PDF herunterladen
Vet Comp Orthop Traumatol 2013; 26(01): 34-41
DOI: 10.3415/VCOT-11-11-0165
Original Research
Schattauer GmbH

Bone marrow concentrate for autologous transplantation in minipigs

Characterization and osteogenic potential of mesenchymal stem cells
M. Herten
1   Department of Orthopaedics, Heinrich-Heine-University, University Hospital, Düsseldorf, Germany
,
J. P. Grassmann
2   Department of Trauma and Hand Surgery, Heinrich-Heine-University, University Hospital, Düsseldorf, Germany
,
M. Sager
3   Central Animal Research Facility, Heinrich-Heine-University, University Hospital, Düsseldorf, Germany
,
L. Benga
3   Central Animal Research Facility, Heinrich-Heine-University, University Hospital, Düsseldorf, Germany
,
J. C. Fischer
4   Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich-Heine-University, University Hospital, Düsseldorf, Germany
,
M. Jäger
5   University Duisburg-Essen, University Hospital, Department of Orthopaedics, Essen, Germany
,
M. Betsch
3   Central Animal Research Facility, Heinrich-Heine-University, University Hospital, Düsseldorf, Germany
,
M. Wild
2   Department of Trauma and Hand Surgery, Heinrich-Heine-University, University Hospital, Düsseldorf, Germany
,
M. Hakimi
2   Department of Trauma and Hand Surgery, Heinrich-Heine-University, University Hospital, Düsseldorf, Germany
,
P. Jungbluth
2   Department of Trauma and Hand Surgery, Heinrich-Heine-University, University Hospital, Düsseldorf, Germany
› Institutsangaben
Weitere Informationen

Publikationsverlauf

Received 06. Dezember 2011

Accepted 16. Juli 2012

Publikationsdatum:
19. Dezember 2017 (online)

Auch verfügbar auf
    Lizenzen und Reprints

Summary

Autologous bone marrow plays an increasing role in the treatment of bone, cartilage and tendon healing disorders. Cell-based therapies display promising results in the support of local regeneration, especially therapies using intra-operative one-step treatments with autologous progenitor cells. In the present study, bone marrow-derived cells were concentrated in a point-of-care device and investigated for their mesenchymal stem cell (MSC) characteristics and their osteogenic potential.

Bone marrow was harvested from the iliac crest of 16 minipigs. The mononucleated cells (MNC) were concentrated by gradient density centrifugation, cultivated, characterized by flow cytometry and stimulated into osteoblasts, adipocytes, and chondrocytes. Cell differentiation was investigated by histological and immunohistological staining of relevant lineage markers. The proliferation capacity was determined via colony forming units of fibroblast and of osteogenic alkaline-phosphatase-positive-cells.

The MNC could be enriched 3.5-fold in nucleated cell concentrate in comparison to bone marrow. Flow cytometry analysis revealed a positive signal for the MSC markers. Cells could be differentiated into the three lines confirming the MSC character. The cellular osteogenic potential correlated significantly with the percentage of newly formed bone in vivo in a porcine metaphyseal long-bone defect model.

This study demonstrates that bone marrow concentrate from minipigs display cells with MSC character and their osteogenic differentiation potential can be used for osseous defect repair in autologous transplantations.

Keywords

Mesenchymal stem cells - minipig - bone marrow concentrate - colony forming units
 

