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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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Heterotopic induction of bone formation by doses of the recombinant human transforming growth factor-β3 (hTGF-β3) osteogenic device in the rectus abdominis of adult baboons Papio ursinus.(A) Induction of a large ossicle after intramuscular implantation of 25 μg hTGF-β3 delivered by the insoluble collagenous matrix as carrier. (B) Corticalization of the ossicle as shown in A by newly formed and mineralized bone in blue surrounding scattered remnants of the collagenous matrix embedded within prominent fibrovascular invasion. (C, D) Histological images of mineralized and corticalized ossicles induced by the 5 μg dose of the hTGF-β3 osteogenic device in the rectus abdominis muscle showing mineralization of the newly formed bone by induction in the rectus abdominis muscle of Papio ursinus. Undecalcified sections cut at 5 βm and stained free-floating with Goldner's trichrome (A, B, C and D, original magnification ×2.8).
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fig01: Heterotopic induction of bone formation by doses of the recombinant human transforming growth factor-β3 (hTGF-β3) osteogenic device in the rectus abdominis of adult baboons Papio ursinus.(A) Induction of a large ossicle after intramuscular implantation of 25 μg hTGF-β3 delivered by the insoluble collagenous matrix as carrier. (B) Corticalization of the ossicle as shown in A by newly formed and mineralized bone in blue surrounding scattered remnants of the collagenous matrix embedded within prominent fibrovascular invasion. (C, D) Histological images of mineralized and corticalized ossicles induced by the 5 μg dose of the hTGF-β3 osteogenic device in the rectus abdominis muscle showing mineralization of the newly formed bone by induction in the rectus abdominis muscle of Papio ursinus. Undecalcified sections cut at 5 βm and stained free-floating with Goldner's trichrome (A, B, C and D, original magnification ×2.8).

Mentions: Implantation of 5 and 25 μg of the hTGF-β3 protein in the rectus abdominis muscle resulted in the induction of large corticalized heterotopic ossicles by day 20 and 30 after implantation (Fig. 1). Doses of 125 μg hTGF-β3 induced large corticalized ossicles embedded within the rectus abdominis muscle (Fig. 2). The large ossicles showed a planar convex geometry extending for several centimetres preferentially along the longitudinal plane of the fascia (Figs. 1A, 2A, C, E). Cut surfaces showed mineralization of the external cortex and were macroscopically brownish-red in colour indicating the induction of bone marrow with endosteal vascularization.


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)

Heterotopic induction of bone formation by doses of the recombinant human transforming growth factor-β3 (hTGF-β3) osteogenic device in the rectus abdominis of adult baboons Papio ursinus.(A) Induction of a large ossicle after intramuscular implantation of 25 μg hTGF-β3 delivered by the insoluble collagenous matrix as carrier. (B) Corticalization of the ossicle as shown in A by newly formed and mineralized bone in blue surrounding scattered remnants of the collagenous matrix embedded within prominent fibrovascular invasion. (C, D) Histological images of mineralized and corticalized ossicles induced by the 5 μg dose of the hTGF-β3 osteogenic device in the rectus abdominis muscle showing mineralization of the newly formed bone by induction in the rectus abdominis muscle of Papio ursinus. Undecalcified sections cut at 5 βm and stained free-floating with Goldner's trichrome (A, B, C and D, original magnification ×2.8).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401141&req=5

fig01: Heterotopic induction of bone formation by doses of the recombinant human transforming growth factor-β3 (hTGF-β3) osteogenic device in the rectus abdominis of adult baboons Papio ursinus.(A) Induction of a large ossicle after intramuscular implantation of 25 μg hTGF-β3 delivered by the insoluble collagenous matrix as carrier. (B) Corticalization of the ossicle as shown in A by newly formed and mineralized bone in blue surrounding scattered remnants of the collagenous matrix embedded within prominent fibrovascular invasion. (C, D) Histological images of mineralized and corticalized ossicles induced by the 5 μg dose of the hTGF-β3 osteogenic device in the rectus abdominis muscle showing mineralization of the newly formed bone by induction in the rectus abdominis muscle of Papio ursinus. Undecalcified sections cut at 5 βm and stained free-floating with Goldner's trichrome (A, B, C and D, original magnification ×2.8).
Mentions: Implantation of 5 and 25 μg of the hTGF-β3 protein in the rectus abdominis muscle resulted in the induction of large corticalized heterotopic ossicles by day 20 and 30 after implantation (Fig. 1). Doses of 125 μg hTGF-β3 induced large corticalized ossicles embedded within the rectus abdominis muscle (Fig. 2). The large ossicles showed a planar convex geometry extending for several centimetres preferentially along the longitudinal plane of the fascia (Figs. 1A, 2A, C, E). Cut surfaces showed mineralization of the external cortex and were macroscopically brownish-red in colour indicating the induction of bone marrow with endosteal vascularization.

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