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Adipose-derived adult stromal cells heal critical-size mouse calvarial defects

Nature Biotechnology volume 22, pages 560–567 (2004)Cite this article

Abstract

In adults and children over two years of age, large cranial defects do not reossify successfully, posing a substantial biomedical burden. The osteogenic potential of bone marrow stromal (BMS) cells has been documented. This study investigates the in vivo osteogenic capability of adipose-derived adult stromal (ADAS) cells, BMS cells, calvarial-derived osteoblasts and dura mater cells to heal critical-size mouse calvarial defects. Implanted, apatite-coated, PLGA scaffolds seeded with ADAS or BMS cells produced significant intramembranous bone formation by 2 weeks and areas of complete bony bridging by 12 weeks as shown by X-ray analysis, histology and live micromolecular imaging. The contribution of implanted cells to new bone formation was 84–99% by chromosomal detection. These data show that ADAS cells heal critical-size skeletal defects without genetic manipulation or the addition of exogenous growth factors.

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Figure 1: X-ray, alizarin red and histological analysis of specimens at 4 weeks.
Figure 2: Proliferation and bone formation of ADS and BMS cells.
Figure 3: Intramembranous versus endochondral bone formation.
Figure 4: Live micro-CT imaging.
Figure 5: Bone metabolic activity of animals implanted with control (no cells) or ADS cell–seeded scaffolds as determined by radiolabeled MDP incorporation overlaid with micro-CT images.

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References

  1. Krebsbach, P.H. et al. Bone formation in vivo: comparison of osteogenesis by transplanted mouse and human marrow stromal fibroblasts. Transplantation 63, 1059–1069 (1997).

    Article  CAS  Google Scholar 

  2. Krebsbach, P.H., Mankani, M.H., Satomura, K., Kuznetsov, S.A. & Robey, P.G. Repair of craniotomy defects using bone marrow stromal cells. Transplantation 66, 1272–1278 (1998).

    Article  CAS  Google Scholar 

  3. Mendes, S.C. et al. Bone tissue-engineered implants using human bone marrow stromal cells: effect of culture conditions and donor age. Tissue Eng. 8, 911–920 (2002).

    Article  CAS  Google Scholar 

  4. Peng, H. et al. Synergistic enhancement of bone formation and healing by stem cell-expressed VEGF and bone morphogenetic protein-4. J. Clin. Invest. 110, 751–759 (2002).

    Article  CAS  Google Scholar 

  5. Vacanti, J.P., Langer, R., Upton, J. & Marler, J.J. Transplantation of cells in matrices for tissue regeneration. Adv. Drug Deliv. Rev. 33, 165–182 (1998).

    Article  Google Scholar 

  6. Wright, V. et al. BMP4-expressing muscle-derived stem cells differentiate into osteogenic lineage and improve bone healing in immunocompetent mice. Mol. Ther. 6, 169–178 (2002).

    Article  CAS  Google Scholar 

  7. Durham, S.R., McComb, J.G. & Levy, M.L. Correction of large (>25 cm(2)) cranial defects with “reinforced” hydroxyapatite cement: technique and complications. Neurosurgery 52, 842–845; discussion 845 (2003).

    Article  Google Scholar 

  8. Harris, S.E. et al. Effects of transforming growth factor beta on bone nodule formation and expression of bone morphogenetic protein 2, osteocalcin, osteopontin, alkaline phosphatase, and type I collagen mRNA in long-term cultures of fetal rat calvarial osteoblasts. J. Bone Miner. Res. 9, 855–863 (1994).

    Article  CAS  Google Scholar 

  9. Dong, J. et al. In vivo evaluation of a novel porous hydroxyapatite to sustain osteogenesis of transplanted bone marrow-derived osteoblastic cells. J. Biomed. Mater. Res. 57, 208–216 (2001).

    Article  CAS  Google Scholar 

  10. Lee, J.Y. et al. Effect of bone morphogenetic protein-2-expressing muscle-derived cells on healing of critical-sized bone defects in mice. J. Bone Joint Surg. Am. 83-A, 1032–1039 (2001).

    Article  CAS  Google Scholar 

  11. Shea, L.D., Wang, D., Franceschi, R.T. & Mooney, D.J. Engineered bone development from a pre-osteoblast cell line on three-dimensional scaffolds. Tissue Eng. 6, 605–617 (2000).

