Hematoma-inspired alginate/platelet releasate/CaPO4 composite: initiation of the inflammatory-mediated response associated with fracture repair in vitro and ex vivo injection delivery

  • Jonathan D. McCanlessEmail author
  • Lisa K. Jennings
  • Joel D. Bumgardner
  • Judith A. Cole
  • Warren O. Haggard


A clinical need continues for consistent bone remodeling within problematic sites such as those of fracture nonunion, avascular necrosis, or irregular bone formations. In attempt to address such needs, a biomaterial system is proposed to induce early inflammatory responses after implantation and to provide later osteoconductive scaffolding for bone regeneration. Biomaterial-induced inflammation would parallel the early stage of hematoma-induced fracture repair and allow scaffold-promoted remodeling of osseous tissue to a healthy state. Initiation of the wound healing cascade by two human concentrated platelet releasate-containing alginate/β-tricalcium phosphate biocomposites has been studied in vitro using the TIB-71™ RAW264.7 mouse monocyte cell line. Inflammatory responses inherent to the base material were found and could be modulated through incorporation of platelet releasate. Differences in hydrogel wt% (2 vs. 8 %) and/or calcium phosphate granule vol.% (20 vs. 10 %) allowed for tuning the response associated with platelet releasate-associated growth factor elution. Tunability from completely suppressing the inflammatory response to augmenting the response was observed through varied elution profiles of both releasate-derived bioagents and impurities inherent to alginate. A 2.5-fold upregulation of inducible-nitric oxide synthase gene expression followed by a tenfold increase in nitrite media levels was induced by inclusion of releasate within the 8 wt%/10 vol.% formulation and was comparable to an endotoxin positive control. Whereas, near complete elimination of inflammation was seen when releasate was included within the 2 wt%/20 vol.% formulation. These in vitro results suggested tunable interactions between the multiple platelet releasate-derived bioagents and the biocomposites for enhancing hematoma-like fracture repair. Additionally, minimally invasive delivery for in situ curing of the implant system via injection was demonstrated in rat tail vertebrae using microcomputed tomography.


Nitric Oxide Alginate iNOS Expression Bone Graft Substitute Monocyte Activation 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



The authors would like to acknowledge Dr. Thomas Sutter and Quynh Tran from the W. Harry Feinstone Center for Genomic Research for use of the iCycler and discussions on qRT-PCR data analysis. We also thank Dr. Richard A. Smith, Dr. Jinsong Huang, and Dr. Anobel Maghsoodpour for help in capturing data and software analysis pertaining to μCT. We also recognize Dr. John L. Williams from The University of Memphis for participation in the first author’s doctoral advisory committee and review of the current work.


