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Sonic hedgehog gene-enhanced tissue engineering for bone regeneration

Abstract

Improved methods of bone regeneration are needed in the craniofacial rehabilitation of patients with significant bone deficits secondary to tumor resection, congenital deformities, and prior to prosthetic dental reconstruction. In this study, a gene-enhanced tissue-engineering approach was used to assess bone regenerative capacity of Sonic hedgehog (Shh)-transduced gingival fibroblasts, mesenchymal stem cells, and fat-derived cells delivered to rabbit cranial bone defects in an alginate/collagen matrix. Human Shh cDNA isolated from fetal lung tissue was cloned into the replication-incompetent retroviral expression vector LNCX, in which the murine leukemia virus retroviral LTR drives expression of the neomycin-resistance gene. The rat β-actin enhancer/promoter complex was engineered to drive expression of Shh. Reverse transcriptase-polymerase chain reaction analysis demonstrated that the transduced primary rabbit cell populations expressed Shh RNA. Shh protein secretion was confirmed by enzyme-linked immunosorbent assay (ELISA). Alginate/ type I collagen constructs containing 2 × 106 Shh-transduced cells were introduced into male New Zealand White rabbit calvarial defects (8 mm). A total of eight groups (N=6) were examined: unrestored empty defects, matrix alone, matrix plus the three cell populations transduced with both control and Shh expression vectors. The bone regenerative capacity of Shh gene enhanced cells was assessed grossly, radiographically and histologically at 6 and 12 weeks postimplantation. After 6 weeks, new full thickness bone was seen emanating directly from the alginate/collagen matrix in the Shh-transduced groups. Quantitative two-dimensional digital analysis of histological sections confirmed statistically significant (P<0.05) amounts of bone regeneration in all three Shh-enhanced groups compared to controls. Necropsy failed to demonstrate any evidence of treatment-related side effects. This is the first study to demonstrate that Shh delivery to bone defects, in this case through a novel gene-enhanced tissue-engineering approach, results in significant bone regeneration. This encourages further development of the Shh gene-enhanced tissue-engineering approach for bone regeneration.

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References

  1. Zellin G . Growth factors and bone regeneration. Swed Dent J 1998; 129: 7–65.

    CAS  Google Scholar 

  2. Ebara S, Nakayama K . Mechanisms for the action of bone morphogenetic proteins and regulation of their activity. Spine 2002; 27: S10–S15.

    Article  PubMed  Google Scholar 

  3. Yoshida K et al. Enhancement by recombinant human bone morphogenetic protein-2 of bone formation by means of porous hydroxyapatite in mandibular bone defects. J Dent Res 1999; 78: 1505–1510.

    Article  CAS  PubMed  Google Scholar 

  4. Miyaji H et al. Hard tissue formation on dentin surfaces applied with recombinant human bone morphogenetic protein-2 in the connective tissue of the palate. J Periodont Res 2002; 37: 204–209.

    Article  CAS  Google Scholar 

  5. Suzuki T et al. Regeneration of defects in the articular cartilage in rabbit temporomandibular joints by bone morphogenetic protein-2. Brit J Oral Maxillofac Surg 2002; 40: 201–206.

    Article  CAS  Google Scholar 

  6. Wozney JM . Overview of bone morphogenetic protein. Spine 2002; 27: S2–S8.

    Article  PubMed  Google Scholar 

  7. Groeneveld EH, Burger EH . Bone morphogenetic proteins in human bone regeneration. Eur J Endocrinol 2000; 142: 9–21.

    Article  CAS  PubMed  Google Scholar 

  8. Valentin-Opran A et al. Clinical evaluation of recombinant bone morphogenetic protein-2. Clin Orthopaed Rel Res 2002; 395: 110–120.

    Article  Google Scholar 

  9. Spinella-Jaegle S et al. Sonic Hedgehog increases the commitment of pluripotent mesenchymal cells into the osteoblastic lineage and abolishes adipocytic differentiation. J Cell Sci 2001; 114: 2085–2094.

