Use of an in vitro model in tissue engineering to study wound repair and differentiation of blastema tissue from rabbit pinna

  • Mohammad Reza Hashemzadeh
  • Nasser Mahdavi-ShahriEmail author
  • Ahmad Reza Bahrami
  • Masoumeh Kheirabadi
  • Fatemeh Naseri
  • Mitra Atighi


Rabbit ear wound repair is an accepted model for studies of tissue regeneration, leading to scar less wound repair. It is believed that a specific tissue, blastema, is responsible for such interesting capacity of tissue regeneration. To test this idea further and to elucidate the cellular events happening during the ear wound repair, we designed some controlled experiments in vitro. Small pieces of the ear were punched and washed immediately with normal saline. The tissues were then cultured in the Dulbecco’s Modified Eagles Medium, supplemented with fetal bovine serum in control group. As a treatment vitamin A and C was used to evaluate the differentiation potency of the tissue. These tissues were fixed, sectioned, stained, and microscopically studied. Micrographs of electron microscopy provided evidences revealing dedifferentiation of certain cells inside the punched tissues after incubation in tissue culture medium. The histological studies revealed that cells of the tissue (i) can undergo cellular proliferation, (ii) differentiate to epithelial, condrogenic, and osteogenic tissues, and (iii) regenerate the wounds. These results could be used for interpretation of the possible events happening during tissue engineering and wound repair in vitro. An important goal of this study is to create a tissue engineering and tissue banking model, so that in the future it could be used in further blastema tissue studies at different levels.


Blastema tissue Rabbit pinna Cell culture Differentiation Wound repair 



The authors are thankful to the Institute of Biotechnology, Ferdowsi University of Mashhad for providing work space and facilities. This project was supported by a grant from the Ferdowsi University of Mashhad.

Supplementary material

11626_2015_9868_MOESM1_ESM.docx (15 kb)
ESM 1 (DOCX 14 kb)


