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Starch-poly-є-caprolactone Microparticles Reduce the Needed Amount of BMP-2

  • Symposium: Tribute to Dr. Marshall Urist: Musculoskeletal Growth Factors
  • Published:
Clinical Orthopaedics and Related Research®

Abstract

BMP-2 is currently administered clinically using collagen matrices often requiring large amounts of BMP-2 due to burst release over a short period of time. We developed and tested a novel injectable drug delivery system consisting of starch-poly-є-caprolactone microparticles for inducing osteogenesis and requiring smaller amounts of BMP-2. We evaluated BMP-2 encapsulation efficiency and the in vitro release profile by enzyme-linked immunosorbent assay. BMP-2 was rapidly released during the first 12 hours, followed by sustained release for up to 10 days. We then evaluated the osteogenic potential of dexamethasone (standard osteogenic induction agent) and BMP-2 after incorporation and during release using an osteo/myoblast cell line (C2C12). Alkaline phosphatase activity was increased by released BMP-2. Mineralization occurred after stimulation with BMP-2-loaded microparticles. A luciferase assay for osteocalcin promoter activity showed high levels of activity upon treatment with BMP-2-loaded microparticles. In contrast, no osteogenesis occurred in C2C12 cells using dexamethasone-loaded microparticles. However, human adipose stem cells exposed to the microparticles produced high amounts of alkaline phosphatase. The data suggest starch-poly-є-caprolactone microparticles are suitable carriers for the incorporation and controlled release of glucocorticoids and growth factors. Specifically, they reduce the amount of BMP-2 needed and allow more sustained osteogenic effects.

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References

  1. Azevedo HS, Gama FM, Reis RL. In vitro assessment of the enzymatic degradation of several starch based biomaterials. Biomacromolecules. 2003;4:1703–1712.

    Article  CAS  PubMed  Google Scholar 

  2. Azevedo HS, Reis RL. Understanding the enzymatic degradation of biodegradable polymers and strategies to control their degradation rate. In: Reis RL, San Roman J, eds. Biodegradable Systems in Tissue Engineering and Regenerative Medicine. Boca Raton, FL: CRC Press; 2005:177–201.

    Google Scholar 

  3. Balmayor ER, Tuzlakoglu K, Azevedo HS, Reis RL. Preparation and characterization of starch-poly-ε-caprolactone microparticles incorporating bioactive agents for drug delivery and tissue engineering applications. Acta Biomater. 2009;5:1035–1045.

    Article  CAS  PubMed  Google Scholar 

  4. Balmayor ER, Tuzlakoglu K, Marques AP, Azevedo HS, Reis RL. A novel enzymatically-mediated drug delivery carrier for bone tissue engineering applications: combining biodegradable starch-based microparticles and differentiation agents. J Mater Sci Mater Med. 2008;19:1617–1623.

    Article  CAS  Google Scholar 

  5. Bastioli C, Bellotti V, Montino A, Tredici GD, Lombi R, Ponti R; Novamont SpA (Milan, IT), assignee. Biodegradable polymeric compositions based on starch and thermoplastic polymers. US patent 5 412 005, May 2, 1995.

  6. Bessa PC, Casal M, Reis RL. Bone morphogenetic proteins in tissue engineering: the road from the laboratory to the clinic, part I (basic concepts). J Tissue Eng Regen Med. 2008;2:1–13.

    Article  CAS  PubMed  Google Scholar 

  7. Bessa PC, Casal M, Reis RL. Bone morphogenetic proteins in tissue engineering: the road from laboratory to clinic, part II (BMP delivery). J Tissue Eng Regen Med. 2008;2:81–96.

    Article  CAS  PubMed  Google Scholar 

  8. Chen D, Zhao M, Mundy GR. Bone morphogenetic proteins. Growth Factors. 2004;22:233–241.

    Article  CAS  PubMed  Google Scholar 

  9. Chen FM, Wu ZF, Sun HH, Wu H, Xin SN, Wang QT, Dong GY, Ma ZW, Huang S, Zhang JJ, Jin Y. Release of bioactive BMP from dextran-derived microspheres: a novel delivery concept. Int J Pharm. 2006;307:23–32.

