Lentsha Nathaniel Ramoshebi

Osteogenic protein-1, a bone morphogenetic protein, induces angiogenesis in the chick chorioallantoic membrane and synergizes with basic fibroblast growth factor and transforming growth factor-β1

Capillary invasion is a vital regulatory signal during bone morphogenesis that is influenced by angiogenic molecules such as fibroblast growth factor (FGF) and some members of the transforming growth factor‐β (TGF‐β) superfamily, including TGF‐βs themselves. Bone morphogenetic proteins (BMPs), which are members of the TGF‐β superfamily, have previously not been shown to possess direct angiogenic properties. Osteogenic protein‐1 (OP‐1; BMP‐7) is a potent regulator of cartilage and bone differentiation in vivo. The osteogenic and angiogenic properties of OP‐1 at both ortho‐ and heterotopic sites in adult chacma baboons (Papio ursinus) are enhanced synergistically by the simultaneous application of relatively low doses of TGF‐β1. The single application of relatively high doses of TGF‐β1 (20 ng), and bFGF (500 ng) or relatively low (100 ng) and high (1,000 ng) doses of OP‐1 in the chick chorioallantoic membrane (CAM) assay elicited a prominent and (for OP‐1) dose‐dependent angiogenic response. The binary application of a relatively low dose of OP‐1 (100 ng) with a relatively low dose of bFGF (100 ng) or with a relatively low (5 ng) or high (20 ng) dose of TGF‐β1 resulted in a synergistic enhancement of the angiogenic response. The angiogenic effect of the relatively low doses of the combined morphogens was distinctly more pronounced than that of the single application of the relatively high doses of the respective factors. The present findings suggest that these morphogens may be deployed in binary combination in order to accentuate experimental angiogenesis. The cooperative interaction of the different morphogens in the CAM assay may provide important biological clues towards the control of clinical angiogenesis. Anat Rec 259:97‐107, 2000.

TISSUE ENGINEERING: TGF-BETA SUPERFAMILY MEMBERS AND DELIVERY SYSTEMS IN BONE REGENERATION

The induction of bone formation requires three parameters that interact in a highly regulated process: soluble osteoinductive signals, capable responding cells, and a supporting matrix substratum or insoluble signal. The use of recombinant and naturally derived bone morphogenetic proteins and transforming growth factor beta(s) (TGF-beta(s)) has increased our understanding of the functions of these morphogens during the induction of endochondral bone formation. In addition, growing understanding of the cellular interactions of living tissues with synthetic biomaterials has led to the in vivo induction of bone formation using porous biomimetic matrices as an alternative to the use of autografts for bone regeneration. This review outlines the basis of bone tissue engineering by members of the TGF-beta superfamily, focusing on their delivery systems and the intrinsic induction of bone formation by specific biomimetic matrices with a defined geometry.

Bone Induction by BMPs/OPs and Related Family Members in Primates: The Critical Role of Delivery Systems

Background: In a series of studies in the primate Papio ursinus, we have examined the capacity of bone morphogenetic proteins (BMPs/OPs) delivered in a variety of biomaterial carrier systems to elicit bone formation in heterotopic and orthotopic sites. In this review, we compare the osteoinductive effects of different biomaterial delivery systems that have or have not been pretreated with BMPs/OPs. In particular, we focus on the geometric induction of bone formation by sintered porous hydroxyapatite (SPHA) discs with concavities on their planar surfaces, which elicit bone formation without exogenously applied BMPs/OPs. Methods: Heterotopic bone formation was examined by bilaterally implanting 100-mg pellets of a collagenous carrier containing BMPs/OPs in the rectus abdominis muscle of the adult baboon. Orthotopic bone formation was examined by implanting 1 g of a collagenous carrier containing BMPs/OPs into two full-thickness critical-sized 25-mm-diameter defects on each side of the calvaria of adult baboons. The BMPs/OPs whose effects were examined included recombinant human osteogenic protein-1 (rhOP-1), recombinant human transforming growth factor-&bgr;1 (rhTGF-&bgr;1), rhTGF-&bgr;2, and porcine platelet derived transforming growth factor-&bgr;1 (pTGF-&bgr;1). Tissue from the rectus abdominis muscle was harvested 30 or 90 days after implantation. Tissue from the orthotopic calvarial model was examined at 1, 3, 6, 9, and 12 months after implantation. To demonstrate the effect of surface geometry on bone induction, hydroxyapatite powders were sintered to form solid discs with a series of concavities on the planar surfaces of the SPHA discs. The discs were either pretreated with exogenous rhOP-1 or not treated with exogenous OP-1. They were then implanted heterotopically or orthotopically into calvarial defects. Bone formation was evaluated histologically in undecalcified sections stained with Goldner’s trichrome stain or 0.1% toluidine blue. Results: Naturally derived BMPs/OPs or rhOP-1 in a collagenous carrier elicit heterotopic bone formation and the complete healing of 25-mm-diameter critical-sized defects by day 90 following implantation. Binary applications of TGF-&bgr;1 together with rhOP-1 in the collagen carrier induced massive endochondral ossicles in heterotopic sites and bone formation in calvarial defects. pTGF-&bgr;1, rhTGF-&bgr;1, and rhTGF-&bgr;2 are powerful inducers of heterotopic endochondral bone formation but elicit limited bone formation in calvarial defects. SPHA discs pretreated with rhOP-1 elicited extensive bone formation in both heterotopic and orthotopic sites. However, SPHA without rhOP-1 also elicited bone formation in heterotopic and orthotopic sites and complete healing of the calvarial defects. Conclusion: We have prepared SPHA discs with concavities on their planar surfaces that induce bone formation in heterotopic or orthotopic critical-sized calvarial defects without exogenously applied BMPs/OPs. This biomaterial induces bone formation by intrinsic osteoinductivity regulated by the geometry of the substratum. The incorporation of specific biological activities into biomaterials by manipulating the geometry of the substratum, defined as geometric induction of bone formation, may make it possible to engineer morphogenetic responses for therapeutic osteogenesis in clinical contexts. Clinical Relevance: We have implemented a clinical trial using naturally derived BMPs/OPs extracted and purified from bovine bone matrices and implanted in craniofacial defects in humans. In addition, the discovery that specific geometric and surface characteristics of sintered hydroxyapatites can induce intrinsic osteoinductivity in primates paves the way for formulation and therapeutic application of porous substrata designed to obtain predictable intrinsic osteoinductivity in clinical contexts.