    CAS  PubMed  Google Scholar 

  10. St-Jacques B et al. Sonic hedgehog signaling is essential for hair development. Curr Biol 1998; 8: 1058–1068.

    Article  CAS  PubMed  Google Scholar 

  11. Peters H, Balling R . Teeth: where and how to make them. Trends Genet 1999; 15: 59–65.

    Article  CAS  PubMed  Google Scholar 

  12. Dassule HR et al. Sonic hedgehog regulates growth and morphogenesis of the tooth. Development 2000; 127: 4775–4785.

    CAS  PubMed  Google Scholar 

  13. Chiang C et al. Cyclopia and defective axial patterning in mice lacking sonic hedgehog gene function. Nature 1996; 383: 407–413.

    Article  CAS  PubMed  Google Scholar 

  14. Hu D, Helms JA . The role of Sonic hedgehog in normal and abnormal craniofacial morphogenesis. Development 1999; 126: 4873–4884.

    CAS  PubMed  Google Scholar 

  15. Nanni L et al. SHH mutation is associated with solitary median maxillary central incisor: a study of 13 patients and review of the literature. Am J Med Genet 2001; 102: 1–10.

    Article  CAS  PubMed  Google Scholar 

  16. Kinto N et al. Fibroblasts expressing sonic hedgehog induce osteoblast differentiation and ectopic bone formation. FEBS Lett 1997; 404: 319–323.

    Article  CAS  PubMed  Google Scholar 

  17. Kato M et al. Identification of Sonic Hedgehog-responsive genes using cDNA microarray. Biochem Biophys Res Comm 2001; 298: 472–478.

    Article  Google Scholar 

  18. Nybakken K, Perrimon N . Hedgehog signal transduction: recent findings. Curr Opin Genet Develop 2002; 12: 503–511.

    Article  CAS  Google Scholar 

  19. Bitgood MJ, McMahon AP . Hedgehog and BMP genes are co-expressed at many diverse sites of cell–cell interaction in the mouse embryo. Dev Biol 1995; 172: 126–138.

    Article  CAS  PubMed  Google Scholar 

  20. Ohsaki K, Osumi N, Nakamura S . Altered whisker patterns induced by ectopic expression of SHH are topographically represented by barrels. Dev Brain Res 2002; 137: 159–170.

    Article  CAS  Google Scholar 

  21. Enamoto-Iwamoto M et al. Hedgehog proteins stimulate chondrogenic cell differentiation and cartilage formation. J Bone Min Res 2000; 15: 1659–1668.

    Article  Google Scholar 

  22. Yuasa T et al. Sonic Hedgehog is involved in osteoblast differentiation by cooperating with BMP-2. J Cell Physiol 2002; 193: 225–232.

    Article  CAS  PubMed  Google Scholar 

  23. Goetz JA, Suber LM, Zeng X, Robbins DJ . Sonic hedgehog as a mediator of long-range signaling. BioEssays 2002; 24: 157–165.

    Article  CAS  PubMed  Google Scholar 

  24. Miller AD, Rosman GJ . Improved retroviral vectors for gene transfer and expression. Biotechniques 1989; 7: 980–990.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Pepicelli CV, Lewis PM, McMahon AP . Sonic hedgehog regulates branching morphogenesis in the mammalian lung. Curr Biol 1998; 8: 1083–1086.

    Article  CAS  PubMed  Google Scholar 

  26. Marigo V et al. Cloning, expression and chromosomal location of SHH and IHH, two human homologues of the Drosophila segment polarity gene Hedgehog. Genomics 1995; 28: 44–51.

    Article  CAS  PubMed  Google Scholar 

  27. Mason JM, Grande DA, Barcia M et al. Expression of human bone morphogenetic protein-7 in primary rabbit periosteal cells: potential utility in gene therapy for bone repair. Gene Therapy 1998; 5: 1098–1104.

    Article  CAS  PubMed  Google Scholar 

  28. Grande DA et al. A dual gene therapy approach to osteochondral defect repair using a bilayer implant containing BMP-7 and IGF-1 transduced periosteal cells. Trans Orthop Res Soc 2001; 26: 294.

    Google Scholar 

  29. Stabler C, Wilks K, Sambanis A, Constantinidis I . The effects of alginate composition on encapsulated BTC3 cells. Biomaterials 2001; 22: 1301–1310.

    Article  CAS  PubMed  Google Scholar 

  30. Fleming JE, Cornell CN, Muschler GF . Bone cells and matrices in orthopedic tissue engineering. Orthop Clin North Am 2000; 31: 357–374.