    Article  CAS  PubMed  Google Scholar 

  10. DeCoster TA, Gehlert R, Mikola EA, Pirela-Cruz MA. Management of posttraumatic segmental bone defects. J Am Acad Orthop Surg. 2004;12:28–38.

    PubMed  Google Scholar 

  11. Demirgoz D, Elvira C, Mano JF, Cunha AM, Piskin E, Reis RL. Chemical modification of starch based biodegradable polymeric blends: effects on water uptake, degradation behaviour and mechanical properties. Polym Degrad Stab. 2000;70:161–170.

    Article  CAS  Google Scholar 

  12. den Boer FC, Wippermann BW, Blokhuis TJ, Patka P, Bakker FC, Haarman H. Healing of segmental bone defects with granular porous hydroxyapatite augmented with recombinant human osteogenic protein-1 or autologous bone marrow. J Orthop Res. 2003;21:521–528.

    Article  Google Scholar 

  13. Gersbach CA, Guldberg RE, Garcia AJ. In vitro and in vivo osteoblastic differentiation of BMP-2- and Runx2-engineered skeletal myoblasts. J Cell Biochem. 2007;100:1324–1336.

    Article  CAS  PubMed  Google Scholar 

  14. Gitelman SE, Kirk M, Ye JQ, Filvaroff EH, Kahn AJ, Derynck R. Vgr-1/BMP-6 induces osteoblastic differentiation of pluripotential mesenchymal cells. Cell Growth Differ. 1995;6:827–836.

    CAS  PubMed  Google Scholar 

  15. Gomes ME, Reis RL, Mikos AG. Injectable polymeric scaffolds for bone tissue engineering. In: Reis RL, San Roman J, eds. Biodegradable Systems in Tissue Engineering and Regenerative Medicine. Boca Raton, FL: CRC Press; 2005:29–38.

    Google Scholar 

  16. Gugala Z, Gogolewski S. Healing of critical-size segmental bone defects in the sheep tibiae using bioresorbable polylactide membranes. Injury. 2002;33(Suppl 2):B71–B76.

    Article  PubMed  Google Scholar 

  17. Huang X, Brazel CS. On the importance and mechanisms of burst release in matrix-controlled drug delivery systems. J Control Release. 2001;73:121–136.

    Article  CAS  PubMed  Google Scholar 

  18. Jorgensen NR, Henriksen Z, Sorensen OH, Civitelli R. Dexamethasone, BMP-2, and 1,25-dihydroxyvitamin D enhance a more differentiated osteoblast phenotype: validation of an in vitro model for human bone marrow-derived primary osteoblasts. Steroids. 2004;69:219–226.

    Article  CAS  PubMed  Google Scholar 

  19. Katagiri T, Yamaguchi A, Ikeda T, Yoshiki S, Wozney JM, Rosen V, Wang EA, Tanaka H, Omura S, Suda T. The non-osteogenic mouse pluripotent cell line, C3H10T1/2, is induced to differentiate into osteoblastic cells by recombinant human bone morphogenetic protein-2. Biochem Biophys Res Commun. 1990;172:295–299.

    Article  CAS  PubMed  Google Scholar 

  20. Katagiri T, Yamaguchi A, Komaki M, Abe E, Takahashi N, Ikeda T, Rosen V, Wozney JM, Fujisawa-Sehara A, Suda T. Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage. J Cell Biol. 1994;127:1755–1766.

    Article  CAS  PubMed  Google Scholar 

  21. Keskin SD, Hasirci V. Controlled release of bioactive agents in gene therapy and tissue engineering. In: Elcin M, ed. Tissue Engineering, Stem Cells and Gene Therapies. New York, NY: Kluwer Academic/Plenum Publishers; 2003:279–296.

