Skip to main content

Nigella Sativa-Coated Hydroxyapatite Scaffolds: Synergetic Cues to Stimulate Myoblasts Differentiation and Offset Infections



At present osteoporosis has come into view as a major health concern. Skeletal diseases typified by weak and fragile bones have imposed threats of fissure. Hydroxyapatite (HAP) is known to induce osteoblast like differentiation and provide mechanical strength, hence, used in bone tissue engineering; whereas, Nigella sativa has also demonstrated potential to treat bone and muscle diseases. This study was aimed to develop potential orthopedic scaffold exploiting natural resources of Saudi Arabia which can be used as prospective tissue engineering implant.


The bone scaffold was developed by grafting biogenic HAP with N. sativa essential oil. N. sativa was applied for boosting osteogenesis and to stimulate antimicrobial potential. Antimicrobial potential was investigated utilizing S. aureus bacteria. Spectroscopic and surface characters of N. sativa grafted HAP scaffolds were analyzed using Fourier-transform infrared spectroscopy, X-ray crystallography and Scanning electron microscopy. To ensure biocompatibility of scaffolds; we selected C2C12 cell-lines; best model to study mechanistic pathways related to osteoblasts and myoblasts differentiation.


Grafting of HAP with N. sativa did not affect typical spherical silhouette of nanoparticles. Characteristically; protein loaded polynucleated myotubes are result of in vitro myogenesis of C2C12 myoblasts in squat serum environment.


It is first study of unique combination of N. sativa and HAP scaffold as a possible candidate of implantation for skeletal muscles regeneration. Outcome of this finding revealed N. sativa grafted HAP enhance differentiation significantly over that of HAP. The proposed scaffold will be an economical natural material for hard and soft tissue engineering and will aid in curing skeletal muscle diseases. Our findings have implications for treatment of muscular/bone diseases.

Graphic Abstract

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7


  1. 1.

    Shuid AN, Ping LL, Muhammad N, Mohamed N, Soelaiman IN. The effects of Labisiapumilavaralata on bone markers and bone calcium in a rat model of post-menopausal osteoporosis. J Ethnopharmacol. 2011;133:538–42.

    Article  Google Scholar 

  2. 2.

    Potu BK, Rao MS, Nampurath GK, Chamallamudi MR, Prasad K, Nayak SR, et al. Evidence-based assessment of antiosteoporotic activity of petroleum-ether extract of Cissusquadrangularis Linn. on ovariectomy-induced osteoporosis. Ups J Med Sci. 2009;114:140–8.

    Article  Google Scholar 

  3. 3.

    Das UN. Nitric oxide as the mediator of the antiosteoporotic actions of estrogen, statins, and essential fatty acids. Exp Biol Med (Maywood). 2002;227:88–93.

    CAS  Article  Google Scholar 

  4. 4.

    Goreja W. Black seed. Nature’s Miracle: Remedy Amazing Herbs Press, New York; 2003. p. 1–64.

    Google Scholar 

  5. 5.

    Cheikh-Rouhou S, Besbes S, Hentati B, Blecker C, Deroanne C, Attia, H. Nigella sativa L.: Chemical composition and physicochemical characteristics of lipid fraction. Food Chem. 2007;101:673–81.

    CAS  Article  Google Scholar 

  6. 6.

    Shuid AN, Mohamed N, Mohamed IN, Othman F, Suhaimi F, Mohd Ramli ES, et al. Nigella sativa: A potential antiosteoporotic agent. Evid Based Complement Alternat Med. 2012;2012:696230.

    Article  Google Scholar 

  7. 7.

    Altan MF, Kanter M, Donmez S, Kartal ME, Buyukbas S. Combination therapy of Nigella sativa and human parathyroid hormone on bone mass, biomechanical behavior and structure in streptozotocin-induced diabetic rats. Acta Histochem. 2007;109:304–14.

    CAS  Article  Google Scholar 

  8. 8.

    Shady AM, Nooh HZ. Effect of black seed (Nigella sativa) on compact bone of streptozotocin induced diabetic rats. Egypt J Histol. 2010;33:168–77.

    Article  Google Scholar 

  9. 9.

    Tahraoui A, El-Hilaly J, Israili ZH, Lyoussi B. Ethnopharmacological survey of plants used in the traditional treatment of hypertension and diabetes in south-eastern Morocco (Errachidia province). J Ethnopharmacol. 2007;110:105–17.

