Modulation of mesenchymal stem cell behavior by nano- and micro-sized β-tricalcium phosphate particles in suspension and composite structures

  • Mollie Smoak
  • Katie Hogan
  • Lisa Kriegh
  • Cong Chen
  • LeKeith B. Terrell
  • Ammar T. Qureshi
  • W. Todd Monroe
  • Jeffrey M. Gimble
  • Daniel J. Hayes
Research Paper


Interest has grown in the use of microparticles and nanoparticles for modifying the mechanical and biological properties of synthetic bone composite structures. Micro- and nano-sized calcium phosphates are of interest for their osteoinductive behavior. Engineered composites incorporating polymers and ceramics, such as poly-l-lactic acid (PLLA) and beta-tricalcium phosphate (β-TCP), for bone tissue regeneration have been well investigated for their proliferative and osteoinductive abilities. Only limited research has been done to investigate the effects of different sizes of β-TCP particles on human mesenchymal stromal cell behavior. As such, the aim of this study was to investigate the modulations of human adipose-derived stem cell (hASCs) behavior within cell/particle and cell/composite systems as functions of particle size, concentration, and exposure time. The incorporation of nanoscale calcium phosphate resulted in improved mechanical properties and osteogenic behavior within the scaffold compared to the microscale calcium phosphate additives. Particle exposure results indicate that cytotoxicity on hASCs correlates inversely with particle size and increases with the increasing exposure time and particle concentration. Composites with increasing β-TCP content, whether microparticles or nanoparticles, were less toxic than colloidal micro- and nano-sized β-TCP particles directly supplied to hASCs. The difference in viability observed as a result of varying exposure route is likely related to the increased cell–particle interactions in the direct exposure compared to the particles becoming trapped within the scaffold/polymer matrix.


Mesenchymal stem cells Cytotoxicity Inflammation Beta-tricalcium phosphate Nano/microparticle Health effects 



The authors would like to thank Dr. Amar Karki and Dr. Dongmei Cao for their assistance in XRD and FIB-SEM characterizations.

Conflict of interests

All authors declare that they have no competing interests with the exception of J.M.G. who is a co-founder and co-owner of LaCell LLC, a biotechnology company focused on the use of hASCs for regenerative medical applications.

Supplementary material

11051_2015_2985_MOESM1_ESM.docx (271 kb)
Supplementary material 1 (DOCX 271 kb)


