We examined the effect of recombinant human osteogenic protein-1 (OP-1, or bone morphogenetic protein-7), a member of the bone morphogenetic protein family, on growth and maturation of day 11, 15 and 17 chick sternal chondrocytes in high density monolayers, suspension and agarose cultures for up to 5 weeks. OP-1 dose-dependently (10-50 ng/ml) promoted chondrocyte maturation associated with enhanced alkaline phosphatase activity, and increased mRNA levels and protein synthesis of type X collagen in both the presence and absence of serum. In serum-free conditions, OP-1 promoted cell proliferation and chondrocyte maturation, without requiring either thyroid hormone or insulin, agents known to support chick chondrocyte differentiation in vitro. When grown in agarose under the same conditions, TGF-beta 1 and retinoic acid neither initiated nor promoted chondrocyte differentiation. The results demonstrate that OP-1, as the sole medium supplement, supports the maturation of embryonic chick sternal chondrocytes in vitro.

REFERENCES

Adams
S. L.
,
Pallante
K. M.
,
Niu
Z.
,
Leboy
P. S.
,
Golden
E. B.
,
Pacifici
M.
(
1991
).
Rapid induction of type X collagen gene expression in cultured chick vertebral chondrocytes.
Exp. Cell Res
193
,
190
197
Asahina
I.
,
Sampath
T. K.
,
Nishimura
I.
,
Hauschka
P. V.
(
1993
).
Human osteogenic protein-1 induces both chondroblastic and osteoblastic differentiation of osteoprogenitor cells derived from newborn rat calvaria.
J. Cell Biol
123
,
921
933
Böhme
K.
,
Conscience-Egli.
M.
,
Tschan
T.
,
Winterhalter
K. H.
,
Bruckner
P.
(
1992
).
Induction of proliferation or hypertrophy ofchondrocytes in serum-free culture: The role of insulin-like growth factor-1, or thyroxine.
J. Cell Biol
116
,
1035
1042
Bonen
D. K.
,
Schmid
T. M.
(
1991
).
Elevated extracellular calcium concentrations induces type X collagen synthesis in chondrocyte cultures.
J. Cell Biol
115
,
1171
1178
Carrington
J. L.
,
Chen
P.
,
Yanagishita
M.
,
Reddi
A. H.
(
1991
).
Osteogenin (bone morphogenetic protein-3) stimulates cartilage formation by chick limb bud cells in vitro.
Dev. Biol
146
,
406
415
Celeste
A. J.
,
Ianazzi
J. A.
,
Taylor
R. C.
,
Hewick
R. M.
,
Rosen
V.
,
Wang
E. A.
,
Wozney
J. M.
(
1990
).
Identification of transforming growth factorfamily members in bone inductive protein purified from bone.
Proc. Nat. Acad. Sci. USA
87
,
9843
9850
Chen
P.
,
Carrington
J. L.
,
Hammonds
R. G.
,
Reddi
A. H.
(
1991
).
Stimulation of chondrogenesis in limb bud mesoderm cells by recombinant human bone morphogenetic protein 2B (BMP-2B) and modulation by transforming growth factor1and 2.
Exp. Cell Res
195
,
509
515
Chen
P.
,
Vukicevic
S.
,
Sampath
T. K.
,
Luyten
F. P.
(
1993
).
Bovine articular chondrocytes do not undergo hypertrophy when cultured in the presence of serum and osteogenic protein-1.
Biochem. Biophys. Res. Commun
197
,
1253
1259
Chen
T. L.
,
Bates
R. L.
,
Dudley
A.
,
Hammonds
R. G.
,
Amento
E. P.
Jr.
(
1991
).
Bone morphogenetic protein-2b stimulation of growth and osteogenic phenotypes in rat osteoblast-like cells: comparison with TGF-1.
J. Bone Miner. Res
6
,
1387
1393
Chomczynski
P.
,
Sacchi
N.
(
1987
).
Single-step method of RNA isolation by acid-guanidinium thiocyanate-phenol-chloroform extraction.
Anal. Biochem
162
,
156
159
Cunningham
N. S.
,
Paralkar
V. M.
,
Reddi
A. H.
(
1992
).
Osteogenin and recombinant bone morphogenetic protein 2B are chemotactic for human monocytes and stimulate transforming growth factor1mRNA expression.
Proc. Nat. Acad. Sci. USA
89
,
11740
11744
Genovese
C.
,
Rowe
D.
,
Kream
B.
(
1984
).
Construction of DNA sequences complementary to rat a1 and a2 collagen mRNA and their use in studying the regulation of type I collagen synhtesis by 1,25-dihydroxy D.
Biochemistry
23
,
6210
6216
Gibson
G. J.
,
Flint
M. H.
(
1985
).
Type X collagen synthesis by chick sternal cartilage and its relationship to endochondral development.
J. Cell Biol
101
,
277
284
Harrison
E. T.
Jr.
,
Luyten
F. P.
,
Reddi
A. H.
(
1991
).
Osteogenin promotes reexpression of cartilage phenotype by dedifferentiated articular chondrocytes in serum-free medium.
