Cell and Tissue Banking

, Volume 14, Issue 2, pp 255–266 | Cite as

Cartilage tissue engineering of nasal septal chondrocyte-macroaggregates in human demineralized bone matrix

  • Juliane LieseEmail author
  • Ulrike Marzahn
  • Karym El Sayed
  • Axel Pruss
  • Andreas Haisch
  • Katharina Stoelzel


Tissue Engineering is an important method for generating cartilage tissue with isolated autologous cells and the support of biomaterials. In contrast to various gel-like biomaterials, human demineralized bone matrix (DBM) guarantees some biomechanical stability for an application in biomechanically loaded regions. The present study combined for the first time the method of seeding chondrocyte-macroaggregates in DBM for the purpose of cartilage tissue engineering. After isolating human nasal chondrocytes and creating a three-dimensional macroaggregate arrangement, the DBM was cultivated in vitro with the macroaggregates. The interaction of the cells within the DBM was analyzed with respect to cell differentiation and the inhibitory effects of chondrocyte proliferation. In contrast to chondrocyte-macroaggregates in the cell-DBM constructs, morphologically modified cells expressing type I collagen dominated. The redifferentiation of chondrocytes, characterized by the expression of type II collagen, was only found in low amounts in the cell-DBM constructs. Furthermore, caspase 3, a marker for apoptosis, was detected in the chondrocyte-DBM constructs. In another experimental setting, the vitality of chondrocytes as related to culture time and the amount of DBM was analyzed with the BrdU assay. Higher amounts of DBM tended to result in significantly higher proliferation rates of the cells within the first 48 h. After 96 h, the vitality decreased in a dose-dependent fashion. In conclusion, this study provides the proof of concept of chondrocyte-macroaggregates with DBM as an interesting method for the tissue engineering of cartilage. The as-yet insufficient redifferentiation of the chondrocytes and the sporadic initiation of apoptosis will require further investigations.


Tissue engineering Human demineralized bone matrix Cartilage Chondrocyte-macroaggregates 



The authors thank Mr. Schurig and Mr. Schweiger for their technical support and for making the DBM available.

Conflict of interest

No competing financial interests exist.


