Ugo Ripamonti

Osteoinduction in porous hydroxyapatite implanted in heterotopic sites of different animal models

Previous studies have demonstrated the induction of bone in coral-derived porous hydroxyapatite when implanted intramuscularly in baboons. This hydroxyapatite-induced bone differentiation model was used to study the effect of different animal species on heterotopic bone formation. Porous hydroxyapatite, obtained after hydrothermal conversion of the calcium carbonate exoskeleton of coral (genus Goniopora), was implanted in the rectus abdominis of adult rabbits, dogs and baboons (Papio ursinus). Specimens were harvested on day 90 after implantation and subjected to histological and histomorphometrical analysis. Minimal amounts of bone formed in hydroxyapatite specimens harvested from rabbits and dogs. Substantial bone differentiation did occur, however, in hydroxyapatite specimens harvested from the rectus abdominis of the baboons. In primates, the porous hydroxyapatite, as used in this study, may act as a solid matrix for adsorption, storage and controlled release of circulating or locally produced bone morphogenetic proteins, which locally initiate bone formation. The results of this study on heterotopic bone formation in porous hydroxyapatite underscore the importance of primate models in biomaterial research, which should be exploited for the formulation of porous substrata with intrinsic osteoinductive activity.

The critical role of geometry of porous hydroxyapatite delivery system in induction of bone by osteogenin, a bone morphogenetic protein.

The collagenous extracellular matrix of bone obtained after dissociative extraction with 4 M guanidine-HCl is an optimal substratum for bone induction by osteogenin, a bone morphogenetic protein. As a proteinaceous substratum, this matrix and other collagen-based materials may be immunogenic. Thus, the search and discovery of a non-immunogenic substratum is a necessary prerequisite for the therapeutic application of the principle of bone induction to skeletal repair. Bovine osteogenin, purified greater than 50,000-fold and with an apparent molecular mass of 28-42 kilodaltons, was delivered into nonresorbable porous hydroxyapatite in granular and disc configuration. A total of 328 preparations were bioassayed for osteogenic activity by subcutaneous implantation into 164 Long-Evans rats. Specimens were harvested at day 7, 11 and 21 after implantation and subjected to alkaline phosphatase activity determination and histologic analysis. Osteogenin combined with discs of porous hydroxyapatite induced in vivo differentiation of the osteogenic phenotype in mesenchymal cells invading the three-dimensional porous space of the inorganic substratum. The geometry of the substratum had a profound influence on bone induction, since the expression of the osteogenic phenotype was solely confined in porous hydroxyapatite with disc configuration. Osteogenin did not induce bone differentiation when combined with granules of porous hydroxyapatite with identical pore dimensions. The finding that the biological activity of osteogenin can be restored and delivered by a substratum with defined geometry other than the insoluble collagenous matrix may form the basis of the potential therapeutic application of bone morphogenetic proteins.

The morphogenesis of bone in replicas of porous hydroxyapatite obtained from conversion of calcium carbonate exoskeletons of coral.

The morphogenesis of bone in a porous hydroxyapatite substratum was studied after intramuscular implantation in adult primates. Replicas of porous hydroxyapatite that had been obtained after hydrothermal conversion of the calcium carbonate exoskeleton of coral (genus Goniopora) were implanted intramuscularly in twenty-four adult male baboons (Papio ursinus). Serial sections from specimens that had been harvested at three, six, and nine months showed that initially the formation of fibrous connective tissue was characterized by a prominent vascular component and by condensations of collagen fibers assembled at the interface of the hydroxyapatite. The morphogenesis of bone was intimately associated with the differentiation of the connective-tissue condensations. Bone formed without an intervening endochondral phase. Although the amount of bone varied considerably, in several specimens extensive bone developed, filling large portions of the porous spaces and culminating in total penetration by bone within the implants. The mean volume fraction composition of the specimens was 20.8 +/- 1.0 per cent (mean and standard error) for bone, 17.3 +/- 1.7 per cent for connective-tissue condensation, 31.9 +/- 1.0 per cent for fibrovascular tissue, 6.4 +/- 0.6 per cent for bone marrow, and 34.6 +/- 0.5 per cent for the hydroxyapatite framework. The amount of bone and marrow increased at each time-period, and the hydroxyapatite framework was significantly reduced between six and nine months. This indicated a moderate biodegradation over time, which was possibly a result of incomplete conversion of carbonate to hydroxyapatite. Linear regression analysis showed a negative correlation between the hydroxyapatite framework and the magnitude of bone formation within the porosities of the hydroxyapatite (p = 0.0001). Biochemical coating of the hydroxyapatite substratum with an allogeneic fibrin-fibronectin protein concentrate prepared from baboon plasma did not significantly increase the amount of bone formation within the porous spaces. The hydroxyapatite substratum may have functioned as a solid-phase domain for anchorage of bone morphogenetic proteins.

