Skip to main content

Advertisement

SpringerLink
Log in

A bioactive nano-calcium phosphate paste for in-situ transfection of BMP-7 and VEGF-A in a rabbit critical-size bone defect: results of an in vivo study

  • Biomaterials Synthesis and Characterization
  • Original Research
  • Published:
Journal of Materials Science: Materials in Medicine Aims and scope Submit manuscript

Abstract

The aim of this study was to prepare an injectable DNA-loaded nano-calcium phosphate paste that is suitable as bioactive bone substitution material. For this we used the well-known potential of calcium phosphate in bone contact and supplemented it with DNA for the in-situ transfection of BMP-7 and VEGF-A in a critical-size bone defect. 24 New Zealand white rabbits were randomly divided into two groups: One group with BMP-7- and VEGF-A-encoding DNA on calcium phosphate nanoparticles and a control group with calcium phosphate nanoparticles only. The bone defect was created at the proximal medial tibia and filled with the DNA-loaded calcium phosphate paste. As control, a bone defect was filled with the calcium phosphate paste without DNA. The proximal tibia was investigated 2, 4 and 12 weeks after the operation. A histomorphological analysis of the dynamic bone parameters was carried out with the Osteomeasure system. The animals treated with the DNA-loaded calcium phosphate showed a statistically significantly increased bone volume per total volume after 4 weeks in comparison to the control group. Additionally, a statistically significant increase of the trabecular number and the number of osteoblasts per tissue area were observed. These results were confirmed by radiological analysis. The DNA-loaded bone paste led to a significantly faster healing of the critical-size bone defect in the rabbit model after 4 weeks. After 12 weeks, all defects had equally healed in both groups. No difference in the quality of the new bone was found. The injectable DNA-loaded calcium phosphate paste led to a faster and more sustained bone healing and induced an accelerated bone formation after 4 weeks. The material was well integrated into the bone defect and new bone was formed on its surface. The calcium phosphate paste without DNA led to a regular healing of the critical-size bone defect, but the healing was slower than the DNA-loaded paste. Thus, the in-situ transfection with BMP-7 and VEGF-A significantly improved the potential of calcium phosphate as pasty bone substitution material.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

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

Similar content being viewed by others

References

  1. Tadic D, Epple M. A thorough physicochemical characterisation of 14 calcium phosphate-based bone substitution materials in comparison to natural bone. Biomaterials. 2004;25:987–94.

    CAS  Google Scholar 

  2. De Long WGJ, Einhorn TA, Koval K, McKee M, Smith W, Sanders R, et al. Bone grafts and bone graft substitutes in orthopaedic trauma surgery. a critical analysis. J Bone Joint Surg Am. 2007;89:649–58.

    Google Scholar 

  3. Kurien T, Pearson RG, Scammell BE. Bone graft substitutes currently available in orthopaedic practice: the evidence for their use. J Bone Joint Surg Am. 2013;95B:583–97.

    Google Scholar 

  4. Ahlmann E, Patzakis M, Roidis N, Shepherd L, Holtom P. Comparison of anterior and posterior iliac crest bone grafts in terms of harvest-site morbidity and functional outcomes. J Bone Joint Surg Am. 2002;84A:716–20.

    Google Scholar 

  5. Arrington ED, Smith WJ, Chambers HG, Bucknell AL, Davino NA. Complications of iliac crest bone graft harvesting. Clin Orthop Relat Res. 1996;329:300–9.

    Google Scholar 

  6. Dimitriou R, Mataliotakis GI, Angoules AG, Kanakaris NK, Giannoudis PV. Complications following autologous bone graft harvesting from the iliac crest and using the RIA: a systematic review. Injury. 2011;42:S3–S15.

    Google Scholar 

  7. Giannoudis PV, Dinopoulos H, Tsiridis E. Bone substitutes: an update. Injury. 2005;36:20–7.

    Google Scholar 

  8. Röder W, Müller H, Müller WE, Merz H. HIV infection in human bone. J Bone Joint Surg Br. 1992;74:179–80.

    Google Scholar 

  9. Vallet-Regi M, González-Calbet JM. Calcium phosphates as substitution of bone tissues. Progr Solid State Chem. 2004;32:1–31.

