June Teare

Transforming Growth Factor-β Isoforms and the Induction of Bone Formation: Implications for Reconstructive Craniofacial Surgery

Craniofacial skeletal reconstruction remains a challenging problem despite major molecular and surgical developments in the understanding of bone formation by induction. The induction of bone formation has been a critical topic of research across the planet. The bone induction principle identified important cues for tissue engineering of bone, namely, osteogenic soluble molecular signals, the bone morphogenetic and osteogenic proteins, and insoluble signals or substrata including biomimetic bioactive matrices and responding stem cells. In primates, and in primates only, the osteogenic soluble molecular signals that initiate the induction of bone formation additionally include the 3 mammalian transforming growth factor-beta (TGF-beta) isoforms, members of the TGF-beta supergene family. The mammalian TGF-beta isoforms, when implanted in the rectus abdominis muscle of the nonhuman primate Papio ursinus, induce rapid and substantial endochondral bone formation resulting in large corticalized ossicles by day 30 after heterotopic implantation; in calvarial defects of the same nonhuman primates, identical or higher doses of the TGF-beta protein do not induce bone formation because of the overexpression of Smad-6 and Smad-7, gene product inhibitors of the TGF-beta signaling pathway. The addition of minced fragments of autogenous rectus abdominis muscle partially restores the osteoinductive activity of the human TGF-beta3 isoform resulting in the induction of bone formation in the treated calvarial defects. Recombinant human TGF-beta3 delivered by Matrigel matrix and implanted in class II and III furcation defects of mandibular molars of P. ursinus induce periodontal tissue regeneration. The addition of minced fragments of autogenous rectus abdominis muscle significantly enhances cementogenesis. This review highlights the induction of bone formation by the osteogenic proteins of the TGF-beta superfamily in the nonhuman primate P. ursinus and reviews combinatorial applications of myoblastic/myogenic stem cell-based therapeutics for bone induction and morphogenesis. The recruitment of myoendothelial cells is also discussed in the light of the intrinsic and spontaneous induction of bone formation by smart biomaterial matrices that induce bone differentiation in heterotopic extraskeletal sites of P. ursinus without the exogenous application of the osteogenic soluble molecular signals of the TGF-beta superfamily.

The induction of endochondral bone formation by transforming growth factor-β3: experimental studies in the non-human primate Papio ursinus

Transforming growth factor‐β3 (TGF‐β3), 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‐β subfamily of proteins. The pleiotropism of the signalling proteins of the TGF‐β superfamily, including the TGF‐β 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‐β3 isoform does not initiate endochondral bone formation. Strikingly and in marked contrast to the rodent bioassay, recombinant human (h)TGF‐β3, when implanted in the rectus abdominis muscle of adult non‐human primates Papio ursinus at doses of 5, 25 and 125 μg 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‐β3 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‐β3 protein is obtained by mixing the hTGF‐β3 device with minced fragments of autogenous rectus abdominis muscle thus adding responding stem cells for further bone induction by the hTGF‐β3 protein. The observed limited bone induction in hTGF‐β3/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‐β signalling pathway. RT‐PCR, Western and Northern blot analyses of tissue specimens generated by the TGF‐β3 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‐β3/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.

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.

Soluble Signals and Insoluble Substrata

The repair and regeneration of bone is a complex process that is temporally and spatially regulated by soluble and insoluble signals (1). The initiation of bone formation during embryonic development and postnatal osteogenesis involves a complex cascade of molecular and morphogenetic processes that ultimately lead to the architectural sculpturing of precisely organized multicellular structures.

Cementogenesis and the induction of periodontal tissue regeneration by the osteogenic proteins of the transforming growth factor‐β superfamily

The antiquity and severity of periodontal diseases are demonstrated by the hard evidence of alveolar bone loss in gnathic remains of the Pliocene/Pleistocene deposits of the Bloubank Valley at Sterkfontein, Swartkrans and Kromdrai in South Africa. Extant Homo has characterized and cloned a superfamily of proteins which include the bone morphogenetic proteins that regulate tooth morphogenesis at different stages of development as temporally and spatially connected events. The induction of cementogenesis, periodontal ligament and alveolar bone regeneration are regulated by the co-ordinated expression of bone morphogenetic proteins. Naturally derived and recombinant human bone morphogenetic proteins induce periodontal tissue regeneration in mammals. Morphological analyses on undecalcified sections cut at 3-6 mum on a series of mandibular molar Class II and III furcation defects induced in the non-human primate Papio ursinus show the induction of cementogenesis. Sharpeys fibers nucleate as a series of composite collagen bundles within the cementoid matrix in close relation to embedded cementocytes. Osteogenic protein-1 and bone morphogenetic protein-2 possess a structure-activity profile, as shown by the morphology of tissue regeneration, preferentially cementogenic and osteogenic, respectively. In Papio ursinus, transforming growth factor-beta(3) also induces cementogenesis, with Sharpeys fibers inserting into newly formed alveolar bone. Capillary sprouting and invasion determine the sequential insertion and alignment of individual collagenic bundles. The addition of responding stem cells prepared by finely mincing fragments of autogenous rectus abdominis muscle significantly enhances the induction of periodontal tissue regeneration when combined with transforming growth factor-beta(3) implanted in Class II and III furcation defects of Papio ursinus.

