Advertisement

Biological Trace Element Research

, Volume 184, Issue 1, pp 136–147 | Cite as

Comparison Study of Bone Defect Healing Effect of Raw and Processed Pyritum in Rats

  • Xingyu Zhu
  • Qianqian Gao
  • Genhua Zhao
  • Heng Wang
  • Ling Liu
  • Zhipeng Chen
  • Yijun Chen
  • Li Wu
  • Zisheng Xu
  • Weidong Li
Article
  • 104 Downloads

Abstract

To evaluate and compare the effect of raw and processed pyritum on tibial defect healing, 32 male Sprague Dawley rats were randomly divided into four groups. After tibial defect, animals were produced and grouped: sham and control group were orally administrated with distilled water (1 mL/100 g), while treatment groups were given aqueous extracts of raw and processed pyritum (1.5 g/kg) for successive 42 days. Radiographic examination showed that bone defect healing effect of the treatment groups was obviously superior compared to that of the control group. Bone mineral density of whole tibia was increased significantly after treating with pyritum. Inductively coupled plasma-optical emission spectrometry showed that the contents of Ca, P, and Mg in callus significantly increased in the treatment groups comparing with the control. Moreover, serological analysis showed that the concentration of serum phosphorus of the treatment groups significantly increased compared with that of the control group. By in vitro study, we have evaluated the effects of drug-containing serum of raw and processed pyritum on osteoblasts. It was manifested that both the drug-containing sera of raw and processed pyritum significantly increased the mRNA levels of alkaline phosphatase and collagen type I. Protein levels of phosphorylated Smad2/3 also increased. The mRNA levels of osteocalcin and transforming growth factor β (TGF-β) type I and II receptors, as well as the protein levels of TGF-β1 in the processed groups, were higher than those in the control. In summary, both raw and processed pyritum-containing sera exhibited positive effects on osteoblasts, which maybe via the TGF-β1/Smad signaling pathway. Notably, the tibia defect healing effect of pyritum was significantly enhanced after processing.

Keywords

Raw pyritum Processed pyritum Bone healing Rat tibia defect TGF-β1/Smad signaling pathway 

Abbreviations

BMD

Bone mineral density

ICP-OES

Inductively coupled plasma-optical emission spectrometer

ALP

Alkaline phosphatase

Col I

Type I collagen

OC

Osteocalcin

TGF-β

Transforming growth factor β

TCM

Traditional Chinese medicine

RT-PCR

Reverse transcription polymerase chain reaction

PTFE

Polytetrafluoroethylene

α-MEM

Alpha modified eagle medium

PBS

Phosphate buffer solution

FBS

Fetal bovine serum

GAPDH

Glyceraldehyde-phosphate dehydrogenase

TBST

Tris-buffered saline Tween

S-Ca

Serum Ca

S-P

Serum P

Notes

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (81373970) and Jiangsu Qinglan Project (2014).

Compliance with Ethical Standards

The study was approved by the Nanjing University of Chinese Medicine Committee on Laboratory Animal Care, and all animals received humane care according to the National Institutes of Health (USA) guidelines. All possible efforts were made to minimize the animals’ suffering and to reduce the number of animals used.

Conflict of Interest

The authors declare that they have no conflicts of interest.