    CAS  Google Scholar 

  10. Navarro M, Michiardi A, Castano O, Planell JA. Biomaterials in orthopaedics. J R Soc Interface. 2008;5:1137–58.

    CAS  Google Scholar 

  11. Bohner M. Design of ceramic-based cements and putties for bone graft substitution. Eur Cell Mater. 2010;20:1–12.

    CAS  Google Scholar 

  12. Amling M, Schilling AF, Pogoda P, Priemel M, Rueger JM. Biomaterials and bone remodeling: the physiologic process required for biologization of bone substitutes. Eur J Trauma. 2006;32:102–6.

    Google Scholar 

  13. Rueger JM. Bone replacement materials—state of the art and the way ahead. Orthopäde. 1998;27:72–9.

    CAS  Google Scholar 

  14. Habraken W, Habibovic P, Epple M, Bohner M. Calcium phosphates in biomedical applications: materials for the future? Mater Today. 2016;19:69–87.

    CAS  Google Scholar 

  15. Arcos D, Boccaccini AR, Bohner M, Diez-Perez A, Epple M, Gomez-Barrena E, et al. The relevance of biomaterials to the prevention and treatment of osteoporosis. Acta Biomater. 2014;10:1793–805.

    CAS  Google Scholar 

  16. Albee F, Morrison H. Studies in bone growth: triple calcium phosphate as a stimulus to osteogenesis. Ann Surg. 1920;71:32–8.

    CAS  Google Scholar 

  17. Bohner M. Resorbable biomaterials as bone graft substitutes. Mater Today. 2010;13:24–30.

    CAS  Google Scholar 

  18. Lee D, Upadhye K, Kumta PN. Nano-sized calcium phosphate (CaP) carriers for non-viral gene delivery. Mater Sci Eng B. 2012;177:289–302.

    CAS  Google Scholar 

  19. Bose S, Tarafder S. Calcium phosphate ceramic systems in growth factor and drug delivery for bone tissue engineering: a review. Acta Biomater. 2012;8:1401–21.

    CAS  Google Scholar 

  20. Sokolova V, Shi Z, Huang S, Du Y, Kopp M, Frede A, et al. Delivery of the TLR ligand poly(I:C) to liver cells in vitro and in vivo by calcium phosphate nanoparticles leads to a pronounced immunostimulation. Acta Biomater. 2017;64:401–10.

    CAS  Google Scholar 

  21. Neuhaus B, Tosun B, Rotan O, Frede A, Westendorf AM, Epple M. Nanoparticles as transfection agents: a comprehensive study with ten different cell lines. RSC Adv. 2016;6:18102–12.

    CAS  Google Scholar 

  22. Hadjicharalambous C, Kozlova D, Sokolova V, Epple M, Chatzinikolaidou M. Calcium phosphate nanoparticles carrying BMP-7 plasmid DNA induce an osteogenic response in MC3T3-E1 pre-osteoblasts. J Biomed Mater Res A. 2015;103:3834–42.

    CAS  Google Scholar 

  23. Zakaria SM, Sharif Zein SH, Othman MR, Yang F, Jansen JA. Nanophase hydroxyapatite as a biomaterial in advanced hard tissue engineering: a review. Tissue Eng B. 2013;19:431–41.

    CAS  Google Scholar 

  24. Mostaghaci B, Loretz B, Haberkorn R, Kickelbick G, Lehr CM. One-step synthesis of nanosized and stable amino-functionalized calcium phosphate particles for DNA transfection. Chem Mater. 2013;25:3667–74.

