Synergistic interaction of bone morphogenetic protein and transforming growth factor-β in bone induction and regeneration
Date
2014-03-13
Authors
Duneas, Nicolaas
Journal Title
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Abstract
Several members of the bone moirphogenetic protein and osteogenic protein
(BMP/OP) and transforming growth factor-13 (TGF-J3) families are molecular
regulators of cartilage and bone regeneration, although their precise mode of signal
transduction and combined interactions are poorly understood. The presence of
several molecular forms suggests multiple functions in vivo as well as synergistic
interactions during both embryonic bone development and regeneration of cartilage
and bone in postfoetal life. Heterotopic and orthotopic implantation o f BMPs/OPs elicit the local differentiation of new bone in a number of animal models studied, whereas reports to date show that implantation o f TGF-Bs in heterotopic sites of the rat results in fibrovascular tissue formation, without evidence of bone formation.Instead, TGF-Bs show limited osteo-chondrogenic activity only when applied orthotopically in some, and not all of the animal models studied. Here, data are presented that show for the first time that, in contrast to previously described animal models, implantation of TGF-13i with a collagenous matrix carrier in the rectus abdominis of the baboon (Papio ursinus) results in de novo bone formation, as evidenced radiographically, histologically and biochemically. TGF-Bj induced endochondral bone at heterotopic sites o f the baboon at doses of 5 jug/lOO mg collagenous matrix as carrier, with an inductive efficiency comparable to single
applications of 5 and 25 fig recombinant human osteogenic protein-1 (rhOP-1), a
morphogen whose osteoinductive activity has previously been demonstrated in a
number of animal models. It was shown further, that rhOP-1 and TGF-Bi (of porcine or recombinant source), interact synergistically to induce massive ossicles in
extraskeletal sites of the primate as early as 14 or 15 days after implantation. Binary
combinations of the morphogens induced ossicles with indeces of bone formation
which were greater than the sum of indeces of single application of each respective
morphogen. By applying isobolographic analysis, a well established pharmacological
mathematical model previously developed for the study of interactions between
pharmacologic agents, it was shown that the interaction between rhOP-1 and TGF-Bi
in bone induction was of the type defined as synergism. The level of tissue induced by
single applications of rhOP-1 (5, 25 and 125 pg/100 mg collagenous matrix) was
raised several-fold by the simultaneous addition of comparatively low doses of TGF-
13i (0.5,1.5 and 5 |ig), which by itself induced bone formation at doses of 5gg/100 mg
collagenous matrix. Combinations of rhOP-1 and TGF-Bi yielded a 2- to 3-fold
increase in cross-sectional area of the newly generated ossicles, with markedly
elevated parameters of bone formation, and corticalisation of the newly formed bone
by day 15, culminating in marrow generation by day 30. The tissue generated by the
combined application of rhOP-1 and TGF-Bi showed distinct morphological
differences when compared with rhOP-1-treated specimens, with large zones of
endochondral development and extensive bone marrow formation. Heterotopic tissue
generated on day 30 by single application o f rhOP-1 or TGF-Bi expressed comparable
levels of OP-1, BMP-3 and type IV collagen mRNA transcripts, whereas TGF-Bi and
type II collagen mRNA expression was 2- to 3-fold higher in TGF-Bi-treated impants,
as determined by Northern analysis. In ossicles generated by rhOP-1 in combination
with TGF-B], mRNA for type II collagen, a marker of chondrogenesis, was increased
in a TGF-13 dose-dependent manner. Type IV collagen mRNA, a marker of angiogenesis, was synergistically upregulated with a 3- to 4-fold increase compared to
ossicles generated by single application of rhOP-1 or TGF-13j. Since angiogenesis is a
prerequisite for osteogenesis, increased angiogenesis may be at least part of the mechanism whereby moiphogen combinations interact synergistically in endochondral bone formation. Single application of pTGF-Bi induced ossicles expressing mRNA for OP-1, BMP-3 and TGF-Bi, showing that osteogenesis elicited by pTGF-Bi proceeds, at least in part, -via the expression of genes of the BMP/OP family. Single applications of rhOP-1 and pTGF-Bi induced ossicles expressing high levels of their own mRNA, suggesting an autoinductive effect of these morphogens in the initiation of the bone differentiation cascade. A t the doses tested, synergy was optimal at a ratio of 1:20 by weight of TGF-B, and rhOP-1 respectively.expressing mRNA for OP-1, BMP-3 and TGF-Bi, showing that osteogenesis elicited by pTGF-Bi proceeds, at least in part, -via the expression of genes of the BMP/OP family. Single applications of rhOP-1 and pTGF-Bi induced ossicles expressing high levels of their own mRNA, suggesting an autoinductive effect of these morphogens in the initiation of the bone differentiation cascade. A t the doses tested, synergy was optimal at a ratio of 1:20 by weight of TGF-B, and rhOP-1 respectively.
Morphogen combinations (5 ug pTGF-Bi with 20 pg rhOP-1, and 5 and 15 jig pTGFBi
with 100 with 100 pg rhOP-1 per gram o f collagenous matrix as carrier) induced
exuberant tissue formation and greater amounts o f osteoid than rhOP-1 alone when
implanted in calvarial defects o f the baboon as evaluated on day 30 and 90, with
displacement of the temporalis muscle above the defects. Since single application o f
TGF-Bi in the primate did not induce bone formation in calvarial defects, whilst it
induces endochondral bone differentiation in heterotopic sites, the data indicate that
that the bone inductive activity of TGF-Bj is site and tissue specific. The findings
presented may provide the basis for synergistic molecular therapeutics for cartilage
and bone regeneration in clinical contexts.