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The induction of endochondral bone formation by transforming growth factor-beta(3): experimental studies in the non-human primate Papio ursinus.

Ripamonti U, Ramoshebi LN, Teare J, Renton L, Ferretti C - J. Cell. Mol. Med. (2008)

Bottom Line: Strikingly and in marked contrast to the rodent bioassay, recombinant human (h)TGF-beta(3), when implanted in the rectus abdominis muscle of adult non-human primates Papio ursinus at doses of 5, 25 and 125 mug per 100 mg of insoluble collagenous matrix as carrier, induces rapid endochondral bone formation resulting in large corticalized ossicles by day 30 and 90.In the same animals, the delivery of identical or higher doses of theTGF-beta(3) protein results in minimal repair of calvarial defects on day 30 with limited bone regeneration across the pericranial aspect of the defects on day 90.RT-PCR, Western and Northern blot analyses of tissue specimens generated by the TGF-beta(3) isoform demonstrate robust expression of Smad-6 and Smad-7 in orthotopic calvarial sites with limited expression in heterotopic rectus abdominis sites.

View Article: PubMed Central - PubMed

Affiliation: Bone Research Unit, Medical Research Council/University of the Witwatersrand, Johannesburg, South Africa. ugo.ripamonti@wits.ac.za

ABSTRACT
Transforming growth factor-beta(3) (TGF-beta(3)), a multi-functional growth modulator of embryonic development, tissue repair and morphogenesis, immunoregulation, fibrosis, angiogenesis and carcinogenesis, is the third mammalian isoform of the TGF-beta subfamily of proteins. The pleiotropism of the signalling proteins of the TGF-beta superfamily, including the TGF-beta proteins per se, are highlighted by the apparent redundancy of soluble molecular signals initiating de novo endochondral bone induction in the primate only. In the heterotopic bioassay for bone induction in the subcutaneous site of rodents, the TGF-beta(3) isoform does not initiate endochondral bone formation. Strikingly and in marked contrast to the rodent bioassay, recombinant human (h)TGF-beta(3), when implanted in the rectus abdominis muscle of adult non-human primates Papio ursinus at doses of 5, 25 and 125 mug per 100 mg of insoluble collagenous matrix as carrier, induces rapid endochondral bone formation resulting in large corticalized ossicles by day 30 and 90. In the same animals, the delivery of identical or higher doses of theTGF-beta(3) protein results in minimal repair of calvarial defects on day 30 with limited bone regeneration across the pericranial aspect of the defects on day 90. Partial restoration of the bone induction cascade by the hTGF-beta(3) protein is obtained by mixing the hTGF-beta(3) device with minced fragments of autogenous rectus abdominis muscle thus adding responding stem cells for further bone induction by the hTGF-beta(3) protein. The observed limited bone induction in hTGF-beta(3)/treated and untreated calvarial defects in Papio ursinus and therefore by extension to Homo sapiens, is due to the influence of Smad-6 and Smad-7 down-stream antagonists of the TGF-beta signalling pathway. RT-PCR, Western and Northern blot analyses of tissue specimens generated by the TGF-beta(3) isoform demonstrate robust expression of Smad-6 and Smad-7 in orthotopic calvarial sites with limited expression in heterotopic rectus abdominis sites. Smad-6 and -7 overexpression in hTGF-beta(3)/treated and untreated calvarial defects may be due to the vascular endothelial tissue of the arachnoids expressing signalling proteins modulating the expression of the inhibitory Smads in pre-osteoblastic and osteoblastic calvarial cell lines controlling the induction of bone in the primate calvarium.

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Expression of the inhibitory Smad-6 and -7 on day 30 (A) and 90 (B), TGF- β1, OP-1 and BMP-3 on day 30 (C) and 90 (D), and expression patterns of TβR I, TβR II, and TβR III on day 30 (E) and 90 (F) after calvarial implantation of inactive collagenous matrix solo as control (0 μg hTGF-β3) and 5, 25 and 125 μg doses of the h hTGF-β3 osteogenic device additionally treated without or with autogenous cellular fragments (B) of rectus abdominis muscle (−C and/or +C). Total RNA from heterotopic or orthotopic samples was reverse-transcribed and amplified with the respective primers. GAPDH expression was assayed as a control. Experiments were performed twice with similar results and representative data shown.
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fig06: Expression of the inhibitory Smad-6 and -7 on day 30 (A) and 90 (B), TGF- β1, OP-1 and BMP-3 on day 30 (C) and 90 (D), and expression patterns of TβR I, TβR II, and TβR III on day 30 (E) and 90 (F) after calvarial implantation of inactive collagenous matrix solo as control (0 μg hTGF-β3) and 5, 25 and 125 μg doses of the h hTGF-β3 osteogenic device additionally treated without or with autogenous cellular fragments (B) of rectus abdominis muscle (−C and/or +C). Total RNA from heterotopic or orthotopic samples was reverse-transcribed and amplified with the respective primers. GAPDH expression was assayed as a control. Experiments were performed twice with similar results and representative data shown.

Mentions: On day 30 (Fig. 6A), heterotopic samples showed no or limited expression of Smad-6 and -7 as compared to significantly elevated expression in orthotopic tissue samples (Fig. 6A). Tissue samples of insoluble collagenous matrix without hTGF-β3 as control also resulted in relative expression of Smad-6 and -7 in orthotopic sites (Fig. 6A).