    CAS  Google Scholar 

  25. Arcos D, Vallet-Regí M. Bioceramics for drug delivery. Acta Mater. 2013;61:890–911.

    CAS  Google Scholar 

  26. Epple M. Review of potential health risks associated with nanoscopic calcium phosphate. Acta Biomater. 2018;77:1–14.

    CAS  Google Scholar 

  27. Marenzana M, Arnett TR. The key role of the blood supply to bone. Bone Res. 2013;1:203–15.

    CAS  Google Scholar 

  28. Kempen DH, Lu L, Heijink A, Hefferan TE, Creemers LB, Maran A, et al. Effect of local sequential VEGF and BMP-2 delivery on ectopic and orthotopic bone regeneration. Biomaterials. 2009;30:2816–25.

    CAS  Google Scholar 

  29. Lieberman JR, Daluiski A, Stevenson S, Wu L, McAllister P, Lee YP, et al. The effect of regional gene therapy with bone morphogenetic protein-2-producing bone-marrow cells on the repair of segmental femoral defects in rats. J Bone Joint Surg Am. 1999;81:905–17.

    CAS  Google Scholar 

  30. Patel ZS, Young S, Tabata Y, Jansen JA, Wong ME, Mikos AG. Dual delivery of an angiogenic and an osteogenic growth factor for bone regeneration in a critical size defect model. Bone. 2008;43:931–40.

    CAS  Google Scholar 

  31. Samee M, Kasugai S, Kondo H, Ohya K, Shimokawa H, Kuroda S. Bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) transfection to human periosteal cells enhances osteoblast differentiation and bone formation. J Pharmacol Sci. 2008;108:18–31.

    CAS  Google Scholar 

  32. Xiao C, Zhou H, Liu G, Zhang P, Fu Y, Gu P, et al. Bone marrow stromal cells with a combined expression of BMP-2 and VEGF-165 enhanced bone regeneration. Biomed Mater. 2011;6:015013.

    Google Scholar 

  33. Lv J, Xiu P, Tan J, Jia Z, Cai H, Liu Z. Enhanced angiogenesis and osteogenesis in critical bone defects by the controlled release of BMP-2 and VEGF: implantation of electron beam melting-fabricated porous Ti6Al4V scaffolds incorporating growth factor-doped fibrin glue. Biomed Mater. 2015;10:035013.

    Google Scholar 

  34. Zhang W, Zhu C, Wu Y, Ye D, Wang S, Zou D, et al. VEGF and BMP-2 promote bone regeneration by facilitating bone marrow stem cell homing and differentiation. Eur Cells Mater. 2014;15:1–12.

    Google Scholar 

  35. Hahn GV, Cohen RB, Wozney JM, Levitz CL, Shore EM, Zasloff MA, et al. A bone morphogenetic protein subfamily: chromosomal localization of human genes for BMP5, BMP6, and BMP7. Genomics. 1992;14:759–62.

    CAS  Google Scholar 

  36. Giannoudis PV, Kanakaris NK, Dimitriou R, Gill I, Kolimarala V, Montgomery RJ. The synergistic effect of autograft and BMP-7 in the treatment of atrophic nonunions. Clin Orthop Relat Res. 2009;467:3239–58.

    Google Scholar 

  37. Reddi AH. Role of morphogenetic proteins in skeletal tissue engineering and regeneration. Nat Biotechnol. 1998;16:247–52.

    CAS  Google Scholar 

  38. Lissenberg-Thunnissen SN, de Gorter DJJ, Sier CFM, Schipper IB. Use and efficacy of bone morphogenetic proteins in fracture healing. Int Orthop. 2011;35:1271–80.

    Google Scholar 

  39. Ong KL, Villarraga ML, Lau E, Carreon LY, Kurtz SM, Glassman SD. Off-label use of bone morphogenetic proteins in the United States using administrative data. Spine. 2010;35:1794–800.

    Google Scholar 

  40. Kanczler JM, Oreffo RO. Osteogenesis and angiogenesis: the potential for engineering bone. Eur Cells Mater. 2008;15:100–14.

    CAS  Google Scholar 

  41. Spies CKG, Schnurer S, Gotterbarm T, Breusch S. The efficacy of Biobon and Ostim within metaphyseal defects using the Gottinger Minipig. Arch Orthop Trauma Surg. 2009;129:979–88.