Exp. Cell Res
192
,
340
345
Harrison
E. T.
Jr.
,
Luyten
F. P.
,
Reddi
A. H.
(
1992
).
Transforming growth factor-beta: its effect on phenotype reexpression by dedifferentiated chondrocytes in the presence and absence of osteogenin.
In Vitro Cell Dev Biol
28
,
445
448
Hiraki
Y.
,
Inoue
H.
,
Shigeno
C.
,
Sanma
Y.
,
Bentz
H.
,
Rosen
D. M.
,
Asada
A.
,
Suzuki
F.
(
1991
).
Bone morphogenetic proteins (BMP-2 and BMP-3) promote growth and expression of the differentiated phenotype of rabbit chondrocytes and osteoblastic MC3T3-E1 in vitro.
J. Bone Miner. Res
6
,
1373
1385
Horton
W. E.
,
Yamada
Y.
,
Hassell
J. R.
(
1987
).
Retinoic acid rapidly reduces cartilage matrix synthesis by altering gene transcription in chondrocytes.
Dev. Biol
123
,
508
516
Iwamoto
M.
,
Golden
E. B. G.
,
Adams
S. L.
,
Noji
S.
,
Pacifici
M.
(
1993
).
Responsiveness to retinoic acid changes during chondrocyte maturation.
Exp. Cell Res
205
,
213
224
Labarca
C.
,
Paigen
K.
(
1980
).
A simple, rapid, and sensitive DNA assay procedure.
Anal. Biochem
102
,
344
352
Leboy
P. S.
,
Vaias
L.
,
Uschmann
B.
,
Golub
E.
,
Adams
S. L.
,
Pacifici
M.
(
1989
).
Ascorbic acid induces alkaline phosphatase, Type X collagen, and calcium deposition in cultured chick chondrocytes.
J. Biol. Chem
264
,
17281
17286
LuValle
P.
,
Ninomiya
Y.
,
Rosenblum
N. D.
,
Olsen
B. R.
(
1988
).
The type X collagen gene.
J. Biol. Chem
263
,
18378
18385
Luyten
F. P.
,
Cunningham
N. S.
,
Ma
S.
,
Muthukumaran
N.
,
Hammonds
R. G.
,
Nevins
W. B.
,
Wood
W. I.
,
Reddi
A. H.
(
1989
).
Purification and partial amino acid sequence of osteogenin, a protein initiating bone differentiation.
J. Biol. Chem
264
,
13377
13380
Luyten
F. P.
,
Yu
M. Y.
,
Vukicevic
S.
,
Hammonds
R. G.
,
Reddi
A. H.
(
1992
).
Natural bovine osteogenin and recombinant human bone morphogenetic protein-2B are equipotent in the maintenance of proteoglycans in bovine articular cartilage explant cultures.
J. Biol. Chem
267
,
3691
3695
Nerlich
A. G.
,
Kirsch
T.
,
Wiest
I.
,
Betz
P.
,
von der Mark.
K.
(
1992
).
Localization of collagen X in human fetal and juvenile articular cartilage and bone.
Histochemistry
98
,
275
281
Oshima
O.
,
Leboy.
P. S.
,
McDonald
S. A.
,
Tuan
R. S.
,
Shapiro
I. M.
(
1989
).
Developmental expression of genes in chick growth cartilage detected by in situ hybridization.
Calc. Tiss. Int
45
,
182
192
Özkaynak
E.
,
Rueger
D. C.
,
Drier
E. A.
,
Corbett
C.
,
Ridge
R. J.
,
Sampath
T. K.
,
Opperman
H.
(
1990
).
OP-1 cDNA encodes an osteogenic protein in the TGF-family.
EMBO J
9
,
2085
2093
Özkaynak
E.
,
Schnegelsberg
P. N. J.
,
Jin
D. F.
,
Clifford
G. M.
,
Warren
F. D.
,
Drier
E. A.
,
Opperman
H.
(
1992
).
Osteogenic protein-2.
J. Biol. Chem
267
,
25220
25227
Pacifici
M.
,
Golden
E. B.
,
Adams
S. L.
,
Shapiro
I. M.
(
1991
).
Cell hypertrophy and type X collagen synthesis in cultured articular chondrocytes.
Exp. Cell Res
192
,
226
270
Pacifici
M.
,
Golden
E. B.
,
Iwamoto
M.
,
Adams.
S. L.
(
1991
).
Retinoic acid treatment induces type X collagen gene expression in cultured chick chondrocytes.
Exp. Cell Res
195
,
38
46
Quarto
R.
,
Campanile
G.
,
Cancedda
R.
,
Dozin
B.
(
1992
).
Thyroid hormone, insulin and glucocorticoids are sufficient to support chondrocyte differentiation to hypertrophy: a serum-free analysis.
J. Cell Biol
119
,
989
995
Reddi
A. H.
,
Huggins
C. B.
(
1972
).
Biochemical sequences in the transformation of normal fibroblast in adolescent rat.
