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BMP-7) in the baboon (Papio ursinus)
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Pergamon
Archs oral Biol. Vol. 41, No. 1, pp. 121-126, 1996 Copyright © 1996ElsevierScienceLtd. All rights reserved Printed in Great Britain 0003-99~9(95)00110-7 0003-9969/96$15.00+ 0.00
SHORT COMMUNICATION INDUCTION OF CEMENTOGENESIS BY RECOMBINANT H U M A N OSTEOGENIC PROTEIN-1 (hOP-1/BMP-7) IN THE BABOON (Papio ursinus) U. RIPAMONTI, x* M. HELIOTIS, l D. C. R U E G E R 2 and T. K. SAMPATH 2 ~Bone Research Laboratory, Medical Research Council/University of the Witwatersrand, Medical School, 2193 Parktown, Johannesburg, South Africa, and 2Creative BioMolecules,Hopkinton, MA 01748, U.S.A. (Accepted 4 September 1995)
Summary--Recombinant human osteogenic protein-1 (hOP-l), a member of the bone morphogenetic protein family, was examined for its etficacy 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/ag of recombinant hOP-1 per g of matrix. After 60 days of healing, histological and histometric analyses on serial, undemineralized sections cut at 7/am 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 Sharpey's 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-l, at the doses tested, induced cementogenesis on surgically denuded root surfaces, indicating a specific function during repair and regeneration of periodontal tissues. Key words: bone morphogenetic proteins, osteogenic protein-1, cementogenesis,periodontal regeneration, primates.
The regulation of periodontal tissue regeneration is still poorly understood. The purification and molecular cloning of the BMP family (BMP-2 to BMP-6 and OP-1 and OP-2) has, however, set the stage for the understanding of the molecular mechanisms underlying bone development and regeneration (Reddi, 1992; Wozney, 1992). The basis of BMPs/OPs as possible therapeutic agents rests on the evidence that the induction and regeneration of cartilage and bone in postnatal models recapitulate events that occur in the normal course of embryonic development (Reddi, 1992). Osteogenesis induced by local therapeutic administration of native or recombinant human (rh) BMPs/OPs exploits a functionally conserved process originally deployed in embryonic development (Reddi, 1992; Ripamonti et al., 1992). On the other hand, the distinct patterns
*To whom all correspondence should be addressed. Abbreviations: BMPs, bone morphogenetic proteins; OPs,
osteogenic proteins.
of expression of BMP/OP transcripts in several developing systems, not limited to cartilage and bone induction, indicate other roles in vertebrate development and organogenesis (for review see Reddi, 1994), as well as novel strategies for therapeutic intervention outside bone. Thus the expression pattern of BMP-2, BMP-4, BMP-3 and OP-1 during tooth morphogenesis (Lyons, Pelton and Hogan, 1990; Vainio et al., 1993; Heikinheimo, 1994; Helder et al., 1996) suggests that specific BMPs/OPs may also be used for repair and regeneration of dentine (Nakashima, 1990; Nakashima, 1994; Rutherford et al., 1993) and periodontal tissues in postnatal life (Bowers et al., 1991; Ripamonti and Reddi, 1994). Whilst BMPs/OPs are strongly expressed during tooth morphogenesis, their in vivo role in periodontal regeneration is not clear. In previous studies in a non-human primate model, highly purified BMP fractions derived from bone matrix induced cementum, periodontal ligament and alveolar bone regeneration (Ripamonti et al., 1994). Moreover, the magnitude and quality of new 121
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connective-tissue attachment formation indicated that members of the BMP/OP family may also initiate cementogenesis and regulate the assembly of a functionally orientated periodontal-ligament system (Ripamonti et al., 1994). The presence of multiple forms of BMPs/OPs may be biologically significant, locally regulating the regeneration of other tissues including the periodontal ligament and cementum. Whether the magnitude of new attachment formation stimulated by hOP-1 in vivo is sufficient to constitute a potential therapeutic effect has not been previously investigated. Preclinical studies on non-human primates are critical to test the efficacy of recombinant molecules under evaluation for regenerative procedures. The experiments described here were designed to test the efficacy of a single application of two doses of hOP-1 in conjunction with a collagenous carrier for periodontal regeneration in furcation defects of the baboon (Papio ur sinus ).