    CAS  Article  Google Scholar 

  10. 10.

    Iconaru SL, Prodan AM, Buton N, Predoi D. Structural characterization and antifungal studies of zinc-doped hydroxyapatite coatings. Molecules. 2017;22:604.

    Article  Google Scholar 

  11. 11.

    Ciobanu CS, Massuyeau F, Constantin LV, Predoi D. Structural and physical properties of antibacterial Ag-doped nano-hydroxyapatite synthesized at 100 C. Nanoscale Res Lett. 2011;6:613.

    Article  Google Scholar 

  12. 12.

    Mondal S, Pal U. 3D hydroxyapatite scaffold for bone regeneration and local drug delivery applications. J Drug Deliv Sci Technol. 2019;53:101131.

    CAS  Article  Google Scholar 

  13. 13.

    Li M, Xiong P, Yan F, Li S, Ren C, Yin Z, et al. An overview of graphene-based hydroxyapatite composites for orthopedic applications. Bioact Mater. 2018;3:1–18.

    Article  Google Scholar 

  14. 14.

    Chouirfa H, Bouloussa H, Migonney V, Falentin-Daudré C. Review of titanium surface modification techniques and coatings for antibacterial applications. Acta Biomater. 2019;83:37–54.

    CAS  Article  Google Scholar 

  15. 15.

    El-Zaeddi H, Martínez-Tomé J, Calín-Sánchez Á, Burló F, Carbonell-Barrachina ÁA. Volatile composition of essential oils from different aromatic herbs grown in mediterranean regions of Spain. Foods. 2016;5:41.

    Article  Google Scholar 

  16. 16.

    Ghosheh OA, Houdi AA, Crooks PA. High performance liquid chromatographic analysis of the pharmacologically active quinones and related compounds in the oil of the black seed (Nigella sativa L.). J Pharm Biomed Anal. 1999;19:757–62.

    CAS  Article  Google Scholar 

  17. 17.

    Amna T, Hassan MS, Sheikh FA, Lee HK, Seo KS, Yoon D, et al. Zinc oxide-doped poly(urethane) spider web nanofibrous scaffold via one-step electrospinning: a novel matrix for tissue engineering. Appl Microbiol Biotechnol. 2013;97:1725–34.

    CAS  Article  Google Scholar 

  18. 18.

    Russell ST, Tisdale MJ. Mechanism of attenuation by beta-hydroxy-beta-methylbutyrate of muscle protein degradation induced by lipopolysaccharide. Mol Cell Biochem. 2009;330:171–9.

    CAS  Article  Google Scholar 

  19. 19.

    Amna T, Hassan MS, Shin WS, Van Ba H, Lee HK, Khil MS, et al. TiO2 nanorods via one-step electrospinning technique: a novel nanomatrix for mouse myoblasts adhesion and propagation. Colloids Surf B Biointerfaces. 2013;101:424–9.

    CAS  Article  Google Scholar 

  20. 20.

    Amna T, Hassan MS, Yousef A, Mishra A, Barakat NA, Khil MS, et al. Inactivation of foodborne pathogens by NiO/TiO 2 composite nanofibers: a novel biomaterial system. Food Bioproc Tech. 2013;6:988–96.

    CAS  Article  Google Scholar 

  21. 21.

    Núñez D, Elgueta E, Varaprasad K, Oyarzún P. Hydroxyapatite nanocrystals synthesized from calcium rich bio-wastes. Mater Lett. 2018;230:64–8.

    Article  Google Scholar 

  22. 22.

    Nivetha K, Prasanna G. GC-MS and FT-IR analysis of Nigella sativa L seeds. Int J Adv Res Biol Sci. 2016;3:45–54.

    CAS  Google Scholar 

  23. 23.

    Rohman A, Man YC. Fourier transform infrared (FTIR) spectroscopy for analysis of extra virgin olive oil adulterated with palm oil. Food Res Int. 2010;43:886–92.

    CAS  Article  Google Scholar 

  24. 24.

    Vongsvivut J, Heraud P, Zhang W, Kralovec JA, McNaughton D, Barrow CJ. Quantitative determination of fatty acid compositions in micro-encapsulated fish-oil supplements using Fourier transform infrared (FTIR) spectroscopy. Food Chem. 2012;135:603–9.