    Google Scholar 

  22. Kim HD, Valentini RF. Human osteoblast response in vitro to platelet-derived growth factor and transforming growth factor-beta delivered from controlled-release polymer rods. Biomaterials. 1997;18:1175–1184.

    Article  CAS  PubMed  Google Scholar 

  23. Kim IS, Song YM, Cho TH, Park YD, Lee KB, Noh I, Weber F, Hwang SJ. In vitro response of primary human bone marrow stromal cells to recombinant human bone morphogenic protein-2 in the early and late stages of osteoblast differentiation. Dev Growth Differ. 2008;50:553–564.

    CAS  PubMed  Google Scholar 

  24. Kim SE, Park JH, Cho YW, Chung H, Jeong SY, Lee EB, Kwon IC. Porous chitosan scaffold containing microspheres loaded with transforming growth factor-beta 1: implications for cartilage tissue engineering. J Control Release. 2003;91:365–374.

    Article  CAS  PubMed  Google Scholar 

  25. Komaki H, Tanaka T, Chazono M, Kikuchi T. Repair of segmental bone defects in rabbit tibiae using a complex of beta-tricalcium phosphate, type I collagen, and fibroblast growth factor-2. Biomaterials. 2006;27:5118–5126.

    Article  CAS  PubMed  Google Scholar 

  26. Krause F, Younger A, Weber M. Recombinant human BMP-2 and allograft compared with autogenous bone graft for reconstruction of diaphyseal tibial fractures with cortical defects. J Bone Joint Surg Am. 2008;90:1168–1169.

    PubMed  Google Scholar 

  27. Laurencin C, Khan Y, El-Amin SF. Bone graft substitutes. Expert Rev Med Devices. 2006;3:49–57.

    Article  CAS  PubMed  Google Scholar 

  28. Laurencin CT, Ambrosio AM, Borden MD, Cooper JA. Tissue engineering: orthopedic applications. Annu Rev Biomed Eng. 1999;1:19–46.

    Article  CAS  PubMed  Google Scholar 

  29. Lewis DH. Controlled release of bioactive agents from lactide/glycolide polymers. In: Chasin M, Langer R, eds. Biodegradable Polymers as Drug Delivery Systems. New York, NY: Marcel Dekker, Inc; 1990:1–42.

    Google Scholar 

  30. Lian JB, Stein GS. Concepts of osteoblast growth and differentiation: basis for modulation of bone cell-development and tissue formation. Crit Rev Oral Biol Med. 1992;3:269–305.

    CAS  PubMed  Google Scholar 

  31. Luppen CA, Chandler RL, Noh T, Mortlock DP, Frenkel B. BMP-2 vs. BMP-4 expression and activity in glucocorticoid-arrested MCM-E1 osteoblasts: Smad signaling, not alkaline phosphatase activity, predicts rescue of mineralization. Growth Factors. 2008;26:226–237.

    Article  CAS  PubMed  Google Scholar 

  32. Marques AP, Reis RL, Hunt JA. The biocompatibility of novel starch-based polymers and composites: in vitro studies. Biomaterials. 2002;23:1471–1478.

    Article  CAS  PubMed  Google Scholar 

  33. Medscape. New bone graft techniques and applications in the spine. Available at: http://cme.medscape.com/viewarticle/443902_2. Accessed May 23, 2009.

  34. Meinel L, Illi OE, Zapf J, Malfanti M, Merkle HP, Gander B. Stabilizing insulin-like growth factor-I in poly(D,L-lactide-co-glycolide) microspheres. J Control Release. 2001;70:193–202.

    Article  CAS  PubMed  Google Scholar 

  35. Mendes SC, Reis RL, Bovell YP, Cunha AM, van Blitterswijk CA, de Bruijn JD. Biocompatibility testing of novel starch-based materials with potential application in orthopaedic surgery: a preliminary study. Biomaterials. 2001;22:2057–2064.

    Article  CAS  PubMed  Google Scholar 

  36. Morsczeck C, Moehl C, Gotz W, Heredia A, Schaffer TE, Eckstein N, Sippel C, Hoffmann KH. In vitro differentiation of human dental follicle cells with dexamethasone and insulin. Cell Biol Int. 2005;29:567–575.

