Repair of bone defects using synthetic mimetics of collagenous extracellular matrices

Abstract

We have engineered synthetic poly(ethylene glycol) (PEG)–based hydrogels as cell-ingrowth matrices for in situ bone regeneration. These networks contain a combination of pendant oligopeptide ligands for cell adhesion (RGDSP) and substrates for matrix metalloproteinase (MMP) as linkers between PEG chains. Primary human fibroblasts were shown to migrate within these matrices by integrin- and MMP-dependent mechanisms. Gels used to deliver recombinant human bone morphogenetic protein-2 (rhBMP-2) to the site of critical- sized defects in rat crania were completely infiltrated by cells and were remodeled into bony tissue within five weeks. Bone regeneration was dependent on the proteolytic sensitivity of the matrices and their architecture. The cell-mediated proteolytic invasiveness of the gels and entrapment of rhBMP-2 resulted in efficient and highly localized bone regeneration.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Scheme for gel preparation.
Figure 2: Three-dimensional fibroblast migration.
Figure 3: rhBMP-2 release triggered by MMP activity.
Figure 4: Bone healing in rat calvaria.
Figure 5: Micro-CT imaging of bone healing.
Figure 6: Effect of gel architecture.

References

  1. 1

    Kingsley, D.M. What do BMPs do in mammals: clues from the mouse short-ear mutation. Trends Genet. 10, 16–21 (1994).

    CAS  Article  Google Scholar 

  2. 2

    Wozney, J.M. & Rosen, V. Bone morphogenetic protein and bone morphogenetic protein gene family in bone formation and repair. Clin. Orthop. 346, 26–37 (1998).

    Article  Google Scholar 

  3. 3

    Schmitt, J.M., Hwang, K., Winn, S.R. & Hollinger, J.O. Bone morphogenetic proteins: an update on basic biology and clinical relevance. J. Orthop. Res. 17, 269–278 (1999).

    CAS  Article  Google Scholar 

  4. 4

    Wozney, J.M. et al. Novel regulators of bone formation: molecular clones and activities. Science 244, 1528–1534 (1988).

    Article  Google Scholar 

  5. 5

    Fiedlaender, G.E. et al. Osteogenic protein-1 (bone morphogenetic protein-7) in the treatment of tibial nonunions. J. Bone Joint Surg. 83A, 151–189 (2001).

    Google Scholar 

  6. 6

    Li, R.H. & Wozney, J.M. Delivering on the promise of bone morphogenetic proteins. Trends Biotechnol. 19, 255–265 (2001).

    CAS  Article  Google Scholar 

  7. 7

    Brekke, J. & Toth, J. Principles of tissue engineering applied to programmable osteogenesis. J. Biomed. Mat. Res. 43, 365–373 (1998).

    Article  Google Scholar 

  8. 8

    Uludag, H. et al. Implantation of recombinant being bone morphogenetic proteins with biomaterial carriers, a correlation between protein pharmacokinetics and osteoinduction in the rat ectopic model. J. Biomed. Mater. Res. 50, 227–238 (2000).

    CAS  Article  Google Scholar 

  9. 9

    Boyne, P.J. Animal studies of application of rhBMP-2 in maxillofacial reconstruction. Bone 19, 83–92 (1996).

    Article  Google Scholar 

  10. 10

    Hollinger, J.O. et al. Recombinant human morphogenetic protein-2 and collagen for bone regeneration. J. Biomed. Mater. Res. (Appl. Biomater.) 43, 356–364 (1998).

    CAS  Article  Google Scholar 

  11. 11

    Ellingsworth, L.R., DeLustro, F., Brennan, J.E., Sawamura, S. & McPherson, J. The human immune response to reconstituted bovine collagen. J. Immunol. 136, 8877–8882 (1986).

    Google Scholar 

  12. 12

    DeLustro, F., Dasch, J., Keefe, J. & Ellingsworth, L. Immune responses to allogeneic and xenogeneic implants of collagen and collagen derivates. Clin. Orthop. 260, 263–279 (1990).