  1. Abdullah B, Shibghatullah AH, Hamid SS, Omar NS, Samsuddin AR (2008) The microscopic biological response of human chondrocytes to bovine bone scaffold. Cell Tissue Bank 10(3):205–213PubMedCrossRefGoogle Scholar
  2. Alexander TH, Sage AB, Chen AC, Schumacher BL, Shelton E, Masuda K, Sah RL, Watson D (2010) Insulin-like growth factor-I and growth differentiation factor-5 promote the formation of tissue-engineered human nasal septal cartilage. Tissue Eng Part C Methods 16(5):1213–1221. doi: 10.1089/ten.TEC.2009.0396 PubMedCrossRefGoogle Scholar
  3. Benya PD, Shaffer JD (1982) Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell 30(1):215–224PubMedCrossRefGoogle Scholar
  4. Bessho K, Tagawa T, Murata M (1990) Purification of rabbit bone morphogenetic protein derived from bone, dentin, and wound tissue after tooth extraction. J Oral Maxillofac Surg 48(2):162–169PubMedCrossRefGoogle Scholar
  5. Bruns J (2003) Tissue engineering—neues zum gewebeersatz im muskel-skelett-system. Steinkopff Verlag, DarmstadtGoogle Scholar
  6. Dieudonne SC, Semeins CM, Goei SW, Vukicevic S, Nulend JK, Sampath TK, Helder M, Burger EH (1994) Opposite effects of osteogenic protein and transforming growth factor beta on chondrogenesis in cultured long bone rudiments. J Bone Miner Res 9(6):771–780PubMedCrossRefGoogle Scholar
  7. Dunham BP, Koch RJ (1998) Basic fibroblast growth factor and insulinlike growth factor I support the growth of human septal chondrocytes in a serum-free environment. Arch Otolaryngol Head Neck Surg 124(12):1325–1330PubMedGoogle Scholar
  8. Glowacki J, Mulliken JB (1985) Demineralized bone implants. Clin Plast Surg 12(2):233–241PubMedGoogle Scholar
  9. Glowacki J, Kaban LB, Murray JE, Folkman J, Mulliken JB (1981) Application of the biological principle of induced osteogenesis for craniofacial defects. Lancet 1(8227):959–962PubMedCrossRefGoogle Scholar
  10. Greene JJ, Watson D (2010) Septal cartilage tissue engineering: new horizons. Facial Plast Surg 26(5):396–404. doi: 10.1055/s-0030-1265019 PubMedCrossRefGoogle Scholar
  11. Haisch A, Loch A, David J, Pruss A, Hansen R, Sittinger M (2000) Preparation of a pure autologous biodegradable fibrin matrix for tissue engineering. Med Biol Eng Comput 38(6):686–689PubMedCrossRefGoogle Scholar
  12. Homicz MR, Chia SH, Schumacher BL, Masuda K, Thonar EJ, Sah RL, Watson D (2003) Human septal chondrocyte redifferentiation in alginate, polyglycolic acid scaffold, and monolayer culture. Laryngoscope 113(1):25–32. doi: 10.1097/00005537-200301000-00005 PubMedCrossRefGoogle Scholar
  13. Homminga GN, Buma P, Koot HW, van der Kraan PM, van den Berg WB (1993) Chondrocyte behavior in fibrin glue in vitro. Acta Orthop Scand 64(4):441–445PubMedCrossRefGoogle Scholar
  14. Hutmacher DW (2000) Scaffolds in tissue engineering bone and cartilage. Biomaterials 21(24):2529–2543PubMedCrossRefGoogle Scholar
  15. Hutmacher DW, Goh JC, Teoh SH (2001) An introduction to biodegradable materials for tissue engineering applications. Ann Acad Med Singapore 30(2):183–191PubMedGoogle Scholar
  16. Iwasaki M, Nakata K, Nakahara H, Nakase T, Kimura T, Kimata K, Caplan AI, Ono K (1993) Transforming growth factor-beta 1 stimulates chondrogenesis and inhibits osteogenesis in high density culture of periosteum-derived cells. Endocrinology 132(4):1603–1608PubMedCrossRefGoogle Scholar
  17. Jäckel M (1998) Die genetische Kontrolle des programmierten Zelltods (Apoptose), vol 46. Springer, HNO, pp 614–625Google Scholar
  18. Jager M, Fischer J, Schultheis A, Lensing-Hohn S, Krauspe R (2006) Extensive H(+) release by bone substitutes affects biocompatibility in vitro testing. J Biomed Mater Res A 76(2):310–322PubMedGoogle Scholar
  19. Kuhls R, Werner-Rustner M, Kuchler I, Soost F (2001) Human demineralised bone matrix as a bone substitute for reconstruction of cystic defects of the lower jaw. Cell Tissue Bank 2(3):143–153PubMedCrossRefGoogle Scholar
  20. Leatherman BD, Dornhoffer JL (2004) The use of demineralized bone matrix for mastoid cavity obliteration. Otol Neurotol 25 (1):22–25; discussion 25–26Google Scholar
  21. Lomas RJ, Gillan HL, Matthews JB, Ingham E, Kearney JN (2001) An evaluation of the capacity of differently prepared demineralised bone matrices (DBM) and toxic residuals of ethylene oxide (EtOx) to provoke an inflammatory response in vitro. Biomaterials 22(9):913–921PubMedCrossRefGoogle Scholar
  22. Maor G, Hochberg Z, Silbermann M (1993) Insulin-like growth factor I accelerates proliferation and differentiation of cartilage progenitor cells in cultures of neonatal mandibular condyles. Acta Endocrinol (Copenh) 128(1):56–64Google Scholar
  23. Martin I, Suetterlin R, Baschong W, Heberer M, Vunjak-Novakovic G, Freed LE (2001) Enhanced cartilage tissue engineering by sequential exposure of chondrocytes to FGF-2 during 2D expansion and BMP-2 during 3D cultivation. J Cell Biochem 83(1):121–128PubMedCrossRefGoogle Scholar