    Google Scholar 

  42. Huber FX, McArthur N, Heimann L, Dingeldein E, Cavey H, Palazzi X, et al. Evaluation of a novel nanocrystalline hydroxyapatite paste Ostim(R) in comparison to Alpha-BSM(R)—more bone ingrowth inside the implanted material with Ostim(R) compared to Alpha BSM(R). BMC Musculoskelet Disord. 2009;10:164.

    Google Scholar 

  43. Carmagnola D, Abati S, Celestino S, Chiapasco M, Bosshardt D, Lang NP. Oral implants placed in bone defects treated with Bio-Oss, Ostim-Paste or PerioGlas: an experimental study in the rabbit tibiae. Clin Oral Implants Res. 2008;19:1246–53.

    Google Scholar 

  44. Klesing J, Chernousova S, Kovtun A, Neumann S, Ruiz L, Gonzalez-Calbet JM, et al. An injectable paste of calcium phosphate nanorods, functionalized with nucleic acids, for cell transfection and gene silencing. J Mater Chem. 2010;20:6144–8.

    CAS  Google Scholar 

  45. Bohner M, Tiainen H, Michel P, Döbelin N. Design of an inorganic dual-paste apatite cement using cation exchange. J Mater Sci Mater Med. 2015;26:1–13.

    CAS  Google Scholar 

  46. Larsson S, Hannink G. Injectable bone-graft substitutes: Current products, their characteristics and indications, and new developments. Injury. 2011;42(Suppl. 2):30–4.

    Google Scholar 

  47. Chernousova S, Klesing J, Soklakova N, Epple M. A genetically active nano-calcium phosphate paste for bone substitution, encoding the formation of BMP-7 and VEGF-A. RSC Adv. 2013;3:11155–61.

    CAS  Google Scholar 

  48. Tenkumo T, Vanegas Sáenz JR, Nakamura K, Shimizu Y, Sokolova V, Epple M, et al. Prolonged release of bone morphogenetic protein-2 in vivo by gene transfection with DNA-functionalized calcium phosphate nanoparticle-loaded collagen scaffolds. Mater Sci Eng C. 2018;92:172–83.

    CAS  Google Scholar 

  49. Dorozhkin SV. Nanosized and nanocrystalline calcium orthophosphates. Acta Biomater. 2010;6:715–34.

    CAS  Google Scholar 

  50. Kesharwani P, Gajbhiye V, Jain NK. A review of nanocarriers for the delivery of small interfering RNA. Biomaterials. 2012;33:7138–50.

    CAS  Google Scholar 

  51. Morille M, Passirani C, Vonarbourg A, Clavreul A, Benoit JP. Progress in developing cationic vectors for non-viral systemic gene therapy against cancer. Biomaterials. 2008;29:3477–96.

    CAS  Google Scholar 

  52. Chernousova S, Epple M. Live-cell imaging to compare the transfection and gene silencing efficiency of calcium phosphate nanoparticles and a liposomal transfection agent. Gene Ther. 2017;24:282–9.

    CAS  Google Scholar 

  53. Klesing J, Chernousova S, Epple M. Freeze-dried cationic calcium phosphate nanorods as versatile carriers of nucleic acids (DNA, siRNA). J Mater Chem. 2012;22:199–204.

    CAS  Google Scholar 

  54. Schlickewei CW, Laaff G, Andresen A, Klatte TO, Rueger JM, Ruesing J, et al. Bone augmentation using a new injectable bone graft substitute by combining calcium phosphate and bisphosphonate as composite—an animal model. J Orthop Surg Res. 2015;10:116.

    Google Scholar 

  55. Dorozhkin SV, Epple M. Biological and medical significance of calcium phosphates. Angew Chem Int Ed. 2002;41:3130–46.

    CAS  Google Scholar 

  56. Tarassoli P, Khan WS, Hughes A, Heidari N. A review of techniques for gene therapy in bone healing. Curr Stem Cell Res Ther. 2013;8:201–9.