  1. Bayreuther K, Francz PI, Gogol J, Hapke C, Maier M, Meinrath HG (1991) Differentiation of primary and secondary fibroblasts in cell culture systems. Mutat Res 256:233–242CrossRefPubMedGoogle Scholar
  2. Berdanier CD (1997) Advanced nutrition micronutrients. CRCGoogle Scholar
  3. Brockes JP, Kumar A (2002) Plasticity and reprogramming of differentiated cells in amphibian regeneration. Nat Rev Mol Cell Biol 3:566–574CrossRefPubMedGoogle Scholar
  4. Cabbabe EB, Korock SW (1986) Wound healing in vitamin C-deficient and nondeficient guinea pig: a pilot study. Ann Plast Surg 17:330–334CrossRefPubMedGoogle Scholar
  5. Combs GF (1992) The vitamins: fundamental aspects in nutrition and health. Academic, San DiegoGoogle Scholar
  6. Corcoran JP, Ferretti P (1999) RA regulation of keratin expression and myogenesis suggests different ways of regenerating muscle in adult amphibian limbs. J Cell Sci 112:1385–1394PubMedGoogle Scholar
  7. Emura M, Mohr U, Riebe M, Aufderheide M, Dungworth DL (1988) Regulation of growth and differentiation by vitamin A in a cloned fetal lung epithelial cell line cultured on collagen gel in hormone-supplemented medium. In Vitro Cell Dev Biol 24:639–48CrossRefPubMedGoogle Scholar
  8. Fröhlich M, Malicev E, Gorensek M, Knezević M, Kregar Velikonja N (2007) Evaluation of rabbit auricular chondrocyte isolation and growth parameters in cell culture. Cell Biol Int 31:620–625CrossRefPubMedGoogle Scholar
  9. Gorensek M, Jaksimović C, Kregar-Velikonja N, Gorensek M, Knezevic M, Jeras M, Pavlovcic V, Cör A (2004) Nucleus pulposus repair with cultured autologous elastic cartilage derived chondrocytes. Cell Mol Biol Lett 9:363–373PubMedGoogle Scholar
  10. Goss RJ (1983) Deer antlers: regeneration, function and evolution. Academic, New YorkGoogle Scholar
  11. Groff JL, Gropper SS (1999) Advanced nutrition and human metabolism. West Publishing, St PaulGoogle Scholar
  12. Hamrick I, Counts SH (2008) Vitamin and mineral supplements. Primary Care: Clin in Office Pract 35:729–747CrossRefGoogle Scholar
  13. Hay ED (1958) The fine structure of blastema cells and differentiating cartilage cells in regenerating limbs of amblystoma larvae. J Biophysic and Biochem Cytol 4:583–592CrossRefGoogle Scholar
  14. Hay ED (1959) Electron microscopic observations of muscle dedifferentiation in regenerating Amblystoma limbs. Dev Biol 1:555–585CrossRefGoogle Scholar
  15. Heldin CH, Westermark B (1999) Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev 79:1283–1316PubMedGoogle Scholar
  16. Hunt TK (1986) Vitamin A and wound healing. J Am Acad Dermatol 15:817–821CrossRefPubMedGoogle Scholar
  17. Kligman LH, Duo CH, Kligman AM (1984) Topical retinoic acid enhances the repair of ultraviolet damaged dermal connective tissue. Connect Tissue Res 12:139–150CrossRefPubMedGoogle Scholar
  18. Langer R, Vacanti JP (1993) Tissue engineering. Science 260:920–926CrossRefPubMedGoogle Scholar
  19. Lanman TH, Ingalls TH (1937) Vitamin C deficiency and wound healing (experimental and clinical study). Ann Surg 105:616–625PubMedCentralCrossRefPubMedGoogle Scholar
  20. Liosner MA, Vorontsova LD (1960) Asexual propagation and regeneration. Pergamon, New YorkGoogle Scholar
  21. Mahmoudi Z, Moghaddam MM, Saeinasab M, Nakhaei-Rad S, Mirahmadi M, Mahdavi-Shahri N, Mahmoudi M, Bahrami AR (2011) Blastema cells derived from rabbit ear show stem cell characteristics. J Cell Mol Res 3:25–30Google Scholar
  22. Mason JB (2007) Vitamins, trace minerals, and other micronutrients. Cecil Medicine. Saunders Elsevier, Philadelphia, chap 237Google Scholar
  23. Morrison JI, Lööf S, He P, Simon A (2006) Salamander limb regeneration involves the activation of a multipotent skeletal muscle satellite cell population. J Cell Biol 172:433–440PubMedCentralCrossRefPubMedGoogle Scholar
  24. Moskalewski S, Adamiec I, Gołaszewska A (1979) Maturation of rabbit auricular chondrocytes grown in vitro in monolayer culture. Am J Anatomy 155:339–348CrossRefGoogle Scholar
  25. Rakel D (2012) Integrative medicine, 3rd edn. Saunders Elsevier, PhiladelphiaGoogle Scholar
  26. Singer AJ, Clark RAF (1999) Cutaneous wound healing. The New Eng J Med 341:738–746CrossRefPubMedGoogle Scholar
  27. Steen TP (1970) Origin and differentiative capacities of cells in the blastema of the regenerating salamander limb. Integr Comparat Biol 10:119–132CrossRefGoogle Scholar
  28. Ten Koppel PG, van Osch GJ, Verwoerd CD, Verwoerd-Verhoef HL (2001) A new in vivo model for testing cartilage grafts and biomaterials: the rabbit pinna punch-hole model. Biomaterials 22:1407–1414CrossRefPubMedGoogle Scholar
  29. Tsao CS (1997) An overview of ascorbic acid chemistry and biochemistry. Marcel Dekker, New YorkGoogle Scholar
  30. Tsonis PA (2000) Regeneration in vertebrates. Dev Biol 221:273–284CrossRefPubMedGoogle Scholar
  31. Tsonis PA (2002) Regenerative biology: the emerging field of tissue repair and restoration. Differentiation 70:397–409CrossRefPubMedGoogle Scholar
  32. Tsonis PA (2004) Stem cells from differentiated cells. Mol interv 4:81–83CrossRefPubMedGoogle Scholar
  33. Uitto J (1979) Collagen polymorphism: isolation and partial characterization of alpha 1(I)-trimer molecules in normal human skin. Arch Biochem Biophys 192:371–379CrossRefPubMedGoogle Scholar
  34. Verlhac V, Gabaudan J (1994) Influence of vitamin C on the immune system of salmonids. Aquaculture Res 25:21–36CrossRefGoogle Scholar
  35. Wahli T, Verlhac V, Girling P, Gabaudan J, Aebischer C (2003) Influence of dietary vitamin C on the wound healing process in rainbow trout (Oncorhynchus mykiss). Aquaculture 225:371–386CrossRefGoogle Scholar
  36. Wallace H (1981) Vertebrate limb regeneration. Wiley, New YorkGoogle Scholar
  37. Wei C, Liu X, Tao J, Wu R, Zhang P, Bian Y, Li Y, Fang F, Zhang Y (2014) Effects of vitamin C on characteristics retaining of in vitro-cultured mouse adipose-derived stem cells. In Vitro Cell Dev Biol Anim 50:75–86CrossRefPubMedGoogle Scholar
  38. Williams-Boyce PK, Daniel JC Jr (1980) Regeneration of rabbit ear tissue. J Exp Zool 212:243–253CrossRefPubMedGoogle Scholar
  39. Williams-Boyce PK, Daniel JCJR (1986) Comparison of ear tissue regeneration in mammals. J Anat 149:55–63PubMedCentralPubMedGoogle Scholar
  40. Yang X, Chen L, Xu X, Li C, Huang C, Deng CX (2001) TGF-β/Smad3 signals repress chondrocyte hypertrophic differentiation and are required for maintaining articular cartilage. J Cell Biol 153:35–46PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2015

Authors and Affiliations

  • Mohammad Reza Hashemzadeh
    • 1
  • Nasser Mahdavi-Shahri
    • 1
    • 2
    Email author
  • Ahmad Reza Bahrami
    • 1
    • 2
  • Masoumeh Kheirabadi
    • 2
  • Fatemeh Naseri
    • 3
  • Mitra Atighi
    • 1
  1. 1.Cell and Molecular Biology Reaserch Group, Institute of BiotechnologyFerdowsi University of MashhadMashhadIran
  2. 2.Department of Biology, Faculty of SciencesFerdowsi University of MashhadMashhadIran
  3. 3.Central LaboratoryFerdowsi University of MashhadMashhadIran

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