  24. Naumann A, Dennis JE, Aigner J, Coticchia J, Arnold J, Berghaus A, Kastenbauer ER, Caplan AI (2004) Tissue engineering of autologous cartilage grafts in three-dimensional in vitro macroaggregate culture system. Tissue Eng 10(11–12):1695–1706PubMedCrossRefGoogle Scholar
  25. Pirsig W, Bean JK, Lenders H, Verwoerd CD, Verwoerd-Verhoef HL (1995) Cartilage transformation in a composite graft of demineralized bovine bone matrix and ear perichondrium used in a child for the reconstruction of the nasal septum. Int J Pediatr Otorhinolaryngol 32(2):171–181PubMedCrossRefGoogle Scholar
  26. Pruss A, Baumann B, Seibold M, Kao M, Tintelnot K, von Versen R, Radtke H, Dorner T, Pauli G, Gobel UB (2001) Validation of the sterilization procedure of allogeneic avital bone transplants using peracetic acid–ethanol. Biologicals 29(2):59–66PubMedCrossRefGoogle Scholar
  27. Richmon JD, Sage AB, Shelton E, Schumacher BL, Sah RL, Watson D (2005) Effect of growth factors on cell proliferation, matrix deposition, and morphology of human nasal septal chondrocytes cultured in monolayer. Laryngoscope 115(9):1553–1560PubMedCrossRefGoogle Scholar
  28. Rotter N, Bucheler M, Haisch A, Wollenberg B, Lang S (2007) Cartilage tissue engineering using resorbable scaffolds. J Tissue Eng Regen Med 1(6):411–416PubMedCrossRefGoogle Scholar
  29. Schulze M, Kuettner KE, Cole AA (2000) Adult human chondrocytes in alginate culture. Preservation of the phenotype for further use in transplantation models. Orthopade 29(2):100–106PubMedGoogle Scholar
  30. Sittinger M, Bujia J, Rotter N, Reitzel D, Minuth WW, Burmester GR (1996) Tissue engineering and autologous transplant formation: practical approaches with resorbable biomaterials and new cell culture techniques. Biomaterials 17(3):237–242PubMedCrossRefGoogle Scholar
  31. ten Koppel PG, van Osch GJ, Verwoerd CD, Verwoerd-Verhoef HL (1998) Efficacy of perichondrium and a trabecular demineralized bone matrix for generating cartilage. Plast Reconstr Surg 102(6):2012–2020; discussion 2021Google Scholar
  32. Urist MR (1965) Bone: formation by autoinduction. Science 150(698):893–899PubMedCrossRefGoogle Scholar
  33. Urist MR, Mikulski AJ (1979) A soluble bone morphogenetic protein extracted from bone matrix with a mixed aqueous and nonaqueous solvent. Proc Soc Exp Biol Med 162(1):48–53PubMedGoogle Scholar
  34. Urist MR, Raskin K, Goltz D, Merickel K (1997) Endogenous bone morphogenetic protein: immunohistochemical localization in repair of a punch hole in the rabbit’s ear. Plast Reconstr Surg 99(5):1382–1389PubMedCrossRefGoogle Scholar
  35. van Osch GJ, ten Koppel PG, van der Veen SW, Poppe P, Burger EH, Verwoerd-Verhoef HL (1999) The role of trabecular demineralized bone in combination with perichondrium in the generation of cartilage grafts. Biomaterials 20(3):233–240PubMedCrossRefGoogle Scholar
  36. van Osch GJ, van der Veen SW, Verwoerd-Verhoef HL (2001) In vitro redifferentiation of culture-expanded rabbit and human auricular chondrocytes for cartilage reconstruction. Plast Reconstr Surg 107(2):433–440PubMedCrossRefGoogle Scholar
  37. von der Mark K, Gauss V, von der Mark H, Muller P (1977) Relationship between cell shape and type of collagen synthesised as chondrocytes lose their cartilage phenotype in culture. Nature 267(5611):531–532PubMedCrossRefGoogle Scholar
  38. Wang JC, Kanim LE, Nagakawa IS, Yamane BH, Vinters HV, Dawson EG (2001) Dose-dependent toxicity of a commercially available demineralized bone matrix material. Spine 26(13):1429–1435; discussion 1435–1426Google Scholar
  39. Zhou S, Yates KE, Eid K, Glowacki J (2005) Demineralized bone promotes chondrocyte or osteoblast differentiation of human marrow stromal cells cultured in collagen sponges. Cell Tissue Bank 6(1):33–44PubMedCrossRefGoogle Scholar
  40. Ziran BH, Smith WR, Morgan SJ (2007) Use of calcium-based demineralized bone matrix/allograft for nonunions and posttraumatic reconstruction of the appendicular skeleton: preliminary results and complications. J Trauma 63(6):1324–1328PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Juliane Liese
    • 1
    Email author
  • Ulrike Marzahn
    • 2
  • Karym El Sayed
    • 2
  • Axel Pruss
    • 3
  • Andreas Haisch
    • 4
  • Katharina Stoelzel
    • 5
  1. 1.Department of General and Visceral SurgeryJohann Wolfgang Goethe UniversityFrankfurtGermany
  2. 2.BiotechnologyBerlinGermany
  3. 3.Tissue Bank, Institute of Transfusion MedicineCharité—Universitätsmedizin BerlinBerlinGermany
  4. 4.Otolaryngology Center BerlinBerlinGermany
  5. 5.Department of Otorhinolaryngology, Head and Neck SurgeryCharité—Universitätsmedizin BerlinBerlinGermany

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