    CAS  Google Scholar 

  57. Im GI. Nonviral gene transfer strategies to promote bone regeneration. J Biomed Mater Res A. 2013;101:3009–18.

    Google Scholar 

  58. Guo X, Huang L. Recent advances in nonviral vectors for gene delivery. Acc Chem Res. 2012;45:971–9.

    CAS  Google Scholar 

  59. Fischer J, Kolk A, Wolfart S, Pautke C, Warnke PH, Plank C, et al. Future of local bone regeneration—protein versus gene therapy. J Craniomaxillofac Surg. 2011;39:54–64.

    CAS  Google Scholar 

  60. Evans CH. Gene therapy for bone healing. Exp Rev Mol Med. 2010;12:e18

    Google Scholar 

  61. Sokolova V, Epple M. Inorganic nanoparticles as carriers of nucleic acids into cells. Angew Chem Int Ed. 2008;47:1382–95.

    CAS  Google Scholar 

  62. Bishop CJ, Majewski RL, Guiriba TRM, Wilson DR, Bhise NS, Quinones-Hinojosa A, et al. Quantification of cellular and nuclear uptake rates of polymeric gene delivery nanoparticles and DNA plasmids via flow cytometry. Acta Biomater. 2016;37:120–30.

    CAS  Google Scholar 

  63. Samal SK, Dash M, Van VS, Kaplan DL, Chiellini E, van Blitterswijk CA, et al. Cationic polymers and their therapeutic potential. Chem Soc Rev. 2012;41:7147–94.

    CAS  Google Scholar 

  64. Pérez-Martínez F, Guerra J, Posadas I, Ceña V. Barriers to non-viral vector-mediated gene delivery in the nervous system. Pharm Res. 2011;28:1843–58.

    Google Scholar 

  65. Wang J, Lu Z, Wientjes MG, Au JLS. Delivery of siRNA therapeutics: barriers and carriers. AAPS J. 2010;12:492–503.

    CAS  Google Scholar 

  66. Pichon C, Billiet L, Midoux P. Chemical vectors for gene delivery: uptake and intracellular trafficking. Curr Opin Biotechnol. 2010;21:640–5.

    CAS  Google Scholar 

  67. Guo J, Fisher KA, Darcy R, Cryan JF, O’Driscoll C. Therapeutic targeting in the silent era: advances in non-viral siRNA delivery. Mol Biosyst. 2010;6:1143–61.

    CAS  Google Scholar 

  68. Wolff JA, Rozema DB. Breaking the bonds: non-viral vectors become chemically dynamic. Mol Ther. 2008;16:8–15.

    CAS  Google Scholar 

  69. Roy I, Stachowiak MK, Bergey EJ. Non-viral gene transfection nanoparticles: function and applications in brain. Nanomedicine. 2008;4:89–97.

    CAS  Google Scholar 

  70. Agarwal A, Mallapragada SK. Synthetic sustained gene delivery systems. Curr Top Med Chem. 2008;8:311–30.

    CAS  Google Scholar 

  71. Li SD, Huang L. Gene therapy progress and prospects: non-viral gene therapy by systemic delivery. Gene Ther. 2006;13:1313–9.

    CAS  Google Scholar 

  72. De Laporte L, Cruz Rea J, Shea LD. Design of modular non-viral gene therapy vectors. Biomaterials. 2006;27:947–54.

    Google Scholar 

  73. Lou J, Xu F, Merkel K, Manske P. Gene therapy: adenovirus-mediated human bone morphogenetic protein-2 gene transfer induces mesenchymal progenitor cell proliferation and differentiation in vitro and bone formation in vivo. J Orthop Res. 1999;17:43–50.

    CAS  Google Scholar 

  74. Keeney M, van den Beucken JJJP, van der Kraan PM, Jansen JA, Pandit A. The ability of a collagen/calcium phosphate scaffold to act as its own vector for gene delivery and to promote bone formation via transfection with VEGF165. Biomaterials. 2010;31:2893–902.

    CAS  Google Scholar 

  75. Li G, Corsi-Payne K, Zheng B, Usas A, Peng H, Huard J. The dose of growth factors influences the synergistic effect of vascular endothelial growth factor on bone morphogenetic protein 4-induced ectopic bone formation. Tissue Eng Part A. 2009;15:2123–33.

