Recombinant human growth/differentiation factor-5 (rhGDF-5) induced bone formation in murine calvariae.
OBJECTIVE
Growth/differentiation factor-5 (GDF-5), a member of the transforming growth factor-beta superfamily, shows a close structural relationship to bone morphogenetic proteins and plays crucial roles in skeletal morphogenesis. Recombinant human (rh) GDF-5 was reported as a suitable factor for enhancing healing in bone defect and inducing ectopic bone formation. The purpose of the present study was to investigate the mechanism of bone formation induced by rhGDF-5 in murine calvariae by radiological, histological and immunohistochemical methods. Cell proliferation was also examined in vitro.


METHODS
Cells including primary osteoblasts, periosteum cells and connective tissue fibroblasts were isolated enzymatically from neonatal murine calvariae or head skin. In the presence or absence of rhGDF-5, cell proliferation was estimated by tetrazolium reduction assay. To examine the mechanism of osteoinduction, rhGDF-5/atelocollagen (AC) composite or 0.01 N HCl/AC composite were injected into murine calvariae subcutaneously. Tissue was examined radiologically, histologically and immunohistochemically.


RESULTS
In the presence of rhGDF-5, proliferation of primary osteoblasts, periosteum cells, and connective tissue fibroblasts was increased significantly in culture. Immunohistochemical observations showed cells at the site injected with rhGDF-5/AC displayed immunoreactivity for proliferating cell nuclear antigen (PCNA). Newly formed bone- and cartilage-like tissue contained chondrocyte osteocyte and osteoclastic cells, and were immunoreactive for both type I and II collagen.


CONCLUSIONS
Exposure to GDF-5 promotes proliferation and differentiation of calvarial cells, which give rise to ectopic bone formation.
Yoshimoto
Takehiko
T
Department of Periodontology, Kagoshima University Graduate School of Medical and Dental Sciences, Japan.
Yamamoto
Matsuo
M
Kadomatsu
Hideshi
H
Sakoda
Kenji
K
Yonamine
Yutaka
Y
Izumi
Yuichi
Y
D000704:Analysis of Variance; D000818:Animals; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D020219:Chondrogenesis / Q000187:drug effects; D005347:Fibroblasts / Q000187:drug effects; D055428:Growth Differentiation Factor 5; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects; D010012:Osteogenesis / Q000187:drug effects*; D010521:Periosteum / Q000166:cytology; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis; D011994:Recombinant Proteins / Q000494:pharmacology; D012535:Scalp / Q000166:cytology; D012886:Skull / Q000166:cytology; D014057:Tomography, X-Ray Computed
D019485:Bone Morphogenetic Proteins; C525893:GDF5 protein, human; C525894:Gdf5 protein, mouse; D055428:Growth Differentiation Factor 5; D018809:Proliferating Cell Nuclear Antigen; D011994:Recombinant Proteins