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Augmentation of the floor of the maxillary sinus with recombinant human bone morphogenetic protein-7: a pilot radiological and histological study in humans
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British Journal of Oral and Maxillofacial Surgery 51 (2013) 247–252
Augmentation of the floor of the maxillary sinus with recombinant human bone morphogenetic protein-7: a pilot radiological and histological study in humans Giuseppe Corinaldesi a , Luigi Piersanti b,∗ , Adriano Piattelli c , Giovanna Iezzi c , Francesco Pieri a , Claudio Marchetti b a b c
University of Bologna, Department of Oral and Dental Sciences, Via San Vitale 59, 40125 Bologna, Italy University of Bologna, Department of Oral and Maxillofacial Surgery, Via Massarenti 9, 40138 Bologna, Italy Dental School University of Chieti-Pescara, Oral Medicine and Pathology, Viale Pindaro 42, 65127 Pescara, Italy
Accepted 10 June 2012 Available online 4 July 2012
Abstract The aim of this study was to evaluate the quantity and quality of bony regeneration after we had used recombinant human bone morphogenetic protein-7 (rhBMP-7 to augment the floor of the maxillary sinus. Nine consecutive patients with bilateral posterior maxillary atrophy who required augmentation of the sinus for interposition of implants were treated simultaneously with rhBMP-7 (Osigraft) with deproteinised bone substitute (0.5 g on the test side) and with deproteinised bone alone (2.0 g on the control side). Computed tomographic images preoperatively, immediately postoperatively, and at 4 months postoperatively showed a mean (SD) postoperative gain of 10.8 (3.0) mm on the test side and of 10.2 (1.8) mm on the control side. Histological and histomorphometric analyses of biopsy specimens showed that there was significantly more new bone on the control side (19.9 (6.8)%) than on the test side (6.6 (4.8)%). In this pilot controlled trial of the use of rhBMP-7, histological analyses showed that it resulted in the formation of less bone than treatment with inorganic bovine hydroxyapatite. © 2012 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Keywords: Human study; Bone regeneration; Sinus lift; Bone morphogenetic protein; BMP-7; rhOP-1; Anorganic bovine bone
Introduction Loss of teeth in the posterior maxilla results in rapid horizontal and vertical resorption of alveolar bone because of the lack of intraosseous stimulation by periodontal ligaments. The absence of maxillary molars leads to increased activity of osteoclasts in Schneider’s membrane, which causes pneumatisation of the sinuses within a few months because of the resorption of bone.1
∗
Corresponding author. Tel.: +39 335 6125333. E-mail addresses: [email protected] (G. Corinaldesi), [email protected] (L. Piersanti), [email protected] (A. Piattelli), [email protected] (G. Iezzi), [email protected] (F. Pieri), [email protected] (C. Marchetti).
Various bone-grafting materials have been evaluated for the augmentation of sinuses,2,3 and alloplasts and xenografts have shown promising results.2 Extensive animal2–6 and human7–9 clinical trials have used bovine bone mineral as a grafting material. All these materials are osteoconductive. In contrast, bone morphogenetic proteins (BMPs) induce mesenchymal stem cells to differentiate into osteoblasts and produce new bone tissue.10 Several recombinant forms of BMP, notably rhBMP2 and rhBMP-7, have been shown to induce bone formation in vivo11,12 and have been tested in clinical trials.13 The use of rhBMP-7, also known as osteogenic protein1 (rhOP-1; INN eptotermin ␣), with type 1 collagen as a carrier (Osigraft; Stryker Biotech, West Lebanon, NH, USA) has been approved in Europe for the treatment of tibial non-union under particular clinical conditions (initial
0266-4356/$ – see front matter © 2012 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bjoms.2012.06.004
248
G. Corinaldesi et al. / British Journal of Oral and Maxillofacial Surgery 51 (2013) 247–252
authorisation 17/05/01, European Union Registration No. EU/1/01/179/001). This product is the first human recombinant BMP approved for clinical use in Europe. Animal studies have shown that rhOP-1 stimulates bony regeneration and reduces healing time when used alone or in combination with osteoconductive biomaterials in sinus augmentation.14 Given the osteoinductive capacity of rhOP-1, the objective of this pilot prospective human control study was to evaluate bone formation in atrophied maxillas that required sinus lifting radiologically, histologically, and histomorphometrically. We compared rhOP-1 with deproteinised bovine bone, with deproteinised bovine bone alone.