  1. Ambard AJ, Mueninghoff L (2006) Calcium phosphate cement: review of mechanical and biological properties. J Prosthodont 15(5):321–328. doi: 10.1111/j.1532-849X.2006.00129.x CrossRefGoogle Scholar
  2. Aubin J (2001) Regulation of osteoblast formation and function. Rev Endocr Metab Disord 2(1):81–94. doi: 10.1023/A:1010011209064 CrossRefGoogle Scholar
  3. Aunoble S, Clément D, Frayssinet P, Harmand MF, Le Huec JC (2006) Biological performance of a new β-TCP/PLLA composite material for applications in spine surgery: in vitro and in vivo studies. J Biomed Mater Res, Part A 78A(2):416–422. doi: 10.1002/jbm.a.30749 CrossRefGoogle Scholar
  4. Bastioli C (ed) (2005) Handbook of biodegradable polymers. Rapra Technology Ltd, ShawburyGoogle Scholar
  5. Blaker J, Maquet V, Jerome R, Boccaccini A, Nazhat S (2005) Mechanical properties of highly porous PDLLA/Bioglass composite foams as scaffolds for bone tissue engineering. Acta Biomater 1(6):643–652. doi: 10.1016/j.actbio.2005.07.003 CrossRefGoogle Scholar
  6. Chai C, Leong KW (2007) Biomaterials approach to expand and direct differentiation of stem cells. Mol Ther 15(3):467–480CrossRefGoogle Scholar
  7. Charles-Harris M, Koch MA, Navarro M, Lacroix D, Engel E, Planell JA (2008) A PLA/calcium phosphate degradable composite material for bone tissue engineering: an in vitro study. J Mater Sci 19(4):1503–1513. doi: 10.1007/s10856-008-3390-9 Google Scholar
  8. Chen C, Garber L, Smoak M, Fargason C, Scherr T, Blackburn C, Hayes DJ (2014) In vitro and In vivo characterization of pentaerythritol triacrylate-co-trimethylolpropane nanocomposite scaffolds as potential bone augments and grafts. Tissue Eng Part A. doi: 10.1089/ten.tea.2014.0018 Google Scholar
  9. Chou Y-F, Chiou W-A, Xu Y, Dunn JCY, Wu BM (2004) effect of pH on the structural evolution of accelerated biomimetic apatite. Biomaterials 25(22):5323–5331. doi: 10.1016/j.biomaterials.2003.12.037 CrossRefGoogle Scholar
  10. El-Ghannam AR (2004) Advanced bioceramic composite for bone tissue engineering: design principles and structure–bioactivity relationship. J Biomed Mater Res 69A:490–501CrossRefGoogle Scholar
  11. Garber L, Chen C, Kilchrist KV, Bounds C, Pojman JA, Hayes D (2013) Thiol-acrylate nanocomposite foams for critical size bone defect repair: a novel biomaterial. J Biomed Mater Res, Part A 101(12):3531–3541. doi: 10.1002/jbm.a.34651 CrossRefGoogle Scholar
  12. Gimble JM, Guilak F (2003) Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy (Taylor & Francis Ltd) 5(5):362–369CrossRefGoogle Scholar
  13. Gunatillake PA, Adhikari R (2003) Biodegradable synthetic polymers for tissue engineering. Eur Cells Mater 5:1–16Google Scholar
  14. Haaparanta A-M, Haimi S, Ellä V, Hopper N, Miettinen S, Suuronen R, Kellomäki M (2010) Porous polylactide/β-tricalcium phosphate composite scaffolds for tissue engineering applications. J Tissue Eng Regen Med 4(5):366–373. doi: 10.1002/term.249 CrossRefGoogle Scholar
  15. Habibovic P, Yuan H, van der Valk CM, Meijer G, van Blitterswijk CA, de Groot K (2005) 3D microenvironment as essential element for osteoinduction by biomaterials. Biomaterials 26(17):3565–3575. doi: 10.1016/j.biomaterials.2004.09.056 CrossRefGoogle Scholar
  16. Hutmacher DW (2000) Scaffolds in tissue engineering bone and cartilage. Biomaterials 21:2529–2543CrossRefGoogle Scholar
  17. Hutmacher DW, Schantz JT, Lam CXF, Tan KC, Lim TC (2007) State of the art and future directions of scaffold-based bone engineering from a biomaterials perspective. J Tissue Eng Regen Med 1(4):245–260. doi: 10.1002/term.24 CrossRefGoogle Scholar
  18. Ignjatovic N, Ninkov P, Ajdukovic Z, Vasiljevic-Radovic D, Uskokovic D (2007) Biphasic calcium phosphate coated with poly-d, l-lactide-co-glycolide biomaterial as a bone substitute. J Eur Ceram Soc 27:1589–1594CrossRefGoogle Scholar
  19. Kamitakahara M, Ohtsuki C, Miyazaki T (2008) Review Paper: behavior of ceramic biomaterials derived from tricalcium phosphate in physiological condition. J Biomater Appl 23(3):197–212. doi: 10.1177/0885328208096798 CrossRefGoogle Scholar
  20. Kang Y, Yin G, Yuan Q, Yao Y, Huang Z, Liao X, Wang H (2008) Preparation of poly(l-lactic acid)/β-tricalcium phosphate scaffold for bone tissue engineering without organic solvent. Mater Lett 62(12–13):2029–2032. doi: 10.1016/j.matlet.2007.11.014 CrossRefGoogle Scholar
  21. Kilroy GE, Foster SJ, Xiying W, Ruiz J, Sherwood S, Heifetz A, Gimble JM (2007) Cytokine profile of human adipose-derived stem cells: expression of angiogenic, hematopoietic, and pro-inflammatory factors. J Cell Physiol 212(3):702–709. doi: 10.1002/jcp.21068 CrossRefGoogle Scholar
  22. Kim J, Taki K, Nagamine S, Ohshima M (2008) Preparation of poly(L-lactic acid) honeycomb monolith structure by unidirectional freezing and freeze-drying. Chem Eng Sci 63(15):3858–3863. doi: 10.1016/j.ces.2008.04.036 CrossRefGoogle Scholar
  23. Kim J-W, Taki K, Nagamine S, Ohshima M (2009) Preparation of porous poly(L-lactic acid) honeycomb monolith structure by phase separation and unidirectional freezing. Langmuir 25(9):5304–5312CrossRefGoogle Scholar