    Article  Google Scholar 

  13. 13

    Boestman, O. Foreign-body reactions to fracture fixation implants of biodegradable synthetic polymers. J. Bone Joint Surg. 73B, 592–596 (1990).

    Article  Google Scholar 

  14. 14

    Meikle, M.C. et al. Effect of poly dl-lactide-co-glycolide implants and xenogeneic bone matrix-derived growth factors on calvarial bone repair in the rabbit. Biomaterials 15, 513–521 (1994).

    CAS  Article  Google Scholar 

  15. 15

    Kenley, R. et al. Osseos regeneration in the rat calvarium using novel delivery systems for recombinant human bone morphogenetic protein-2 (rhBMP-2). J. Biomed. Mater. Res. 28, 1139–1147 (1994).

    CAS  Article  Google Scholar 

  16. 16

    Zegzula, D., Buck, D.C., Brekke, J., Wozney, J.M. & Hollinger, J.O. Bone formation with use of rhBMP-2 (recombinant human bone morphogenetic protein-2). J. Bone Joint Surg. 79A, 1778–1790 (1997).

    Article  Google Scholar 

  17. 17

    Ertel, S.I. Evaluation of poly(d-carbonate), a tyrosine-derived degradable polymer, for orthopedic applications. J. Biomed. Mater. Res. 29, 1337–1348 (1995).

    CAS  Article  Google Scholar 

  18. 18

    Anseth, K.S., Sharstri, V.R. & Langer, R. Photopolymerizable degradable polyanhydrides with osteocompatibility. Nat. Biotechnol. 17, 156–159 (1999).

    CAS  Article  Google Scholar 

  19. 19

    Zhu, G., Mallery, S.R. & Schwendeman, S.P. Stabilization of proteins encapsulated in injectable poly(lactide-co-glycolide). Nat. Biotechnol. 18, 52–57 (1999).

    Article  Google Scholar 

  20. 20

    Saito, N. et al. A biodegradable polymer as a cytokine delivery system for inducing bone formation. Nat. Biotechnol. 19, 332–335 (2001).

    CAS  Article  Google Scholar 

  21. 21

    Hubbell, J.A. Bioactive biomaterials. Curr. Opin. Biotechnol. 10, 123–129 (1999).

    CAS  Article  Google Scholar 

  22. 22

    Griffith, L.G. & Naughton, G. Tissue engineering—current challenges and expanding opportunities. Science 295, 1009–1014 (2002).

    CAS  Article  Google Scholar 

  23. 23

    Pratt, A.B. & Hubbell, J.A. Cell-responsive Synthetic Biomaterials Formed In Situ (California Institute of Technology, Pasadena, CA, 2001).

    Google Scholar 

  24. 24

    Halstenberg, S., Panitch, A., Rizzi, S., Hall, H. & Hubbell, J.A. Biologically engineered protein-graft-poly(ethylene glycol) hydrogels: a cell adhesive and plasmin-degradable biosynthetic material for tissue repair. Biomacromolecules 3, 710–723 (2002).

    CAS  Article  Google Scholar 

  25. 25

    Gobin, A.S. & West, J.L. Cell migration through defined, synthetic ECM analogs. FASEB 16, 751–753 (2002).

    CAS  Article  Google Scholar 

  26. 26

    Clark, R.A.F. Molecular and Cellular Biology of Wound Repair (Plenum Press, NY, 1996).

    Google Scholar 

  27. 27

    Woessner, J.F. & Nagase, H. Matrix Metalloproteinases and TIMPs (Oxford Univ. Press, New York, 2000).

    Google Scholar 

  28. 28

    Sternlicht, M.D. & Werb, Z. How matrix metalloproteinases regulate cell behavior. Annu. Rev. Cell Dev. Biol. 17, 463–516 (2001).

    CAS  Article  Google Scholar 

  29. 29

    Vu, T.H. & Werb, Z. Matrix metalloproteinases: effectors of development and normal physiology. Genes Dev. 14, 2123–2133 (2000).

    CAS  Article  Google Scholar 

  30. 30

    West, J.L. & Hubbell, J.A. Separation of the arterial wall from blood contact using hydrogel barriers reduces intimal thickening after balloon injury in the rat: the roles of medial and luminal factors in arterial healing. Proc. Natl. Acad. Sci. USA. 93, 13188–13193 (1996).