    Article  CAS  Google Scholar 

  12. Muller-Mai, C.M., Stupp, S.I., Voigt, C. & Gross, U. Nanoapatite and organoapatite implants in bone: histology and ultrastructure of the interface. J. Biomed. Mater. Res. 29, 9–18 (1995).

    Article  CAS  Google Scholar 

  13. Reddi, A. Bioceramics, cells and signals in tissue engineering. Bioceramics, Proceedings of the International Symposium on Ceramics in Medicine 11, 9–11 (1998).

    Google Scholar 

  14. Hench, L.L. Bioceramics. J. Am. Ceramic Soc. 81, 1705–1728 (1998).

    Article  CAS  Google Scholar 

  15. Bourgeois, B. et al. Calcium-deficient apatite: a first in vivo study concerning bone ingrowth. J. Biomed. Mater. Res. 65A, 402–408 (2003).

    Article  CAS  Google Scholar 

  16. Neo, M. et al. A comparative study of ultrastructures of the interfaces between four kinds of surface-active ceramic and bone. J. Biomed. Mater. Res. 26, 1419–1432 (1992).

    Article  CAS  Google Scholar 

  17. van Blitterswijk, C.A., Hesseling, S.C., Grote, J.J., Koerten, H.K. & de Groot, K. The biocompatibility of hydroxyapatite ceramic: a study of retrieved human middle ear implants. J. Biomed. Mater. Res. 24, 433–453 (1990).

    Article  CAS  Google Scholar 

  18. Le Huec, J.C., Schaeverbeke, T., Clement, D., Faber, J. & Le Rebeller, A. Influence of porosity on the mechanical resistance of hydroxyapatite ceramics under compressive stress. Biomaterials 16, 113–118 (1995).

    Article  CAS  Google Scholar 

  19. Sarkar, B.K. Fatigue of brittle materials—a critical appraisal. Bull. Mater. Sci. 18, 755–772 (1995).

    Article  CAS  Google Scholar 

  20. Frazza, E.J. & Schmitt, E.E. A new absorbable suture. J. Biomed. Mater. Res. 5, 43–58 (1971).

    Article  CAS  Google Scholar 

  21. Rosa, A.L., Beloti, M.M., Van Noort, R., Hatton, P.V. & Devlin, A.J. Surface topography of hydroxyapatite affects ROS17/2.8 cells response. Pesqui. Odontol. Bras. 16, 209–215 (2002).

    Article  Google Scholar 

  22. Okumura, A. et al. Substrate affects the initial attachment and subsequent behavior of human osteoblastic cells (Saos-2). Biomaterials 22, 2263–2271 (2001).

    Article  CAS  Google Scholar 

  23. Zhao, F. et al. [Tissue engineering study on chitosan-gelatin/hydroxyapatite composite scaffolds–osteoblasts culture]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 16, 130–133 (2002).

    Google Scholar 

  24. Steiner, C., Elixhauser, A. & Schnaier, J. The healthcare cost and utilization project: an overview. Eff. Clin. Pract. 5, 143–151 (2002).

    PubMed  Google Scholar 

  25. Hong, L. et al. Bone regeneration at rabbit skull defects treated with transforming growth factor-beta1 incorporated into hydrogels with different levels of biodegradability. J. Neurosurg. 92, 315–325. (2000).

    Article  CAS  Google Scholar 

  26. Buttery, L.D. et al. Differentiation of osteoblasts and in vitro bone formation from murine embryonic stem cells. Tissue Eng. 7, 89–99 (2001).

    Article  CAS  Google Scholar 

  27. Gundle, R., Joyner, C.J. & Triffitt, J.T. Human bone tissue formation in diffusion chamber culture in vivo by bone-derived cells and marrow stromal fibroblastic cells. Bone 16, 597–601 (1995).

    Article  CAS  Google Scholar 

  28. Haynesworth, S.E., Goshima, J., Goldberg, V.M. & Caplan, A.I. Characterization of cells with osteogenic potential from human marrow. Bone 13, 81–88 (1992).

    Article  CAS  Google Scholar 

  29. Johnstone, B., Hering, T.M., Caplan, A.I., Goldberg, V.M. & Yoo, J.U. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp. Cell. Res. 238, 265–272 (1998).