Periodontal tissue regeneration by recombinant human transforming growth factor-β3 in Papio ursinus

BACKGROUND AND OBJECTIVE Osteogenic proteins of the transforming growth factor-beta superfamily induce periodontal tissue regeneration in animal models, including primates. To our knowledge, no studies have been performed in periodontal regeneration using the transforming growth factor-beta 3 isoform. In the present study, recombinant human transforming growth factor-beta 3 was examined for its ability to induce periodontal tissue regeneration in the nonhuman primate, Papio ursinus. MATERIAL AND METHODS Class II furcation defects were surgically created bilaterally in the maxillary and mandibular molars of four adult baboons. Heterotopic ossicles, for transplantation to selected furcation defects, were induced within the rectus abdominis muscle by recombinant human transforming growth factor-beta 3. Forty days later, the periodontal defects were implanted with recombinant human transforming growth factor-beta 3 in Matrigel as the delivery system, with recombinant human transforming growth factor-beta 3 plus minced muscle tissue in Matrigel, or with the harvested recombinant human transforming growth factor-beta 3-induced ossicles. Sixty days after periodontal implantation, the animals were killed and the specimens harvested. Histological analysis on undecalcified sections measured the area and volume of new alveolar bone and the coronal extension of newly formed alveolar bone and cementum. RESULTS Morphometric analyses showed pronounced periodontal regeneration in experimental defects compared with controls. Substantial regeneration was observed in defects implanted with fragments of heterotopically induced ossicles and with recombinant human transforming growth factor-beta 3 plus minced muscle tissue. CONCLUSION Recombinant human transforming growth factor-beta 3 in Matrigel significantly enhanced periodontal tissue regeneration in the nonhuman primate, P. ursinus.

Transforming Growth Factor-β Supports the Rapid Morphogenesis of Heterotopic Endochondral Bone Initiated by Human Osteogenic Protein-1 via the Synergistic Upregulation of Molecular Markers

Members of the transforming growth factor-β (TGF-β) superfamily of proteins, the bone morphogenetic proteins (BMPs) and the TGF-β isoforms, are involved in the coordination of cartilage and bone differentiation both in embryonic development and in postnatal life. Both osteogenic protein-1 (OP-1) and TGF-β1 have been shown to be potent regulators and inducers of heterotopic endochondral bone induction in non-human primates. In marked contrast, TGF-β1 does not induce heterotopic endochondral bone in rodents. In the primate, the osteogenic properties of OP-1 are synergistically enhanced by the combined administration of TGF-β1. The binary application of OP-1 (0.1, 0.3, 1.0 and 3.0μg) and TGF-β1 (0.01, 0.03 and 0.1 μg) to 25 mg of guanidinium-inactivated insoluble collagenous bone matrix as carrier in the rodent heterotopic bioassay for 7, 12 and 21 days resulted in a classical synergistic, dose-dependent and temporal up-regulation of OP-1-induced endochondral bone formation. There were significant increases in alkaline phosphatase activity (day 12) and calcium content (days 12 and 21). mRNA expression of OP-1, TGF-β1, BMP-3 and collagens type II and IV, markers of bone formation, showed an up-regulation of the genes (days 12 and 21) by the binary applications of the morphogens. Histologically, single applications of OP-1 elicited a dose dependent induction of endochondral bone formation while the binary applications resulted in a temporal acceleration of the morphogenetic cascade. The optimal ratio of OP-1/TGF-β1 was 30:1 by weight for endochondral bone formation and expression of molecular markers. The present data provides insights to the mechanisms of synergistic molecular therapeutics for endochondral bone formation in clinical contexts.