Patients and methods Between October 2007 and December 2008, three men and six women (mean age 50 years, range 39–61) who required bilateral augmentation of the maxillary sinus floor to allow rehabilitation with osseointegrated implants were recruited to participate in this study that was approved by the Ethics Committee of S. Orsola-Malpighi Hospital, Bologna, Italy (No. 56/2007/U/Sper 15.05.2007). Participants had no clinical or radiological signs of active or chronic sinus conditions, and no clinical history of cardiac, haemorrhagic, haematological, or kidney disease. The following criteria were used to exclude patients from the study: residual maxillary atrophy of more than 5 mm (the distance between the sinus floor and the alveolar ridge); tobacco misuse (smoking more than 15 cigarettes/day); known hypersensitivity to the active component or to collagen; poor oral hygiene (plaque index for dentate patients 25% or less); pregnancy; current infection at the reconstruction site or ongoing systemic infection; uncontrolled diabetes mellitus or other serious metabolic disease; HIV or hepatitis B or C infection; cancer; history of chemotherapy, radiotherapy, or immunosuppression during the past 5 years; diagnosed autoimmune disorders, current or past use of drugs that may affect bone metabolism or tissue regeneration and repair (such as bisphosphonates); and current therapeutic regimen of steroids or non-steroidal anti-inflammatory drugs. Computed tomographic (CT) images were used to evaluate the vertical height of the sinus obtained postoperatively and variation in this value at the time of insertion of the implant. The quality of the bone was evaluated histologically and histomorphometrically 4 months postoperatively. In particular, the percentages of new bone tissue, soft tissue, and residual Bio-Oss particles were measured with respect to the total area of the sample examined. Clinical evaluation was used to monitor treatment-related adverse events. All operations were done by the same surgeon (GC) under general anaesthesia with nasal endotracheal intubation. Standard surgical access to the lateral maxillary wall was achieved through a mucosal crestal incision and release of vestibular incisions anteriorly and posteriorly. A bony
window was designed on the lateral wall of the maxillary sinus using piezosurgery (Mectron S.p.A., Carasco, Italy). After the sinus membrane had been dissected and raised, block randomisation was used to designate a test side and a control side for each patient. On the test side, the maxillary sinus region was filled after the membrane had been raised with one vial of Osigraft (eptotermin ␣ 3.5 mg in collagen 1 g) that had been reconstituted with saline solution 2.5 ml and sodium chloride 9 mg/ml for injection. The dosage of eptotermin ␣ 3.5 mg was chosen because in previous case reports 2.5 mg had been used with inconsistent bony regeneration in the sinus. The lateral wall was filled with inorganic bovine hydroxyapatite 0.5 g (Bio-Oss; Geistlich Biomaterials, Wolhousen, Switzerland) to maintain the graft in the site and completely fill the sinus. The control site was grafted with inorganic bovine hydroxyapatite 2 g (Bio-Oss; Geistlich Biomaterials) reconstituted with saline solution 2.5 ml. Both sinus grafts were covered with resorbable collagen membrane (Bio-Guide; Geistlich Biomaterials), and primary closure of the soft tissue was by non-absorbable sutures. Ceftriaxone 1 g (Rocefin; Roche S.p.A., Milan, Italy) was given twice daily for a week as prophylaxis against infection. CT evaluations were made preoperatively, immediately postoperatively, and 4 months later to facilitate the planned position of the implant. Surgical augmentation was evaluated by comparing preoperative (T0) and immediately postoperative (T1) CT scans. This calculation was based on height, measured from the lowest point of the alveolar ridge to the maxillary sinus floor, using a line parallel to the hypothetical position of the implant as a reference. The extent of resorption was evaluated at the time of insertion of the implant. This was expressed as a percentage: (T1 − T2) × 100: (T1 − T0), where (T1 − T2) indicated the amount of resorbed graft as a percentage of the total size of the initial graft (T1 − T0) (Table 1). Implants were positioned 4 months postoperatively, and at this time a 3-mm trephine-bur biopsy specimen was taken from the buccal side of the bone. Specimens were stored immediately in 10% buffered formalin and processed to obtain thinly ground sections (Precise 1 automated system; Assing, Rome, Italy). Each specimen was dehydrated in an ascending series of alcohol rinses and embedded in a glycol methacrylate resin (Technovit 7200 VLC; Kulzer, Wehrheim, Germany). After polymerisation, the specimen was sectioned longitudinally along the major axis with a high-precision diamond disc at about 150 m and ground down to about 30 m. Three slides were prepared and stained with basic fuchsin and toluidine blue. Histomorphometric examination was made with a light microscope (Laborlux S; Leitz, Wetzlar, Germany) connected to a high-resolution video camera (KY-F55B 3-CCD; JVC Italy S.p.A., Segrate, Italy) and interfaced with a monitor and personal computer (Pentium III 1200 MMX; Intel, Santa Clara, CA, USA). This optical system was associated with a digitising pad (Matrix Vision GmbH, Oppenweiler, Germany) and a histometry software package
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Table 1 Radiological evaluation with computed tomography of the sinus. Test side (Osigraft, rh-OP-1), control side (Bio-Oss).