The induction of endochondral bone formation by transforming growth factor-beta(3): experimental studies in the non-human primate Papio ursinus.

Ripamonti U, Ramoshebi LN, Teare J, Renton L, Ferretti C - J. Cell. Mol. Med. (2008)

Expression of the inhibitory Smad-6 and -7 on day 30 (A) and 90 (B), TGF- β1, OP-1 and BMP-3 on day 30 (C) and 90 (D), and expression patterns of TβR I, TβR II, and TβR III on day 30 (E) and 90 (F) after calvarial implantation of inactive collagenous matrix solo as control (0 μg hTGF-β3) and 5, 25 and 125 μg doses of the h hTGF-β3 osteogenic device additionally treated without or with autogenous cellular fragments (B) of rectus abdominis muscle (−C and/or +C). Total RNA from heterotopic or orthotopic samples was reverse-transcribed and amplified with the respective primers. GAPDH expression was assayed as a control. Experiments were performed twice with similar results and representative data shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4401141&req=5

fig06: Expression of the inhibitory Smad-6 and -7 on day 30 (A) and 90 (B), TGF- β1, OP-1 and BMP-3 on day 30 (C) and 90 (D), and expression patterns of TβR I, TβR II, and TβR III on day 30 (E) and 90 (F) after calvarial implantation of inactive collagenous matrix solo as control (0 μg hTGF-β3) and 5, 25 and 125 μg doses of the h hTGF-β3 osteogenic device additionally treated without or with autogenous cellular fragments (B) of rectus abdominis muscle (−C and/or +C). Total RNA from heterotopic or orthotopic samples was reverse-transcribed and amplified with the respective primers. GAPDH expression was assayed as a control. Experiments were performed twice with similar results and representative data shown.
Mentions: On day 30 (Fig. 6A), heterotopic samples showed no or limited expression of Smad-6 and -7 as compared to significantly elevated expression in orthotopic tissue samples (Fig. 6A). Tissue samples of insoluble collagenous matrix without hTGF-β3 as control also resulted in relative expression of Smad-6 and -7 in orthotopic sites (Fig. 6A).

Bottom Line: Strikingly and in marked contrast to the rodent bioassay, recombinant human (h)TGF-beta(3), when implanted in the rectus abdominis muscle of adult non-human primates Papio ursinus at doses of 5, 25 and 125 mug per 100 mg of insoluble collagenous matrix as carrier, induces rapid endochondral bone formation resulting in large corticalized ossicles by day 30 and 90.In the same animals, the delivery of identical or higher doses of theTGF-beta(3) protein results in minimal repair of calvarial defects on day 30 with limited bone regeneration across the pericranial aspect of the defects on day 90.RT-PCR, Western and Northern blot analyses of tissue specimens generated by the TGF-beta(3) isoform demonstrate robust expression of Smad-6 and Smad-7 in orthotopic calvarial sites with limited expression in heterotopic rectus abdominis sites.

View Article: PubMed Central - PubMed

Affiliation: Bone Research Unit, Medical Research Council/University of the Witwatersrand, Johannesburg, South Africa. ugo.ripamonti@wits.ac.za

ABSTRACT
Transforming growth factor-beta(3) (TGF-beta(3)), a multi-functional growth modulator of embryonic development, tissue repair and morphogenesis, immunoregulation, fibrosis, angiogenesis and carcinogenesis, is the third mammalian isoform of the TGF-beta subfamily of proteins. The pleiotropism of the signalling proteins of the TGF-beta superfamily, including the TGF-beta proteins per se, are highlighted by the apparent redundancy of soluble molecular signals initiating de novo endochondral bone induction in the primate only. In the heterotopic bioassay for bone induction in the subcutaneous site of rodents, the TGF-beta(3) isoform does not initiate endochondral bone formation. Strikingly and in marked contrast to the rodent bioassay, recombinant human (h)TGF-beta(3), when implanted in the rectus abdominis muscle of adult non-human primates Papio ursinus at doses of 5, 25 and 125 mug per 100 mg of insoluble collagenous matrix as carrier, induces rapid endochondral bone formation resulting in large corticalized ossicles by day 30 and 90. In the same animals, the delivery of identical or higher doses of theTGF-beta(3) protein results in minimal repair of calvarial defects on day 30 with limited bone regeneration across the pericranial aspect of the defects on day 90. Partial restoration of the bone induction cascade by the hTGF-beta(3) protein is obtained by mixing the hTGF-beta(3) device with minced fragments of autogenous rectus abdominis muscle thus adding responding stem cells for further bone induction by the hTGF-beta(3) protein. The observed limited bone induction in hTGF-beta(3)/treated and untreated calvarial defects in Papio ursinus and therefore by extension to Homo sapiens, is due to the influence of Smad-6 and Smad-7 down-stream antagonists of the TGF-beta signalling pathway. RT-PCR, Western and Northern blot analyses of tissue specimens generated by the TGF-beta(3) isoform demonstrate robust expression of Smad-6 and Smad-7 in orthotopic calvarial sites with limited expression in heterotopic rectus abdominis sites. Smad-6 and -7 overexpression in hTGF-beta(3)/treated and untreated calvarial defects may be due to the vascular endothelial tissue of the arachnoids expressing signalling proteins modulating the expression of the inhibitory Smads in pre-osteoblastic and osteoblastic calvarial cell lines controlling the induction of bone in the primate calvarium.

Show MeSH
Related in: MedlinePlus