Novel osteoconductive β-tricalcium phosphate/poly(L-lactide-co-e-caprolactone) scaffold for bone regeneration: a study in a rabbit calvarial defect

  • Hanna PihlmanEmail author
  • Pauli Keränen
  • Kaarlo Paakinaho
  • Jere Linden
  • Markus Hannula
  • Iida-Kaisa Manninen
  • Jari Hyttinen
  • Mikko Manninen
  • Outi Laitinen-Vapaavuori
Tissue Engineering Constructs and Cell Substrates Original Research
Part of the following topical collections:
  1. Tissue Engineering Constructs and Cell Substrates


The advantages of synthetic bone graft substitutes over autogenous bone grafts include abundant graft volume, lack of complications related to the graft harvesting, and shorter operation and recovery times for the patient. We studied a new synthetic supercritical CO2 –processed porous composite scaffold of β-tricalcium phosphate and poly(L-lactide-co-caprolactone) copolymer as a bone graft substitute in a rabbit calvarial defect. Bilateral 12 mm diameter critical size calvarial defects were successfully created in 18 rabbits. The right defect was filled with a scaffold moistened with bone marrow aspirate, and the other was an empty control. The material was assessed for applicability during surgery. The follow-up times were 4, 12, and 24 weeks. Radiographic and micro-CT studies and histopathological analysis were used to evaluate new bone formation, tissue ingrowth, and biocompatibility. The scaffold was easy to shape and handle during the surgery, and the bone-scaffold contact was tight when visually evaluated after the implantation. The material showed good biocompatibility and its porosity enabled rapid invasion of vasculature and full thickness mesenchymal tissue ingrowth already at four weeks. By 24 weeks, full thickness bone ingrowth within the scaffold and along the dura was generally seen. In contrast, the empty defect had only a thin layer of new bone at 24 weeks. The radiodensity of the material was similar to the density of the intact bone. In conclusion, the new porous scaffold material, composed of microgranular β-TCP bound into the polymer matrix, proved to be a promising osteoconductive bone graft substitute with excellent handling properties.



This study was financially supported by the Finnish Funding Agency for Technology and Innovation (40326/13) and by grants from the Finnish Foundation of Veterinary Research and The Finnish Veterinary Foundation. The authors kindly acknowledge DVM Mikael Morelius for the help with surgical procedures.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Calori GM, Mazza E, Colombo M, Ripamonti C. The use of bone-graft substitutes in large bone defects: any specific needs? Injury. 2011;42:56–63.CrossRefGoogle Scholar
  2. 2.
    Moore WR, Graves SE, Bain GI. Synthetic bone graft substitutes. Anz J Surg. 2001;71:354–61.CrossRefGoogle Scholar
  3. 3.
    Myeroff C, Archdeacon M. Autogenous bone graft: donor sites and techniques. J Bone Jt Surg. 2011;93:2227–36.CrossRefGoogle Scholar
  4. 4.
    Fillingham Y, Jacobs J. Bone grafts and their substitutes. Bone Join J. 2016;98(1 Suppl A):6–9.CrossRefGoogle Scholar
  5. 5.
    Schnee CL, Freese A, Weil RJ, Marcotte PJ. Analysis of harvest morbidity and radiographic outcome using autograft for anterior cervical fusion. Spine. 1997;22:2222–7.CrossRefGoogle Scholar
  6. 6.
    Sawin PD, Traynelis VC, Menezes AH. A comparative analysis of fusion rates and donor-site morbidity for autogeneic rib and iliac crest bone grafts in posterior cervical fusions. J Neurosurg. 1998;88:255–65.CrossRefGoogle Scholar
  7. 7.
    Ebraheim NA, Elgafy H, Xu R. Bone-graft harvesting from iliac and fibular donor sites: techniques and complications. J Am Acad Orthop Surg. 2001;9:210–8.CrossRefGoogle Scholar
  8. 8.
