European Journal of Trauma and Emergency Surgery

, Volume 37, Issue 6, pp 635–644 | Cite as

Mesenchymal stem cell (MSC) and endothelial progenitor cell (EPC) growth and adhesion in six different bone graft substitutes

  • J. Schultheiss
  • C. Seebach
  • D. Henrich
  • K. Wilhelm
  • J. H. Barker
  • J. Frank
Original Article



Several different synthetic and allograft bone graft substitutes are used clinically to treat large bone defects. In contrast to the “gold standard” of autologous bone grafts, these do not contain bone-forming (MSC) or vessel-forming (EPC) cells. In order to achieve the same level of success enjoyed by autologous bone grafts, they must be compatible with mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC). In a previous study, we seeded MSC onto six different bone graft substitutes and then measured the cell adhesion, viability, differentiation, and morphology. In the present study, we seeded both MSC and EPC onto the same six bone graft substitutes and measured the same parameters.


In vitro, 125,000 MSC and 125,000 EPC were seeded onto Chronos®, Vitoss®, Actifuse®, Biobase®, Cerabone®, and Tutoplast®. Cell adhesion (fluorescence microscopy) and viability (MTT assay) were measured on days 2, 6, and 10. Osteogenic (cbfa-1, alkaline phosphatase [ALP], osteocalcin, collagen-1 alpha [Col1A]) and endothelial (von Willebrand factor [vWF], vascular endothelial growth factor [VEGF], kinase domain receptor [KDR]) gene expression were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR). Morphology was described by scanning electron microscopy (SEM) at day 2.


MSC adhered significantly better to Tutoplast®, Chronos®, Actifuse®, and Biobase®. EPC adhered better to Actifuse®, Chronos®, Biobase®, and Tutoplast®. Viability increased over time when seeded on Tutoplast® and Chronos®. Osteogenic and endothelial gene expression were detectable at day 10 in cells seeded on Chronos®, Actifuse®, and Tutoplast®. The best morphology of MSC and EPC was found on Tutoplast®, Chronos®, Actifuse®, and Biobase®.


When bone graft substitutes are used to help fill large defects, it is important that their interaction with these cells be supportive of bone healing.


Biomaterials Scaffolds Bone graft substitutes Osteoconduction Ceramics Mesenchymal stem cell Endothelial progenitor cell Cell adhesion Bone tissue engineering 