rh-OP-1 1 2 3 4 5 6 7 8 9
Preoperatively
Postoperatively
At 4 months
Resorption at 4 months
Percentage resorption
3.6 4.7 4.4 1.9 2.8 4.3 3.9 3.3 3.6
14.6 10.6 13.6 12.7 15.1 15.2 13.2 12.9 13.2
12.3 8.9 12.7 11.9 14.6 14.7 12.1 11.8 12.7
−2.3 −1.7 −0.9 −0.8 −0.5 −0.5 −1.1 −1.1 −0.5
−15.8 −16.0 −6.6 −6.3 −3.3 −3.3 −8.3 −8.5 −3.8 −8.0 (4.9)
Mean (SD) Bio-Oss 1 2 3 4 5 6 7 8 9
2.6 4.6 1.2 2.3 3.2 4.1 3.2 2.9 4.1
15.4 10.6 12.9 16.4 18.9 15.8 14.1 14.6 13.3
Mean (SD)
with image-capturing capabilities (Image-Pro Plus 4.5; Media Cybernetics Inc., Immagini & Computer Snc., Milan, Italy). Histomorphometric measurements were made to calculate the percentages (the area fractions) of newly formed bone, residual graft materials, and soft-tissue components (connective tissue, or bone marrow, or both). We used the Statistical Package for the Social Sciences (version 11, SPSS Inc., Chicago, IL) to assess the significance of any differences between the test and control groups in the height of alveolar bone below the sinuses and the radiological and histomorphometric results. Group means (SD) were calculated for each variable measured. We used a splitmouth design to assess the significance of the differences between the experimental groups using Student’s t test for paired samples. Probabilities of less than 0.05 were accepted as significant.
Results Clinical and radiological results CT scans showed that the mean (SD) basal bone height was 3.1 (1.0) mm (range 1.9–4.7) on the right side and 2.8 (1.1) mm (range 1.5–4.5) on the left. The height of residual alveolar bone below the sinuses between the test and control side did not differ significantly (p = 0.3). Radiological examination showed that the mean gain in height after the sinus lift was 12.2 (4.2) mm (range = 18.3–5.4) on the test side and 11.9 (2.8) mm (range = 15.7–5.6) on the control side. The mean resorption
12.6 9.3 11.9 15.7 16.6 15.1 13 13.7 12.4
−2.8 −1.3 −1 −0.7 −2.3 −0.7 −1.1 −0.9 −0.9
−18.2 −12.3 −7.8 −4.3 −12.2 −4.4 −7.8 −6.2 −6.8 −8.9 (4.5)
volume 4 months postoperatively was 1.4 (1.2) mm (range 4.3–0.4) on the test side and 1.7 (1.0) mm (range 3.8–0.7) on the control side (Table 2). There were no major complications. Four patients developed small tears of Schneider’s membrane (test side: case 2; and control side: cases 1, 3, and 4) and were treated with resorbable membrane (Bio-Guide; Geistlich Biomaterials). There was no evidence of adverse local or systemic effects at 10 days, 3 weeks, 6 weeks, and 3 months postoperatively. Histological and histomorphometric observations All histological sections in the test group showed formation of new bone only at the most coronal parts of the biopsy specimen, which corresponded to the buccal portion of the augmented sinus. The new bone was primarily lamellar, and was in close contact with the remaining particles of hydroxyapatite. The apical portions of the biopsy specimens that contained only rhOP-1 differed; all these portions were filled with remnants of collagen carrier embedded in loose connective tissue, with no appreciable formation of bone in or around them (Figs. 1 and 2). In contrast, the biopsy specimens of the control sites showed newly formed lamellar bone surrounding particles of hydroxyapatite along their entire lengths. Similar particles were in contact with biological fluids and spaces in marrow in only a few areas. There were no inflammatorycell infiltrates or foreign-body reactions, no gaps at the bone–hydroxyapatite interface, and the bone was always in close contact with the particles. No particles of hydroxyapatite had resorbed (Figs. 3 and 4).