    Dimitriou R, Mataliotakis GI, Angoules AG, Kanakaris NK, Giannoudis PV. Complications following autologous bone graft harvesting from the iliac crest and using the RIA: a systematic review. Injury. 2011;42:3–15.CrossRefGoogle Scholar
  9. 9.
    Bizenjima T, Takeuchi T, Seshima F, Saito A. Effect of poly(lactide‐co‐glycolide) (PLGA)‐coated beta‐tricalcium phosphate on the healing of rat calvarial bone defects: a comparative study with pure‐phase beta‐tricalcium phosphate. Clin Oral Implants Res. 2016;27:1360–7.CrossRefGoogle Scholar
  10. 10.
    Hak DJ. The use of osteoconductive bone graft substitutes in orthopaedic trauma. J Am Acad Orthop Surg. 2007;15:525–36.CrossRefGoogle Scholar
  11. 11.
    Thaler M, Lechner R, Gstöttner M, Kobel C, Bach C. The use of beta-tricalcium phosphate and bone marrow aspirate as a bone graft substitute in posterior lumbar interbody fusion. Eur Spine J. 2013;22:1173–82.CrossRefGoogle Scholar
  12. 12.
    Albee FH. Studies in bone growth: triple calcium phosphate as a stimulus to osteogenesis. Ann Surg. 1920;71:32–9.CrossRefGoogle Scholar
  13. 13.
    Zerbo IR, Zijderveld SA, de Boer A, Bronckers AL, de Lange G, ten Bruggenkate CM, et al. Histomorphometry of human sinus floor augmentation using a porous beta-tricalcium phosphate: a prospective study. Clin Oral Implants Res. 2004;15:724–32.CrossRefGoogle Scholar
  14. 14.
    Zijderveld S, Zerbo I, van den Bergh J, Schulten E, ten Bruggenkate C. Maxillary sinus floor augmentation using a β-tricalcium phosphate (Cerasorb) alone compared to autogenous bone grafts. Int J Oral Maxillofac Implants. 2005;20:432–40.Google Scholar
  15. 15.
    Ghanaati S, Barbeck M, Orth C, Willershausen I, Thimm BW, Hoffmann C, et al. Influence of β-tricalcium phosphate granule size and morphology on tissue reaction in vivo. Acta Biomater. 2010;6:4476–87.CrossRefGoogle Scholar
  16. 16.
    Zerbo IR, Bronckers AL, de Lange G, Burger EH. Localisation of osteogenic and osteoclastic cells in porous β-tricalcium phosphate particles used for human maxillary sinus floor elevation. Biomaterials . 2005;26:1445–51.CrossRefGoogle Scholar
  17. 17.
    Honda M, Yada T, Ueda M, Kimata K. Cartilage formation by cultured chondrocytes in a new scaffold made of poly(L-lactide-ϵ-caprolactone) sponge. J Oral Maxillofac Surg. 2000;58:767–75.CrossRefGoogle Scholar
  18. 18.
    Honda M, Morikawa N, Hata K, Yada T, Morita S, Ueda M, et al. Rat costochondral cell characteristics on poly (l-lactide-co- ε-caprolactone) scaffolds. Biomaterials . 2003;24:3511–9.CrossRefGoogle Scholar
  19. 19.
    Jeong SI, Kim SH, Kim YH, Kim B, Kang SW, Kwon JH, et al. In vivo biocompatibilty and degradation behavior of elastic poly(l-lactide- co- ε-caprolactone) scaffolds. Biomaterials . 2004;25:5939–46.CrossRefGoogle Scholar
  20. 20.
    Nandi SK, Roy S, Mukherjee P, Kundu B, De DK, Basu D. Orthopaedic applications of bone graft & graft substitutes: a review. Indian J Med Res. 2010;132:15–30.Google Scholar
  21. 21.
    Hernigou P, Desroches A, Queinnec S, Flouzat Lachaniette C, Poignard A, Allain J, et al. Morbidity of graft harvesting versus bone marrow aspiration in cell regenerative therapy. Int Orthop. 2014;38:1855–60.CrossRefGoogle Scholar
  22. 22.