    CAS  Article  Google Scholar 

  25. 25.

    Guillen MD, Cabo N. Characterization of edible oils and lard by Fourier transform infrared spectroscopy. Relationships between composition and frequency of concrete bands in the fingerprint region. J Am Oil Chem Soc. 1997;74:1281–6.

    CAS  Article  Google Scholar 

  26. 26.

    Nurrulhidayah AF, Che Man YB, Al-Kahtani HA, Rohman A. Application of FTIR spectroscopy coupled with chemometrics for authentication of Nigella sativa seed oil. Spectrosc. 2011;25:243–50.

    CAS  Article  Google Scholar 

  27. 27.

    Alkhatib H, Mohamed F, Doolaanea AA. ATR-FTIR and spectroscopic methods for analysis of black seed oil from alginate beads. Int J App Pharm. 2018;10:147–52.

    CAS  Article  Google Scholar 

  28. 28.

    McMahon DK, Anderson PA, Nassar R, Bunting JB, Saba Z, Oakeley AE, et al. C2C12 cells: biophysical, biochemical, and immunocytochemical properties. Am J Physiol. 1994;266:C1795–802.

    CAS  Article  Google Scholar 

  29. 29.

    Bi P, Ramirez-Martinez A, Li H, Cannavino J, McAnally JR, Shelton JM, et al. Control of muscle formation by the fusogenic micropeptide myomixer. Science. 2017;356:323–7.

    CAS  Article  Google Scholar 

  30. 30.

    Bajaj P, Reddy B Jr, Millet L, Wei C, Zorlutuna P, Bao G, et al. Patterning the differentiation of C2C12 skeletal myoblasts. Integr Biol (Camb). 2011;3:897–909.

    CAS  Article  Google Scholar 

  31. 31.

    Yaffe D, Saxel O. Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature. 1977;270:725–7.

    CAS  Article  Google Scholar 

  32. 32.

    Tavakkoli A, Mahdian V, Razavi BM, Hosseinzadeh H. Review on clinical trials of black seed (Nigella sativa) and its active constituent, thymoquinone. J Pharmacopuncture. 2017;20:179–93.

    Article  Google Scholar 

  33. 33.

    Benhaddou-Andaloussi A, Martineau LC, Spoor D, Vuong T, Leduc C, Joly E, et al. Antidiabetic activity of Nigella sativa. Seed extract in cultured pancreatic β-cells, skeletal muscle cells, and adipocytes. Pharm Biol. 2008;46:96–104.

    Article  Google Scholar 

  34. 34.

    Cazzola M, Ferraris S, Allizond V, Bertea CM, Novara C, Cochis A, et al. Grafting of the peppermint essential oil to a chemically treated Ti6Al4V alloy to counteract the bacterial adhesion. Surf Coat Technol. 2019;378:125011.

    CAS  Article  Google Scholar 

  35. 35.

    Veliça P, Bunce CM. A quick, simple and unbiased method to quantify C2C12 myogenic differentiation. Muscle Nerve. 2011;44:366–70.

    Article  Google Scholar 

  36. 36.

    Hieter P, Griffiths T. Polyploidy–more is more or less. Science. 1999;285:210–1.

    CAS  Article  Google Scholar 

  37. 37.

    Burattini S, Ferri P, Battistelli M, Curci R, Luchetti F, Falcieri E. C2C12 murine myoblasts as a model of skeletal muscle development: morpho-functional characterization. Eur J Histochem. 2004;48:223–34.

    CAS  PubMed  Google Scholar 

Download references

Author information



Corresponding authors

Correspondence to Touseef Amna or M. Shamshi Hassan.

Ethics declarations

Conflict of interest

The authors declare no conflicts of interest.

Ethical statement

There is no use of animal models for the experiments. All authors approve the submission.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Amna, T., Alghamdi, A.A.A., Shang, K. et al. Nigella Sativa-Coated Hydroxyapatite Scaffolds: Synergetic Cues to Stimulate Myoblasts Differentiation and Offset Infections. Tissue Eng Regen Med 18, 787–795 (2021).

Download citation


  • N. sativa
  • Differentiation
  • Hydroxyapatite
  • Myoblasts
  • Antibacterial