Hyaluronan-based heparin-incorporated hydrogels for generation of axially vascularized bioartificial bone tissues: in vitro and in vivo evaluation in a PLDLLA–TCP–PCL-composite system

  • Subha N. Rath
  • Galyna Pryymachuk
  • Oliver A. Bleiziffer
  • Christopher X. F. Lam
  • Andreas Arkudas
  • Saey T. B. Ho
  • Justus P. Beier
  • Raymund E. Horch
  • Dietmar W. Hutmacher
  • Ulrich Kneser
Article

Abstract

Smart matrices are required in bone tissue-engineered grafts that provide an optimal environment for cells and retain osteo-inductive factors for sustained biological activity. We hypothesized that a slow-degrading heparin-incorporated hyaluronan (HA) hydrogel can preserve BMP-2; while an arterio–venous (A–V) loop can support axial vascularization to provide nutrition for a bio-artificial bone graft. HA was evaluated for osteoblast growth and BMP-2 release. Porous PLDLLA–TCP–PCL scaffolds were produced by rapid prototyping technology and applied in vivo along with HA-hydrogel, loaded with either primary osteoblasts or BMP-2. A microsurgically created A–V loop was placed around the scaffold, encased in an isolation chamber in Lewis rats. HA-hydrogel supported growth of osteoblasts over 8 weeks and allowed sustained release of BMP-2 over 35 days. The A–V loop provided an angiogenic stimulus with the formation of vascularized tissue in the scaffolds. Bone-specific genes were detected by real time RT-PCR after 8 weeks. However, no significant amount of bone was observed histologically. The heterotopic isolation chamber in combination with absent biomechanical stimulation might explain the insufficient bone formation despite adequate expression of bone-related genes. Optimization of the interplay of osteogenic cells and osteo-inductive factors might eventually generate sufficient amounts of axially vascularized bone grafts for reconstructive surgery.

Abbreviations

HA

Hyaluronic acid/hyaluronan hydrogel

BMP

Bone morphogenetic protein

CT

Computerized tomography

A–V

Arterio–venous

PLDLLA

Poly(L-lactide-co-D,L-lactide)

PCL

Poly(ε-caprolactone)

TCP

β-Tri-calcium phosphate

Notes

Acknowledgments

This study was supported by research grants from the Deutsche Forschungsgemeinschaft (DFG) (KN 578/2-1) and the Xue Hong and Hans Georg Geis Foundation. The authors thank Dr. Andreas Hess, Institute of Experimental and Clinical Pharmacology and Toxicology for helping in micro-CT scanning and Prof. Peter Greil and Mr. Peter Reinhard for production of the Teflon chambers.