    Article  CAS  PubMed  Google Scholar 

  37. Patel ZS, Yamamoto M, Ueda H, Tabata Y, Mikos AG. Biodegradable gelatin microparticles as delivery systems for the controlled release of bone morphogenetic protein-2. Acta Biomater. 2008;4:1126–1138.

    Article  CAS  PubMed  Google Scholar 

  38. Perka C, Schultz O, Spitzer RS, Lindenhayn K, Burmester GR, Sittinger M. Segmental bone repair by tissue-engineered periosteal cell transplants with bioresorbable fleece and fibrin scaffolds in rabbits. Biomaterials. 2000;21:1145–1153.

    Article  CAS  PubMed  Google Scholar 

  39. Perry CR. Bone repair techniques, bone graft, and bone graft substitutes. Clin Orthop Relat Res. 1999;360:71–86.

    Article  PubMed  Google Scholar 

  40. Reis RL, Cunha AM. Starch Polymers. In: Buschow KHJ, Cahn RW, Flemings MC, Ilschner B, Kramer EJ, Mahajan S, Veyssiere P, eds. Encyclopedia of Materials: Science and Technology. Vol 11. New York, NY: Elsevier Ltd; 2001:8810–8816.

    Google Scholar 

  41. Reis RL, Mendes SC, Cunha AM, Bevis MJ. Processing and in vitro degradation of starch/EVOH thermoplastic blends. Polym Int. 1997;43:347–352.

    Article  CAS  Google Scholar 

  42. Rickard DJ, Sullivan TA, Shenker BJ, Leboy PS, Kazhdan I. Induction of rapid osteoblast differentiation in rat bone-marrow stromal cell-cultures by dexamethasone and BMP-2. Dev Biol. 1994;161:218–228.

    Article  PubMed  Google Scholar 

  43. Rose FR, Hou Q, Oreffo RO. Delivery systems for bone growth factors: the new players in skeletal regeneration. J Pharm Pharmacol. 2004;56:415–427.

    Article  CAS  PubMed  Google Scholar 

  44. Salehian B, Mahabadi V, Bilas J, Taylor WE, Ma K. The effect of glutamine on prevention of glucocorticoid-induced skeletal muscle atrophy is associated with myostatin suppression. Metabolism. 2006;55:1239–1247.

    Article  CAS  PubMed  Google Scholar 

  45. Salgado AJ, Coutinho OP, Reis RL. Novel starch-based scaffolds for bone tissue engineering: cytotoxicity, cell culture, and protein expression. Tissue Eng. 2004;10:465–474.

    Article  CAS  PubMed  Google Scholar 

  46. Sampath TK, Maliakal JC, Hauschka PV, Jones WK, Sasak H, Tucker RF, White KH, Coughlin JE, Tucker MM, Pang RH, et al. Recombinant human osteogenic protein-1 (hOP-1) induces new bone formation in vivo with a specific activity comparable with natural bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro. J Biol Chem. 1992;267:20352–20362.

    CAS  PubMed  Google Scholar 

  47. Silva GA, Coutinho OP, Ducheyne P, Shapiro IM, Reis RL. Starch-based microparticles as vehicles for the delivery of active platelet-derived growth factor. Tissue Eng. 2007;13:1259–1268.

    Article  CAS  PubMed  Google Scholar 

  48. te Pas MFW, de Jong PR, Verburg FJ. Glucocorticoid inhibition of C2C12 proliferation rate and differentiation capacity in relation to mRNA levels of the MRF gene family. Mol Biol Rep. 2000;27:87–98.

    Article  CAS  PubMed  Google Scholar 

  49. Uludag H, Gao T, Porter TJ, Friess W, Wozney JM. Delivery systems for BMPs: factors contributing to protein retention at an application site. J Bone Joint Surg Am. 2001;83(Suppl 1 Pt 2):S128–S135.