Complete Regeneration of Bone in the Baboon by Recombinant Human Osteogenic Protein-1 (hOP-1, Bone Morphogenetic Protein-7)

We examined the efficacy of a single application of recombinant human osteogenic protein-1 (hOP-1, bone morphogenetic protein-7) for its ability to regenerate large calvarial defects in adult male baboons (Papio ursinus). Recombinant hOP-1, in conjunction with baboon or bovine guanidinium-extracted insoluble collagenous bone matrix (0.1, 0.5 and 2.5 mg per g of collagenous matrix as carrier), was implanted in 46 calvarial defects surgically prepared in 14 baboons, whilst 18 defects were implanted with the carrier matrix without hOP-1. Specimens were harvested on d 15, 30, 90 and 365 and subjected to histomorphometry on serial undecalcified sections cut at 7 microm to study the temporal sequence of tissue morphogenesis after the single application of hOP-1. Histological analysis indicated that the induction of new bone formation proceeded from the periphery to the central core of hOP-1 treated specimens after rapid angiogenesis and mesenchymal cell migration in apposition to the collagenous matrix. Whilst chondrogenesis was limited, newly formed bone has already filled with fully differentiated bone marrow elements as early as d 15, even with the 0.1 mg dose of hOP-1. On d 30 and 90, doses of 0.1 and 0.5 mg of hOP-1 showed greater amounts of bone than controls, and on d 90, they induced complete regeneration of the defects. Doses of 2.5 mg hOP-1 per g of matrix induced extensive osteogenesis initially with heterotopic ossification and displacement of the temporalis muscle above the defects. One year after implantation of hOP-1 there was restoration of the internal and external cortices of the calvaria. These results show that hOP-1 induces complete regeneration of calvarial bone in the adult primate, and suggest that the optimal activity of hOP-1 to achieve regeneration is between 100 and 500 microg of hOP-1 per g of matrix. These results in the primate may form the scientific basis for future clinical applications of hOP-1.

Tissue Engineering, Morphogenesis, and Regeneration of the Periodontal Tissues By Bone Morphogenetic Proteins

Tissue engineering is the emerging field of science developing techniques for fabrication of new tissues for replacement based on principles of cell and developmental biology and biomaterials. Morphogenesis is the cascade of pattern formation and the attainment of form of the various organs and the organism as a whole. The periodontium consist of the periodontal ligament, cementum, and alveolar bone. Bone has considerable potential for regeneration and therefore is a prototypic model for tissue engineering. The three main ingredients for tissue engineering are regulatory signals, responding stem cells, and extracellular matrix. Recent advances in molecular biology of the bone morphogenetic proteins (BMPs) have set the stage for tissue engineering of bone and related tissues, including the periodontium. Bone-derived BMPs, with a collagenous matrix as carrier, induced cementum and alveolar bone regeneration in surgically created furcation defects in the primate. It is noteworthy that there was morphogenesis of periodontal ligament and a faithful insertion of Sharpeys fibers into cementum. In the same furcation model, recombinant human osteogenic protein-1 (rhOP-1, also known as BMP-7), in conjunction with the collagenous carrier, induced extensive cementogenesis with insertion of Sharpeys fibers into the newly formed cementum. The observation that BMPs induce cementogenesis and periodontal ligament formation indicates that these proteins may have multiple functions in vivo not limited to cartilage and bone induction. The rapid advances in the molecular biology of BMPs and their receptors bode well for novel strategies to engineer the regeneration of the periodontal tissues.

Recombinant Transforming Growth Factor‐β1 Induces Endochondral Bone in the Baboon and Synergizes with Recombinant Osteogenic Protein‐1 (Bone Morphogenetic Protein‐7) to Initiate Rapid Bone Formation