A Macroporous Bioreactor Super Activated by the Recombinant Human Transforming Growth Factor-β3

Macroporous single phase hydroxyapatite (HA) and biphasic HA/β-tricalcium phosphate with 33% post-sinter hydroxyapatite (HA/β-TCP) were combined with 25 or 125 μg recombinant human transforming growth factor-β3 (hTGF-β3) to engineer a super activated bioreactor implanted in orthotopic calvarial and heterotopic rectus abdominis muscle sites and harvested on day 30 and 90. Coral-derived calcium carbonate fully converted (100%) and partially converted to 5 and 13% hydroxyapatite/calcium carbonate (5 and 13% HA/CC) pre-loaded with 125 and 250 μg hTGF-β3, and 1:5 and 5:1 binary applications of hTGF-β3: hOP-1 by weight, were implanted in the rectus abdominis and harvested on day 20 and 30, respectively, to monitor spatial/temporal morphogenesis by high doses of hTGF-β3. Bone formation was assessed on decalcified paraffin-embedded sections by measuring the fractional volume of newly formed bone. On day 30 and 90, single phase HA implants showed greater amounts of bone when compared to biphasic specimens; 5 and 13% HA/CC pre-loaded with 125 and 250 μg hTGF-β3 showed substantial induction of bone formation; 250 μg hTGF-β3 induced as yet unreported massive induction of bone formation as early as 20 days prominently outside the profile of the macroporous constructs. The induction of bone formation is controlled by the implanted ratio of the recombinant morphogens, i.e., the 1:5 hTGF-β3:hOP-1 ratio by weight was greater than the inverse ratio. The unprecedented tissue induction by single doses of 250 μg hTGF-β3 resulting in rapid bone morphogenesis of vast mineralized ossicles with multiple trabeculations surfaced by contiguous secreting osteoblasts is the novel molecular and morphological frontier for the induction of bone formation in clinical contexts.

Synergistic induction of periodontal tissue regeneration by binary application of human osteogenic protein-1 and human transforming growth factor-β3 in Class II furcation defects of Papio ursinus

BACKGROUND AND OBJECTIVE Binary applications of recombinant human osteogenic protein-1 (hOP-1) and transforming growth factor-β3 (hTGF-β3) synergize to induce pronounced bone formation. To induce periodontal tissue regeneration, binary applications of hOP-1 and hTGF-β(3) were implanted in Class II furcation defects of the Chacma baboon, Papio ursinus. MATERIAL AND METHODS Defects were created bilaterally in the furcation of the first and second mandibular molars of three adult baboons. Single applications of 25 μg hOP-1 and 75 μg hTGF-β(3) in Matrigel(®) matrix were compared with 20:1 binary applications, i.e. 25 μg hOP-1 and 1.25 μg hTGF-β(3). Morcellated fragments of autogenous rectus abdominis striated muscle were added to binary applications. Sixty days after implantation, the animals were killed and the operated tissues harvested en bloc. Undecalcified sections were studied by light microscopy, and regenerated tissue was assessed by measuring volume and height of newly formed alveolar bone and cementum. RESULTS The hOP-1 and hTGF-β(3) induced periodontal tissue regeneration and cementogenesis. Qualitative morphological analysis of binary applications showed clear evidence for considerable periodontal tissue regeneration. Quantitatively, the differences in the histomorphometric values did not reach statistical significance for the group size chosen for this primate study. The addition of morcellated muscle fragments did not enhance tissue regeneration. Binary applications showed rapid expansion of the newly formed bone against the root surfaces following fibrovascular tissue induction in the centre of the treated defects. CONCLUSION Binary applications of hOP-1 and hTGF-β(3) in Matrigel(®) matrix in Class II furcation defects of P. ursinus induced substantial periodontal tissue regeneration, which was tempered, however, by the anatomy of the furcation defect model, which does not allow for the rapid growth and expansion of the synergistic induction of bone formation, particularly when additionally treated with responding myoblastic stem cells.

Induction of cementogenesis and periodontal ligament regeneration by the bone morphogenetic proteins

The complex tissue morphologies of the periodontal tissues, the locking of the teeth into the alveolar bone with the overlying gingival tissues, and the temporo-mandibular joint with the associated powerful masticatory muscles are a superb example of design architecture and engineering.