In the assay for heterotopic bone induction in rodents [1–6], neither naturally-derived nor recombinant TGF-β isoforms initiate endochondral bone formation [24–27]. In marked contrast to the rodent bioassay, the TGF-β isoforms so far tested in our laboratories have shown a marked site and tissue-specific endochondral osteoinductivity in the pri
Our studies in the primate Papio ursinus have shown differential osteogenic responses following heterotopic and orthotopic implantation of TGF-β isoforms. Whereas TGF-β1 and TGF-β2 induced substantial endochondral bone formation in the rectus abdominis muscle (Fig. 1C) resulting in large corticalized ossicles by day 90 post implantation [9, 10, 21, 22], repair of calvarial defects was poor  (Fig. 2A), restricted to limited bone regeneration at the pericranial perimeter only 90 days post-implan-tation [2, 9, 10, 21] (Fig. 2B).
Given the ubiquitous nature of TGF-β family members, receptors are expressed on all cell types examined to date  and the pleiotropic nature of their activity within the responding cell, it is unsurprising that the TGF-β signalling pathway is strictly regulated. We now know many of the complex mechanisms controlling TGF-β signal transduction to ensure specificity of response by the target cell, and regulation is at multiple levels, from the cell surface to within the nucleus .
The Smad pathway is the major intracellular signalling system activated by TGF-β superfamily members. Over expression of Smad6 and Smad7, inhibitors of the vertebrate Smad-based TGF-β signalling pathway [31–34], would mechanistically explain the observed site and tissue specificity of endochondral osteoinductivity of the TGF-β isoforms in the primates . Suppression of TGF-β-initiated signal transduction by two inhibitory Smad proteins Smad6 and Smad7 represents a negative autocrine feedback loop since their expression is rapidly induced by TGF-β itself .
Using Northern blot analysis and RT (reverse transcription)-PCR, we have shown mRNA of Smad6 and Smad7 to be expressed where TGF-β-induced bone formation was limited, but almost absent heterotopically (Fig. 3A), in sites of exuberant new bone formation by induction. [2, 10, 23]. These exciting findings in the adult non-human primate highlight the importance of the inhibitory Smads in regulating de novo bone formation in clinical contexts.
Limited bone formation in orthotopic bony sites by the TGF-β isoforms so far tested in our laboratories is potentially due to decreased osteoblastic proliferation and differentiation. Expression of the inhibitory Smad6 and Smad7, as later shown by RTPCR, is clearly suggested by the morphologic analysis of calvarial specimens treated with 100µg hTGF-β2 and harvested on day 90 post-implanta-tion. The examined specimens showed bone formation at both interfacial regions that seemed, at least morphologically, inhibited to proceed or grow centripetally with the generation of a rather substantial fibrogenic response between the inactive particles of the collagenous matrix [21, 23] (Fig. 2B).
The present data indicate that the observed morphological effects of Smad6 and Smad7 on osteoblasts synthesis and bone matrix formation by induction are due to its specific inhibition of the TGF-β and BMPs/OPs signalling pathway in orthotopic sites.
It is worth noting that tissues generated via induction by TGF-β1 and β2 isoforms expressed OP-1 and BMP-3 mRNAs synthesis (Fig. 3B), indicating that bone formation induced by TGF-βs in the rectus abdominis muscle requires, at least in part, synthesis of members of the BMPs-OPs family [10, 21, 22].
mate Papio ursinus
. Remarkably this occurs in primates only [2, 10, 12, 20–23] (Fig. 1C