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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 regeneration and induction of bone formation in calvarial defects implanted with doses of the transforming growth factor-β3 (hTGF-β3) osteogenic device without (A) and with minced fragments of autogenous rectus abdominis muscle (E, F and G) harvested 90 days post-implantation. (A) Induction of bone in a calvarial defect implanted with 125 μg of the hTGF-β3 osteogenic device showing bone formation predominantly on the pericranial aspect of the specimen with lack of bone formation at the endocranial dural aspect of the specimen (arrows). Arrows in B, C and D point to the inhibition of bone formation within the fibrogenic collagenous matrix facing the newly formed bone pericranially.(E, F and G). Addition to the hTGF-β3 osteogenic device of autogenous minced fragments of rectus abdominis muscle induces partial restoration of the biological activity of 125 μg of the hTGF-β3 osteogenic device with large islands of newly formed bone in specimens harvested on day 90 after implantation. Undecalcified sections cut at 5 μm stained free-floating with Goldner's trichrome. (A, E, F and G) original magnification ×1.8; (B, C and D) original magnification ×7.
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fig04: Morphology of calvarial regeneration and induction of bone formation in calvarial defects implanted with doses of the transforming growth factor-β3 (hTGF-β3) osteogenic device without (A) and with minced fragments of autogenous rectus abdominis muscle (E, F and G) harvested 90 days post-implantation. (A) Induction of bone in a calvarial defect implanted with 125 μg of the hTGF-β3 osteogenic device showing bone formation predominantly on the pericranial aspect of the specimen with lack of bone formation at the endocranial dural aspect of the specimen (arrows). Arrows in B, C and D point to the inhibition of bone formation within the fibrogenic collagenous matrix facing the newly formed bone pericranially.(E, F and G). Addition to the hTGF-β3 osteogenic device of autogenous minced fragments of rectus abdominis muscle induces partial restoration of the biological activity of 125 μg of the hTGF-β3 osteogenic device with large islands of newly formed bone in specimens harvested on day 90 after implantation. Undecalcified sections cut at 5 μm stained free-floating with Goldner's trichrome. (A, E, F and G) original magnification ×1.8; (B, C and D) original magnification ×7.

Mentions: Orthotopic tissue sections prepared from calvarial specimens harvested on day 90 occasionally showed mineralized bone across the defect confined to the pericranial aspect (Fig. 4A). The presence of inhibitory binding proteins and/or the expression of inhibitory Smads has been suggested by the morphological analysis of calvarial specimens treated with varying doses of the TGF-β3 combined with ICBM as carrier, that is, the TGF-β3 osteogenic device. Morphological analysis showed that bone formed essentially at the pericranial surface of the implant below the temporalis muscle on day 90. Moreover, the bone that had formed at both interfacial regions seemed to be, at least morphologically, inhibited to proceed centripetally with the generation of a substantial fibrogenic response between the inactive particles of the collagenous matrix subjacent to the newly formed bone pericranially (see Fig. 4B and D). It was thus mandatory to study the expression of the inhibitory Smad-6 and -7 proteins in orthotopic calvarial defects to mechanistically unravel the segregated osteogenic induction by the hTGF-β3 isoform in calvarial defects.


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 regeneration and induction of bone formation in calvarial defects implanted with doses of the transforming growth factor-β3 (hTGF-β3) osteogenic device without (A) and with minced fragments of autogenous rectus abdominis muscle (E, F and G) harvested 90 days post-implantation. (A) Induction of bone in a calvarial defect implanted with 125 μg of the hTGF-β3 osteogenic device showing bone formation predominantly on the pericranial aspect of the specimen with lack of bone formation at the endocranial dural aspect of the specimen (arrows). Arrows in B, C and D point to the inhibition of bone formation within the fibrogenic collagenous matrix facing the newly formed bone pericranially.(E, F and G). Addition to the hTGF-β3 osteogenic device of autogenous minced fragments of rectus abdominis muscle induces partial restoration of the biological activity of 125 μg of the hTGF-β3 osteogenic device with large islands of newly formed bone in specimens harvested on day 90 after implantation. Undecalcified sections cut at 5 μm stained free-floating with Goldner's trichrome. (A, E, F and G) original magnification ×1.8; (B, C and D) original magnification ×7.
© Copyright Policy
Related In: Results  -  Collection

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

fig04: Morphology of calvarial regeneration and induction of bone formation in calvarial defects implanted with doses of the transforming growth factor-β3 (hTGF-β3) osteogenic device without (A) and with minced fragments of autogenous rectus abdominis muscle (E, F and G) harvested 90 days post-implantation. (A) Induction of bone in a calvarial defect implanted with 125 μg of the hTGF-β3 osteogenic device showing bone formation predominantly on the pericranial aspect of the specimen with lack of bone formation at the endocranial dural aspect of the specimen (arrows). Arrows in B, C and D point to the inhibition of bone formation within the fibrogenic collagenous matrix facing the newly formed bone pericranially.(E, F and G). Addition to the hTGF-β3 osteogenic device of autogenous minced fragments of rectus abdominis muscle induces partial restoration of the biological activity of 125 μg of the hTGF-β3 osteogenic device with large islands of newly formed bone in specimens harvested on day 90 after implantation. Undecalcified sections cut at 5 μm stained free-floating with Goldner's trichrome. (A, E, F and G) original magnification ×1.8; (B, C and D) original magnification ×7.
Mentions: Orthotopic tissue sections prepared from calvarial specimens harvested on day 90 occasionally showed mineralized bone across the defect confined to the pericranial aspect (Fig. 4A). The presence of inhibitory binding proteins and/or the expression of inhibitory Smads has been suggested by the morphological analysis of calvarial specimens treated with varying doses of the TGF-β3 combined with ICBM as carrier, that is, the TGF-β3 osteogenic device. Morphological analysis showed that bone formed essentially at the pericranial surface of the implant below the temporalis muscle on day 90. Moreover, the bone that had formed at both interfacial regions seemed to be, at least morphologically, inhibited to proceed centripetally with the generation of a substantial fibrogenic response between the inactive particles of the collagenous matrix subjacent to the newly formed bone pericranially (see Fig. 4B and D). It was thus mandatory to study the expression of the inhibitory Smad-6 and -7 proteins in orthotopic calvarial defects to mechanistically unravel the segregated osteogenic induction by the hTGF-β3 isoform in calvarial defects.

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