Tissue segregation restores the induction of bone formation by the mammalian transforming growth factor-β3 in calvarial defects of the non-human primate Papio ursinus

Abstract

A diffusion molecular hypothesis from the dura and/or the leptomeninges below that would control the induction of calvarial membranous bone formation by the recombinant human transforming growth factor-β3 (hTGF-β3) was investigated. Coral-derived calcium carbonate-based macroporous constructs (25 mm diameter; 3.5/4 mm thickness) with limited hydrothermal conversion to hydroxyapatite (7% HA/CC) were inserted into forty calvarial defects created in 10 adult Chacma baboons Papio ursinus. In 20 defects, an impermeable nylon foil membrane (SupraFOIL®) was inserted between the cut endocranial bone and the underlying dura mater. Twenty of the macroporous constructs were preloaded with hTGF-β3 (125 μg in 1000 μl 20 mM sodium succinate, 4% mannitol pH4.0), 10 of which were implanted into defects segregated by the SupraFOIL® membrane, and 10 into non-segregated defects. Tissues were harvested on day 90, processed for decalcified and undecalcified histology and quantitative real-time polymerase chain reaction (qRT-PCR). Segregated untreated macroporous specimens showed a reduction of bone formation across the macroporous spaces compared to non-segregated constructs. qRT-PCR of segregated untreated specimens showed down regulation of osteogenic protein-1 (OP-1), osteocalcin (OC), bone morphogenetic protein-2 (BMP-2), RUNX-2 and inhibitor of DNA binding-2 and -3 (ID2,ID3) and up regulation of TGF-β3, a molecular signalling pathway inhibiting the induction of membranous bone formation. Non-segregated hTGF-β3/treated constructs also showed non-osteogenic expression profiles when compared to non-segregated untreated specimens. Segregated hTGF-β3/treated 7% HA/CC constructs showed significantly greater induction of bone formation across the macroporous spaces and, compared to non-segregated hTGF-β3/treated constructs, showed up regulation of OP-1, OC, BMP-2, RUNX-2, ID2 and ID3. Similar up-regulated expression profiles were seen for untreated non-segregated constructs. TGF-β signalling via ID genes creates permissive or refractory micro-environments that regulate the induction of calvarial bone formation which is controlled by the exogenous hTGF-β3 upon segregation of the calvarial defects. The dura is the common regulator of the induction of calvarial bone formation modulated by the presence or absence of the SupraFOIL® membrane with or without hTGF-β3.