In three adult male baboons, selected and prepared as described by Ripamonti et al. (1994), mucoperiosteal flaps were raised and 12 class II furcation defects were surgically prepared in the first and second mandibular molars. Exposed roots were curetted to remove periodontal ligament fibres and cementum, and notched with small burs at the level of the residual bony housing (Ripamonti et al., 1994). The depth of each furcation defect extended for at least 10-12 mm buccolingually as measured from the buccal entrance of the exposed furcations. Recombinant hOP-1 was prepared as described by Sampath et al. (1992). hOP-1 in 50% acetonitrile, 0.1% trifluoroacetic acid was combined with bovine insoluble collagenous matrix as carrier, prepared after dissociative extraction of demineralized bone matrix (Sampath and Reddi, 1981), and lyophilized. Two doses of hOP-1 were used: 100 and 500#g per g of carrier matrix. Approximately 200mg of carrier matrix was used per furcation defect. Portions of bovine collagenous matrix, inactive after extraction of osteogenic proteins, were used as control without the addition of hOP- 1. The flaps were closely readapted with resorbable sutures and a plaque control regimen was instituted weekly for the duration of the experiment. Perfusion and preparation of tissue for histology on day 60 after surgery were as described by Ripamonti et al. (1994). In brief,
specimen blocks, each including the first and second molars with surrounding bone and soft tissues, were embedded, undemineralized, in methlymethacrylate, and trimmed along the buccal aspect until the notches on the roots could be detected. Serial sections, including dentine and associated periodontal tissues, were then cut at 7 # m in the mesiodistal plane throughout the entire buccolingual extension of each furcation defect using tungsten-carbide knives mounted on a Reichert-Jung Policut-S microtome. Every 14th section, approx. 100 p m apart, was stained using the free-floating method with the Goldner's trichrome stain for undemineralized bone. Four step-serial sections, representing the buccal (one section), internal (two sections) and central (one section) regions of the buccal half of the defects were selected for histometry. The buccal half of the defects was determined by cutting the specimens until the mesiodistal fossa was reached. At this level, serial sections were representative of the central region of the defects. Sections representing the buccal, internal and central regions were approx. 600/~m apart from each other. The two sections representing the internal region were 200 # m apart. Sections from the different specimens were identified by levels of serial sectioning and matched by the size of the root canals and the pulp chambers (Ripamonti et al., 1994). Histometric measurements were made with a calibrated micrometer mounted in a Reichert Univar microscope at x40 (Ripamonti et al., 1994) (Table 1). Representative photomicrographs of hOP-Itreated specimens are shown in Figs 1-3. Results of histometric analysis are presented in Table 1. Both histological and histometric analyses point to the striking induction of cementogenesis by both doses of hOP-1 with the collagenous carrier. Deposition of organized and highly cellular mineralized tissue (interpreted as cementum) had occurred on the exposed roots, often extending to the fornix of the treated furcations (Figs 1-3). Control specimens showed limited regeneration (Table 1), scattered remnants of the collagenous carrier, and apical migration of the junctional epithelium. The preparation and analysis of undemineralized sections cut at 7 # m allowed the differentiation of mineralized versus as yet not mineralized cementum, or cementoid (Fig. 2) (Ripamonti et al., 1994). Within the central regions of the exposed furcations, substantial amounts of remaining collage-
Table 1. Histometric analysis of periodontal regeneration in 12 furcation defects surgically prepared in 3 adult baboons, blCBM: bovine insoluble collagenous matrix carrier; hOP-l: recombinant human osteogenic protein (100 and 500 #g per g of collagenous carrier); N--F: extent of furcation exposure--from the apical border of the notch (N) in the distal and mesial root to the fornix (F) of the furcation; N--NC: new cementum formation (NC)-from N to the coronal termination of the newly formed cementum along the distal and mesial roots blCBM solo (n = 2)
100 #g hOP-1 (n = 4)
500pg hOP-1 (n = 6)
Distal
N--F N--NC
9.4 ___0.1 2.6 __+0.2
NS p < 0.01
9.3 ___0.2 6.2 ___0.5
NS NS
8.8 + 0.2 6.2 + 0.3
9.3 + 0.1 2.8 __+0.I
NS p < 0.01
9.3 ___0.1 6.1 ___0.8
NS NS
8.8 __.0.3 6.7 _.+0.3
Mesial
N--F N--NC
Values (in mm) are means + SE. Levels of significance were determined using the Statistical Analysis System (1989) General Linear Models procedure with multiple interactions.
Cementogenesis by osteogenic protein-1
2 Fig. 1. Photomicrographs of furcation defects treated with 100/~g of hOP-1 per g of cartier. (A) Low-power view showing cementogenesis from the notches (white arrows) in the root surfaces, and extending to the fornix of the exposed furcation (arrow). There is separation between the residual alveolar bony housing and the tissue that had formed within the furcation, x 10. (B) Higher magnification of previous section (arrow area in A). Formation of Sharpey's fibres inserting into cementoid and mineralized cementum (in blue) in proximity to the fornix of the furcation, x 200. (C) High-power view of an adjacent section to (A) (open arrows area in A) but without artefactual separation of the dentine--cementum tissue complex: regenerated cellular and mineralized cementum with inserted connective tissue fibres, x 200. Fig. 2. Induction of cementogenesis by hOP-1. (A) Deposition of highly cellular and as yet to be mineralized cementoid along a denuded root surface in the coronal area of a furcation defect treated with 500 ~g of hOP-1 per g of carrier. Arrow indicates mineralized cemental matrix in more apical region of the defect, x 100. (B) Detail of newly deposited highly cellular cementoid. Whilst non-mineralized cementoid faced a highly vascular but non-orientated connective tissue space, regions displaying mineralized cementum showed the insertion of functionally orientated Sharpey's fibres penetrating the cementoid seam and the subjacent mineralized cemental layer. × 200. (C and D) Details of newly formed cementum. Note mineralized cementum (open arrows in C) and deposition of highly cellular cementoid in more coronal direction, close to the fornix of the furcation. Closed arrow points to loci of nascent mineralization within the non-mineralized phase of the newly deposited cemental matrix (detailed in D). x 100 and x 200, respectively.