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Table 2 Mean (SD) histomorphometric data 4 months after sinus augmentation with Osigraft, rh-OP1, combined with bovine bone hydroxyapatite (HA) in the buccal portion of the augmented sinuses, or hydroxyapatite alone. Variable
rhOP-1-HA (test)
HA (control)
p value
Percentage of newly formed bone Percentage of remaining HA particles Percentage of soft tissue components
6.55 (4.75) 27.66 (4.74) 65.77 (6.9)
19.88 (6.79) 43 (4.89) 37.11 (5.03)
0.0003 0.0004 <0.0001
Fig. 1. Test site: a limited amount of newly formed bone was present in only the most lateral portion of the specimen in close proximity to the bovine hydroxyapatite particles (H). There were no signs of bony formation in the remaining internal portion where the collagen particles were completely covered by connective tissue (acid fuchsin and toluidine blue, original magnification 12×). Fig. 4. Control site: high-powered image of a single bovine hydroxyapatite particle (H) in close and tight contact with newly formed bone. There were no epithelial cells, connective tissue, or inflammatory cell infiltrates present at the bone/biomaterial interface (acid fuchsin and toluidine blue, original magnification 200×).
Table 2 shows the histomorphometric results for the two groups. Control sites (19.9 (6.8%)) had significantly more newly formed bone than did test sites (6.6 (4.8)%, p = 0.0003).
Discussion
Fig. 2. Test site: high-powered image of residual collagen particles (*) completely encapsulated in dense connective tissue with no inflammatory infiltrate (acid fuchsin and toluidine blue, original magnification 100×).
Fig. 3. Control site: bovine hydroxyapatite particles (H) surrounded and joined by newly formed trabecular bone (red) along the entire length of the specimen. There were no osteoclasts or macrophages present (acid fuchsin and toluidine blue, original magnification 20×).
The purpose of this in vivo pilot study was to evaluate the healing response and bony formation in the maxillary sinus stimulated by rhOP-1 in a collagen carrier with the use of rh-BMP-7 both radiographically and histologically. Reconstituted rhOP-1 allowed adequate dimensions of sinus lift, and the membrane had not collapsed after 4 months. The two sides did not differ significantly in the mean quantity of graft resorption, so rhOP-1 maintained membrane-lifting to a degree similar to that of other grafting materials.15 Although no patient developed an adverse reaction between the time of operation and placement of the implant, histological and histomorphometric analyses showed unfavourable results on the test side. No specimen showed histological signs of an inflammatory reaction, but the high percentage of soft tissue may have restricted osteoconduction. Significantly more soft tissue was present in sinuses augmented with BMP-7 than in sinuses grafted with anorganic bovine bone. Histological analyses showed that the collagen particles were completely covered by, and encapsulated within, dense connective tissue with no inflammatory
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infiltrate. Histological examination showed no initiation of bony formation at any test site, indicating that a longer healing time before the biopsy specimen was taken would not have changed the results. This contrasts with the use of rhOP-1 in cases such as the treatment of non-union of long bones, in which there was a significant progression in the success rate at follow-up intervals of 4–5, 7–8, and 9 months or more.16 Animal studies using rhOP-1 have reported significantly more newly formed bone at test sites than at control sites, with the exception of a single non-human primate model in which such a difference was not found. Two of these studies showed that only higher doses were able to increase the amount of newly formed bone significantly. Dose-dependent effects have been reported with the use of rhOP-1, with the following (highest) doses producing the best results: rhOP-1 1000 g in an inorganic bovine hydroxyapatite block to prefabricate vascularised bone grafts in miniature pigs17 ; rhOP-10.5 mg in inorganic bovine hydroxyapatite to treat calvarial defects in baboons18 ; rhOP-1 2.5 mg for augmentation of the maxillary sinus in chimpanzees19 ; and rhOP-1 2.5 mg in collagen matrix for augmentation of the sinus in non-human primates, which demonstrably increased bone mineral density.20 Human studies of raising and augmenting of the maxillary sinus have produced inconsistent results of formation of bone with the use of rhOP-1 2.5 mg in a collagen carrier. In these case reports the histological analysis of bony formation