Several members of the bone morphogenetic protein (BMP) and transforming growth factor‐β (TGF‐β) families are molecular regulators of cartilage and bone regeneration, although their actual roles and combined interactions in skeletal repair are poorly understood. The presence of several molecular forms suggests multiple functions in vivo as well as synergistic interactions during both embryonic bone development and regeneration of cartilage and bone in postfetal life. Here we show for the first time that recombinant human transforming growth factor‐β1 (TGF‐β1) induces endochondral bone formation in extraskeletal sites of adult baboons. We also show that TGF‐β1 and recombinant human osteogenic protein‐1 (OP‐1, bone morphogenetic protein‐7) synergize in inducing large ossicles in extraskeletal sites of the primate as early as 15 days after implantation. A single application of OP‐1, in conjunction with an insoluble collagenous matrix as carrier (5, 25, and 125 μg/100 mg of carrier matrix) induced bone differentiation in the rectus abdominis of the baboon. This level of tissue induction was raised several‐fold by the simultaneous addition of comparatively low doses of TGF‐β1 (0.5, 1.5, and 5 μg), which by itself induces bone formation in the rectus abdominis at doses of 5 μg/100 mg of carrier matrix. Combinations of OP‐1 and TGF‐β1 yielded a 2‐ to 3‐fold increase in cross‐sectional area of the newly generated ossicles, with markedly elevated key parameters of bone formation, and corticalization of the newly formed bone by day 15, culminating in bone marrow generation by day 30. The tissue generated by the combined application of OP‐1 and TGF‐β1 showed distinct morphological differences when compared with OP‐1–treated specimens, with large zones of endochondral development and extensive bone marrow formation. At the doses tested, synergy was optimal at a ratio of 1:20 by weight of TGF‐β1 and OP‐1, respectively. These results provide evidence for a novel function of TGF‐β1 in the primate and the scientific basis for synergistic molecular therapeutics for the rapid regeneration of cartilage and bone.

Osteogenin, a bone morphogenetic protein, adsorbed on porous hydroxyapatite substrata, induces rapid bone differentiation in calvarial defects of adult primates.

Osteogenin, a bone morphogenetic protein, in conjunction with insoluble collagenous bone matrix initiates local endochondral bone differentiation by induction in vivo. This study, by exploiting the affinity of native osteogenin for hydroxyapatite, was designed to construct a delivery system for the expression of the biologic activity of osteogenin in nonhealing calvarial defects of adult primates. After exposure of the calvaria, 64 cranial defects, 25 mm in diameter, were prepared in 16 adult male baboons (Papio ursinus). Defects were implanted with disks of porous nonresorbable and resorbable hydroxyapatite substrata obtained after hydrothermal conversion of calcium carbonate exoskeletons of corals. In each animal, one disk of each hydroxyapatite preparation was treated with osteogenin isolated and purified from baboon bone matrix after sequential chromatography on heparin-Sepharose, hydroxyapatite, and Sephacryl S-200 gel filtration columns. The remaining two defects were implanted with one disk of each hydroxyapatite preparation without osteogenin as control. Histomorphometry on decalcified sections prepared on days 30 and 90 showed superior osteogenesis in osteogenin-treated nonresorbable hydroxyapatite specimens as compared with controls. On day 90, substantial bone formation also had occurred in control nonresorbable hydroxyapatite specimens. On day 90, but not on day 30, significantly greater amounts of bone had formed in osteogenin-treated resorbable specimens as compared with resorbable controls. Overall, resorbable substrata performed poorly when compared with nonresorbable substrata, perhaps due to a premature dissolution of the implants. These results provide evidence that the biologic activity of osteogenin can be restored and delivered by a substratum other than the organic collagenous matrix, inducing rapid bone differentiation in calvarial defects of adult nonhuman primates. The adsorption strategy of osteogenin on porous inorganic nonimmunogenic substrata may help to design appropriate osteogenic delivery systems for craniofacial and orthopedic applications in humans.

Induction of cementogenesis by recombinant human osteogenic protein-1 (hOP-1/BMP-7) in the baboon (Papio ursinus)

Recombinant human osteogenic protein-1 (hOP-1), a member of the bone morphogenetic protein family, was examined for its efficacy in periodontal regeneration. Twelve furcation defects, surgically prepared in the first and second mandibular molars, were treated with bovine insoluble collagenous matrix in conjunction with 0.0 (control), 100 and 500 mu g of recombinant hOP-1 per g of matrix. After 60 days of healing, histological and histometric analyses on serial, undemineralized sections cut at 7 mu m showed substantial cementogenesis on the exposed dentine of furcations treated with both doses of hOP-1 (p < 0.01 vs control). Foci of nascent mineralization were seen within the newly deposited cementoid along the coronal areas of hOP-1-treated defects. Within the furcations, there were substantial amounts of residual collagenous carrier, interspersed with a mineralized matrix having histological features of cementum. This mineralized cementum-like material was predominantly deposited around the carrier, and blended into newly formed cementum along the root surfaces. In the apical area, the cementum-like material and the remaining alveolar bony housing were not connected; indeed the two components were separated by a fibrovascular tissue that had numerous features of the periodontal ligament space. Formation and insertion of Sharpeys fibres into newly formed root cementum were also observed. It is likely that the expression of specific cell phenotypes by hOP-1 is regulated, in part, by the extracellular matrix microenvironment, including dentine. Thus, exposed dentine, in the presence of exogenous hOP-1 at the doses tested, may preferentially modulate the expression of the cementogenic phenotype. These findings in a non-human primate show that hOP-1, at the doses tested, induced cementogenesis on surgically denuded root surfaces, indicating a specific function during repair and regeneration of periodontal tissues.