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Fig. 3. Caption opposite.
3
Cementogenesis by osteogenic protein-I nous carrier were enclosed by a mineralized matrix that extended and blended into the newly deposited cementum along the root surfaces (Figs 1A, 3B and 3C). This newly formed mineralized tissue in direct apposition to the collagenous carrier showed limited vascular invasion, and an almost complete absence of capillaries in instances of substantial matrix deposition and mineralization in close proximity to the cementum deposited on the exposed dentine (Figs 3B and C). Importantly, there was separation and not fusion of the mineralized tissue under discussion with the remaining alveolar bone in the apical region of the furcations (Figs 1A and 3A). Indeed, the two components were separated by a vascular fibrous tissue resembling a periodontal ligament space (Fig. 3D). Whilst there are no structural markers that unequivocally differentiate cementum from bone, the histological analysis suggests that the mineralized tissue that occupies the furcation area, in direct apposition to the remnants of the collagenous carrier, is predominantly cementum rather than alveolar bone. Notable distinct features of cementum are avascularity and lack of significant remodelling (MacNeil and Somerman, 1993). Both features may help to explain the persistence of implanted collagenous matrix. It is noteworthy that implantation of 100 and 500#g of hOP-1 in conjunction with an identical preparation of bovine carrier resulted in rapid bone formation and concomitant incorporation and dissolution of the carrier by day 30 in calvarial defects of adult baboons (U. Ripamonti, B. van den Heever, M. Tucker, T. K. Sampath, D. Rueger and A. H. Reddi, unpublished observations). In the present study, the separation between tissues (alveolar bone and the cementum-like material in the furcation) was maintained by a fibrovascular tissue that shared numerous features of a periodontal ligament space (Fig. 3D). Conceptually, the formation of an additional periodontal ligament is because the biochemical and positional information (including characteristics of both substrata) are, on one side, those of alveolar bone, and on the other, those of cementum. A pertinent analogy to this observation is the fibrovascular space that constantly delineates and characterizes cemental growths such as cementomas. In such instances, fusion between the cementoma and the surrounding alveolar bone never occurs, and the presence of the fibrous space is typically seen on radiographs as a radiolucent ring around the lesion (Lucas, 1984).
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Within the limits of the histological and histometric analyses, the present experiment in non-human primates demonstrates substantial induction of cementogenesis by a single application of hOP-1 in conjunction with a collagenous matrix on surgically denuded root surfaces. The regeneration of periodontal ligament and the insertion of functionally orientated Sharpey's fibres into cementum were also observed. As hOP-I in conjunction with the bovine collagenous matrix induces bone formation in bone defects of the cranial and appendicular skeleton (for review see Ripamonti and Reddi, 1995), it is likely that the expression of specific cell phenotypes by hOP-I is regulated, in part, by extracellular matrix substrata. Thus, in a periodontal defect, the presence of exposed dentine may preferentially modulate the expression of the cementogenic phenotype on readily available cell populations (precementoblasts and their progenitors) from the ligament space. The critical regulatory role of extracellular matrix substrata in the phenotypic expression of a variety of cells has been reported previously (Reddi, 1992; Reddi, 1994). The expression of different phenotypes and the generation of cementum or alveolar bone by BMPs/OPs may depend on whether a common lineage of progenitor cells, residing in the periodontal ligament (Melcher et al., 1986; Melcher et al., 1987), attach to exposed dentinal substrata or stay in the alveolar bony side of the periodontal ligament space. The distinct patterns of BMP/OP expression, not limited to skeletal elements, point to multiple and specialized roles in development and thus regeneration. Moreover, limited promiscuity amongst receptors in binding BMP-4 and OP-I also suggests that BMPs may have different functions in vivo (ten Dijke et al., 1994). An important question is whether the presence of multiple forms of BMP/OP have a therapeutic significance, hOP-l, BMP-2 and BMP-4 are equally capable, after a single application, of induction of bone as well as reparative dentinogenesis in postnatal animal models (Wang et al., 1990; Hammonds et al., 1991; Sampath et al., 1992; Rutherford et al., 1993; Nakashima, 1994), raising important questions on the biological significance of this apparent redundancy (Reddi, 1992; Reddi, 1994). Our findings show that doses of 100 and 500 pg hOP-1 per g of carrier induce cementogenesis on surgically denuded root surfaces, indicating a specific function during repair and regeneration of periodontal tissues. The finding of cementum deposition with inserted
Fig. 3. Photomicrographs of furcation defects treated with 500/~m of hOP-1. (A) Low-power view showing extensive cementogenesis on the mesial root (arrows) up to the fornix of the furcation. Note cementation of the residual collagenous carrier within the fureation by a mineralized matrix in direct apposition to the distal root and to the furcation fornix. As in Fig. 1A, separation and not fusion between the residual bony housing and the mineralized matrix--collagenous carrier tissue complex within the furcation. × 10. (B) Detail of previous section showing limited vascular invasion within the mineralized matrix in close apposition to dentine, and the residual collagenous carrier, x 40. (C) Adjacent section, approx. 600/am apart from (B), showing areas of focal fusion between cementum formed along exposed dentine and the mineralized tissue enveloping the residual carrier, x 40. (D) Detail of the fibrovascular space generated between the residual alveolar bony housing and the mineralized matrix within the furcation: prominent capillary network, and assembly of a periodontal ligament-like structure with Sharpey's fibres inserting into the cementum-like material (black arrows). White arrows point to osteoid seams (in orange-red) and populated by contiguous osteoblasts, lining mineralized bone (in blue), x 100.
