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Plasma clots gelled by different amounts of calcium for stem cell delivery

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Abstract

Purpose

Freshly prepared autologous plasma clots may serve as a carrier matrix for expanded multipotent mesenchymal stromal cells (MSCs) or bone marrow cells. By varying the calcium concentration, plasma clots with different properties can be produced. The purpose of this in vitro study was to determine the optimal calcium concentrations for the clotting process, intra-clot cell viability, and clot lysis.

Methods

Different plasma clots were prepared by adding an equal volume of RPMI1640 (with or without MSCs) to citrate plasma (either containing platelets or platelet-free). Clotting was initiated by the addition of CaCl2 (10 g/100 ml H2O, 10 % solution). The final concentration of CaCl2 ranged from 1 to 10 % by volume of plasma. Viability and distribution of the MSCs were analysed by calcein-AM/propidium iodide staining. MSC-embedded plasma clots were dissolved with trypsin (0.25 %), and recovered cells were further incubated for 1 week under cell culture conditions.

Results

The viability of MSCs embedded in clots formed by the addition of 1–8 % by volume CaCl2 was not affected by incubation of up to 1 week. In contrast, clots produced by higher volumes of CaCl2 solutions (9–10 % by volume of plasma) showed decreased numbers of viable cells. Intra-clot cell proliferation was highest in clots produced by addition of 5 % CaCl2 by plasma volume. Osteocalcin release was not influenced in platelet-free plasma but decreased in platelet-containing plasma. Morphological analysis of stained recovered MSCs revealed that lysis of the plasma clot did not affect cell morphology or subsequent spontaneous proliferation.

Conclusions

Clot formation and clot stability can be controlled by changing the concentration of CaCl2 added to plasma. The addition of 5 % CaCl2 produced a plasma clot with optimal results for stem cell delivery.

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References

  1. Nodarian T, Sariali E, Khiami F, Pascal-Mousselard H, Catonne Y (2010) Iliac crest bone graft harvesting complications: a case of liver herniation. Orthop Traumatol Surg Res. doi:10.1016/j.otsr.2010.03.016

  2. Arrington ED, Smith WJ, Chambers HG, Bucknell AL, Davino NA (1996) Complications of iliac crest bone graft harvesting. Clin Orthop Relat Res 329:300–309

    Article  PubMed  Google Scholar 

  3. Fowler BL, Dall BE, Rowe DE (1995) Complications associated with harvesting autogenous iliac bone graft. Am J Orthop (Belle Mead NJ) 24(12):895–903

    CAS  Google Scholar 

  4. Trombi L, Mattii L, Pacini S, D’Alessandro D, Battolla B, Orciuolo E, Buda G, Fazzi R, Galimberti S, Petrini M (2008) Human autologous plasma-derived clot as a biological scaffold for mesenchymal stem cells in treatment of orthopedic healing. J Orthop Res 26(2):176–183. doi:10.1002/jor.20490

    Article  PubMed  Google Scholar 

  5. Ho W, Tawil B, Dunn JC, Wu BM (2006) The behavior of human mesenchymal stem cells in 3D fibrin clots: dependence on fibrinogen concentration and clot structure. Tissue Eng 12(6):1587–1595. doi:10.1089/ten.2006.12.1587

    Article  PubMed  CAS  Google Scholar 

  6. Granero-Molto F, Weis JA, Miga MI, Landis B, Myers TJ, O’Rear L, Longobardi L, Jansen ED, Mortlock DP, Spagnoli A (2009) Regenerative effects of transplanted mesenchymal stem cells in fracture healing. Stem Cells 27(8):1887–1898. doi:10.1002/stem.103

    Article  PubMed  CAS  Google Scholar 

  7. Yamada Y, Ueda M, Naiki T, Takahashi M, Hata K, Nagasaka T (2004) Autogenous injectable bone for regeneration with mesenchymal stem cells and platelet-rich plasma: tissue-engineered bone regeneration. Tissue Eng 10(5–6):955–964. doi:10.1089/1076327041348284