    CAS  Google Scholar 

  76. Kolk A, Handschel J, Drescher W, Rothamel D, Kloss F, Blessmann M, et al. Current trends and future perspectives of bone substitute materials—from space holders to innovative biomaterials. J Craniomaxillofac Surg. 2012;40:706–18.

    Google Scholar 

  77. Wegman F, Bijenhof A, Schuijff L, Oner FC, Dhert WJA, Alblas J. Osteogenic differentiation as a result of BMP-2 plasmid DNA based gene therapy in vitro and in vivo. Eur Cell Mater. 2011;21:230–42.

    CAS  Google Scholar 

  78. Zhang C, Wang KZ, Qiang H, Tang YL, Li QA, Li MA, et al. Angiopoiesis and bone regeneration via co-expression of the hVEGF and hBMP genes from an adeno-associated viral vector in vitro and in vivo. Acta Pharmacol Sin. 2010;31:821–30.

    CAS  Google Scholar 

  79. Koh JT, Zhao Z, Wang Z, Lewis IS, Krebsbach PH, Franceschi RT. Combinatorial gene therapy with BMP2/7 enhances cranial bone regeneration. J Dent Res. 2008;87:845–9.

    CAS  Google Scholar 

  80. Laschke MW, Witt K, Pohlemann T, Menger MD. Injectable nanocrystalline hydroxyapatite paste for bone substitution: in vivo analysis of biocompatibility and vascularization. J Biomed Mater Res B. 2007;82B:494–505.

    CAS  Google Scholar 

  81. Detsch R, Hagmeyer D, Neumann M, Schaefer S, Vortkamp A, Wuelling M, et al. The resorption of nanocrystalline calcium phosphates by osteoclast-like cells. Acta Biomater. 2010;6:3223–33.

    CAS  Google Scholar 

  82. Tenkumo T, Vanegas Saenz JR, Takada Y, Takahashi M, Rotan O, Sokolova V, et al. Gene transfection of human mesenchymal stem cells with a nano-hydroxyapatite-collagen scaffold containing DNA-functionalized calcium phosphate nanoparticles. Genes Cells. 2016;21:682–95.

    CAS  Google Scholar 

Download references

Acknowledgements

Wolfgang Lehmann and Matthias Epple thank the Deutsche Forschungsgemeinschaft (DFG) for funding within the projects EP 22/39-1 and LE 1298/3-1.

Author information

Authors and Affiliations

  1. Department of Trauma, Hand and Reconstructive Surgery, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246, Hamburg, Germany

    Carsten Schlickewei, Till O. Klatte, Yasmin Wildermuth, Georg Laaff & Johannes M. Rueger

  2. Inorganic Chemistry and Center for Nanointegration Duisburg-Essen (CeNIDE), University of Duisburg-Essen, Universitaetsstr. 5-7, 45117, Essen, Germany

    Johannes Ruesing, Svitlana Chernousova & Matthias Epple

  3. Department of Trauma, Orthopaedics and Plastic Surgery, University Hospital Göttingen, Robert-Koch-Str. 40, 37075, Göttingen, Germany

    Wolfgang Lehmann

Authors
  1. Carsten Schlickewei
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Till O. Klatte
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yasmin Wildermuth
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Georg Laaff
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Johannes M. Rueger
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Johannes Ruesing
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Svitlana Chernousova
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Wolfgang Lehmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Matthias Epple
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Carsten Schlickewei or Matthias Epple.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Animal studies

All institutional and national guidelines for the care and use of laboratory animals were followed.

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

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Schlickewei, C., Klatte, T.O., Wildermuth, Y. et al. A bioactive nano-calcium phosphate paste for in-situ transfection of BMP-7 and VEGF-A in a rabbit critical-size bone defect: results of an in vivo study. J Mater Sci: Mater Med 30, 15 (2019). https://doi.org/10.1007/s10856-019-6217-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s10856-019-6217-y

Search

Navigation