References

  1. 1.
    Pneumaticos SG, Triantafyllopoulos GK, Basdra EK, Papavassiliou AG. Segmental bone defects: from cellular and molecular pathways to the development of novel biological treatments. J Cell Mol Med. 2010. doi: 10.1111/j.1582-4934.2010.01062.x.
  2. 2.
    Scheufler O, Schaefer DJ, Jaquiery C, Braccini A, Wendt DJ, Gasser JA, et al. Spatial and temporal patterns of bone formation in ectopically pre-fabricated, autologous cell-based engineered bone flaps in rabbits. J Cell Mol Med. 2008;12(4):1238–49. doi: 10.1111/j.1582-4934.2008.00137.x.CrossRefGoogle Scholar
  3. 3.
    Arkudas A, Tjiawi J, Bleiziffer O, Grabinger L, Polykandriotis E, Beier JP, et al. Fibrin gel-immobilized VEGF and bFGF efficiently stimulate angiogenesis in the AV loop model. Mol Med. 2007;13(9–10):480–7.Google Scholar
  4. 4.
    Reddi A. Bone morphogenetic proteins: from basic science to clinical applications. J Bone Joint Surg J. 2001;83(Suppl 1, Part 1):S1.Google Scholar
  5. 5.
    Arkudas A, Beier J, Heidner K, Tjiawi J, Polykandriotis E, Srour S, et al. Axial prevascularization of porous matrices using an arteriovenous loop promotes survival and differentiation of transplanted autologous osteoblasts. Tissue Eng. 2007;13(7):1549–60.CrossRefGoogle Scholar
  6. 6.
    Kneser U, Schaefer DJ, Polykandriotis E, Horch RE. Tissue engineering of bone: the reconstructive surgeon’s point of view. J Cell Mol Med. 2006;10(1):7–19.CrossRefGoogle Scholar
  7. 7.
    Beier J, Horch R, Hess A, Arkudas A, Heinrich J, Loew J, et al. Axial vascularization of a large volume calcium phosphate ceramic bone substitute in the sheep AV loop model. J Tissue Eng Regen Med. 2010;4(3):216–23.CrossRefGoogle Scholar
  8. 8.
    Ai-Aql ZS, Alagl AS, Graves DT, Gerstenfeld LC, Einhorn TA. Molecular mechanisms controlling bone formation during fracture healing and distraction osteogenesis. J Dent Res. 2008;87(2):107–18.CrossRefGoogle Scholar
  9. 9.
    Reddi A. Role of morphogenetic proteins in skeletal tissue engineering and regeneration. Nat Biotechnol. 1998;16(3):247–52.CrossRefGoogle Scholar
  10. 10.
    Eyckmans J, Roberts SJ, Schrooten J, Luyten FP. A clinically relevant model of osteoinduction: a process requiring calcium phosphate and BMP/Wnt signaling. J Cell Mol Med. 2009. doi: 10.1111/j.1582-4934.2009.00807.x.
  11. 11.
    Terheyden H, Menzel C, Wang H, Springer IN, Rueger DR, Acil Y. Prefabrication of vascularized bone grafts using recombinant human osteogenic protein-1–part 3: dosage of rhOP-1, the use of external and internal scaffolds. Int J Oral Maxillofac Surg. 2004;33(2):164–72.CrossRefGoogle Scholar
  12. 12.
    Patel VV, Zhao L, Wong P, Pradhan BB, Bae HW, Kanim L, et al. An in vitro and in vivo analysis of fibrin glue use to control bone morphogenetic protein diffusion and bone morphogenetic protein–stimulated bone growth. Spine J. 2006;6(4):397–403.CrossRefGoogle Scholar
  13. 13.
    Seeherman H, Wozney J, Li R. Bone morphogenetic protein delivery systems. Spine (Phila Pa 1976). 2002;27(16 Suppl 1):S16–23.CrossRefGoogle Scholar
  14. 14.
    Yamamoto M, Takahashi Y, Tabata Y. Controlled release by biodegradable hydrogels enhances the ectopic bone formation of bone morphogenetic protein. Biomaterials. 2003;24(24):4375–83.CrossRefGoogle Scholar
  15. 15.
    Anitua E, Sánchez M, Orive G, Andia I. Delivering growth factors for therapeutics. Trends Pharmacol Sci. 2008;29(1):37–41.CrossRefGoogle Scholar
  16. 16.
    Meyer R Jr, Gruber H, Howard B, Tabor O Jr, Murakami T, Kwiatkowski T, et al. Safety of recombinant human bone morphogenetic protein-2 after spinal laminectomy in the dog. Spine. 1999;24(8):747.CrossRefGoogle Scholar
  17. 17.
    Bishop G, Einhorn T. Current and future clinical applications of bone morphogenetic proteins in orthopaedic trauma surgery. Int Orthop. 2007;31(6):721–7.CrossRefGoogle Scholar
  18. 18.
    Pike DB, Cai S, Pomraning KR, Firpo MA, Fisher RJ, Shu XZ, et al. Heparin-regulated release of growth factors in vitro and angiogenic response in vivo to implanted hyaluronan hydrogels containing VEGF and bFGF. Biomaterials. 2006;27(30):5242–51. doi: 10.1016/j.biomaterials.2006.05.018.CrossRefGoogle Scholar
  19. 19.
    Zhao B, Katagiri T, Toyoda H, Takada T, Yanai T, Fukuda T, et al. Heparin potentiates the in vivo ectopic bone formation induced by bone morphogenetic protein-2. J Biol Chem. 2006;281(32):23246.CrossRefGoogle Scholar
  20. 20.
    Zein I, Hutmacher DW, Tan KC, Teoh SH. Fused deposition modeling of novel scaffold architectures for tissue engineering applications. Biomaterials. 2002;23(4):1169–85.CrossRefGoogle Scholar
  21. 21.