    Article  PubMed  CAS  Google Scholar 

  8. Bruder SP, Kraus KH, Goldberg VM, Kadiyala S (1998) The effect of implants loaded with autologous mesenchymal stem cells on the healing of canine segmental bone defects. J Bone Joint Surg Am 80(7):985–996

    PubMed  CAS  Google Scholar 

  9. Quarto R, Mastrogiacomo M, Cancedda R, Kutepov SM, Mukhachev V, Lavroukov A, Kon E, Marcacci M (2001) Repair of large bone defects with the use of autologous bone marrow stromal cells. N Engl J Med 344(5):385–386. doi:10.1056/NEJM200102013440516

    Article  PubMed  CAS  Google Scholar 

  10. Hernigou P, Mathieu G, Poignard A, Manicom O, Beaujean F, Rouard H (2006) Percutaneous autologous bone-marrow grafting for nonunions. Surgical technique. J Bone Joint Surg Am 88(Suppl 1 Pt 2):322–327. doi:10.2106/JBJS.F.00203

    PubMed  Google Scholar 

  11. Hernigou P, Poignard A, Beaujean F, Rouard H (2005) Percutaneous autologous bone-marrow grafting for nonunions. Influence of the number and concentration of progenitor cells. J Bone Joint Surg Am 87(7):1430–1437. doi:10.2106/JBJS.D.02215

    Article  PubMed  Google Scholar 

  12. Garnavos C, Mouzopoulos G, Morakis E (2010) Fixed intramedullary nailing and percutaneous autologous concentrated bone-marrow grafting can promote bone healing in humeral-shaft fractures with delayed union. Injury 41(6):563–567. doi:10.1016/j.injury.2009.08.003

    Article  PubMed  Google Scholar 

  13. Ben-Ari A, Rivkin R, Frishman M, Gaberman E, Levdansky L, Gorodetsky R (2009) Isolation and implantation of bone marrow-derived mesenchymal stem cells with fibrin micro beads to repair a critical-size bone defect in mice. Tissue Eng Part A 15(9):2537–2546. doi:10.1089/ten.tea.2008.0567

    Article  PubMed  CAS  Google Scholar 

  14. Trombi L, D’Alessandro D, Pacini S, Fiorentino B, Scarpellini M, Fazzi R, Galimberti S, Guazzini S, Petrini M (2008) Good manufacturing practice-grade fibrin gel is useful as a scaffold for human mesenchymal stromal cells and supports in vitro osteogenic differentiation. Transfusion 48(10):2246–2251. doi:10.1111/j.1537-2995.2008.01829.x

    Article  PubMed  Google Scholar 

  15. Zhu SJ, Choi BH, Jung JH, Lee SH, Huh JY, You TM, Lee HJ, Li J (2006) A comparative histologic analysis of tissue-engineered bone using platelet-rich plasma and platelet-enriched fibrin glue. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 102(2):175–179. doi:10.1016/j.tripleo.2005.08.034

    Article  PubMed  Google Scholar 

  16. Bensaid W, Triffitt JT, Blanchat C, Oudina K, Sedel L, Petite H (2003) A biodegradable fibrin scaffold for mesenchymal stem cell transplantation. Biomaterials 24(14):2497–2502

    Article  PubMed  CAS  Google Scholar 

  17. Wolberg AS, Campbell RA (2008) Thrombin generation, fibrin clot formation and hemostasis. Transfus Apher Sci 38(1):15–23. doi:10.1016/j.transci.2007.12.005

    Article  PubMed  Google Scholar 

  18. Farrior E, Ladner K (2011) Platelet gels and hemostasis in facial plastic surgery. Facial Plast Surg 27(4):308–314. doi:10.1055/s-0031-1283050

    Article  PubMed  CAS  Google Scholar 

  19. Dohan Ehrenfest DM, Bielecki T, Mishra A, Borzini P, Inchingolo F, Sammartino G, Rasmusson L, Everts PA (2012) In search of a consensus terminology in the field of platelet concentrates for surgical use: platelet-rich plasma (PRP), platelet-rich fibrin (PRF), fibrin gel polymerization and leukocytes. Curr Pharm Biotechnol 13(7):1131–1137