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Sharpey's fibres in the absence of a concomitant growth o f alveolar bone suggests that alveolar bone regeneration is not a requirement for cementogenesis to occur. A l t h o u g h the expression patterns of B M P / O P transcripts during root morphogenesis are not yet available, it is likely that the developmental stages of cementum and periodontal ligament formation are also regulated by sequential expression of some of the B M P / O P genes. Very recently, it was shown that rhBMP-2 induced substantial alveolar bone regeneration in a canine model. Cementogenesis, however, was limited when compared to the extent of bone regeneration (Sigurdsson et al., 1995). It is noteworthy that highly purified osteogenic preparations (containing several B M P / O P members) induced cementum, periodontal ligament and alveolar bone regeneration (Ripamonti et al., 1994). Future research must focus on optimal doses and molecular combinations, developing a structure-activity profile amongst members of the B M P / O P family. Acknowledgements--This work is supported by the South
African Medical Research Council, the University of the Witwatersrand, Johannesburg, and in part by a grant from Creative BioMolecules, Hopkinton, MA. We thank Ms Barbara van den Heever for the superb histological preparation of undemineralized sections. REFERENCES
Bowers G., Felton F., Middleton C., Glynn D., Sharp S., Mellonig J., Corio R., Emerson J., Park S., Suzuki J., Ma S., Romberg E. and Reddi A. H. (1991) Histologic comparison of regeneration in human intrabony defects when osteogenin is combined with demineralized freezedried bone allograft and with purified bovine collagen. J. Periodontol. 62, 690-702. Hammonds R. G., Schwall R., Dudley A., Berkemeier L., Lai C., Lee J., Cunningham N., Reddi A. H., Wood W.I. and Mason A. J. (1991) Bone inducing activity of mature BMP-2b produced from a hybrid BMP-2a/2b precursor. Mol. Endocrinol. 5, 149-155. Heikinheimo K. (I 994) Stage-specific expression of decapentaplegic-Vg-related genes 2, 4, and 6 (bone morphogenetic proteins 2, 4, and 6) during human tooth morphogenesis. J. dent. Res. 73, 590-597. Helder M. N., Vukicevic S., Ozkaynak E., Luyten F. P., Wrltgens J. H. M., Burger E. H., Oppermann H. and Bronckers A. L. J. J. (1996) Developmental expression of bone morphogenetic proteins 7 (BMP-7) and 3 (BMP-3) in tooth germs of the embryonic and newborn mouse. J. dent. Res. (in press). Lucas R. B. (1984) Pathology o f Tumours o f the Oral Tissues, 4th edn, p. 102. Churchill Livingstone, London. Lyons K. M., Pelton R. W. and Hogan B. L. M. (1990) Organogenesis and pattern formation in the mouse: RNA distribution patterns suggest a role for bone morphogenetic protein-2A (BMP-2A). Development 1119, 833-844, MacNeil R. L. and Somerman M. J. (1993) Molecular factors regulating development and regeneration of cementum. J. perio. Res. 28, 550-559. Melcher A. H., Cheong T., Cox J., Nemeth E. and Shiga A. (1986) Synthesis of cementum-like tissue in vitro by cells cultured by bone: a light and electron microscope study. J. perio. Res. 21, 592-612. Melcher A. H., McCulloeh C. A. G., Cheong T., Nemeth E.
