Molecular expression patterns during hTGF-β3 induced bone formation

Tagliatti, Nicolas
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Bone induction involves the enhanced differentiation of osteoblasts and osteoclasts from specific stem cells in response to specific morphogens and occurs within a defined differentiation programme. Contrary to that of rodents, lagomorphs and canines, the recombinant human transforming growth factor-β3 (hTGF- β3) induces endochondral bone formation in the non-human primate Chacma baboon Papio ursinus. Coral-derived 7% hydroxyapatite/calcium carbonate (CC/HA) preloaded with 125 µg hTGF-β3 were implanted in the rectus abdominis muscle of 4 adult Papio ursinus. These constructs along with surrounding muscle tissue were harvested on days 17, 19, 25, 27 and 30 after heterotopic implantation. Tissues were homogenised, and RNA extracted from both the coral-derived devices and the surrounding muscle tissue. Following a reverse transcription step, the extracts were analysed by quantitative reverse-transcriptase polymerase chain reaction to study the temporal changes in gene expression for members of the Transforming Growth Factor-β (TGFβ), Bone Morphogenic Protein (BMP) and Fibroblast Growth Factor (FGF) families and their receptors, WNT signalling, Sma and mad (SMAD) families along with collagen type I (Col1), Noggin, POU Class 5 Homeobox 1 (POU5F1) and SRY (Sex Determining Region Y)-Box 2 (SOX-2). The rapid induction of bone formation was sustained and highlighted by β-catenin and Col1 overexpression at days 19 and 23, with down-regulation of CD-10, CryαB, Noggin, POU5F1 and FGF-1, -2 and their receptors. The SMAD signalling cascade is prominent with the up-regulation of SMAD-2 and -4 and the down-regulation of SMAD-5. Stemness genes, such as SOX-2, were over-expressed while CD-10 and CD-92 were down-regulated. BMP-6 was down-regulated with up-regulation of BMP-2 and -9. The study shows that implantation of 125 µg hTGF-β3 preloaded coral-derived calcium phosphate macroporous bioreactors induces bone formation in heterotopic sites of the rectus abdominus muscle by day 19 by the temporal expression of a range of genes implicated in the bone induction cascade.
A research submitted in fulfilment for the degree of Masters of Science Dissertation to the Faculty of Health Sciences, School of Clinical Medicine, University of the Witwatersrand, Johannesburg, 2019