    PubMed  Google Scholar 

  20. Ferris D, Frisbie D, Kisiday J, McIlwraith CW (2012) In vivo healing of meniscal lacerations using bone marrow-derived mesenchymal stem cells and fibrin glue. Stem Cells Int 2012:691605. doi:10.1155/2012/691605

    PubMed  Google Scholar 

  21. Schildhauer TA, Seybold D, Geßmann J, Muhr G, Köller M (2007) Fixation of porous calcium phosphate with expanded bone marrow cells using an autologous plasma clot. Mat-wiss u Werkstofftech 38(12):1012–1014. doi:10.1002/mawe.200700239

    Article  CAS  Google Scholar 

  22. Weisel JW (2007) Structure of fibrin: impact on clot stability. J Thromb Haemost 5(Suppl 1):116–124. doi:10.1111/j.1538-7836.2007.02504.x

    Article  PubMed  CAS  Google Scholar 

  23. Wolberg AS (2010) Plasma and cellular contributions to fibrin network formation, structure and stability. Haemophilia 16(Suppl 3):7–12. doi:10.1111/j.1365-2516.2010.02253.x

    Article  PubMed  CAS  Google Scholar 

  24. Rock G, Neurath D, Lu M, Alharbi A, Freedman M (2006) The contribution of platelets in the production of cryoprecipitates for use in a fibrin glue. Vox Sang 91(3):252–255. doi:10.1111/j.1423-0410.2006.00788.x

    Article  PubMed  CAS  Google Scholar 

  25. Sanchez AR, Sheridan PJ, Kupp LI (2003) Is platelet-rich plasma the perfect enhancement factor? A current review. Int J Oral Maxillofac Implants 18(1):93–103

    PubMed  Google Scholar 

  26. Slater M, Patava J, Kingham K, Mason RS (1995) Involvement of platelets in stimulating osteogenic activity. J Orthop Res 13(5):655–663. doi:10.1002/jor.1100130504

    Article  PubMed  CAS  Google Scholar 

  27. Catelas I, Sese N, Wu BM, Dunn JC, Helgerson S, Tawil B (2006) Human mesenchymal stem cell proliferation and osteogenic differentiation in fibrin gels in vitro. Tissue Eng 12(8):2385–2396. doi:10.1089/ten.2006.12.2385

    Article  PubMed  CAS  Google Scholar 

  28. Isogai N, Landis WJ, Mori R, Gotoh Y, Gerstenfeld LC, Upton J, Vacanti JP (2000) Experimental use of fibrin glue to induce site-directed osteogenesis from cultured periosteal cells. Plast Reconstr Surg 105(3):953–963

    Article  PubMed  CAS  Google Scholar 

  29. Lee OK (2008) Fibrin glue as a vehicle for mesenchymal stem cell delivery in bone regeneration. J Chin Med Assoc 71(2):59–61. doi:10.1016/S1726-4901(08)70075-3

    Article  PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of General and Trauma Surgery, BG University Hospital Bergmannsheil, Ruhr University Bochum, Bürkle-de-la-Camp-Platz 1, 44789, Bochum, Germany

    Jan Gessmann, Dominik Seybold & Thomas Armin Schildhauer

  2. Department of Surgical Research, BG University Hospital Bergmannsheil, Ruhr University Bochum, Bürkle-de-la-Camp-Platz 1, 44789, Bochum, Germany

    Elvira Peter & Manfred Köller

Authors
  1. Jan Gessmann
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  2. Dominik Seybold
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  3. Elvira Peter
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  4. Thomas Armin Schildhauer
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Correspondence to Jan Gessmann.

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Gessmann, J., Seybold, D., Peter, E. et al. Plasma clots gelled by different amounts of calcium for stem cell delivery. Langenbecks Arch Surg 398, 161–167 (2013). https://doi.org/10.1007/s00423-012-1015-8

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  • DOI: https://doi.org/10.1007/s00423-012-1015-8

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