A method for toxicological evaluation of biomaterials based on colony formation of V79 cells

  • Yoshihiko Kotoura
  • Takao Yamamuro
  • Jitsuhiko Shikata
  • Yoshiaki Kakutani
  • Toshiaki Kitsugi
  • Hiroya Tanaka
Original Articles


This report describes a method for cytotoxicity screening of biomaterials based on colony formation of V79 cells. For this test, two metals (titanium and nickel), two ceramics (alumina ceramic and tricalcium phosphate), and two types of polymeric material [high density polyethylene (HDP) and polyvinylchloride (PVC)] were used. Each metal and ceramic was cast into a disk and semidisk 49 mm in diameter and 1 to 2 mm thick. The HDP was molded into a petri dish and PVC was used as a thin film. The materials were sterilized by heating or with ethylene oxide and placed in plastic petri dishes, after which 8 ml cell suspension containing 100 cells were added to each dish. After 1 week, the colonies formed on the materials were fixed, stained, and then the number of colonies was counted. Titanium, alumina ceramic, and HDP showed no differences from the controls in terms of colonies. On the disks and the semidisks of nickel and tricalcium phosphate and on the thin disks of PVC, however, no colonies were detected. The V79 cells used in this experiment showed a rapid and logarithmically stable growth curve and such a high rate of colony formation as to form visible noticeable colonies, and were therefore suitable cells for screening test the cytotoxicity of biomaterials. Unlike other previously reported methods of in vitro cytotoxicity testing, this method permits assay of colonies formed from a single cell after proliferation directly on the materials. Moreover, the test with semidisks permits simple screening to assess the cytotoxicity is caused by either the chemical substances or the physical properties of the materials. Furthermore, since colonies of V79 cells are formed even on metals, it is generally easy to evaluate quantitatively the cytotoxicity of solid materials. As it has more advantages than other methods, this screening method appears suitable for the cytotoxicity test of biomaterials.


Colony Formation Ethylene Oxide High Density Polyethylene Tricalcium Phosphate High Density Polyethylene 
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Dieser Bericht beschreibt eine Methode zur zytotoxischen Bestimmung von Biomaterialien mittels Kolonienbildung von V79-Zellen. Für den Test wurden zwei metallische (Titan und Nickel), zwei keramische (Aluminiumoxidkeramik und Tricalciumphosphatkeramik) und zwei plastische („High density polyethylene” und „Polyvinylchroride”) Materialien verwendet. Die metallischen und keramischen Materialien wurden jeweils in Form von Scheiben und Halbscheiben von 49 mm Durchmesser und 1–2 mm Stärke gegossen. HDP wurde wie Petrischale geformt und PVC wurde mit Form von dünnen rundlichem Film getestet. Nach der Hitzesterilisation oder Gassterilisation mit Äthylenoxid wurden die Materialien in Petrischalen aus Plastik gelegt. Danach wurde in jede Schale 8 Milliliter Zellsuspension mit 100 Zellen gegeben. Nach einer Woche wurden die Kolonien, die sich auf den Materialien gebildet hatten, fixiert, gefärbt und gezählt. Titan, Aluminiumoxidkeramik und HDP wiesen bezüglich Kolonienbildung keinen Unterschied zu den Kontrollen auf. Hingegen konnten auf den Scheiben und Halbscheiben aus Nickel und Tricalciumphosphatkeramik und auf den Filmen von PVC keine Kolonien festgestellt werden. Die hier verwendeten V79-Zellen zeichnen sich durch ein rapides und stetiges logarithmisches Wachstum aus. Die Rate der Kolonienbildung ist dermaßen hoch, daß sich nach einer Woche mit bloßem Auge erkennbare Kolonien gebildet hatten. Daher eignen sich diese Zellen für den Test zur Bestimmung der Zytotoxität von Biomaterialien. Im Unterschied zu anderen berichteten Methoden erlaubt die hier beschriebene die Untersuchung von Kolonien, die sich aus einer einzigen Zelle direkt auf dem Material ausbreiten. Zudem erlaubt der Test mit den Halbscheiben einen einfachen Rückschluß auf die Ursache der Zytotoxität: sind es die chemischen Substanzen oder die physikalischen Eigenschaften des Materials. Da sich V79-