and Shiga A. (1987) Cells from bone synthesize cementum-like and bony-like tissue in vitro and may migrate into periodontal ligament in vivo. J. perio. Res. 22, 246-247. Nakashima M. (1990) The induction of reparative dentine in the amputated dental pulp of the dog by bone morphogenetic protein. Archs oral Biol. 35, 493-497. Nakashima M. (1994) Induction of dentine in amputated pulp of dogs by recombinant human bone morphogenetic proteins-2 and -4 with collagen matrix. Archs oral Biol. 39, 1085-1089. Reddi A. H. (1992) Regulation of cartilage and bone differentiation by bone morphogenetic proteins. Curr. Opin. cell Biol. 4, 850-855. Reddi A. H. (1994) Bone and cartilage differentiation. Curr. Opin. gen. Dev. 4, 737-744. Ripamonti U., Ma S., Cunningham N., Yeates L. and Reddi A. H. (1992) Initiation of bone regeneration in adult baboons by osteogenin, a bone morphogenetic protein. Matrix 12, 369-380. Ripamonti U., Heliotis M., van den Heever B. and Reddi A. H. (1994) Bone morphogenetic proteins induce periodontal regeneration in the baboon (Papio ursinus). J. perio. Res. 29, 439-445. Ripamonti U. and Reddi A. H. (1994) Periodontal regeneration: potential role of bone morphogenetic proteins. J. perio. Res. 29, 225-235. Ripamonti U. and Reddi A. H. (1995) Bone morphogenetic proteins: applications in plastic and reconstructive surgery. Adv. plast, reconstr. Surg. 11, 47-73. Rutherford R. B., Wahle J., Tucker M., Rueger D. and Charette M. (1993) Induction of reparative dentine formation in monkeys by recombinant human osteogenic protein-1. Archs oral Biol. 38, 571-576. Sampath T. K. and Reddi A. H. (1981) Dissociative extraction and reconstitution of extracellular matrix components involved in local bone differentiation. Proc. Natn. Acad. Sci. U.S.A. 78, 7599-7603. Sampath T. K., Maliakal J. C., Hauschka P. V., Jones W. K., Sasak H., Tucker R. F., White K. H., Coughlin J. E., Tucker M. M., Pang R. H. L., Corbett C., t)zkaynak E., Oppermann H. and Rueger D. C. (1992) Recombinant human osteogenic protein-1 (hOP-l) induces new bone formation in vivo with a specific activity comparable with natural bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro. J. biol. Chem. 267, 20352-20362. Sigurdsson T. J., Lee M. B., Kubota K., Turek T. J., Wozney J. M. and Wikesj6 U. M. E. (1995) Periodontal repair in dogs: recombinant human bone morphogenetic protein-2 significantly enhances periodontal regeneration. J. Periodontol. 66, 131-138. Statistical Analysis System (1989) S A S / S T A T S User's Guide, Version 6, 4th ed, Vol 2, pp. 891 996. SAS Institute Inc., Cary, NC. ten Dijke P., Yamashita H., Sampath T. K., Reddi A. H., Estevez M., Riddle D. L., Ichijo H., Heldin C.-H. and Miyazono K. (1994) Identification of type I receptors for osteogenic protein-I and bone morphogenetic protein-4. J. biol. Chem. 269, 16985-16988. Vainio S., Karavanova I., Jowett A. and Thesleff I. (1993) Identification of BMP-4 as a signal mediating secondary induction between epithelial and mesenchymal tissues during early tooth development. Cell 75, 45-58. Wang E. A., Rosen V., D'Alessandro J. S., Bauduy M., Cordes P., Harada T., Israel D. I., Hewick R. M., Kerns K. M., LaPan P., Luxenberg D. P., McQuaid D., Moutsatsos I. K., Nove J. and Wozney J. M. (1990) Recombinant human bone morphogenetic protein induces bone formation. Proc. Natn. Acad. Sci. U.S.A. 87, 2220 2224. Wozney J. M. (1992) The bone morphogenetic protein family and osteogenesis. Molec. rep. Dev. 32, 160 167.
Pergamon
Archs oral Biol. Vol. 41, No. 1, pp. 121-126, 1996 Copyright © 1996ElsevierScienceLtd. All rights reserved Printed in Great Britain 0003-99~9(95)00110-7 0003-9969/96$15.00+ 0.00
SHORT COMMUNICATION INDUCTION OF CEMENTOGENESIS BY RECOMBINANT H U M A N OSTEOGENIC PROTEIN-1 (hOP-1/BMP-7) IN THE BABOON (Papio ursinus) U. RIPAMONTI, x* M. HELIOTIS, l D. C. R U E G E R 2 and T. K. SAMPATH 2 ~Bone Research Laboratory, Medical Research Council/University of the Witwatersrand, Medical School, 2193 Parktown, Johannesburg, South Africa, and 2Creative BioMolecules,Hopkinton, MA 01748, U.S.A. (Accepted 4 September 1995)
Summary--Recombinant human osteogenic protein-1 (hOP-l), a member of the bone morphogenetic protein family, was examined for its etficacy 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/ag of recombinant hOP-1 per g of matrix. After 60 days of healing, histological and histometric analyses on serial, undemineralized sections cut at 7/am 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 Sharpey's 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-l, at the doses tested, induced cementogenesis on surgically denuded root surfaces, indicating a specific function during repair and regeneration of periodontal tissues. Key words: bone morphogenetic proteins, osteogenic protein-1, cementogenesis,periodontal regeneration, primates.