  1. 1.
    Dimitriou R, Jones E, McGonagle D, Giannoudis PV. Bone regeneration: current concepts and future directions. BMC Med. 2011;9(66):1–10.Google Scholar
  2. 2.
    Bauer TW. An overview of the histology of skeletal substitute materials. Arch Pathol Lab Med. 2007;131(2):217–24.Google Scholar
  3. 3.
    Kolk A, Handschel J, Drescher W, Rothamel D, Kloss F, Blessmann M, et al. Current trends and future perspectives of bone substitute materials—from space holders to innovative biomaterials. J Craniomaxillofac Surg. 2012. [Epub ahead of print].Google Scholar
  4. 4.
    Janicki P, Schmidmaier G. What should be the characteristics of the ideal bone graft substitute? Combining scaffolds with growth factors and/or stem cells. Injury. 2011;42(Suppl 2):S77–81.CrossRefGoogle Scholar
  5. 5.
    Kalfas IH. Principles of bone healing. Neurosurg Focus. 2001;10(4);E1:1–4.CrossRefGoogle Scholar
  6. 6.
    Kolar P, Schmidt-Bleek K, Schell H, Gaber T, Toben D, Schmidmaier G, et al. The early fracture hematoma and its potential role in fracture healing. Tissue Eng B. 2010;16(4):427–34.CrossRefGoogle Scholar
  7. 7.
    Sun Y, Feng Y, Zhang CQ, Chen SB, Cheng XG. The regenerative effect of platelet-rich plasma on healing in large osteochondral defects. Int Orthop. 2010;34(4):589–97.CrossRefGoogle Scholar
  8. 8.
    Burg KJ, Porter S, Kellam JF. Biomaterial developments for bone tissue engineering. Biomaterials. 2000;21(23):2347–59.CrossRefGoogle Scholar
  9. 9.
    Williams DF. On the mechanisms of biocompatibility. Biomaterials. 2008;29(20):2941–53.CrossRefGoogle Scholar
  10. 10.
    El-Sharkawy H, Kantarci A, Deady J, Hasturk H, Liu H, Alshahat M, et al. Platelet-rich plasma: growth factors and pro- and anti-inflammatory properties. J Periodontol. 2007;78(4):661–9.CrossRefGoogle Scholar
  11. 11.
    Mehta S, Watson JT. Platelet rich concentrate: basic science and current clinical applications. J Orthop Trauma. 2008;22(6):432–8.CrossRefGoogle Scholar
  12. 12.
    McCanless JD, Jennings LK, Cole JA, Bumgardner JD, Haggard WO. In vitro differentiation and biocompatibility of mesenchymal stem cells on a novel platelet releasate-containing injectable composite. J Biomed Mater Res A. 2012;100(1):220–9.Google Scholar
  13. 13.
    McCanless JD, Jennings LK, Cole JA, Bumgardner JD, Haggard WO. Induction of the early inflammatory-mediated cellular responses of fracture healing in vitro using platelet releasate-containing alginate/CaPO4 biomaterials for early osteoarthritis prevention. J Biomed Mater Res A. 2012;100(5):1107–14.Google Scholar
  14. 14.
    Haggard WO, McCanless JD, Inventors; University of Memphis, Assignee. Provisional patent application: Biomaterial composition and method of use, 6 May 2011.Google Scholar
  15. 15.
    Beamer B, Hettrich C, Lane J. Vascular endothelial growth factor: an essential component of angiogenesis and fracture healing. HSS J. 2010;6(1):85–94.Google Scholar
  16. 16.
    Ma PX. Alginate for tissue engineering. In: Ma PX, Elisseeff J, editors. Scaffolding in tissue engineering. Boca Raton: CRC Press; 2006. p. 13–25.Google Scholar
  17. 17.
    Nunamaker EA, Purcell EK, Kipke DR. In vivo stability and biocompatibility of implanted calcium alginate disks. J Biomed Mater Res A. 2007;83(4):1128–37.Google Scholar
  18. 18.
    Kong H, Mooney DJ. Polysaccharide-based hydrogels in tissue engineering. In: Dumitriu S, editor. Polysaccharides: structural diversity and functional versatility. 2nd ed. New York: CCR Press; 2005. p. 817–38.Google Scholar
  19. 19.
    White MM, Jennings LK. Appendix. Platelet protocols: research and clinical laboratory procedures. San Diego: Academic Press; 1999. p. 99–101.Google Scholar
  20. 20.
    Nicolin V, Ponti C, Narducci P, Grill V, Bortul R, Zweyer M, et al. Different levels of the neuronal nitric oxide synthase isoform modulate the rate of osteoclastic differentiation of TIB-71 and CRL-2278 RAW 264.7 murine cell clones. Anat Rec A. 2005;286(2):945–54.Google Scholar
  21. 21.
    Lock EF, Ziemiecke R, Marron J, Dittmer DP. Efficiency clustering for low-density microarrays and its application to QPCR. BMC Bioinform. 2010;11(386):1–8.Google Scholar
  22. 22.
    McCanless J, Cole J, Jennings L, Bumgardner J, Haggard W, editors. Biomaterial-assessed fracture healing as a model for the early prevention of joint degeneration: in vitro osteochondrogenesis, angiogenesis, immune response, and ex vivo injection delivery (in review). 58th Annual Meeting of the Orthopaedic Research Society, San Franscisco, CA; 2012.Google Scholar
  23. 23.
    Goldring SR. The role of bone in osteoarthritis pathogenesis. Rheum Dis Clin N Am. 2008;34(3):561–71.CrossRefGoogle Scholar