  1. 1.
    Barriga A, Díaz-De-Rada P, Barroso JL, Alfonso M, Lamata M, Hernáez S, Beguiristáin JL, San-Julián M, Villas C. Frozen cancellous bone allografts: positive cultures of implanted grafts in posterior fusions of the spine. Eur Spine J. 2004;13(2):152–6.PubMedCrossRefGoogle Scholar
  2. 2.
    Togawa D, Bauer TW, Lieberman IH, Sakai H. Lumbar intervertebral body fusion cages: histological evaluation of clinically failed cages retrieved from humans. J Bone Joint Surg Am. 2004;86:70–9.PubMedGoogle Scholar
  3. 3.
    Van Heest A, Swiontkowski M. Bone-graft substitutes. Lancet. 1999;353:SI28–9.PubMedGoogle Scholar
  4. 4.
    Nelson ER, Huang Z, Ma T, Lindsey D, Jacobs C, Smith RL, Goodman SB. New bone formation by murine osteoprogenitor cells cultured on corticocancellous allograft bone. J Orthop Res. 2008;26(12):1660–4.PubMedCrossRefGoogle Scholar
  5. 5.
    Giannoudis PV, Einhorn TA, Marsh D. Fracture healing: the diamond concept. Injury. 2007;38:S3–6.CrossRefGoogle Scholar
  6. 6.
    Petite H, Viateau V, Bensaïd W, Meunier A, de Pollak C, Bourguignon M, Oudina K, Sedel L, Guillemin G. Tissue-engineered bone regeneration. Nat Biotechnol. 2000;18:959–63.PubMedCrossRefGoogle Scholar
  7. 7.
    Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284:143–7.PubMedCrossRefGoogle Scholar
  8. 8.
    Bruder SP, Fink DJ, Caplan AI. Mesenchymal stem cells in bone development, bone repair, and skeletal regeneration therapy. J Cell Biochem. 1994;56:283–94.PubMedCrossRefGoogle Scholar
  9. 9.
    Quarto R, Thomas D, Liang CT. Bone progenitor cell deficits and the age-associated decline in bone repair capacity. Calcif Tissue Int. 1995;56:123–9.PubMedCrossRefGoogle Scholar
  10. 10.
    Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997;275:964–7.PubMedCrossRefGoogle Scholar
  11. 11.
    Henrich D, Seebach C, Kaehling C, Scherzed A, Wilhelm K, Tewksbury R, Powerski M, Marzi I. Simultaneous cultivation of human endothelial-like differentiated precursor cells and human marrow stromal cells on beta-tricalcium phosphate. Tissue Eng Part C Methods. 2009;15(4):551–60.PubMedCrossRefGoogle Scholar
  12. 12.
    Arkudas A, Beier JP, Heidner K, Tjiawi J, Polykandriotis E, Srour S, Sturzl M, Horch RE, Kneser U. Axial prevascularization of porous matrices using an arteriovenous loop promotes survival and differentiation of transplanted autologous osteoblasts. Tissue Eng. 2007;13:1549–60.PubMedCrossRefGoogle Scholar
  13. 13.
    Seebach C, Henrich D, Kähling C, Wilhelm K, Tami AE, Alini M, Marzi I. Endothelial progenitor cells and mesenchymal stem cells seeded onto beta-TCP granules enhance early vascularization and bone healing in a critical-sized bone defect in rats. Tissue Eng Part A. 2010;16(6):1961–70.PubMedCrossRefGoogle Scholar
  14. 14.
    Zhang SJ, Zhang H, Wei YJ, Su WJ, Liao ZK, Hou M, Zhou JY, Hu SS. Adult endothelial progenitor cells from human peripheral blood maintain monocyte/macrophage function throughout in vitro culture. Cell Res. 2006;16:577–84.PubMedCrossRefGoogle Scholar
  15. 15.
    Fernandez Pujol B, Lucibello FC, Zuzarte M, Lütjens P, Müller R, Havemann K. Dendritic cells derived from peripheral monocytes express endothelial markers and in the presence of angiogenic growth factors differentiate into endothelial-like cells. Eur J Cell Biol. 2001;80:99–110.PubMedCrossRefGoogle Scholar
  16. 16.
    Rehman J, Li J, Orschell CM, March KL. Peripheral blood “endothelial progenitor cells” are derived from monocyte/macrophages and secrete angiogenic growth factors. Circulation. 2003;107:1164–9.PubMedCrossRefGoogle Scholar
  17. 17.
    Grellier M, Ferreira-Tojais N, Bourget C, Bareille R, Guillemot F, Amédée J. Role of vascular endothelial growth factor in the communication between human osteoprogenitors and endothelial cells. J Cell Biochem. 2009;106:390–8.PubMedCrossRefGoogle Scholar
  18. 18.
    Assmus B, Schächinger V, Teupe C, Britten M, Lehmann R, Döbert N, Grünwald F, Aicher A, Urbich C, Martin H, Hoelzer D, Dimmeler S, Zeiher AM. Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI). Circulation. 2002;106:3009–17.PubMedCrossRefGoogle Scholar