References

  1. 1.
    Einhorn TA, Gerstenfeld LC (2015) Fracture healing: mechanisms and interventions. Nat Rev Rheumatol 11(1):45–54.  https://doi.org/10.1038/nrrheum.2014.164 CrossRefPubMedGoogle Scholar
  2. 2.
    Majeed SA (2015) Fracture healing: mechanisms and therapeutics. Osteoporos Int 26:S421–S422Google Scholar
  3. 3.
    Marsell R, Einhorn TA (2011) The biology of fracture healing. Injury 42(6):551–555.  https://doi.org/10.1016/j.injury.2011.03.031 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Corrarino JE (2015) Fracture repair: mechanisms and management. JNP—J Nurse Pract 11(10):960–967.  https://doi.org/10.1016/j.nurpra.2015.07.009 CrossRefGoogle Scholar
  5. 5.
    Aubin JE (1998) Advances in the osteoblast lineage. Biochem Cell Biol 76(6):899–910.  https://doi.org/10.1139/o99-005 CrossRefPubMedGoogle Scholar
  6. 6.
    Messer JG, Kilbarger AK, Erikson KM, Kipp DE (2009) Iron overload alters iron-regulatory genes and proteins, down-regulates osteoblastic phenotype, and is associated with apoptosis in fetal rat calvaria cultures. Bone 45(5):972–979.  https://doi.org/10.1016/j.bone.2009.07.073 CrossRefPubMedGoogle Scholar
  7. 7.
    Ochiai H, Okada S, Saito A, Hoshi K, Yamashita H, Takato T, Azuma T (2012) Inhibition of insulin-like growth factor-1 (IGF-1) expression by prolonged transforming growth factor-beta1 (TGF-beta1) administration suppresses osteoblast differentiation. J Biol Chem 287(27):22654–22661.  https://doi.org/10.1074/jbc.M111.279091 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Laurent P, Camps J, About I (2012) Biodentine(TM) induces TGF-beta1 release from human pulp cells and early dental pulp mineralization. Int Endod J 45(5):439–448.  https://doi.org/10.1111/j.1365-2591.2011.01995.x CrossRefPubMedGoogle Scholar
  9. 9.
    Sowa H, Kaji H, Yamaguchi T, Sugimoto T, Chihara K (2002) Smad3 promotes alkaline phosphatase activity and mineralization of osteoblastic MC3T3-E1 cells. J Bone Miner Res 17(7):1190–1199.  https://doi.org/10.1359/jbmr.2002.17.7.1190 CrossRefPubMedGoogle Scholar
  10. 10.
    Janssens K, ten Dijke P, Janssens S, Van Hul W (2005) Transforming growth factor-beta1 to the bone. Endocr Rev 26(6):743–774.  https://doi.org/10.1210/er.2004-0001 CrossRefPubMedGoogle Scholar
  11. 11.
    Wildemann B, Schmidmaier G, Brenner N (2004) Quantification, localization, and expression of IGF-I and TGF-beta1 during growth factor-stimulated fracture healing. Calcif Tissue Int 74(4)Google Scholar
  12. 12.
    Wong RWK, Rabie ABM (2006) Traditional Chinese medicines and bone formation—a review. J Oral Maxillofac Surg 64(5):828–837.  https://doi.org/10.1016/j.joms.2006.01.017 CrossRefPubMedGoogle Scholar
  13. 13.
    Peng LH, Ko CH, Siu SW, Koon CM, Yue GL, Cheng WH, Lau TW, Han QB, Ng KM, Fung KP, Lau CBS, Leung PC (2010) In vitro & in vivo assessment of a herbal formula used topically for bone fracture treatment. J Ethnopharmacol 131(2):282–289.  https://doi.org/10.1016/j.jep.2010.06.039 CrossRefPubMedGoogle Scholar
  14. 14.
    Liu L, Zhao GH, Gao QQ, Chen YJ, Chen ZP, Xu ZS, Li WD (2017) Changes of mineralogical characteristics and osteoblast activities of raw and processed pyrites. RSC Adv 7(45):28373–28382.  https://doi.org/10.1039/c7ra03970k CrossRefGoogle Scholar
  15. 15.
    Hwang J, Do Hur S, Seo YB (2004) Mineralogical and chemical changes in pyrite after traditional processing for use in medicines. Am J Chin Med 32(6):907–919.  https://doi.org/10.1142/s0192415x0400251x CrossRefPubMedGoogle Scholar
  16. 16.
    Wu HH, C.J. (2012) The processing of Chinese material. Press of People’s Medical Publishing House, BeijingGoogle Scholar
  17. 17.
    Genhua Z, Zebin W, Qianqian G, Zhipeng C, Baochang C, Weidong L (2015) Studies on pyritum before and after processing in promoting fracture healing and its mechanism. Tradis Chin Drug Res Clin Pharmacol 26(4):481–485Google Scholar
  18. 18.
    Pang Rj (2007) Bioleaching pyritum and testing the efficacy action of its leachate. Dissertation, Lan zhou UniversityGoogle Scholar
  19. 19.
    Mao BF (2009) Experimental investigation on Chinese medicine pyritum with low frequency ultrasound treatment for heal fracture. Dissertation, Liaoning University of Traditional Chinese MedicineGoogle Scholar
  20. 20.
    Kim JM, Lee JH, Lee GS, Noh EM, Song HK, Gu DR, Kim SC, Lee SH, Kwon KB, Lee YR (2017) Sophorae Flos extract inhibits RANKL-induced osteoclast differentiation by suppressing the NF-kappaB/NFATc1 pathway in mouse bone marrow cells. BMC Complement Altern Med 17(1):164.  https://doi.org/10.1186/s12906-016-1550-x
  21. 21.
    Mackenzie EL, Iwasaki K, Tsuji Y (2008) Intracellular iron transport and storage: from molecular mechanisms to health implications. Antioxid Redox Signal 10(6):997–1030.  https://doi.org/10.1089/ars.007.1893