in 4 patients showed new bone in only one sinus at 6 months postoperatively.21,22 Because the dose is important for the successful use of BMP-2, and dose-dependent results have been published,23,24 we used rhOp-1 3.5 mg (1400 g) in an absorbable collagen carrier in the present study. As described previously for the use of BMP-2, the dose and the selection of carrier also presented challenges in the use of BMP-7. Although human clinical studies have evaluated the use of BMP-2 with a self-containing absorbable collagen sponge carrier in the treatment of bony defects, collagen carriers have been studied more extensively. Animal studies have documented inadequate regeneration of bone with the use of collagen alone as a substitute bone graft.25 Collagen may not have sufficiently effective scaffolding capabilities to support the rhBMP-7-induced formation of bone in sinus defects. Absorbable collagen may not provide the three-dimensional structure needed to support the formation of new bone, and non-resorbable materials with greater stability may be more effective. The observations of this pilot study should be interpreted with caution because of the limited size of the histological sample and the prevalence of female patients, with possible diminished regenerative capabilities as a result of the effects of their hormones. In conclusion, the use of rhOP-1 for augmentation of the sinuses gave poor results, with less new bone formed than with inorganic bovine hydroxyapatite. The correct dose and method of use of rhOP-1 should be assessed before it is used further in the augmentation of human sinuses.
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Conflict of interest statement The authors disclose any financial and personal relationship with other people or organisations that could inappropriately influence their work. The vials of Osigraft (eptotermin 3.5 mg in collagen 1 g) used for this study were kindly donated by Stryker Biotech, West Lebanon, NH, USA.
Ethical approval This work was approved by the Ethical Committee of S. Orsola-Malpighi Hospital, Bologna, Italy (No. 56/2007/U/ Sper 15.05.2007) and all the subjects gave informed consent to the work.
References 1. Galindo-Moreno P, Avila G, Fernández-Barbero JE, Aguilar M, Sánchez-Fernández E, Cutando A, et al. Evaluation of sinus floor elevation using a composite bone graft mixture. Clinical Oral Implants Research 2007;18:376–82. 2. Jensen SS, Aaboe M, Pinholt EM, Hjørting-Hansen E, Melsen F, Ruyter IE. Tissue reaction and material characteristics of four bone substitutes. The International Journal of Oral & Maxillofacial Implants 1996;11:55–66. 3. Kirsch A, Ackermann KL, Hürzeler MB, Hutmacher D. Sinus grafting with porous hydroxyapatite. In: Jensen OT, editor. The sinus bone graft. Chicago: Quintessence; 1999. p. 79–94. 4. Pinholt EM, Ruyter IE, Haanaes HR, Bang G. Chemical, physical, and histologic studies on four commercial apatites used for alveolar ridge augmentation. Journal of Oral and Maxillofacial Surgery 1992;50:859–67. 5. Cohen RE, Mullarky RH, Noble B, Comeau RL, Neiders ME. Phenotypic characterization of mononuclear cells following anorganic bovine bone implantation in rats. Journal of Periodontology 1994;65: 1008–15. 6. Berglundh T, Lindhe J. Healing around implants placed in bone defects treated with Bio Oss. An experimental study in the dog. Clinical Oral Implants Research 1997;8:117–24. 7. Callan DP, Rohrer MD. Use of bovine-derived hydroxyapatite in the treatment of edentulous ridge defects: a human clinical and histologic case report. Journal of Periodontology 1993;64:575–82. 8. Camelo M, Nevins ML, Schenk RK, Simion M, Rasperini G, Lynch SE, et al. Clinical, radiographic and histologic evaluation of human periodontal defects treated with Bio-Oss and Bio-Gide. The International Journal of Periodontics & Restorative Dentistry 1998;18:321–31. 9. Artzi Z, Nemcovsky CE, Dayan D. Bovine-HA spongiosa blocks and immediate implant placement in sinus augmentation procedures. Histopathological and histomorphometric observations on different histological stainings in 10 consecutive patients. Clinical Oral Implants Research 2002;13:420–7. 10. Urist MR. Bone: formation by autoinduction. Science 1965;150:893–9. 11. Heckman JD, Boyan BD, Aufdemorte TB, Abbott JT. The use of bone morphogenetic protein in the treatment of non-union in a canine model. The Journal of Bone and Joint Surgery 1991;73A:750–64. 12. Gerhart TN, Kirker-Head CA, Kriz MJ, Holtrop ME, Hennig GE, Hipp J, et al. Healing segmental femoral defects in sheep using recombinant human bone morphogenetic protein. Clinical Orthopaedics and Related Research 1993;293:317–26.