  24. Lewinski N, Colvin V, Drezek R (2008) Cytotoxicity of nanoparticles. Small 4(1):26–49CrossRefGoogle Scholar
  25. Ma PX, Zhang R, Xiao G, Franceschi R (2001) Engineering new bone tissue in vitro on highly porous poly (a-hydroxyl acids)/hydroxyapatite composite scaffolds. J Biomed Mater Res 54(2):284–293CrossRefGoogle Scholar
  26. Maquet V, Boccaccini AR, Pravata L, Notingher I, Jérôme R (2003) Preparation, characterization, and in vitro degradation of bioresorbable and bioactive composites based on bioglass-filled polylactide foams. J Biomed Mater Res 66A:335–346CrossRefGoogle Scholar
  27. Marino G, Rosso F, Cafiero G, Tortora C, Moraci M, Barbarisi M, Barbaris A (2010) b-Tricalcium phosphate 3D scaffold promote alone osteogenic differentiation of human adipose stem cells: in vitro study. J Mater Sci Mater Med 21:353–363CrossRefGoogle Scholar
  28. Panetta JN, Gupta MD, Longaker MT (2010) Bone regeneration and repair. Curr Stem Cell Res Ther 5(2):122–128. doi: 10.2174/157488810791268618 CrossRefGoogle Scholar
  29. Qureshi AT, Monroe WT, Dasa V, Gimble JM, Hayes DJ (2013) miR-148b–Nanoparticle conjugates for light mediated osteogenesis of human adipose stromal/stem cells. Biomaterials 34(31):7799–7810CrossRefGoogle Scholar
  30. Rezwan K, Chen Q, Blaker J, Boccaccini A (2006) Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering. Biomaterials 27(18):3413–3431. doi: 10.1016/j.biomaterials.2006.01.039 CrossRefGoogle Scholar
  31. Roether JA, Boccaccini AR, Hench LL, Maquet V, Gautier S, Jerome R (2002) Development and in vitro characterisation of novel bioresorbable and bioactive composite materials based on polylactide foams and bioglasss for tissue engineering applications. Biomaterials 23:3871–3878CrossRefGoogle Scholar
  32. Schugens C, Maquet V, Grandfils C, Jerome R, Teyssie P (1996) Biodegradable and macroporous polylactide implants for cell transplantation: 1. Preparation of macroporous polylactide supports by solid-liquid phase separation. Polymer 37(6):1027–1038CrossRefGoogle Scholar
  33. Shora L, Guceria S, Wen X, Gandhi M, Sun W (2007) Fabrication of three-dimensional polycaprolactone/hydroxyapatite tissue scaffolds and osteoblast-scaffold interactions in vitro. Biomaterials 28:5291–5297CrossRefGoogle Scholar
  34. Smoak M, Chen C, Qureshi A, Garber L, Pojman JA, Janes ME, Hayes DJ (2014) Antimicrobial cytocompatible pentaerythritol triacrylate-co-trimethylolpropane composite scaffolds for orthopaedic implants. J Appl Polym Sci. doi: 10.1002/app.41099 Google Scholar
  35. Sui G, Yang X, Mei F, Hu X, Chen G, Deng X, Ryu S (2007) Poly-L-lactic acid/hydroxyapatite hybrid membrane for bone tissue regeneration. J Biomed Mater Res, Part A 82A(2):445–454. doi: 10.1002/jbm.a.31166 CrossRefGoogle Scholar
  36. Sun F, Zhou H, Lee J (2011) Various preparation methods of highly porous hydroxyapatite/polymer nanoscale biocomposites for bone regeneration. Acta Biomater 7(11):3813–3828. doi: 10.1016/j.actbio.2011.07.002 CrossRefGoogle Scholar
  37. Thamaraiselvi TV, Rajeswari S (2004) Biological evaluation of bioceramic materials—a review. Trends Biomater Artif Org 18(1):9–17Google Scholar
  38. Uskokovic V, Uskokovic DP (2011) Review: nanosized hydroxyapatite and other calcium phosphates: chemistry of formation and application as drug and gene delivery agents. J Biomed Mater Res Part B: Appl Biomater 96B:152–191CrossRefGoogle Scholar
  39. Wang H, Li Y, Zuo Y, Li J, Ma S, Cheng L (2007) Biocompatibility and osteogenesis of biomimetic nano-hydroxyapatite/polyamide composite scaffolds for bone tissue engineering. Biomaterials 28(22):3338–3348. doi: 10.1016/j.biomaterials.2007.04.014 CrossRefGoogle Scholar
  40. Wei G, Ma PX (2004) Structure and properties of nano-hydroxyapatite/polymer composite scaffolds for bone tissue engineering. Biomaterials 25:4749–4757CrossRefGoogle Scholar
  41. Yu G, Wu X, Dietrich MA, Polk P, Scott LK, Ptitsyn AA, Gimble JM (2010) Yield and characterization of subcutaneous human adipose-derived stem cells by flow cytometric and adipogenic mRNA analyzes. Cytotherapy 12(4):538–546. doi: 10.3109/14653241003649528 CrossRefGoogle Scholar
  42. Zanetti AS, McCandless GT, Chan JY, Gimble JM, Hayes DJ (2013) In vitro human adipose-derived stromal/stem cells osteogenesis in akermanite: poly-ε-caprolactone scaffolds. J Biomater App. doi: 10.1177/0885328213490974 Google Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Mollie Smoak
    • 1
  • Katie Hogan
    • 1
  • Lisa Kriegh
    • 1
  • Cong Chen
    • 1
  • LeKeith B. Terrell
    • 1
  • Ammar T. Qureshi
    • 1
  • W. Todd Monroe
    • 1
  • Jeffrey M. Gimble
    • 2
  • Daniel J. Hayes
    • 1
  1. 1.Department of Biological and Agricultural EngineeringLouisiana State University and LSU AgCenterBaton RougeUSA
  2. 2.Center for Stem Cell Research & Regenerative MedicineTulane University School of MedicineNew OrleansUSA

Personalised recommendations