  24. 24.
    Motamedi K, Seeger LL. Benign bone tumors. Radiol Clin N Am. 2011;49(6):1115–34, v.Google Scholar
  25. 25.
    Schmidhammer R, Zandieh S, Mittermayr R, Pelinka LE, Leixnering M, Hopf R, et al. Assessment of bone union/nonunion in an experimental model using microcomputed technology. J Trauma. 2006;61(1):199–205.CrossRefGoogle Scholar
  26. 26.
    Mora R, Paley D. Nonunion of the long bones: diagnosis and treatment with compression–distraction techniques. New York: Springer; 2006.CrossRefGoogle Scholar
  27. 27.
    Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature. 2003;423(6937):337–42.CrossRefGoogle Scholar
  28. 28.
    Gordon S, Taylor PR. Monocyte and macrophage heterogeneity. Nat Rev Immunol. 2005;5(12):953–64.CrossRefGoogle Scholar
  29. 29.
    Tanaka T, Saito M, Chazono M, Kumagae Y, Kikuchi T, Kitasato S, et al. Effects of alendronate on bone formation and osteoclastic resorption after implantation of beta-tricalcium phosphate. J Biomed Mater Res. 2010;93(2):469–74.Google Scholar
  30. 30.
    Semple JW, Italiano JE Jr, Freedman J. Platelets and the immune continuum. Nat Rev Immunol. 2011;11(4):264–74.CrossRefGoogle Scholar
  31. 31.
    Brass L. Understanding and evaluating platelet function. Hematol Am Soc Hematol Educ Program. 2010;2010:387–96.CrossRefGoogle Scholar
  32. 32.
    Leslie M. Cell biology. Beyond clotting: the powers of platelets. Science (New York, NY). 2010;328(5978):562–4.Google Scholar
  33. 33.
    dos Santos EA, Farina M, Soares GA, Anselme K. Surface energy of hydroxyapatite and beta-tricalcium phosphate ceramics driving serum protein adsorption and osteoblast adhesion. J Mater Sci Mater Med. 2008;19(6):2307–16.CrossRefGoogle Scholar
  34. 34.
    Aslam R, Speck ER, Kim M, Crow AR, Bang KW, Nestel FP, et al. Platelet Toll-like receptor expression modulates lipopolysaccharide-induced thrombocytopenia and tumor necrosis factor-alpha production in vivo. Blood. 2006;107(2):637–41.CrossRefGoogle Scholar
  35. 35.
    Shiraki R, Inoue N, Kawasaki S, Takei A, Kadotani M, Ohnishi Y, et al. Expression of Toll-like receptors on human platelets. Thromb Res. 2004;113(6):379–85.CrossRefGoogle Scholar
  36. 36.
    Haselmayer P, Grosse-Hovest L, von Landenberg P, Schild H, Radsak MP. TREM-1 ligand expression on platelets enhances neutrophil activation. Blood. 2007;110(3):1029–35.CrossRefGoogle Scholar
  37. 37.
    Washington AV, Gibot S, Acevedo I, Gattis J, Quigley L, Feltz R, et al. TREM-like transcript-1 protects against inflammation-associated hemorrhage by facilitating platelet aggregation in mice and humans. J Clin Investig. 2009;119(6):1489–501.CrossRefGoogle Scholar
  38. 38.
    Mitani T, Terashima M, Yoshimura H, Nariai Y, Tanigawa Y. TGF-beta1 enhances degradation of IFN-gamma-induced iNOS protein via proteasomes in RAW 264.7 cells. Nitric Oxide. 2005;13(1):78–87.CrossRefGoogle Scholar
  39. 39.
    Gruber R, Karreth F, Fischer MB, Watzek G. Platelet-released supernatants stimulate formation of osteoclast-like cells through a prostaglandin/RANKL-dependent mechanism. Bone. 2002;30(5):726–32.CrossRefGoogle Scholar
  40. 40.
    Cuetara BL, Crotti TN, O’Donoghue AJ, McHugh KP. Cloning and characterization of osteoclast precursors from the RAW264.7 cell line. In Vitro Cell Dev Biol Anim. 2006;42(7):182–8.CrossRefGoogle Scholar
  41. 41.
    Gurevitch O, Slavin S, Resnick I, Khitrin S, Feldman A. Mesenchymal progenitor cells in red and yellow bone marrow. Folia Biol (Praha). 2009;55(1):27–34.Google Scholar
  42. 42.
    Muguruma Y, Yahata T, Miyatake H, Sato T, Uno T, Itoh J, et al. Reconstitution of the functional human hematopoietic microenvironment derived from human mesenchymal stem cells in the murine bone marrow compartment. Blood. 2006;107(5):1878–87.CrossRefGoogle Scholar
  43. 43.
    Wang ML, Massie J, Perry A, Garfin SR, Kim CW. A rat osteoporotic spine model for the evaluation of bioresorbable bone cements. Spine J. 2007;7(4):466–74.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Jonathan D. McCanless
    • 1
    Email author
  • Lisa K. Jennings
    • 2
    • 3
  • Joel D. Bumgardner
    • 1
  • Judith A. Cole
    • 4
  • Warren O. Haggard
    • 1
  1. 1.Biomedical Engineering DepartmentHerff College of Engineering, The University of MemphisMemphisUSA
  2. 2.Department of Internal MedicineCollege of Medicine, The University of Tennessee Health Science CenterMemphisUSA
  3. 3.Vascular Biology Center of ExcellenceThe University of Tennessee Health Science CenterMemphisUSA
  4. 4.Department of Biological SciencesCollege of Arts and Sciences, The University of MemphisMemphisUSA

Personalised recommendations