  • References

  • 1 Dominici M, Le Blanc K, Mueller I. et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006; 8: 315-317.
    CrossrefPubMedGoogle Scholar
  • 2 Friedenstein AJ, Petrakova KV, Kurolesova AI. et al. Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation 1968; 6: 230-247.
    CrossrefPubMedGoogle Scholar
  • 3 Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997; 276: 71-74.
    CrossrefPubMedGoogle Scholar
  • 4 Caplan AI. Mesenchymal stem cells. J Orthop Res 1991; 9: 641-650.
    CrossrefPubMedGoogle Scholar
  • 5 Haynesworth SE, Goshima J, Goldberg VM. et al. Characterization of cells with osteogenic potential from human marrow. Bone 1992; 13: 81-88.
    CrossrefPubMedGoogle Scholar
  • 6 Bennett JH, Joyner CJ, Triffitt JT. et al. Adipocytic cells cultured from marrow have osteogenic potential. J Cell Sci 1991; 99: 131-139.
    PubMedGoogle Scholar
  • 7 Beresford JN, Bennett JH, Devlin C. et al. Evidence for an inverse relationship between the differentiation of adipocytic and osteogenic cells in rat marrow stromal cell cultures. J Cell Sci 1992; 102: 341-351.
    CrossrefPubMedGoogle Scholar
  • 8 Yoo JU, Barthel TS, Nishimura K. et al. The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells. J Bone Joint Surg Am 1998; 80: 1745-1757.
    CrossrefPubMedGoogle Scholar
  • 9 Johnstone B, Hering TM, Caplan AI. et al. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res 1998; 238: 265-272.
    CrossrefPubMedGoogle Scholar
  • 10 Wakitani S, Saito T, Caplan AI. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve 1995; 18: 1417-1426.
    CrossrefPubMedGoogle Scholar
  • 11 Jiang Y, Jahagirdar BN, Reinhardt RL. et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002; 418: 41-49.
    CrossrefPubMedGoogle Scholar
  • 12 Kawada H, Fujita J, Kinjo K. et al. Nonhematopoietic mesenchymal stem cells can be mobilized and differentiate into cardiomyocytes after myocardial infarction. Blood 2004; 104: 3581-3587.
    CrossrefPubMedGoogle Scholar
  • 13 Young RG, Butler DL, Weber W. et al. Use of mesenchymal stem cells in a collagen matrix for Achilles tendon repair. J Orthop Res 1998; 16: 406-413.
    CrossrefPubMedGoogle Scholar
  • 14 Caplan AI, Dennis JE. Mesenchymal stem cells as trophic mediators. J Cell Biochem 2006; 98: 1076-1084.
    CrossrefPubMedGoogle Scholar
  • 15 Le Blanc K. Immunomodulatory effects of fetal and adult mesenchymal stem cells. Cytotherapy 2003; 5: 485-489.
    CrossrefPubMedGoogle Scholar
  • 16 da Silva Meirelles L, Chagastelles PC, Nardi NB. Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci 2006; 119: 2204-2213.
    CrossrefPubMedGoogle Scholar
  • 17 Bianco P, Riminucci M, Gronthos S. et al. Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells 2001; 19: 180-192.
    CrossrefPubMedGoogle Scholar
  • 18 Connolly JF, Guse R, Tiedeman J. et al. Autologous marrow injection as a substitute for operative grafting of tibial nonunions. Clin Orthop Relat Res 1991; 259-270.
    PubMedGoogle Scholar
  • 19 Jäger M, Hernigou P, Zilkens C. et al. Cell therapy in bone healing disorders. Orthop Rev 2010; 2: 79-87.
    PubMedGoogle Scholar
  • 20 Owen ME, Cave J, Joyner CJ. Clonal analysis in vitro of osteogenic differentiation of marrow CFU-F. J Cell Sci 1987; 87: 731-738.
    PubMedGoogle Scholar
  • 21 Castro-Malaspina H, Gay RE, Resnick G. et al. Characterization of human bone marrow fibroblast colony-forming cells (CFU-F) and their progeny. Blood 1980; 56: 289-301.
    PubMedGoogle Scholar
  • 22 Hernigou P, Poignard A, Beaujean F. et al. Percutaneous autologous bone-marrow grafting for nonunions. Influence of the number and concentration of progenitor cells. J Bone Joint Surg Am 2005; 87: 1430-1437.
    PubMedGoogle Scholar