    Lam C, Olkowski R, Swieszkowski W, Tan K, Gibson I, Hutmacher D. Mechanical and in vitro evaluations of composite PLDLLA/TCP scaffolds for bone engineering. Virtual Phys Prototyp. 2008;3(4):193–7.CrossRefGoogle Scholar
  22. 22.
    Yang J, Wan Y, Tu C, Cai Q, Bei J, Wang S. Enhancing the cell affinity of macroporous poly(L-lactide) cell scaffold by a convenient surface modification method. Polym Int. 2003;52(12):1892–9.CrossRefGoogle Scholar
  23. 23.
    Kneser U, Stangenberg L, Ohnolz J, Buettner O, Stern-Straeter J, Mobest D, et al. Evaluation of processed bovine cancellous bone matrix seeded with syngenic osteoblasts in a critical size calvarial defect rat model. J Cell Mol Med. 2006;10(3):695–707.CrossRefGoogle Scholar
  24. 24.
    Kneser U, Polykandriotis E, Ohnolz J, Heidner K, Grabinger L, Euler S, et al. Engineering of vascularized transplantable bone tissues: induction of axial vascularization in an osteoconductive matrix using an arteriovenous loop. Tissue Eng. 2006;12(7):1721–31.CrossRefGoogle Scholar
  25. 25.
    Bolland BJRF, Kanczler JM, Dunlop DG, Oreffo ROC. Development of in vivo μCT evaluation of neovascularisation in tissue engineered bone constructs. Bone. 2008;43(1):195–202.CrossRefGoogle Scholar
  26. 26.
    Arkudas A, Pryymachuk G, Hoereth T, Beier J, Polykandriotis E, Bleiziffer O, et al. Dose-Finding Study of Fibrin Gel-Immobilized Vascular Endothelial Growth Factor 165 and Basic Fibroblast Growth Factor in the Arteriovenous Loop Rat Model. Tissue Eng A. 2009;15(9):2501–11.CrossRefGoogle Scholar
  27. 27.
    Talwar R, Di Silvio L, Hughes FJ, King GN. Effects of carrier release kinetics on bone morphogenetic protein-2-induced periodontal regeneration in vivo. J Clin Periodontol. 2001;28(4):340–7.CrossRefGoogle Scholar
  28. 28.
    Seeherman H, Li R, Bouxsein M, Kim H, Li X, Smith-Adaline E, et al. rhBMP-2/calcium phosphate matrix accelerates osteotomy-site healing in a nonhuman primate model at multiple treatment times and concentrations. J Bone Joint Surg. 2006;88(1):144.CrossRefGoogle Scholar
  29. 29.
    Rothstein S, Federspiel W, Little S. A unified mathematical model for the prediction of controlled release from surface and bulk eroding polymer matrices. Biomaterials. 2009;30(8):1657–64.CrossRefGoogle Scholar
  30. 30.
    Adams JR, Sander G, Byers S. Expression of hyaluronan synthases and hyaluronidases in the MG63 osteoblast cell line. Matrix Biol. 2006;25(1):40–6. doi: 10.1016/j.matbio.2005.08.007.CrossRefGoogle Scholar
  31. 31.
    Kakudo N, Kusumoto K, Wang YB, Iguchi Y, Ogawa Y. Immunolocalization of vascular endothelial growth factor on intramuscular ectopic osteoinduction by bone morphogenetic protein-2. Life Sci. 2006;79(19):1847–55. doi: 10.1016/j.lfs.2006.06.033.CrossRefGoogle Scholar
  32. 32.
    Toole BP, Hascall VC. Hyaluronan and tumor growth. Am J Pathol. 2002;161(3):745–7.CrossRefGoogle Scholar
  33. 33.
    Mazumdar J, Dondeti V, Simon M. Hypoxia-inducible factors in stem cells and cancer. J Cell Mol Med. 2009;13(11–12):4319–28.CrossRefGoogle Scholar
  34. 34.
    Jeon O, Song S, Kang S, Putnam A, Kim B. Enhancement of ectopic bone formation by bone morphogenetic protein-2 released from a heparin-conjugated poly(l-lactic-co-glycolic acid) scaffold. Biomaterials. 2007;28(17):2763–71.CrossRefGoogle Scholar
  35. 35.
    Inoda H, Yamamoto G, Hattori T. rh-BMP2-induced ectopic bone for grafting critical size defects: a preliminary histological evaluation in rat calvariae. Int J Oral Maxillofac Surg. 2007;36(1):39–44.CrossRefGoogle Scholar
  36. 36.
    Murakami T, Saito A, Hino S, Kondo S, Kanemoto S, Chihara K, et al. Signalling mediated by the endoplasmic reticulum stress transducer OASIS is involved in bone formation. Nat Cell Biol. 2009;11(10):1205–11. doi: 10.1038/ncb1963.CrossRefGoogle Scholar
  37. 37.
    Lamoureux F, Baud’huin M, Duplomb L, Heymann D, Redini F. Proteoglycans: key partners in bone cell biology. BioEssays. 2007;29(8).Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Subha N. Rath
    • 1
    • 2
  • Galyna Pryymachuk
    • 1
  • Oliver A. Bleiziffer
    • 1
  • Christopher X. F. Lam
    • 2
  • Andreas Arkudas
    • 1
  • Saey T. B. Ho
    • 3
  • Justus P. Beier
    • 1
  • Raymund E. Horch
    • 1
  • Dietmar W. Hutmacher
    • 4
  • Ulrich Kneser
    • 1
  1. 1.Department of Plastic and Hand SurgeryUniversity of Erlangen Medical CenterErlangenGermany
  2. 2.Division of BioengineeringNational University of SingaporeSingaporeSingapore
  3. 3.Graduate Programme in Bioengineering, Yong Loo Lin School of MedicineNational University of SingaporeSingaporeSingapore
  4. 4.Faculty of Engineering, Faculty of Science, Institute of Health and Biomedical InnovationQueensland University of TechnologyBrisbaneAustralia

Personalised recommendations