  22. 22.
    Bassett CAL (1962) Current concepts of bone formation. J Bone Joint Surg 44(6):1217–1219Google Scholar
  23. 23.
    Nagata M, Lonnerdal B (2011) Role of zinc in cellular zinc trafficking and mineralization in a murine osteoblast-like cell line. J Nutr Biochem 22(2):172–178.  https://doi.org/10.1016/j.jnutbio.2010.01.003 CrossRefPubMedGoogle Scholar
  24. 24.
    Yusa K, Yamamoto O, Fukuda M, Koyota S, Koizumi Y, Sugiyama T (2011) In vitro prominent bone regeneration by release zinc ion from Zn-modified implant. Biochem Biophys Res Commun 412(2):273–278.  https://doi.org/10.1016/.bbrc.2011.07.082 CrossRefPubMedGoogle Scholar
  25. 25.
    Qiao YQ, Zhang WJ, Tian P, Meng FH, Zhu HQ, Jiang XQ, Liu XY, Chu PK (2014) Stimulation of bone growth following zinc incorporation into biomaterials. Biomaterials 35(25):6882–6897.  https://doi.org/10.1016/j.biomaterials.2014.04.101 CrossRefPubMedGoogle Scholar
  26. 26.
    Wang J MX-y, Feng Y-f, Ma T-c, Lei W, Wang L (2015) Promotive effect of magnesium ions on viability and differentiation of osteoblasts and the underlying mechanism. Prog Mod Biomed 15(15):2836–2839Google Scholar
  27. 27.
    Hu YC, Cheng HL, Hsieh BS, Huang LW, Huang TC, Chang KL (2012) Arsenic trioxide affects bone remodeling by effects on osteoblast differentiation and function. Bone 50(6):1406–1415.  https://doi.org/10.1016/j.bone.2012.03.012 CrossRefPubMedGoogle Scholar
  28. 28.
    Cui C, Wang S, Myneni VD, Hitomi K, Kaartinen MT (2014) Transglutaminase activity arising from Factor XIIIA is required for stabilization and conversion of plasma fibronectin into matrix in osteoblast cultures. Bone 59:127–138.  https://doi.org/10.1016/j.bone.2013.11.006 CrossRefPubMedGoogle Scholar
  29. 29.
    Foster LJ, Zeemann PA, Li C, Mann M, Jensen ON, Kassem M (2005) Differential expression profiling of membrane proteins by quantitative proteomics in a human mesenchymal stem cell line undergoing osteoblast differentiation. Stem Cells 23(9):1367–1377.  https://doi.org/10.1634/stemcells.2004-0372 CrossRefPubMedGoogle Scholar
  30. 30.
    Yoshikawa Y, Kode A, Xu L, Mosialou I, Silva BC, Ferron M, Clemens TL, Economides AN, Kousteni S (2011) Genetic evidence points to an osteocalcin-independent influence of osteoblasts on energy metabolism. J Bone Miner Res 26(9):2012–2025.  https://doi.org/10.1002/jbmr.417 CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Franceschi R (1999) The developmental control of osteoblast-specific gene expression: role of specific transcription factors and the extracellular matrix environment. Crit Rev Oral Biol Med 10(1):40–57CrossRefPubMedGoogle Scholar
  32. 32.
    Renny T, Franceschi BSI (1992) Relationship between collagen synthesis and expression of the osteoblast phenotype in MC3T3-E1 cells. J Bone Miner Res 7(2):235–246Google Scholar
  33. 33.
    Franceschi RT, Ge C, Xiao G, Roca H, Jiang D (2009) Transcriptional regulation of osteoblasts. Cells Tissues Organs 189(1–4):144–152.  https://doi.org/10.1159/000151747 CrossRefPubMedGoogle Scholar
  34. 34.
    Kristensen HB, Andersen TL, Marcussen N, Rolighed L, Delaisse JM (2014) Osteoblast recruitment routes in human cancellous bone remodeling. Am J Pathol 184(3):778–789.  https://doi.org/10.1016/j.ajpath.2013.11.022 CrossRefPubMedGoogle Scholar
  35. 35.
    Mu Y, Gudey SK, Landstrom M (2012) Non-Smad signaling pathways. Cell Tissue Res 347(1):11–20.  https://doi.org/10.1007/s00441-011-1201-y CrossRefPubMedGoogle Scholar
  36. 36.
    Chen M, Lv Z, Jiang S (2011) The effects of triptolide on airway remodelling and transforming growth factor-beta(1)/Smad signalling pathway in ovalbumin-sensitized mice. Immunology 132(3):376–384.  https://doi.org/10.1111/j.1365-2567.2010.03392.x CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Oryan A, Alidadi S, Bigham-Sadegh A, Moshiri A (2017) Effectiveness of tissue engineered based platelet gel embedded chitosan scaffold on experimentally induced critical sized segmental bone defect model in rat. Injury 48:1466–1474.  https://doi.org/10.1016/j.injury.2017.04.044 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  • Xingyu Zhu
    • 1
  • Qianqian Gao
    • 1
  • Genhua Zhao
    • 1
  • Heng Wang
    • 1
  • Ling Liu
    • 1
  • Zhipeng Chen
    • 1
    • 2
  • Yijun Chen
    • 3
  • Li Wu
    • 1
  • Zisheng Xu
    • 4
  • Weidong Li
    • 1
    • 2
  1. 1.College of PharmacyNanjing University of Chinese MedicineNanjingPeople’s Republic of China
  2. 2.Engineering Center of State Ministry of Education for Standardization of Chinese Medicine ProcessingNanjing University of Chinese MedicineNanjingChina
  3. 3.Modern Analysis Center of Nanjing UniversityNanjingChina
  4. 4.Wuhu Pure Sunshine Natural Medicine Company LimitedWuhuPeople’s Republic of China

Personalised recommendations