflammation. Photomed Laser Surg 28(1):69–74. doi: 10.1089/pho.2008.2301 CrossRefPubMedGoogle Scholar
  4. 4.
    da Silva JP, da Silva MA, Almeida AP, Lombardi Junior I, Matos AP (2010) Laser therapy in the tissue repair process: a literature review. Photomed Laser Surg 28(1):17–21. doi: 10.1089/pho.2008.2372 CrossRefPubMedGoogle Scholar
  5. 5.
    Oliveira FA, Matos AA, Santesso MR, Tokuhara CK, Leite AL, Bagnato VS, Machado MA, Peres-Buzalaf C, Oliveira RC (2016) Low intensity lasers differently induce primary human osteoblast proliferation and differentiation. J Photochem Photobiol B 163:14–21. doi: 10.1016/j.jphotobiol.2016.08.006 CrossRefPubMedGoogle Scholar
  6. 6.
    Petri AD, Teixeira LN, Crippa GE, Beloti MM, de Oliveira PT, Rosa AL (2010) Effects of low-level laser therapy on human osteoblastic cells grown on titanium. Braz Dent J 21(6):491–498CrossRefPubMedGoogle Scholar
  7. 7.
    Feng J, Zhang Y, Xing D (2012) Low-power laser irradiation (LPLI) promotes VEGF expression and vascular endothelial cell proliferation through the activation of ERK/Sp1 pathway. Cell Signal 24(6):1116–1125. doi: 10.1016/j.cellsig.2012.01.013 CrossRefPubMedGoogle Scholar
  8. 8.
    Kawasaki K, Shimizu N (2000) Effects of low-energy laser irradiation on bone remodeling during experimental tooth movement in rats. Lasers Surg Med 26(3):282–291CrossRefPubMedGoogle Scholar
  9. 9.
    Alfredo PP, Bjordal JM, Dreyer SH, Meneses SR, Zaguetti G, Ovanessian V, Fukuda TY, Junior WS, Lopes Martins RA, Casarotto RA, Marques AP (2012) Efficacy of low level laser therapy associated with exercises in knee osteoarthritis: a randomized double-blind study. Clin Rehabil 26(6):523–533. doi: 10.1177/0269215511425962 CrossRefPubMedGoogle Scholar
  10. 10.
    Takeda Y (1988) Irradiation effect of low-energy laser on alveolar bone after tooth extraction. Experimental study in rats. Int J Oral Maxillofac Surg 17(6):388–391CrossRefPubMedGoogle Scholar
  11. 11.
    Yaakobi T, Maltz L, Oron U (1996) Promotion of bone repair in the cortical bone of the tibia in rats by low energy laser (He-Ne) irradiation. Calcif Tissue Int 59(4):297–300CrossRefPubMedGoogle Scholar
  12. 12.
    Ingham PW, McMahon AP (2001) Hedgehog signaling in animal development: paradigms and principles. Genes Dev 15(23):3059–3087. doi: 10.1101/gad.938601 CrossRefPubMedGoogle Scholar
  13. 13.
    Huangfu D, Anderson KV (2006) Signaling from Smo to Ci/Gli: conservation and divergence of Hedgehog pathways from Drosophila to vertebrates. Development 133(1):3–14. doi: 10.1242/dev.02169 CrossRefPubMedGoogle Scholar
  14. 14.
    Long F (2011) Building strong bones: molecular regulation of the osteoblast lineage. Nat Rev Mol Cell Biol 13(1):27–38. doi: 10.1038/nrm3254 CrossRefPubMedGoogle Scholar
  15. 15.
    Long F, Chung UI, Ohba S, McMahon J, Kronenberg HM, McMahon AP (2004) Ihh signaling is directly required for the osteoblast lineage in the endochondral skeleton. Development 131(6):1309–1318. doi: 10.1242/dev.01006 CrossRefPubMedGoogle Scholar
  16. 16.
    Alman BA (2015) The role of hedgehog signalling in skeletal health and disease. Nat Rev Rheumatol 11(9):552–560. doi: 10.1038/nrrheum.2015.84 CrossRefPubMedGoogle Scholar
  17. 17.