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

The spontaneous induction of bone formation in heterotopic rectus abdominis and orthotopic calvarial sites by coral-derived biomimetic matrices of different chemical compositions was investigated in a long-term study in the non-human primate Papio ursinus. Coral-derived calcium carbonate constructs were converted to hydroxyapatite by hydrothermal exchange. Limited conversion produced hydroxyapatite/calcium carbonate (HA/CC) constructs of 5% and 13% hydroxyapatite. Rods of 20 mm in length and 7 mm in diameter were implanted in heterotopic rectus abdominis sites; discs 25 mm in diameter were implanted in orthotopic calvarial defects of six adult non-human primates P. ursinus. Heterotopic samples also included fully converted hydroxyapatite replicas sintered at 1100 degrees C. To further enhance spontaneous osteoinductive activity, fully converted hydroxyapatite replicas were coated with the synthetic peptide P15 known to increase the adhesion of fibroblasts to anorganic bovine mineral. Bone induction was assessed at 60, 90 and 365 days by histological examination, alkaline phosphatase and osteocalcin expression, as well as by the expression of BMP-7, GDF-10 and collagen type IV mRNAs. Induction of bone occurred in the concavities of the matrices at all time points. At 365 days, bone marrow was evident in the P15-coated and uncoated implants. Resorption of partially converted calcium carbonate/hydroxyapatite was apparent, as well as remodeling of the newly formed bone. Northern blot analyses of samples from heterotopic specimens showed high levels of expression of BMP-7 and collagen type IV mRNA in all specimen types at 60 days, correlating with the induction of the osteoblastic phenotype in invading fibrovascular cells. Orthotopic specimens showed prominent bone formation across the different implanted constructs. The concavities of the matrices biomimetize the remodeling cycle of the osteonic primate cortico-cancellous bone and promote the ripple-like cascade of the induction of bone formation. This study demonstrates for the first time that partially converted HA/CC constructs also induce spontaneous differentiation of bone, albeit only seen one year post-implantation.

Transforming growth factor-beta 1: induction of bone morphogenetic protein genes expression during endochondral bone formation in the baboon, and synergistic interaction with osteogenic protein-1 (BMP-7).

Certain members of the bone morphogenetic protein (BMP) and transforming growth factor-beta (TGF-beta) families are inducers of endochondral bone formation in vivo. TGF-betas, however, do not initiate bone formation when implanted in heterotopic (extraskeletal) sites of rodents. Here we show that platelet-derived porcine TGF-beta 1 (pTGF-beta 1) induces endochondral bone in heterotopic sites of the baboon (Papio ursinus) at doses of 5 microgram per 100 mg of guanidinium-inactivated collagenous bone matrix as carrier, with an inductive efficiency comparable to 5 and 25 micrograms of recombinant osteogenic protein-1 (hOP-1, BMP-7), a well characterized inducer of bone formation. We further demonstrate that pTGF-beta 1 and hOP-1 interact synergistically to induce large ossicles in the rectus abdominis of the primate as evaluated by key parameters of bone formation on day 14 and 30. Tissue generated on day 30 by 5 microgram pTGF-beta 1 or 25 micrograms hOP-1 induced comparable expression levels of OP-1, BMP-3 and type IV collagen mRNA transcripts, whereas TGF-beta 1 and type II collagen expression was 2 to 3 fold higher in pTGF-beta 1-treated implants, as determined by Northern analysis. In ossicles generated by 25 micrograms hOP-1 in combination with relatively low doses of pTGF-beta 1 (0.5, 1.5 and 5 micrograms), type II collagen expression increased in a pTGF-beta 1 dose-dependent manner, whilst type IV collagen was synergistically upregulated with a 3 to 4 fold increase compared to ossicles generated by a single application of 5 micrograms pTGF-beta 1 or 25 micrograms hOP-1. Morphogen combinations (5 micrograms pTGF-beta 1 with 20 micrograms hOP-1, and 5 and 15 micrograms pTGF-beta 1 with 100 micrograms hOP-1 per g of collagenous matrix as carrier) induced exuberant tissue formation and greater amounts of osteoid than hOP-1 alone when implanted in calvarial defects of the baboon as evaluated. on day 30 and 90, with displacement of the temporalis muscle above the defects. Since a single application of TGF-beta 1 in the primate did not induce bone formation in calvarial defects, whilst it induces endochondral bone differentiation in heterotopic sites, our data indicate that the bone inductive activity of TGF-beta 1 is site and tissue specific. mRNA expression of multiple members of the TGF-beta superfamily suggests complex autocrine and paracrine activities of the ligands and different signalling pathways on responding cells during the cascade of endochondral bone formation in the primate. The present findings may provide the basis for synergistic molecular therapeutics for cartilage and bone regeneration in clinical contexts.