    PubMed  Google Scholar 

  50. Urist MR, Silverman BF, Büring K, Dubuc FL, Rosenberg JM. The bone induction principle. Clin Orthop Relat Res. 1967;53:243–283.

    Article  CAS  PubMed  Google Scholar 

  51. Wang SB, Chen AZ, Weng LJ, Chen MY, Xie XL. Effect of drug-loading methods on drug load, encapsulation efficiency and release properties of alginate/poly-L-arginine/chitosan ternary complex microcapsules. Macromol Biosci. 2004;4:27–30.

    Article  PubMed  Google Scholar 

  52. Wildemann B, Kadow-Romacker A, Pruss A, Haas NP, Schmidmaier G. Quantification of growth factors in allogenic bone grafts extracted with three different methods. Cell Tissue Bank. 2007;8:107–114.

    Article  CAS  PubMed  Google Scholar 

  53. Yamaguchi A, Katagiri T, Ikeda T, Wozney JM, Rosen V, Wang EA, Kahn AJ, Suda T, Yoshiki S. Recombinant human bone morphogenetic protein-2 stimulates osteoblastic maturation and inhibits myogenic differentiation in vitro. J Cell Biol. 1991;113:681–687.

    Article  CAS  PubMed  Google Scholar 

  54. Yamaguchi A, Komori T, Suda T. Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1. Endocr Rev. 2000;21:393–411.

    Article  CAS  PubMed  Google Scholar 

  55. Yamamoto N, Furuya K, Hanada K. Progressive development of the osteoblast phenotype during differentiation of osteoprogenitor cells derived from fetal rat calvaria: model for in vitro bone formation. Biol Pharm Bull. 2002;25:509–515.

    Article  CAS  PubMed  Google Scholar 

  56. Yeo Y, Park K. Control of encapsulation efficiency and initial burst in polymeric microparticle systems. Arch Pharm Res. 2004;27:1–12.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We thank Ms. Daniela Dopler for expert technical assistance during the in vitro cell culture experiments.

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Authors and Affiliations

  1. 3B’s Research Group–Biomaterials, Biodegradables and Biomimetics, Department of Polymer Engineering, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, S. Cláudio do Barco, 4806-909, Caldas das Taipas, Guimarães, Portugal

    E. R. Balmayor MSc, H. S. Azevedo PhD & R. L. Reis PhD, DSc

  2. IBB–Institute for Biotechnology and Bioengineering, PT Government Associated Laboratory, Guimarães, Portugal

    E. R. Balmayor MSc, H. S. Azevedo PhD & R. L. Reis PhD, DSc

  3. Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Austrian Cluster for Tissue Regeneration, AUVA Research Centre, Vienna, Austria

    E. R. Balmayor MSc, G. A. Feichtinger MSc & M. van Griensven MD, PhD

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  1. E. R. Balmayor MSc
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  2. G. A. Feichtinger MSc
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  3. H. S. Azevedo PhD
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  4. M. van Griensven MD, PhD
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  5. R. L. Reis PhD, DSc
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Correspondence to E. R. Balmayor MSc.

Additional information

One or more authors have received funding from a personal grant of Marie Curie Host Fellowships for Early Stage Research Training (EST) “Alea Jacta EST” (MEST-CT-2004-008104) (ERB) and from the European NoE EXPERTISSUES (NMP3-CT-2004-500283) (RLR).

This work was performed at 3B′s Research Group–Biomaterials, Biodegradables and Biomimetics (material development and characterization, as well as in vitro BMP-2 release studies including the testing of encapsulation efficiency) and at the Ludwig Boltzmann Institute for Experimental and Clinical Traumatology (in vitro cell studies).

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Balmayor, E.R., Feichtinger, G.A., Azevedo, H.S. et al. Starch-poly-є-caprolactone Microparticles Reduce the Needed Amount of BMP-2. Clin Orthop Relat Res 467, 3138–3148 (2009). https://doi.org/10.1007/s11999-009-0954-z

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  • DOI: https://doi.org/10.1007/s11999-009-0954-z

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