    Nakamura T, Aikawa T, Iwamoto-Enomoto M, Iwamoto M, Higuchi Y, Pacifici M, Kinto N, Yamaguchi A, Noji S, Kurisu K, Matsuya T (1997) Induction of osteogenic differentiation by hedgehog proteins. Biochem Biophys Res Commun 237(2):465–469CrossRefPubMedGoogle Scholar
  18. 18.
    Enomoto-Iwamoto M, Nakamura T, Aikawa T, Higuchi Y, Yuasa T, Yamaguchi A, Nohno T, Noji S, Matsuya T, Kurisu K, Koyama E, Pacifici M, Iwamoto M (2000) Hedgehog proteins stimulate chondrogenic cell differentiation and cartilage formation. J Bone Miner Res 15(9):1659–1668. doi: 10.1359/jbmr.2000.15.9.1659 CrossRefPubMedGoogle Scholar
  19. 19.
    Tim CR, Pinto KN, Rossi BR, Fernandes K, Matsumoto MA, Parizotto NA, Renno AC (2014) Low-level laser therapy enhances the expression of osteogenic factors during bone repair in rats. Lasers Med Sci 29(1):147–156. doi: 10.1007/s10103-013-1302-9 CrossRefPubMedGoogle Scholar
  20. 20.
    Ko CY, Kang H, Ryu Y, Jung B, Kim H, Jeong D, Shin HI, Lim D, Kim HS (2013) The effects of minimally invasive laser needle system on suppression of trabecular bone loss induced by skeletal unloading. Lasers Med Sci 28(6):1495–1502. doi: 10.1007/s10103-013-1265-x CrossRefPubMedGoogle Scholar
  21. 21.
    Aleksic V, Aoki A, Iwasaki K, Takasaki AA, Wang CY, Abiko Y, Ishikawa I, Izumi Y (2010) Low-level Er:YAG laser irradiation enhances osteoblast proliferation through activation of MAPK/ERK. Lasers Med Sci 25(4):559–569. doi: 10.1007/s10103-010-0761-5 CrossRefPubMedGoogle Scholar
  22. 22.
    Kesper DA, Didt-Koziel L, Vortkamp A (2010) Gli2 activator function in preosteoblasts is sufficient to mediate Ihh-dependent osteoblast differentiation, whereas the repressor function of Gli2 is dispensable for endochondral ossification. Dev Dyn 239(6):1818–1826. doi: 10.1002/dvdy.22301 CrossRefPubMedGoogle Scholar
  23. 23.
    Warzecha J, Gottig S, Bruning C, Lindhorst E, Arabmothlagh M, Kurth A (2006) Sonic hedgehog protein promotes proliferation and chondrogenic differentiation of bone marrow-derived mesenchymal stem cells in vitro. J Orthop Sci 11(5):491–496. doi: 10.1007/s00776-006-1058-1 CrossRefPubMedGoogle Scholar
  24. 24.
    Yang J, Andre P, Ye L, Yang YZ (2015) The Hedgehog signalling pathway in bone formation. Int J Oral Sci 7(2):73–79. doi: 10.1038/ijos.2015.14 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Goetz SC, Ocbina PJ, Anderson KV (2009) The primary cilium as a Hedgehog signal transduction machine. Methods Cell Biol 94:199–222. doi: 10.1016/S0091-679X(08)94010-3 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag London Ltd. 2017

Authors and Affiliations

  • Qiushi Li
    • 1
  • Yingxin Chen
    • 1
  • Shujun Dong
    • 1
  • Shujie Liu
    • 1
  • Xiaodan Zhang
    • 2
  • Xi Si
    • 3
  • Yanmin Zhou
    • 4
    Email author
  1. 1.VIP Integrated Department, School and Hospital of StomatologyJilin UniversityChangchunChina
  2. 2.Department of Oral Health ScienceAffiliated Stomatological Hospital of Harbin Medical UniversityHarbinChina
  3. 3.Department of Oral MedicineHainan Medical UniversityHaikouChina
  4. 4.Department of Oral Implantology, School and Hospital of StomatologyJilin UniversityChangchunChina

Personalised recommendations