The regulation of periodontal tissue regeneration is still poorly understood. The purification and molecular cloning of the BMP family (BMP-2 to BMP-6 and OP-1 and OP-2) has, however, set the stage for the understanding of the molecular mechanisms underlying bone development and regeneration (Reddi, 1992; Wozney, 1992). The basis of BMPs/OPs as possible therapeutic agents rests on the evidence that the induction and regeneration of cartilage and bone in postnatal models recapitulate events that occur in the normal course of embryonic development (Reddi, 1992). Osteogenesis induced by local therapeutic administration of native or recombinant human (rh) BMPs/OPs exploits a functionally conserved process originally deployed in embryonic development (Reddi, 1992; Ripamonti et al., 1992). On the other hand, the distinct patterns
*To whom all correspondence should be addressed. Abbreviations: BMPs, bone morphogenetic proteins; OPs,
osteogenic proteins.
of expression of BMP/OP transcripts in several developing systems, not limited to cartilage and bone induction, indicate other roles in vertebrate development and organogenesis (for review see Reddi, 1994), as well as novel strategies for therapeutic intervention outside bone. Thus the expression pattern of BMP-2, BMP-4, BMP-3 and OP-1 during tooth morphogenesis (Lyons, Pelton and Hogan, 1990; Vainio et al., 1993; Heikinheimo, 1994; Helder et al., 1996) suggests that specific BMPs/OPs may also be used for repair and regeneration of dentine (Nakashima, 1990; Nakashima, 1994; Rutherford et al., 1993) and periodontal tissues in postnatal life (Bowers et al., 1991; Ripamonti and Reddi, 1994). Whilst BMPs/OPs are strongly expressed during tooth morphogenesis, their in vivo role in periodontal regeneration is not clear. In previous studies in a non-human primate model, highly purified BMP fractions derived from bone matrix induced cementum, periodontal ligament and alveolar bone regeneration (Ripamonti et al., 1994). Moreover, the magnitude and quality of new 121
122
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connective-tissue attachment formation indicated that members of the BMP/OP family may also initiate cementogenesis and regulate the assembly of a functionally orientated periodontal-ligament system (Ripamonti et al., 1994). The presence of multiple forms of BMPs/OPs may be biologically significant, locally regulating the regeneration of other tissues including the periodontal ligament and cementum. Whether the magnitude of new attachment formation stimulated by hOP-1 in vivo is sufficient to constitute a potential therapeutic effect has not been previously investigated. Preclinical studies on non-human primates are critical to test the efficacy of recombinant molecules under evaluation for regenerative procedures. The experiments described here were designed to test the efficacy of a single application of two doses of hOP-1 in conjunction with a collagenous carrier for periodontal regeneration in furcation defects of the baboon (Papio ur sinus ).
In three adult male baboons, selected and prepared as described by Ripamonti et al. (1994), mucoperiosteal flaps were raised and 12 class II furcation defects were surgically prepared in the first and second mandibular molars. Exposed roots were curetted to remove periodontal ligament fibres and cementum, and notched with small burs at the level of the residual bony housing (Ripamonti et al., 1994). The depth of each furcation defect extended for at least 10-12 mm buccolingually as measured from the buccal entrance of the exposed furcations. Recombinant hOP-1 was prepared as described by Sampath et al. (1992). hOP-1 in 50% acetonitrile, 0.1% trifluoroacetic acid was combined with bovine insoluble collagenous matrix as carrier, prepared after dissociative extraction of demineralized bone matrix (Sampath and Reddi, 1981), and lyophilized. Two doses of hOP-1 were used: 100 and 500#g per g of carrier matrix. Approximately 200mg of carrier matrix was used per furcation defect. Portions of bovine collagenous matrix, inactive after extraction of osteogenic proteins, were used as control without the addition of hOP- 1. The flaps were closely readapted with resorbable sutures and a plaque control regimen was instituted weekly for the duration of the experiment. Perfusion and preparation of tissue for histology on day 60 after surgery were as described by Ripamonti et al. (1994). In brief,
specimen blocks, each including the first and second molars with surrounding bone and soft tissues, were embedded, undemineralized, in methlymethacrylate, and trimmed along the buccal aspect until the notches on the roots could be detected. Serial sections, including dentine and associated periodontal tissues, were then cut at 7 # m in the mesiodistal plane throughout the entire buccolingual extension of each furcation defect using tungsten-carbide knives mounted on a Reichert-Jung Policut-S microtome. Every 14th section, approx. 100 p m apart, was stained using the free-floating method with the Goldner's trichrome stain for undemineralized bone. Four step-serial sections, representing the buccal (one section), internal (two sections) and central (one section) regions of the buccal half of the defects were selected for histometry. The buccal half of the defects was determined by cutting the specimens until the mesiodistal fossa was reached. At this level, serial sections were representative of the central region of the defects. Sections representing the buccal, internal and central regions were approx. 600/~m apart from each other. The two sections representing the internal region were 200 # m apart. Sections from the different specimens were identified by levels of serial sectioning and matched by the size of the root canals and the pulp chambers (Ripamonti et al., 1994). Histometric measurements were made with a calibrated micrometer mounted in a Reichert Univar microscope at x40 (Ripamonti et al., 1994) (Table 1). Representative photomicrographs of hOP-Itreated specimens are shown in Figs 1-3. Results of histometric analysis are presented in Table 1. Both histological and histometric analyses point to the striking induction of cementogenesis by both doses of hOP-1 with the collagenous carrier. Deposition of organized and highly cellular mineralized tissue (interpreted as cementum) had occurred on the exposed roots, often extending to the fornix of the treated furcations (Figs 1-3). Control specimens showed limited regeneration (Table 1), scattered remnants of the collagenous carrier, and apical migration of the junctional epithelium. The preparation and analysis of undemineralized sections cut at 7 # m allowed the differentiation of mineralized versus as yet not mineralized cementum, or cementoid (Fig. 2) (Ripamonti et al., 1994). Within the central regions of the exposed furcations, substantial amounts of remaining collage-