  19. 19.
    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.PubMedCrossRefGoogle Scholar
  20. 20.
    Eggli PS, Müller W, Schenk RK. Porous hydroxyapatite and tricalcium phosphate cylinders with two different pore size ranges implanted in the cancellous bone of rabbits. A comparative histomorphometric and histologic study of bony ingrowth and implant substitution. Clin Orthop Relat Res. 1988;232:127–38.PubMedGoogle Scholar
  21. 21.
    Hing KA, Best SM, Tanner KET, Bonfield W, Revell PA. Mediation of bone ingrowth in porous hydroxyapatite bone graft substitutes. J Biomed Mater Res A. 2004;68:187–200.PubMedCrossRefGoogle Scholar
  22. 22.
    Lawrence BJ, Madihally SV. Cell colonization in degradable 3D porous matrices. Cell Adh Migr. 2008;2:9–16.PubMedCrossRefGoogle Scholar
  23. 23.
    Guillotin B, Bourget C, Remy-Zolgadri M, Bareille R, Fernandez P, Conrad V, Amédée-Vilamitjana J. Human primary endothelial cells stimulate human osteoprogenitor cell differentiation. Cell Physiol Biochem. 2004;14:325–32.PubMedCrossRefGoogle Scholar
  24. 24.
    Rouwkema J, Westerweel PE, de Boer J, Verhaar MC, van Blitterswijk CA. The use of endothelial progenitor cells for prevascularized bone tissue engineering. Tissue Eng Part A. 2009;15(8):2015–27.PubMedCrossRefGoogle Scholar
  25. 25.
    Carson JS, Bostrom MP. Synthetic bone scaffolds and fracture repair. Injury. 2007;38(Suppl 1):S33–7.CrossRefGoogle Scholar
  26. 26.
    Seebach C, Schultheiss J, Wilhelm K, Frank J, Henrich D. Comparison of six bone-graft substitutes regarding to cell seeding efficiency, metabolism and growth behaviour of human mesenchymal stem cells (MSC) in vitro. Injury. 2010;41(7):731–8.PubMedCrossRefGoogle Scholar
  27. 27.
    Seebach C, Henrich D, Tewksbury R, Wilhelm K, Marzi I. Number and proliferative capacity of human mesenchymal stem cells are modulated positively in multiple trauma patients and negatively in atrophic nonunions. Calcif Tissue Int. 2007;80(4):294–300.PubMedCrossRefGoogle Scholar
  28. 28.
    Henrich D, Hahn P, Wahl M, Wilhelm K, Dernbach E, Dimmeler S, Marzi I. Serum derived from multiple trauma patients promotes the differentiation of endothelial progenitor cells in vitro: possible role of transforming growth factor-beta1 and vascular endothelial growth factor165. Shock. 2004;21:13–6.PubMedCrossRefGoogle Scholar
  29. 29.
    Hofmann A, Konrad L, Gotzen L, Printz H, Ramaswamy A, Hofmann C. Bioengineered human bone tissue using autogenous osteoblasts cultured on different biomatrices. J Biomed Mater Res A. 2003;67:191–9.PubMedCrossRefGoogle Scholar
  30. 30.
    Daculsi G, Passuti N. Effect of the macroporosity for osseous substitution of calcium phosphate ceramics. Biomaterials. 1990;11:86–7.PubMedGoogle Scholar
  31. 31.
    Klenke FM, Liu Y, Yuan H, Hunziker EB, Siebenrock KA, Hofstetter W. Impact of pore size on the vascularization and osseointegration of ceramic bone substitutes in vivo. J Biomed Mater Res A. 2008;85(3):777–86.PubMedGoogle Scholar
  32. 32.
    Groessner-Schreiber B, Tuan RS. Enhanced extracellular matrix production and mineralization by osteoblasts cultured on titanium surfaces in vitro. J Cell Sci. 1992;101:209–17.PubMedGoogle Scholar
  33. 33.
    Lim JY, Shaughnessy MC, Zhou Z, Noh H, Vogler EA, Donahue HJ. Surface energy effects on osteoblast spatial growth and mineralization. Biomaterials. 2008;29(12):1776–84.PubMedCrossRefGoogle Scholar
  34. 34.
    Bowers KT, Keller JC, Randolph BA, Wick DG, Michaels CM. Optimization of surface micromorphology for enhanced osteoblast responses in vitro. Int J Oral Maxillofac Implants. 1992;7:302–10.PubMedGoogle Scholar
  35. 35.
    Kakiuchi M, Ono K. Preparation of bank bone using defatting, freeze-drying and sterilisation with ethylene oxide gas. Part 2. Clinical evaluation of its efficacy and safety. Int Orthop. 1996;20:147–52.PubMedCrossRefGoogle Scholar
  36. 36.
