Differential effect of BMP4 on NIH/3T3 and C2C12 cells: implications for endochondral bone formation.
After intramuscular implantation, BMP4-expressing NIH/3T3 fibroblasts and BMP4-expressing C2C12 myoblasts can promote ectopic cartilage and bone formation. Fibroblasts tend to undergo chondrogenesis, whereas myoblasts primarily undergo osteogenesis. These results suggest that endochondral bone formation may involve different cell types, a finding that could have major implications for the tissue engineering of bone and cartilage.


BACKGROUND
The delivery of BMP4 through cell-based gene therapy can trigger ectopic endochondral bone formation in skeletal muscle. We hypothesized that, when stimulated with or transduced to express BMP4, different types of cells residing within skeletal muscle might participate in different stages of endochondral bone formation.


METHODS
We compared the responses of a fibroblast cell line (NIH/3T3), a myoblast cell line (C2C12), primary fibroblasts, and primary myoblasts to BMP4 stimulation in vitro. We then transduced the four cell populations to express BMP4 and compared their ability to promote ectopic endochondral bone formation in skeletal muscle.


RESULTS
Under the influence of BMP4 in vitro and in vivo, NIH/3T3 cells differentiated toward both chondrogenic and osteogenic lineages, whereas most C2C12 cells underwent primarily osteogenic differentiation. NIH/3T3 cells genetically modified to express BMP4 induced delayed but more robust cartilage formation than did genetically modified C2C12 cells, which promoted rapid ossification. These differences in terms of the timing and amount of cartilage and bone formation persisted even after we introduced a retrovirus encoding dominant negative Runx2 (DNRunx2) into the C2C12 cells, which interferes with the function of Runx2. Superior osteogenic potential was also displayed by the primary myoblasts in vitro and in vivo compared with the primary fibroblasts. The different proliferation abilities and differentiation potentials exhibited by these cells when influenced by BMP4 may at least partially explain the differing roles that BMP4-expressing myogenic cells and BMP4-expressing fibroblastic cells play in endochondral bone formation.


CONCLUSIONS
Our findings suggest that the process of endochondral bone formation in skeletal muscle after delivery of BMP4 involves different cell types, including fibroblastic cells, which are more involved in the chondrogenic phases, and myoblastic cells, which are primarily involved in osteogenesis. These findings could have important implications for the development of tissue engineering applications focused on bone and cartilage repair.
Li
Guangheng
G
Growth and Development Laboratory, Children's Hospital of Pittsburgh, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15213-2582, USA.
Peng
Hairong
H
Corsi
Karin
K
Usas
Arvydas
A
Olshanski
Anne
A
Huard
Johnny
J
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D018952:Antigens, CD34 / Q000096:biosynthesis; D000950:Antigens, Ly / Q000096:biosynthesis; D001681:Biological Assay; D015153:Blotting, Western; D015519:Bone Density; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000378:metabolism*; D001842:Bone and Bones / Q000378:metabolism / Q000473:pathology*; D002356:Cartilage / Q000166:cytology / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D019070:Cell Lineage; D049109:Cell Proliferation; D019902:Chondrocytes / Q000166:cytology; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics; D003893:Desmin / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005799:Genes, Dominant; D007150:Immunohistochemistry; D007763:Lac Operon; D008565:Membrane Proteins / Q000096:biosynthesis; D051379:Mice; D018482:Muscle, Skeletal / Q000166:cytology; D009132:Muscles / Q000166:cytology; D032446:Myoblasts / Q000378:metabolism; D041681:NIH 3T3 Cells; D010011:Osteocytes / Q000166:cytology; D010012:Osteogenesis; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D023822:Tissue Engineering; D014746:Vimentin / Q000378:metabolism
D018952:Antigens, CD34; D000950:Antigens, Ly; C525810:Bmp4 protein, mouse; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D050659:Core Binding Factor Alpha 1 Subunit; D003893:Desmin; C074650:Ly6a protein, mouse; D008565:Membrane Proteins; D014746:Vimentin; D000469:Alkaline Phosphatase