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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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Morphology of calvarial repair and induction of bone formation by doses of the hTGF-β3 osteogenic device without (left column) and with (right column) minced fragments of autogenous rectus abdominis muscle harvested 30 days after implantation.(A, C, E, G and I) Lack of bone induction and differentiation in calvarial defects harvested on day 30 after implantation of 25 (A), 125 (C, E, G) and 250 (I) μg of the hTGF-β3 osteogenic device without the addition of minced cellular fragments of autogenous rectus abdominis muscle. Blue arrow in (G) indicates a very mall island of mineralized bone (in blue) located in the pericranial area of the specimen.(B) Lack of bone differentiation in a calvarial defect implanted with insoluble collagenous bone matrix solo as control 30 days after implantation.(D) Higher power view of (C) illustrating an interfacial region with newly formed mineralized bone at the level of the craniotomy only (dark blue arrows) blending into the remnants of the collagenous matrix. A prominent fibrous layer (light blue arrow) inhibits the induction of bone formation from the margin of the craniotomy.(F, H, J) Calvarial defects harvested on day 30 after implantation of 125 (F and H) and 250 (J) μg the hTGF-β3 osteogenic device with the addition of minced fragments of autogenous rectus abdominis muscle. Partial restoration of the biological activity and induction of islands of newly formed mineralized bone in blue (arrows) within the implanted hTGF-β3 osteogenic device. Undecalcified sections cut at 5 μm stained free-floating with Goldner's trichrome.(A, B, C, E, F, G, H, I, J) original magnification ×1.2 (D) original magnification ×7.
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fig03: Morphology of calvarial repair and induction of bone formation by doses of the hTGF-β3 osteogenic device without (left column) and with (right column) minced fragments of autogenous rectus abdominis muscle harvested 30 days after implantation.(A, C, E, G and I) Lack of bone induction and differentiation in calvarial defects harvested on day 30 after implantation of 25 (A), 125 (C, E, G) and 250 (I) μg of the hTGF-β3 osteogenic device without the addition of minced cellular fragments of autogenous rectus abdominis muscle. Blue arrow in (G) indicates a very mall island of mineralized bone (in blue) located in the pericranial area of the specimen.(B) Lack of bone differentiation in a calvarial defect implanted with insoluble collagenous bone matrix solo as control 30 days after implantation.(D) Higher power view of (C) illustrating an interfacial region with newly formed mineralized bone at the level of the craniotomy only (dark blue arrows) blending into the remnants of the collagenous matrix. A prominent fibrous layer (light blue arrow) inhibits the induction of bone formation from the margin of the craniotomy.(F, H, J) Calvarial defects harvested on day 30 after implantation of 125 (F and H) and 250 (J) μg the hTGF-β3 osteogenic device with the addition of minced fragments of autogenous rectus abdominis muscle. Partial restoration of the biological activity and induction of islands of newly formed mineralized bone in blue (arrows) within the implanted hTGF-β3 osteogenic device. Undecalcified sections cut at 5 μm stained free-floating with Goldner's trichrome.(A, B, C, E, F, G, H, I, J) original magnification ×1.2 (D) original magnification ×7.

Mentions: On day 20 and 30 after implantation, control calvarial defects treated with collagenous matrix without hTGF-β3 showed minimal osteogenesis confined to the margins of the defects only (Fig. 3A). Doses of the recombinant hTGF-β3 protein (25, 125 and 250 μg) also failed to regenerate bone in the treated calvarial defects (Figs. 3C, E, G and I). Newly formed bone was strictly confined to the margins of the craniotomies (Fig. 3D) occasionally extending pericranially (Figs. 3C and D). A single specimen treated with 125 μg hTGF-β3 showed an island of newly formed mineralized bone just below the temporalis muscle on day 30 (Fig. 3G).


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)

Morphology of calvarial repair and induction of bone formation by doses of the hTGF-β3 osteogenic device without (left column) and with (right column) minced fragments of autogenous rectus abdominis muscle harvested 30 days after implantation.(A, C, E, G and I) Lack of bone induction and differentiation in calvarial defects harvested on day 30 after implantation of 25 (A), 125 (C, E, G) and 250 (I) μg of the hTGF-β3 osteogenic device without the addition of minced cellular fragments of autogenous rectus abdominis muscle. Blue arrow in (G) indicates a very mall island of mineralized bone (in blue) located in the pericranial area of the specimen.(B) Lack of bone differentiation in a calvarial defect implanted with insoluble collagenous bone matrix solo as control 30 days after implantation.(D) Higher power view of (C) illustrating an interfacial region with newly formed mineralized bone at the level of the craniotomy only (dark blue arrows) blending into the remnants of the collagenous matrix. A prominent fibrous layer (light blue arrow) inhibits the induction of bone formation from the margin of the craniotomy.(F, H, J) Calvarial defects harvested on day 30 after implantation of 125 (F and H) and 250 (J) μg the hTGF-β3 osteogenic device with the addition of minced fragments of autogenous rectus abdominis muscle. Partial restoration of the biological activity and induction of islands of newly formed mineralized bone in blue (arrows) within the implanted hTGF-β3 osteogenic device. Undecalcified sections cut at 5 μm stained free-floating with Goldner's trichrome.(A, B, C, E, F, G, H, I, J) original magnification ×1.2 (D) original magnification ×7.
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Related In: Results  -  Collection

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fig03: Morphology of calvarial repair and induction of bone formation by doses of the hTGF-β3 osteogenic device without (left column) and with (right column) minced fragments of autogenous rectus abdominis muscle harvested 30 days after implantation.(A, C, E, G and I) Lack of bone induction and differentiation in calvarial defects harvested on day 30 after implantation of 25 (A), 125 (C, E, G) and 250 (I) μg of the hTGF-β3 osteogenic device without the addition of minced cellular fragments of autogenous rectus abdominis muscle. Blue arrow in (G) indicates a very mall island of mineralized bone (in blue) located in the pericranial area of the specimen.(B) Lack of bone differentiation in a calvarial defect implanted with insoluble collagenous bone matrix solo as control 30 days after implantation.(D) Higher power view of (C) illustrating an interfacial region with newly formed mineralized bone at the level of the craniotomy only (dark blue arrows) blending into the remnants of the collagenous matrix. A prominent fibrous layer (light blue arrow) inhibits the induction of bone formation from the margin of the craniotomy.(F, H, J) Calvarial defects harvested on day 30 after implantation of 125 (F and H) and 250 (J) μg the hTGF-β3 osteogenic device with the addition of minced fragments of autogenous rectus abdominis muscle. Partial restoration of the biological activity and induction of islands of newly formed mineralized bone in blue (arrows) within the implanted hTGF-β3 osteogenic device. Undecalcified sections cut at 5 μm stained free-floating with Goldner's trichrome.(A, B, C, E, F, G, H, I, J) original magnification ×1.2 (D) original magnification ×7.
Mentions: On day 20 and 30 after implantation, control calvarial defects treated with collagenous matrix without hTGF-β3 showed minimal osteogenesis confined to the margins of the defects only (Fig. 3A). Doses of the recombinant hTGF-β3 protein (25, 125 and 250 μg) also failed to regenerate bone in the treated calvarial defects (Figs. 3C, E, G and I). Newly formed bone was strictly confined to the margins of the craniotomies (Fig. 3D) occasionally extending pericranially (Figs. 3C and D). A single specimen treated with 125 μg hTGF-β3 showed an island of newly formed mineralized bone just below the temporalis muscle on day 30 (Fig. 3G).

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