    Luvizuto ER, Queiroz TP, Margonar R, Panzarini SR, Hochuli-Vieira E, Okamoto T, et al. Osteoconductive properties of β-tricalcium phosphate matrix, polylactic and polyglycolic acid gel, and calcium phosphate cement in bone defects. J Craniofac Surg. 2012;23:e430–3.CrossRefGoogle Scholar
  23. 23.
    Tanuma Y, Matsui K, Kawai T, Matsui A, Suzuki O, Kamakura S. et al.Comparison of bone regeneration between octacalcium phosphate/collagen composite and β-tricalcium phosphate in canine calvarial defect.Oral Surg Oral Med Oral Pathol Oral Radiol. 2013;115:9–17.CrossRefGoogle Scholar
  24. 24.
    Lappalainen O, Karhula S, Haapea M, Kyllönen L, Haimi S, Miettinen S, et al. Bone healing in rabbit calvarial critical-sized defects filled with stem cells and growth factors combined with granular or solid scaffolds. Childs Nerv Syst. 2016;32:681–8.CrossRefGoogle Scholar
  25. 25.
    Hernandes C. J. Cancellous bone. In: Murphy W, Black J, Hastings G, editors. Handbook of biomaterial properties. New York: Springer-Verlag; 2016. pp 15–21.Google Scholar
  26. 26.
    Tsuruga E, Takita H, Itoh H, Wakisaka Y, Kuboki Y. Pore size of porous hydroxyapatite as the cell-substratum controls BMP-induced osteogenesis. J Biochem. 1997;121:317–24.CrossRefGoogle Scholar
  27. 27.
    Karageorgiou V, Kaplan D. Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials . 2005;26:5474–91.CrossRefGoogle Scholar
  28. 28.
    Gosain AK, Santoro TD, Song L, Capel CC, Sudhakar PV, Matloub HS. Osteogenesis in calvarial defects: contribution of the dura, the pericranium, and the surrounding bone in adult versus infant animals. Plast Reconstr Surg. 2003;112:515–27.CrossRefGoogle Scholar
  29. 29.
    Lappalainen O, Karhula SS, Haapea M, Kauppinen S, Finnilä M, Saarakkala S, et al. Micro-CT analysis of bone healing in rabbit calvarial critical-sized defects with solid bioactive glass, tricalcium phosphate granules or autogenous bone. J Oral Macillofac Res. 2016;7:e4.Google Scholar
  30. 30.
    Jan A, Sándor G, Brkovic B, Peel S, Kim YD, Xiao W, et al. Effect of hyperbaric oxygen on demineralized bone matrix and biphasic calcium phosphate bone substitutes. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2010;109:59–66.CrossRefGoogle Scholar
  31. 31.
    Ghanaati S, Barbeck M, Willershausen I, Thimm B, Stuebinger S, Korzinskas T, et al. Nanocrystalline hydroxyapatite bone substitute leads to sufficient bone tissue formation already after 3 months: histological and histomorphometrical analysis 3 and 6 months following human sinus cavity augmentation. Clin Implant Dent Relat Res. 2013;15:883–92.CrossRefGoogle Scholar
  32. 32.
    Miron RJ, Zohdi H, Fujioka-Kobayashi M, Bosshardt DD. Giant cells around bone biomaterials: osteoclasts or multi-nucleated giant cells? Acta Biomater. 2016;46:15–28.CrossRefGoogle Scholar
  33. 33.
    Barbeck M, Booms P, Unger R, Hoffmann V, Sader R, Kirkpatrick CJ, et al. Multinucleated giant cells in the implant bed of bone substitutes are foreign body giant cells—new insights into the material‐mediated healing process. J Biomed Mater Res A. 2017;105:1105–11.CrossRefGoogle Scholar
  34. 34.
