Bone induction by recombinant human osteogenic protein-1 (hOP-1, BMP-7) in the primate Papio ursinus with expression of mRNA of gene products of the TGF-? superfamily

Abstract

Predictable bone induction in clinical contexts requires information on the expression and cross regulation of gene products of the transforming growth factor‐β (TGF‐β) superfamily elicited by single applications of each recombinant human bone morphogenetic/ostegenic proteins (BMPs/OPs). Using the calvarium and the rectus abdominis muscle of adult baboons Papio ursinus as a model for tissue induction and morphogenesis, this study investigated the induction of bone morphogenesis by γ‐irradiated hOP‐1 delivered by γ‐irradiated bovine insoluble collagenous bone matrix, the hOP‐1 osteogenic device, for bone induction in heterotopic and orthotopic sites of the primate Papio ursinus and the expression patterns of OP‐1, collagen type IV, BMP‐3 and TGFβ1mRNAs elicited by increasing single applications of doses of the hOP‐1 osteogenic devices (0.1, 0.5 and 2.5 mg hOP‐1/g of matrix) applied heterotopically in the rectus abdominis muscle and orthotopically in 48 calvarial defects of 12 adult baboons. Histology and histomorphometry on serial undecalcified sections prepared from the specimens harvested on day 15, 30 and 90 showed that all the doses of the hOP‐1 osteogenic device induced bone formation culminating in complete calvarial regeneration by day 90. Type IV collagen mRNA expression, a marker of angiogenesis, was strongly expressed in both heterotopic and orthotopic tissues. High levels of expression of OP‐1 mRNA demonstrated autoinduction of OP‐1 mRNAs. Expression levels of BMP‐3 mRNA varied from tissues induced in heterotopic vs. orthotopic sites with high expression in rapidly forming heterotopic ossicles together with high expression of type IV collagen mRNA. The temporal and spatial expressions of TGF‐β1 mRNA indicate a specific temporal transcriptional window during which expression of TGF‐β1 is mandatory for successful and optimal osteogenesis. The induction of bone by hOP‐1 in Papio ursinus develops as a mosaic structure with distinct spatial and temporal patterns of gene expression of members of the TGF‐β superfamily that singly, synergistically and synchronously initiate and maintain tissue induction and morphogenesis.

Soluble osteogenic molecular signals and the induction of bone formation

“…BMP-3, OP-1, GDF-10 and TGF-b1 have been shown to be expressed in ossicles de novo generated after local applications of hTGF-b1 and hTGF-b2 [69,70]. Expression of OP-1, type IV collagen, BMP-3 and TGF-b1 mRNAs as shown by Northern blot analyses, is initiated upon implantation of doses of hOP-1 in both heterotopic and orthotopic sites of the non-human primate P. ursinus [71]. The studied mRNAs show a temporal and spatial pattern of expression indicating progressing stages of osteogenic differentiation during the induction of bone formation by the implanted hOP-1 osteogenic devices [71].…”

“…Expression of OP-1, type IV collagen, BMP-3 and TGF-b1 mRNAs as shown by Northern blot analyses, is initiated upon implantation of doses of hOP-1 in both heterotopic and orthotopic sites of the non-human primate P. ursinus [71]. The studied mRNAs show a temporal and spatial pattern of expression indicating progressing stages of osteogenic differentiation during the induction of bone formation by the implanted hOP-1 osteogenic devices [71]. High levels of expression of OP-1 mRNA demonstrated autoinduction of OP-1 mRNA during tissue induction by hOP-1 [71].…”

The induction of bone formation by coral-derived calcium carbonate/hydroxyapatite constructs

“…cDNA probes for g-actin, type IV collagen and OP-1 were as described [27,28]; the probe for type IV collagen a2 encoded most of the collagenous domain and part of the non-collagenous domain [27] and the probe for OP-1 comprised a 679 bp fragment of OP-1 (pÖ 320) corresponding to amino acids of the N-terminal part of the mature polypeptide [28]. The cDNA probe for growth and differentiation factor-10 (GDF-10) [30] was kindly provided by N. Cunningham (formerly of Johns Hopkins University, Maryland, USA).…”

“…Hybridisation was at 68 C, and the membranes were washed twice for 15 min at room temperature with 150 ml of 2Â SSC, 0.1% SDS followed by the stringency wash at 68 C with 0.1Â SSC, 0.1% SDS. The hybridised membranes were exposed to Kodak film (Biomax MS) with intensified screens for 15-75 h at À70 C. Signals were quantified relative to g-actin blots by densitometric analysis (Gel Documentation and Analysis System, Syngene, U.K.) [28].…”

“…Samples of 100-150 mg from quadruplicate specimens of harvested tissue specimens from the rectus abdominis muscle on days 60, 90 and 365 before bilateral carotid perfusion were pooled and crushed to a fine powder with mortar and pestle pre-cooled at À70 C. RNA was extracted as previously described [27,28]. RNA was separated by formaldehyde agarose gel electrophoresis and transferred to hybond N þ (Amersham, UK) nylon membrane using standard protocols [29].…”