    CAS  Article  Google Scholar 

  31. 31

    Weber, F.E., Eyrich, G., Graetz, K.W., Maly, F.E. & Sailer, H.F. Slow and continuous application of human recombinant bone morphogenetic protein via biodegradable poly(lactide-co-glycolide) foamspheres. Int. J. Oral Maxillofac. Surg. 31, 60–65 (2002).

    CAS  Article  Google Scholar 

  32. 32

    Ruppert, R., Hoffmann, E. & Sebald, W. Human bone morphogenetic protein 2 contains a heparin-binding site which modifies its biological activity. Eur. J. Biochem. 237, 295–302 (1996).

    CAS  Article  Google Scholar 

  33. 33

    Patel, S., Cudney, B. & MacPherson, A. Polymeric precipitants for the crystallinization of macromolecules. Biochem. Biophys. Res. Commun. 207, 819–828 (1995).

    CAS  Article  Google Scholar 

  34. 34

    Uludang, H. et al. rhBMP-collagen sponges as osteoinductive devices: effects of in vitro sponge characteristics and protein pi on in vivo rhBMP pharmacokinetics. Ann. New York Acad. Sci. 875, 369–378 (1999).

    Article  Google Scholar 

  35. 35

    Elbert, D.L., Pratt, A.B., Lutolf, M.P., Halstenberg, S. & Hubbell, J.A. Protein delivery from materials formed by self-selective conjugate addition reactions. J. Contr. Rel. 76, 11–25 (2001).

    CAS  Article  Google Scholar 

  36. 36

    Weber, F.E. et al. Disulfide bridge conformers of mature BMP are inhibitors for heterotopic ossification. Biochem. and Biophys. Res. Commun. 268, 554–558 (2001).

    Article  Google Scholar 

  37. 37

    European Agency for the Evaluation of Medicinal Products. European Public Assessment Report: osteogenic protein 1 (Cpmp/0393/01). http://www.eudra.org/ humandocs/humans/epar/osteogenicprot1/osteogenicprot1.htm (2001).

  38. 38

    Lutolf, M.P., Tirelli, N., Cerritelli, S., Cavalli, L. & Hubbell, J.A. Systematic modulation of Michael-type reactivity of thiols through the use of charged amino acids. Bioconj. Chem. 12, 1051 (2001).

    CAS  Article  Google Scholar 

  39. 39

    Muller, R. & Ruegsegger, P. Micro-tomographic imaging for the nondestructive evaluation of trabecular bone architecture. Stud. Health Technol. Infor. 40, 61–79 (1997).

    CAS  Google Scholar 

  40. 40

    Odgaard, A. & Gundersen, H.J. Quantification of connectivity in cancellous bone, with special emphasis on 3-D reconstructions. Bone 14, 173–182 (1993).

    CAS  Article  Google Scholar 

  41. 41

    Muller, R., Hildebrand, T. & Ruegsegger, P. Noninvasive bone-biopsy—a new method to analyze and display the 3-dimensional structure of trabecular bone. Phys. Med. Biol. 39, 145–164 (1994).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

Funding of this study was provided by the Swiss National Science Foundation (NFP46 grant 58681), the Swiss Federal Agency for Education and Science (01.0224), and the European Union Framework 5 Program (C5RD-CT-2000-00267). We thank A. Zisch and G. Raeber for helpful discussions.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Jeffrey A. Hubbell.

Ethics declarations

Competing interests

The materials employed in this study are the subject of patents that are held by the Swiss Federal Institute of Technology and the University of Zurich. These patents have been licensed by Kuros Biosurgery AG, in which the Swiss Federal Institute of Technology, the University of Zurich, and some of the authors hold equity, and by which some of the authors are employed as consultants.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lutolf, M., Weber, F., Schmoekel, H. et al. Repair of bone defects using synthetic mimetics of collagenous extracellular matrices. Nat Biotechnol 21, 513–518 (2003). https://doi.org/10.1038/nbt818

Download citation

Further reading