    Article  CAS  Google Scholar 

  30. Wakitani, S., Saito, T. & Caplan, A.I. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve 18, 1417–1426 (1995).

    Article  CAS  Google Scholar 

  31. Jaiswal, N., Haynesworth, S.E., Caplan, A.I. & Bruder, S.P. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro . J. Cell Biochem. 64, 295–312 (1997).

    Article  CAS  Google Scholar 

  32. Toquet, J. et al. Osteogenic potential in vitro of human bone marrow cells cultured on macroporous biphasic calcium phosphate ceramic. J. Biomed. Mater. Res. 44, 98–108 (1999).

    Article  CAS  Google Scholar 

  33. Im, G.I., Kim, D.Y., Shin, J.H., Hyun, C.W. & Cho, W.H. Repair of cartilage defect in the rabbit with cultured mesenchymal stem cells from bone marrow. J. Bone Joint Surg. Br. 83, 289–294 (2001).

    Article  CAS  Google Scholar 

  34. Halvorsen, Y.D. et al. Extracellular matrix mineralization and osteoblast gene expression by human adipose tissue-derived stromal cells. Tissue Eng. 7, 729–741 (2001).

    Article  CAS  Google Scholar 

  35. Tholpady, S.S., Katz, A.J. & Ogle, R.C. Mesenchymal stem cells from rat visceral fat exhibit multipotential differentiation in vitro . Anat. Rec. 272A, 398–402 (2003).

    Article  Google Scholar 

  36. Zuk, P.A. et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 7, 211–228 (2001).

    Article  CAS  Google Scholar 

  37. Zuk, P.A. et al. Human adipose tissue is a source of multipotent stem cells. Mol. Biol. Cell 13, 4279–4295 (2002).

    Article  CAS  Google Scholar 

  38. Mizuno, H. et al. Myogenic differentiation by human processed lipoaspirate cells. Plast. Reconstr. Surg. 109, 199–209 (2002).

    Article  Google Scholar 

  39. Ashjian, P.H. et al. In vitro differentiation of human processed lipoaspirate cells into early neural progenitors. Plast. Reconstr. Surg. 111, 1922–1931 (2003).

    Article  Google Scholar 

  40. Wickham, M.Q., Erickson, G.R., Gimble, J.M., Vail, T.P. & Guilak, F. Multipotent stromal cells derived from the infrapatellar fat pad of the knee. Clin. Orthop. 412, 196–212 (2003).

    Article  Google Scholar 

  41. Halvorsen, Y.D. et al. Thiazolidinediones and glucocorticoids synergistically induce differentiation of human adipose tissue stromal cells: biochemical, cellular, and molecular analysis. Metabolism 50, 407–413 (2001).

    Article  CAS  Google Scholar 

  42. Erickson, G.R. et al. Chondrogenic potential of adipose tissue-derived stromal cells in vitro and in vivo . Biochem. Biophys. Res. Commun. 290, 763–769 (2002).

    Article  CAS  Google Scholar 

  43. Safford, K.M. et al. Neurogenic differentiation of murine and human adipose-derived stromal cells. Biochem. Biophys. Res. Commun. 294, 371–379 (2002).

    Article  CAS  Google Scholar 

  44. Dragoo, J.L. et al. Bone induction by BMP-2 transduced stem cells derived from human fat. J. Orthop. Res. 21, 622–629 (2003).

    Article  CAS  Google Scholar 

  45. Lee, J.A. et al. Biological alchemy: engineering bone and fat from fat-derived stem cells. Ann. Plast. Surg. 50, 610–617 (2003).

    Article  Google Scholar 

  46. Cowan, C.M., Quarto, N., Warren, S.M., Salim, A. & Longaker, M.T. Age-related changes in the biomolecular mechanisms of calvarial osteoblast biology affect FGF-2 signaling and osteogenesis. J. Biol. Chem. 278, 32005–32013 (2003).

    Article  CAS  Google Scholar 

  47. Fong, K.D. et al. New strategies for craniofacial repair and replacement: a brief review. J. Craniofac. Surg. 14, 333–339 (2003).

    Article  Google Scholar 

  48. Mulliken, J.B. & Glowacki, J. Induced osteogenesis for repair and construction in the craniofacial region. Plast. Reconstr. Surg. 65, 553–560 (1980).