Table 1. Histometric analysis of periodontal regeneration in 12 furcation defects surgically prepared in 3 adult baboons, blCBM: bovine insoluble collagenous matrix carrier; hOP-l: recombinant human osteogenic protein (100 and 500 #g per g of collagenous carrier); N--F: extent of furcation exposure--from the apical border of the notch (N) in the distal and mesial root to the fornix (F) of the furcation; N--NC: new cementum formation (NC)-from N to the coronal termination of the newly formed cementum along the distal and mesial roots blCBM solo (n = 2)
100 #g hOP-1 (n = 4)
500pg hOP-1 (n = 6)
Distal
N--F N--NC
9.4 ___0.1 2.6 __+0.2
NS p < 0.01
9.3 ___0.2 6.2 ___0.5
NS NS
8.8 + 0.2 6.2 + 0.3
9.3 + 0.1 2.8 __+0.I
NS p < 0.01
9.3 ___0.1 6.1 ___0.8
NS NS
8.8 __.0.3 6.7 _.+0.3
Mesial
N--F N--NC
Values (in mm) are means + SE. Levels of significance were determined using the Statistical Analysis System (1989) General Linear Models procedure with multiple interactions.
Cementogenesis by osteogenic protein-1
2 Fig. 1. Photomicrographs of furcation defects treated with 100/~g of hOP-1 per g of cartier. (A) Low-power view showing cementogenesis from the notches (white arrows) in the root surfaces, and extending to the fornix of the exposed furcation (arrow). There is separation between the residual alveolar bony housing and the tissue that had formed within the furcation, x 10. (B) Higher magnification of previous section (arrow area in A). Formation of Sharpey's fibres inserting into cementoid and mineralized cementum (in blue) in proximity to the fornix of the furcation, x 200. (C) High-power view of an adjacent section to (A) (open arrows area in A) but without artefactual separation of the dentine--cementum tissue complex: regenerated cellular and mineralized cementum with inserted connective tissue fibres, x 200. Fig. 2. Induction of cementogenesis by hOP-1. (A) Deposition of highly cellular and as yet to be mineralized cementoid along a denuded root surface in the coronal area of a furcation defect treated with 500 ~g of hOP-1 per g of carrier. Arrow indicates mineralized cemental matrix in more apical region of the defect, x 100. (B) Detail of newly deposited highly cellular cementoid. Whilst non-mineralized cementoid faced a highly vascular but non-orientated connective tissue space, regions displaying mineralized cementum showed the insertion of functionally orientated Sharpey's fibres penetrating the cementoid seam and the subjacent mineralized cemental layer. × 200. (C and D) Details of newly formed cementum. Note mineralized cementum (open arrows in C) and deposition of highly cellular cementoid in more coronal direction, close to the fornix of the furcation. Closed arrow points to loci of nascent mineralization within the non-mineralized phase of the newly deposited cemental matrix (detailed in D). x 100 and x 200, respectively.
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Fig. 3. Caption opposite.
3
Cementogenesis by osteogenic protein-I nous carrier were enclosed by a mineralized matrix that extended and blended into the newly deposited cementum along the root surfaces (Figs 1A, 3B and 3C). This newly formed mineralized tissue in direct apposition to the collagenous carrier showed limited vascular invasion, and an almost complete absence of capillaries in instances of substantial matrix deposition and mineralization in close proximity to the cementum deposited on the exposed dentine (Figs 3B and C). Importantly, there was separation and not fusion of the mineralized tissue under discussion with the remaining alveolar bone in the apical region of the furcations (Figs 1A and 3A). Indeed, the two components were separated by a vascular fibrous tissue resembling a periodontal ligament space (Fig. 3D). Whilst there are no structural markers that unequivocally differentiate cementum from bone, the histological analysis suggests that the mineralized tissue that occupies the furcation area, in direct apposition to the remnants of the collagenous carrier, is predominantly cementum rather than alveolar bone. Notable distinct features of cementum are avascularity and lack of significant remodelling (MacNeil and Somerman, 1993). Both features may help to explain the persistence of implanted collagenous matrix. It is noteworthy that implantation of 100 and 500#g of hOP-1 in conjunction with an identical preparation of bovine carrier resulted in rapid bone formation and concomitant incorporation and dissolution of the carrier by day 30 in calvarial defects of adult baboons (U. Ripamonti, B. van den Heever, M. Tucker, T. K. Sampath, D. Rueger and A. H. Reddi, unpublished observations). In the present study, the separation between tissues (alveolar bone and the cementum-like material in the furcation) was maintained by a fibrovascular tissue that shared numerous features of a periodontal ligament space (Fig. 3D). Conceptually, the formation of an additional periodontal ligament is because the biochemical and positional information (including characteristics of both substrata) are, on one side, those of alveolar bone, and on the other, those of cementum. A pertinent analogy to this observation is the fibrovascular space that constantly delineates and characterizes cemental growths such as cementomas. In such instances, fusion between the cementoma and the surrounding alveolar bone never occurs, and the presence of the fibrous space is typically seen on radiographs as a radiolucent ring around the lesion (Lucas, 1984).