    Article  PubMed  Google Scholar 

  31. Rudert M . Histological evaluation of osteochondral defects; consideration of animal models with emphasis on the rabbit, experimental setup, follow-up and applied methods. Cells Tissue Organs 2002; 171: 229–240.

    Article  Google Scholar 

  32. Hollinger JO, Kleinschmidt JC . The critical size defect as an experimental model to test bone repair materials. J Craniofacial Surg 1990; 1: 60–68.

    Article  CAS  Google Scholar 

  33. Tsuchida H et al. Engineered allogeneic mesenchymal stem cells repair femoral segmental defect in rats. J Orthoped Res 2003; 21: 44–53.

    Article  Google Scholar 

  34. Frame JW . A convenient animal model for testing bone substitute materials. J Oral Surg 1980; 38: 176–180.

    CAS  PubMed  Google Scholar 

  35. Gosain AK et al. Osteogenesis in cranial defects: reassessment of the concept of critical size and the expression of TGF-[beta] isoforms. Plast Reconsrtuct Surg 2000; 106: 360–371.

    Article  CAS  Google Scholar 

  36. Kramer IR, Killey HC, Wright HC . A histological and radiological comparison of the healing of defects in the rabbit calvarium with and without implanted heterogenous anorganic bone. Arch Oral Biol 1968; 13: 1095–1106.

    Article  CAS  PubMed  Google Scholar 

  37. Breitbart AS et al. Gene-enhanced tissue engineering: applications for bone healing using cultured periosteal cells transduced retrovirally with the BMP-7 gene. Ann Plast Surg 1999; 42: 488–495.

    Article  CAS  PubMed  Google Scholar 

  38. Diduch DR, Jordan LC, Mierisch CM, Balian G . Marrow stromal cells embedded in alginate for repair of osteochondral defects. J Arthroscop Relat Surg 2000; 16: 571–577.

    Article  CAS  Google Scholar 

  39. Milla E et al. Poly(L-lactide) acid/alginate composite membranes for guided tissue regeneration. J Biomed Mater Res 2001; 57: 248–257.

    Article  Google Scholar 

  40. Loebsack A et al. In vivo characterization of a porous hydrogel material for use as a tissue bulking agent. J Biomed Mater Res 2001; 57: 575–581.

    Article  CAS  PubMed  Google Scholar 

  41. Shang Q et al. Tissue-engineered bone repair of sheep cranial defects with autologous bone marrow stromal cells. J Craniofac Surg 2001; 12: 586–593.

    Article  CAS  PubMed  Google Scholar 

  42. Miralles G et al. Sodium alginate sponges with or without sodium hyaloronate: in vitro engineering of cartilage. J Biomed Mater Res 2001; 57: 268–278.

    Article  CAS  PubMed  Google Scholar 

  43. Murphy MG et al. The effects of rhBMP-2 on human osteosarcoma cells and gingival fibroblasts in vitro. J Oral Implantol 2001; 27: 16–24.

    Article  CAS  PubMed  Google Scholar 

  44. Krebsbach PH, Gu K, Franceschi RT, Rutherford RB . Gene therapy-directed osteogenesis: BMP-7 transduced human fibroblasts form bone in vivo. Hum Gene Ther 2000; 11: 1201–1210.

    Article  CAS  PubMed  Google Scholar 

  45. Zuk PA et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 2001; 7: 211–228.

    Article  CAS  PubMed  Google Scholar 

  46. Gysin R et al. Ex vivo gene therapy with stromal cells transduced with a retroviral vector containing the BMP4 gene completely heals critical size calvarial defect in rats. Gene Therapy 2002; 9: 991–999.

    Article  CAS  PubMed  Google Scholar 

  47. Markowitz D, Goff S, Bank A . A safe packaging line for gene transfer: separating viral genes on two different plasmids. J Virol 1988; 62: 1120–1124.

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Miller AD, Buttimore C . Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol 1986; 6: 2895–2902.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank Yana Moses for preparation of the histological sections. This research was supported in part by Public Health Service Grant R41 DE-015430 to JMM from the National Institutes of Health (NIDCR).

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Edwards, P., Ruggiero, S., Fantasia, J. et al. Sonic hedgehog gene-enhanced tissue engineering for bone regeneration. Gene Ther 12, 75–86 (2005). https://doi.org/10.1038/sj.gt.3302386

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