    Article  CAS  Google Scholar 

  49. Bostrom, R. & Mikos, A. (eds.) Tissue Engineering of Bone, vol. 1 (Birkhauser, Boston, 1997).

  50. Lieberman, J.R., Daluiski, A. & Einhorn, T.A. The role of growth factors in the repair of bone. Biology and clinical applications. J. Bone Joint Surg. Am. 84-A, 1032–1044 (2002).

    Article  Google Scholar 

  51. Bradley, J.P. et al. Studies in cranial suture biology: in vitro cranial suture fusion. Cleft Palate Craniofac. J. 33, 150–156 (1996).

    Article  CAS  Google Scholar 

  52. Bradley, J.P., Levine, J.P., McCarthy, J.G. & Longaker, M.T. Studies in cranial suture biology: regional dura mater determines in vitro cranial suture fusion. Plast. Reconstr. Surg. 100, 1091–1099 (1997).

    Article  CAS  Google Scholar 

  53. Opperman, L.A., Passarelli, R.W., Morgan, E.P., Reintjes, M. & Ogle, R.C. Cranial sutures require tissue interactions with dura mater to resist osseous obliteration in vitro . J. Bone Miner. Res. 10, 1978–1987 (1995).

    Article  CAS  Google Scholar 

  54. Opperman, L.A. et al. Dura mater secretes soluble heparin-binding factors required for cranial suture morphogenesis. In vitro cell dev. biol. 32, 150–158 (1996).

    Article  Google Scholar 

  55. Mehrara, B.J. et al. Basic fibroblast growth factor and transforming growth factor beta-1 expression in the developing dura mater correlates with calvarial bone formation. Plast. Reconstr. Surg. 104, 435–444 (1999).

    Article  CAS  Google Scholar 

  56. Greenwald, J.A. et al. Regional differentiation of cranial suture-associated dura mater in vivo and in vitro: implications for suture fusion and patency. J. Bone Miner. Res. 15, 2413–2430 (2000).

    Article  CAS  Google Scholar 

  57. Jiang, Y. et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418, 41–49 (2002).

    Article  CAS  Google Scholar 

  58. Medvinsky, A. & Smith, A. Stem cells: Fusion brings down barriers. Nature 422, 823–825 (2003).

    Article  CAS  Google Scholar 

  59. Wang, X. et al. Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature 422, 897–901 (2003).

    Article  CAS  Google Scholar 

  60. Vassilopoulos, G., Wang, P.R. & Russell, D.W. Transplanted bone marrow regenerates liver by cell fusion. Nature 422, 901–904 (2003).

    Article  CAS  Google Scholar 

  61. Wiedmann-Al-Ahmad, M., Gutwald, R., Lauer, G., Hubner, U. & Schmelzeisen, R. How to optimize seeding and culturing of human osteoblast-like cells on various biomaterials. Biomaterials 23, 3319–3328 (2002).

    Article  CAS  Google Scholar 

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Acknowledgements

This work has been supported by a grant from the National Institutes of Health (R01DE-14526) and the Oak foundation to M.T.L. The authors would like to thank Randall P. Nacamuli for his scientific and editorial assistance. We also acknowledge Koji Iwata and Michael Goris for their assistance with small animal imaging.

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Authors and Affiliations

  1. The Department of Surgery, Stanford University School of Medicine, Stanford University, 257 Campus Drive, Stanford, 94305, California, USA

    Catherine M Cowan, Yun-Ying Shi, Oliver O Aalami, Romy Thomas, Natalina Quarto & Michael T Longaker

  2. The Department of Bioengineering, University of California, Los Angeles, Los Angeles, California, USA

    Yu-Fen Chou & Benjamin Wu

  3. The Department of Radiology, Division of Nuclear Medicine, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, 94305, California, USA

    Carina Mari

  4. The Department of Pediatrics, Stanford University School of Medicine, Stanford University, 318 Campus Drive, Stanford, 94305, California, USA

    Christopher H Contag

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Cowan, C., Shi, YY., Aalami, O. et al. Adipose-derived adult stromal cells heal critical-size mouse calvarial defects. Nat Biotechnol 22, 560–567 (2004). https://doi.org/10.1038/nbt958

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