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Within the limits of the histological and histometric analyses, the present experiment in non-human primates demonstrates substantial induction of cementogenesis by a single application of hOP-1 in conjunction with a collagenous matrix on surgically denuded root surfaces. The regeneration of periodontal ligament and the insertion of functionally orientated Sharpey's fibres into cementum were also observed. As hOP-I in conjunction with the bovine collagenous matrix induces bone formation in bone defects of the cranial and appendicular skeleton (for review see Ripamonti and Reddi, 1995), it is likely that the expression of specific cell phenotypes by hOP-I is regulated, in part, by extracellular matrix substrata. Thus, in a periodontal defect, the presence of exposed dentine may preferentially modulate the expression of the cementogenic phenotype on readily available cell populations (precementoblasts and their progenitors) from the ligament space. The critical regulatory role of extracellular matrix substrata in the phenotypic expression of a variety of cells has been reported previously (Reddi, 1992; Reddi, 1994). The expression of different phenotypes and the generation of cementum or alveolar bone by BMPs/OPs may depend on whether a common lineage of progenitor cells, residing in the periodontal ligament (Melcher et al., 1986; Melcher et al., 1987), attach to exposed dentinal substrata or stay in the alveolar bony side of the periodontal ligament space. The distinct patterns of BMP/OP expression, not limited to skeletal elements, point to multiple and specialized roles in development and thus regeneration. Moreover, limited promiscuity amongst receptors in binding BMP-4 and OP-I also suggests that BMPs may have different functions in vivo (ten Dijke et al., 1994). An important question is whether the presence of multiple forms of BMP/OP have a therapeutic significance, hOP-l, BMP-2 and BMP-4 are equally capable, after a single application, of induction of bone as well as reparative dentinogenesis in postnatal animal models (Wang et al., 1990; Hammonds et al., 1991; Sampath et al., 1992; Rutherford et al., 1993; Nakashima, 1994), raising important questions on the biological significance of this apparent redundancy (Reddi, 1992; Reddi, 1994). Our findings show that doses of 100 and 500 pg hOP-1 per g of carrier induce cementogenesis on surgically denuded root surfaces, indicating a specific function during repair and regeneration of periodontal tissues. The finding of cementum deposition with inserted
Fig. 3. Photomicrographs of furcation defects treated with 500/~m of hOP-1. (A) Low-power view showing extensive cementogenesis on the mesial root (arrows) up to the fornix of the furcation. Note cementation of the residual collagenous carrier within the fureation by a mineralized matrix in direct apposition to the distal root and to the furcation fornix. As in Fig. 1A, separation and not fusion between the residual bony housing and the mineralized matrix--collagenous carrier tissue complex within the furcation. × 10. (B) Detail of previous section showing limited vascular invasion within the mineralized matrix in close apposition to dentine, and the residual collagenous carrier, x 40. (C) Adjacent section, approx. 600/am apart from (B), showing areas of focal fusion between cementum formed along exposed dentine and the mineralized tissue enveloping the residual carrier, x 40. (D) Detail of the fibrovascular space generated between the residual alveolar bony housing and the mineralized matrix within the furcation: prominent capillary network, and assembly of a periodontal ligament-like structure with Sharpey's fibres inserting into the cementum-like material (black arrows). White arrows point to osteoid seams (in orange-red) and populated by contiguous osteoblasts, lining mineralized bone (in blue), x 100.
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Sharpey's fibres in the absence of a concomitant growth o f alveolar bone suggests that alveolar bone regeneration is not a requirement for cementogenesis to occur. A l t h o u g h the expression patterns of B M P / O P transcripts during root morphogenesis are not yet available, it is likely that the developmental stages of cementum and periodontal ligament formation are also regulated by sequential expression of some of the B M P / O P genes. Very recently, it was shown that rhBMP-2 induced substantial alveolar bone regeneration in a canine model. Cementogenesis, however, was limited when compared to the extent of bone regeneration (Sigurdsson et al., 1995). It is noteworthy that highly purified osteogenic preparations (containing several B M P / O P members) induced cementum, periodontal ligament and alveolar bone regeneration (Ripamonti et al., 1994). Future research must focus on optimal doses and molecular combinations, developing a structure-activity profile amongst members of the B M P / O P family. Acknowledgements--This work is supported by the South
African Medical Research Council, the University of the Witwatersrand, Johannesburg, and in part by a grant from Creative BioMolecules, Hopkinton, MA. We thank Ms Barbara van den Heever for the superb histological preparation of undemineralized sections. REFERENCES
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