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  1. 1.
    Aoki Y, Sasaki T, Susuda K, Tsuge H, Kanno T (1982) The effect of biomaterials on rabbit cultured cartilage cell. Orthopaedic Ceramic Implants (Proc of Japanese Society of Orthopaedic Ceramic Implants) 2:27–33Google Scholar
  2. 2.
    Autian J (1977) Toxicological evaluation of biomaterials: Primary acute toxicity screening program. Artif Organs 1:53–60Google Scholar
  3. 3.
    Bearden LJ, Cooke FW (1980) Growth inhibition of cultured fibroblasts by cobalt and nickel. J Biomed Mater Res 14:289–309Google Scholar
  4. 4.
    Cameron HU, Macnab I, Pilliar RM (1977) Evaluation of a biodegradable ceramic. J Biomed Mater Res 11:179–186Google Scholar
  5. 5.
    Chawla AS (1982) Toxicity evaluation of a novel filler free silicone rubber biomaterial by cell culture techniques. J Biomed Mater Res 16:501–508Google Scholar
  6. 6.
    Elkind MM, Sutton H (1960) Radiation response of mammalian cells grown in culture. I. Repair of X-ray damage in surviving Chinese hamster cells. Radiat Res 13:556–593Google Scholar
  7. 7.
    Ford DK, Yerganian G (1958) Observations on the chromosomes of Chinese hamster cells in tissue culture. J Natl Cancer Inst 21:393–425Google Scholar
  8. 8.
    Homsy CA (1970) Bio-compatibility in selection of materials for implantation. J Biomed Mater Res 4:341–356Google Scholar
  9. 9.
    Johnson HJ, Northup SJ, Seagraves PA, Gravin PJ, Wallin RF (1983) Biocompatibility test procedures for materials evaluation in vitro. I. Comparative test system sensitivity. J Biomed Mater Res 17:571–586Google Scholar
  10. 10.
    Kakiuchi M, Takaoka K, Shimuzu N, Yoshikawa H, Ono K (1982) Evaluation of biocompatibility of implant materials in cell culture system. Orthopaedic Ceramic Implants (Proc of Japanese Society of Orthopaedic Ceramic Implants) 2:39–43Google Scholar
  11. 11.
    Kawahara H, Yamagami A, Nakamura M, Jr (1968) Biological testing of dental materials by means of tissue culture. Int Dent J 18:443–467Google Scholar
  12. 12.
    Nakamura M, Kawahara H, Kawada Y, Hikari S, Sakaguchi Y, Yokoyama A, Nakamura H (1983) Long-term bicompatibility test of Fe-Cr alloy. Proceedings 5th annual meeting of Japanese Society for Biomaterials 5:1–2 (in Japanese)Google Scholar
  13. 13.
    Pappas AM, Cohen J (1968) Toxicity of metal particles in tissue culture. J Bone Joint Surg [Am] 50:535–547Google Scholar
  14. 14.
    Schachtschabel DO, Blencke BA (1976) Effect of pulverized implantation materials (plastic and glass ceramic) on growth and metabolism of mammalian cell cultures. Eur Surg Res 8:71–80Google Scholar
  15. 15.
    Sharefkin JB, Lather C, Smith M, Rich NM (1983) Endothelial cell labeling with indium-III-oxine as a marker of cell attachment to bioprosthetic surfaces. J Biomed Mater Res 17:345–357Google Scholar
  16. 16.
    Taylor JA, Abodeely RA, Fuson RL (1973) Rapid screening of biomedical polymers by two methods of tissue culture. Trans Am Soc Artif Intern Organs 19:175–178Google Scholar
  17. 17.
    Ulreich JB, Chvapil M (1981) A quantitative microassay for in-vitro toxicity testing of biomaterials. J Biomed Mater Res 15:913–922Google Scholar
  18. 18.
    Wilsnack RE (1976) Quantitative cell culture biocompatibility testing of medical devices and correlation to animal tests. Biomat Med Devices Artif Organs 4:235–261Google Scholar
  19. 19.
    Wilson J, Pigott GH, Schoen EF, Hench LL (1981) Toxicology and biocompatibility of bioglasses. J Biomed Mater Res 15:805–817Google Scholar

Copyright information

© Springer-Verlag 1985

Authors and Affiliations

  • Yoshihiko Kotoura
    • 1
  • Takao Yamamuro
    • 1
  • Jitsuhiko Shikata
    • 1
  • Yoshiaki Kakutani
    • 1
  • Toshiaki Kitsugi
    • 1
  • Hiroya Tanaka
    • 1
  1. 1.Department of Orthopedic Surgery, Faculty of MedicineKyoto UniversitySakyoku, KyotoJapan

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