  • 23 Solchaga LA, Penick KJ, Welter JF. Chondrogenic differentiation of bone marrow-derived mesenchymal stem cells: tips and tricks. Methods Mol Biol 2011; 698: 253-278.
    PubMedGoogle Scholar
  • 24 Wheeler DL, Cross AR, Eschbach EJ. et al. Grafting of massive tibial subchondral bone defects in a caprine model using beta-tricalcium phosphate versus autograft. J Orthop Trauma 2005; 19: 85-91.
    CrossrefPubMedGoogle Scholar
  • 25 Eley KA, Witherow H, Hayward R. et al. The evaluation of bony union after frontofacial distraction. J Craniofac Surg 2009; 20: 275-278.
    CrossrefPubMedGoogle Scholar
  • 26 Jungbluth P, Hakimi M, Grassmann P. et al. The progress of early phase bone healing using porous granules produced from calcium phosphate cement. Eur J Med Res 2010; 15: 196-203.
    PubMedGoogle Scholar
  • 27 Jungbluth P, Wild M, Grassmann JP. et al. Platelet-rich plasma on calcium phosphate granules promote metaphyseal bone healing in mini-pigs. J Orthop Res 2010; 28: 1448-1455.
    CrossrefPubMedGoogle Scholar
  • 28 Hakimi M, Jungbluth P, Sager M. et al. Combined use of platelet-rich plasma and autologous bone grafts in the treatment of long bone defects in mini-pigs. Injury 2010; 41: 717-723.
    CrossrefPubMedGoogle Scholar
  • 29 Thoesen MS, Berg-Foels WS, Stokol T. et al. Use of a centrifugation-based, point-of-care device for production of canine autologous bone marrow and platelet concentrates. Am J Vet Res 2006; 67: 1655-1661.
    CrossrefPubMedGoogle Scholar
  • 30 Jäger M, Herten M, Fochtmann U. et al. Bridging the gap: Bone marrow aspiration concentrate reduces autologous bone grafting in osseous defects. J Orthop Res 2011; 29: 173-180.
    CrossrefPubMedGoogle Scholar
  • 31 Hermann PC, Huber SL, Herrler T. et al. Concentration of bone marrow total nucleated cells by a point-of-care device provides a high yield and preserves their functional activity. Cell Transplant 2008; 16: 1059-1069.
    PubMedGoogle Scholar
  • 32 Agay D, Scherthan H, Forcheron F. et al. Multipotent mesenchymal stem cell grafting to treat cutaneous radiation syndrome: development of a new minipig model. Exp Hematol 2010; 38: 945-956.
    CrossrefPubMedGoogle Scholar
  • 33 Chang C, Niu D, Zhou H. et al. Mesenchymal stroma cells improve hyperglycemia and insulin deficiency in the diabetic porcine pancreatic microenvironment. Cytotherapy 2008; 10: 796-805.
    CrossrefPubMedGoogle Scholar
  • 34 Muschler GF, Nitto H, Boehm CA. et al. Age- and gender-related changes in the cellularity of human bone marrow and the prevalence of osteoblastic progenitors. J Orthop Res 2001; 19: 117-125.
    CrossrefPubMedGoogle Scholar
  • 35 Janicki P, Boeuf S, Steck E. et al. Prediction of in vivo bone forming potency of bone marrow-derived human mesenchymal stem cells. Eur Cell Mater 2011; 21: 488-507.
    PubMedGoogle Scholar
  • 36 Seeger FH, Tonn T, Krzossok N. et al. Cell isolation procedures matter: a comparison of different isolation protocols of bone marrow mononuclear cells used for cell therapy in patients with acute myocardial infarction. Eur Heart J 2007; 28: 766-772.
    CrossrefPubMedGoogle Scholar
  • 37 Turan RG, Bozdag-Turan I, Ortak J. et al. Improvement of cardiac function by intracoronary freshly isolated bone marrow cells transplantation in patients with acute myocardial infarction. Circ J 2011; 75: 683-691.
    CrossrefPubMedGoogle Scholar
  • 38 Iba O, Matsubara H, Nozawa Y. et al. Angiogenesis by implantation of peripheral blood mononuclear cells and platelets into ischemic limbs. Circulation 2002; 106: 2019-2025.
    CrossrefPubMedGoogle Scholar
  • 39 Massberg S, Konrad I, Schurzinger K. et al. Platelets secrete stromal cell-derived factor 1alpha and recruit bone marrow-derived progenitor cells to arterial thrombi in vivo. J Exp Med 2006; 203: 1221-1233.
    CrossrefPubMedGoogle Scholar