Proc. Nat. Acad. Sci. USA
69
,
1601
1605
Ripamonti
U.
,
Ma
S. S.
,
van den Heever
B.
,
Reddi
A. H.
(
1992
).
Osteogenin, a bone morphogenetic protein, absorbed on porous hydroxyapatite substrate, induces rapid bone differentiation in calvarial defects of adult primates.
Plast. Reconstr. Surg
90
,
382
393
Sampath
T. K.
,
Coughlin
J. E.
,
Whetstone
R. M.
,
Banach
D.
,
Corbett
C.
,
Ridge
R. J.
,
Özkaynak
E.
,
Opperman
H.
,
Rueger
D. C.
(
1990
).
Bovine osteogenic protein is composed of dimers of OP-1 and BMP-2A, two members of the transforming growth factor-superfamily.
J. Biol. Chem
265
,
13198
13205
Sampath
T. K.
,
Maliakal
J. C.
,
Hauschka
P. V.
,
Jones
W. K.
,
Sasak
H.
,
Tucker
R. F.
,
White
K. H.
,
Coughlin
J. E.
,
Tucker
M. M.
,
Pang
R. H. L.
,
Corbett
C.
,
Özkaynak
E.
,
Oppermann
H.
,
Rueger
D. C.
(
1992
).
Recombinant human osteogenic protein (hOP-1) induces new boneformation in vivo with specific activity comparable to natural bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro.
J. Biol. Chem
267
,
20352
20362
Schmid
T. M.
,
Pope
R. G.
,
Linsenmayer
T. F.
(
1990
).
Hypertrophic cartilage matrix. Type X collagen, supramolecular assembly, and calcification. Ann.
NY Acad. Sci
508
,
64
73
Solursh
M.
,
Jensen
K. L.
,
Reiter
R. S.
,
Schmid
T. M.
,
Linsenmayer
T. F.
(
1986
).
Environmental regulation of type X collagen production by cultures of limb mesenchyme, mesectoderm, and sternal chondrocytes.
Dev. Biol
117
,
90
101
Thies
R. S.
,
Bauduy
M.
,
Ashton
B. A.
,
Kurtzberg
L.
,
Wozney
J. M.
,
Rosen
V.
(
1992
).
Recombinant human BMP-2 induces osteoblastic differentiation in W-20-17 stromal cells.
Endocrinology
130
,
1318
1314
Urist
M. R.
(
1965
).
Bone formation by autoinduction.
Science
150
,
893
899
Vukicevic
S.
,
Luyten
F. P.
,
Reddi
A. H.
(
1989
).
Stimulation of the expression of osteogenic and chondrogenic phenotypes in vitro by osteogenin.
Proc. Nat. Acad. Sci. USA
86
,
8793
8797
Vukicevic
S.
,
Luyten
F. P.
,
Reddi
A. H.
(
1990
).
Osteogenin inhibits proliferation and stimulates differentiation in mouse osteoblast like cells (MC3T3-E1).
Biochem. Biophys. Res. Commun
166
,
750
756
Vukicevic
S.
,
Latin
V.
,
Chen
P.
,
Batorsky
R.
,
Reddi
A. H.
,
Sampath
T. K.
(
1994
).
Localization of OP-1 during human embryonic development: high affinity binding to basement membranes.
Biochem. Biophys. Res. Commun
198
,
693
700
Wang
E.
,
Rosen
V.
,
Cordes
P.
,
Hewick
R. M.
,
Kriz
M. J.
,
Luxenberg
D. P.
,
Sibley
B. S.
,
Wozney
J. M.
(
1988
).
Purification and characterization of other distinct bone-inducing factors.
Proc. Nat. Acad. Sci. USA
85
,
9484
8488
Wozney
J. M.
,
Rosen
V.
,
Celeste
A. J.
,
Mitsock
L. M.
,
Whitters
M. J.
,
Kriz
R. W.
,
Hewick
R. M.
,
Wang
E. A.
(
1988
).
Novel regulators of bone formation: Molecular clones and activities.
Science
242
,
1528
1533
Yamaguchi
A.
,
Katagiri
T.
,
Ikeda
T.
,
Wozney
J. M.
,
Rosen
V.
,
Wang
E. A.
,
Kahn
A. J.
,
Suda
T.
,
Yoshiki
S.
(
1991
).
Recombinant human bone morphogenetic protein-2 stimulates osteoblastic maturation and inhibits myogenic differentiation in vitro.
J. Cell Biol
113
,
681
687
Young
M. F.
,
Vogeli
G.
,
Nunez
A. M.
,
Fernandez
M. P.
,
Sullivan
M.
,
Sobel
M. E.
(
1984
).
Isolation of cDNA and genomic DNA clones encoding type II collagen.
Nucl. Acids Res
12
,
4207
4228
Yun Tso
J.
,
Sun
X. H.
,
Kao
T. H.
,
Reece
K. S.
,
Wu
R.
(
1985
).
Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNA: genomic complexity and molecular evolution of the gene.
Nucl. Acids Res
13
,
2485
2502
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