    Peltola M, Kinnunen I, Aitasalo K. Reconstruction of orbital wall defects with bioactive glass plates. J Oral Macillofac Surg. 2008;66:639–46.CrossRefGoogle Scholar
  35. 35.
    van Haaren EH, Smit TH, Phipps K, Wuisman PI, Blunn G, Heyligers IC. Tricalcium-phosphate and hydroxyapatite bone-graft extender for use in impaction grafting revision surgery. J Bone Jt Surg Br. 2005;87:267–71.CrossRefGoogle Scholar
  36. 36.
    Oakley J, Kuiper JH. Factors affecting the cohesion of impaction bone graft. J Bone Jt Surg [Br]. 2006;88:828–31.CrossRefGoogle Scholar
  37. 37.
    Schroeder C, Grupp T, Fritz B, Schilling C, Chevalier Y, Utzschneider S, et al. The influence of third-body particles on wear rate in unicondylar knee arthroplasty: a wear simulator study with bone and cement debris. J Mater Sci Mater Med. 2013;24:1319–25.CrossRefGoogle Scholar
  38. 38.
    Sanda M, Shiota M, Fujii M, Kon K, Fujimori T, Kasugai S. Capability of new bone formation with a mixture of hydroxyapatite and beta‐tricalcium phosphate granules. Clin Oral Implants Res. 2015;26:1369–74.CrossRefGoogle Scholar
  39. 39.
    Schmitz JP, Hollinger JO. The critical size defect as an experimental model for craniomandibulofacial nonunions. Clin Orthop Relat Res. 1986;205:299–308.Google Scholar
  40. 40.
    Fok T, Jan A, Peel S, Evans AW, Clokie C, Sándor G. Hyperbaric oxygen results in increased vascular endothelial growth factor (VEGF) protein expression in rabbit calvarial critical-sized defects. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2008;105:417–22.CrossRefGoogle Scholar
  41. 41.
    Jan A, Sándor G, Iera D, Mhawi A, Peel S, Evans AW, et al. Hyperbaric oxygen results in an increase in rabbit calvarial critical sized defects. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006;101:144–9.CrossRefGoogle Scholar
  42. 42.
    Cooper GM, Mooney MP, Gosain AK, Campbell PG, Losee JE, Huard J. Testing the critical size in calvarial bone defects: revisiting the concept of a critical-size defect. Plast Reconstr Surg. 2010;125:1685–92.CrossRefGoogle Scholar
  43. 43.
    Borie E, Fuentes R, del Sol M, Oporto G, Engelke W. The influence of FDBA and autogenous bone particles on regeneration of calvaria defects in the rabbit: a pilot study. Ann Anat. 2011;193:412–7.CrossRefGoogle Scholar
  44. 44.
    Pelegrine AA, Aloise AC, Zimmermann A, Mello e Oliveira R, Ferreira LM. Repair of critical‐size bone defects using bone marrow stromal cells: a histomorphometric study in rabbit calvaria. Part I: Use of fresh bone marrow or bone marrow mononuclear fraction. Clin Oral Implants Res. 2014;25:567–72.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Hanna Pihlman
    • 1
    Email author
  • Pauli Keränen
    • 1
  • Kaarlo Paakinaho
    • 2
    • 3
  • Jere Linden
    • 1
  • Markus Hannula
    • 4
  • Iida-Kaisa Manninen
    • 5
  • Jari Hyttinen
    • 2
  • Mikko Manninen
    • 3
  • Outi Laitinen-Vapaavuori
    • 1
  1. 1.Faculty of Veterinary MedicineUniversity of HelsinkiHelsinkiFinland
  2. 2.Faculty of Medicine and Life Sciences, BiomeditechUniversity of TampereTampereFinland
  3. 3.Orton Orthopaedic HospitalHelsinkiFinland
  4. 4.Faculty of Biomedical Science and EngineeringTampere University of TechnologyTampereFinland
  5. 5.Muonio Health CenterMuonioFinland

Personalised recommendations