    Kakiuchi M, Ono K, Nishimura A, Shiokawa H. Preparation of bank bone using defatting, freeze-drying and sterilisation with ethylene oxide gas. Part 1. Experimental evaluation of its efficacy and safety. Int Orthop. 1996;20:142–6.PubMedCrossRefGoogle Scholar
  37. 37.
    Arinzeh TL, Tran T, Mcalary J, Daculsi G. A comparative study of biphasic calcium phosphate ceramics for human mesenchymal stem-cell-induced bone formation. Biomaterials. 2005;26:3631–8.PubMedCrossRefGoogle Scholar
  38. 38.
    Mastrogiacomo M, Muraglia A, Komlev V, Peyrin F, Rustichelli F, Crovace A, Cancedda R. Tissue engineering of bone: search for a better scaffold. Orthod Craniofac Res. 2005;8(4):277–84.PubMedCrossRefGoogle Scholar
  39. 39.
    Dalby MJ, Di Silvio L, Harper EJ, Bonfield W. Increasing hydroxyapatite incorporation into poly(methylmethacrylate) cement increases osteoblast adhesion and response. Biomaterials. 2002;23(2):569–76.PubMedCrossRefGoogle Scholar
  40. 40.
    Matsuda T, Davies JE. The in vitro response of osteoblasts to bioactive glass. Biomaterials. 1987;8:275–84.PubMedCrossRefGoogle Scholar
  41. 41.
    Kubarev OL, Komlev VS, Maitz M, Barinov SM. Bioactive composite ceramics in the hydroxyapatite–tricalcium phosphate system. Dokl Chem. 2007;413:72–4.CrossRefGoogle Scholar
  42. 42.
    Schmal H, Niemeyer P, Roesslein M, Hartl D, Loop T, Südkamp NP, Stark GB, Mehlhorn AT. Comparison of cellular functionality of human mesenchymal stromal cells and PBMC. Cytotherapy. 2007;9:69–79.PubMedCrossRefGoogle Scholar
  43. 43.
    Vogel JP, Szalay K, Geiger F, Kramer M, Richter W, Kasten P. Platelet-rich plasma improves expansion of human mesenchymal stem cells and retains differentiation capacity and in vivo bone formation in calcium phosphate ceramics. Platelets. 2006;17:462–9.PubMedCrossRefGoogle Scholar
  44. 44.
    Vermeulen P, Dickens S, Degezelle K, Van den Berge S, Hendrickx B, Vranckx JJ. A plasma-based biomatrix mixed with endothelial progenitor cells and keratinocytes promotes matrix formation, angiogenesis, and reepithelialization in full-thickness wounds. Tissue Eng Part A. 2009;15:1533–42.PubMedCrossRefGoogle Scholar
  45. 45.
    Ivarsson M, McWhirter A, Borg TK, Rubin K. Type I collagen synthesis in cultured human fibroblasts: regulation by cell spreading, platelet-derived growth factor and interactions with collagen fibers. Matrix Biol. 1998;16:409–25.PubMedCrossRefGoogle Scholar
  46. 46.
    Warstat K, Meckbach D, Weis-Klemm M, Hack A, Klein G, de Zwart P, Aicher WK. TGF-beta enhances the integrin alpha2beta1-mediated attachment of mesenchymal stem cells to type I collagen. Stem Cells Dev. 2010;19(5):645–56.PubMedCrossRefGoogle Scholar
  47. 47.
    Usami K, Mizuno H, Okada K, Narita Y, Aoki M, Kondo T, Mizuno D, Mase J, Nishiguchi H, Kagami H, Ueda M. Composite implantation of mesenchymal stem cells with endothelial progenitor cells enhances tissue-engineered bone formation. J Biomed Mater Res A. 2009;90:730–41.PubMedGoogle Scholar
  48. 48.
    Geuze RE, Wegman F, Oner FC, Dhert WJ, Alblas J. Influence of endothelial progenitor cells and platelet gel on tissue-engineered bone ectopically in goats. Tissue Eng Part A. 2009;15:3669–77.PubMedCrossRefGoogle Scholar
  49. 49.
    Matsumoto T, Kawamoto A, Kuroda R, Ishikawa M, Mifune Y, Iwasaki H, Miwa M, Horii M, Hayashi S, Oyamada A, Nishimura H, Murasawa S, Doita M, Kurosaka M, Asahara T. Therapeutic potential of vasculogenesis and osteogenesis promoted by peripheral blood CD34-positive cells for functional bone healing. Am J Pathol. 2006;169:1440–57.PubMedCrossRefGoogle Scholar
  50. 50.
    Timmermans F, Plum J, Yöder MC, Ingram DA, Vandekerckhove B, Case J. Endothelial progenitor cells: identity defined? J Cell Mol Med. 2009;13:87–102.PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • J. Schultheiss
    • 1
  • C. Seebach
    • 1
  • D. Henrich
    • 1
  • K. Wilhelm
    • 1
  • J. H. Barker
    • 1
  • J. Frank
    • 1
  1. 1.Department of Trauma, Hand and Reconstructive SurgeryJohann-Wolfgang-Goethe UniversityFrankfurtGermany

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