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13. Valentin-Opran A, Wozney J, Csimma C, Lilly L, Riedel GE. Clinical evaluation of recombinant human bone morphogenetic protein-2. Clinical Orthopaedics and Related Research 2002;395:110–20. 14. Jung RE, Thoma DS, Hammerle CH. Assessment of the potential of growth factors for localized alveolar ridge augmentation: a systematic review. Journal of Clinical Periodontology 2008;35:255–81. 15. Chiapasco M, Zaniboni M, Boisco M. Augmentation procedures for the rehabilitation of deficient edentulous ridges with oral implants. Clinical Oral Implants Research 2006;17(Suppl. 2):136–59. 16. Ronga M, Baldo F, Zappalà G, Cherubino P, BMP-7 Italian Observational Study (BIOS) Group. Recombinant human bone morphogenetic protein-7 for treatment of long bone non-union: an observational, retrospective, non-randomized study of 105 patients. Injury 2006;37(Suppl. 3):S51–6 [Erratum: Injury 2007;38:1224]. 17. Terheyden H, Menzel C, Wang H, Springer IN, Rueger DR, Acil Y. Prefabrication of vascularized bone grafts using recombinant human osteogenic protein-1—Part 3: Dosage of rhOP-1, the use of external and internal scaffolds. International Journal of Oral and Maxillofacial Surgery 2004;33:164–72. 18. Ripamonti U, Crooks J, Rueger DC. Induction of bone formation by recombinant human osteogenic protein-1 and sintered porous hydroxyapatite in adult primates. Plastic and Reconstructive Surgery 2001;107:977–88. 19. McAllister BS, Margolin MD, Cogan AG, Taylor M, Wollins J. Residual lateral wall defects following sinus grafting with recombinant human osteogenic protein-1 or Bio-Oss in the chimpanzee. The International Journal of Periodontics & Restorative Dentistry 1998;18: 227–39.
20. Margolin MD, Cogan AG, Taylor M, Buck D, McAllister TN, Toth C, et al. Maxillary sinus augmentation in the non-human primate: a comparative radiographic and histologic study between recombinant human osteogenic protein-1, and natural bone mineral. Journal of Periodontology 1998;69:911–9. 21. Groeneveld EH, van den Bergh JP, Holzmann P, ten Bruggenkate CM, Tuinzing DB, BurgerF E.H. Histomorphometrical analysis of bone formed in human maxillary sinus floor elevations grafted with OP-1 device, demineralised bone matrix, or autogenous bone. Comparison with non-grafted sites in a series of case reports. Clinical Oral Implants Research 1999;10:499–509. 22. Van den Bergh JP, ten Bruggenkate CM, Groeneveld HH, Burger EH, Tuinzing DB. Recombinant human bone morphogenetic protein7 in maxillary sinus floor elevation surgery in 3 patients compared to autogenous bone grafts. A clinical pilot study. Journal of Clinical Periodontology 2000;27:627–36. 23. Boyne PJ, Lilly LC, Marx RE, Moy PK, Nevins M, Spagnoli DB, et al. De novo bone induction by recombinant human bone morphogenetic protein-2 (rhBMP-2) in maxillary sinus floor augmentation. Journal of Oral and Maxillofacial Surgery 2005;63:1693–707. 24. Fiorellini JP, Howell TH, Cochran D, Malmquist J, Lilly LC, Spagnoli D, et al. Randomized study evaluating recombinant human bone morphogenetic protein-2 for extraction socket augmentation. Journal of Periodontology 2005;76:605–13. 25. Barboza EP, Duarte ME, Geolás L, Sorensen RG, Riedel GE, Wikesjö UM. Ridge augmentation following implantation of recombinant human bone morphogenetic protein-2 in the dog. Journal of Periodontology 2000;71:488–96.
British Journal of Oral and Maxillofacial Surgery 51 (2013) 247–252
Augmentation of the floor of the maxillary sinus with recombinant human bone morphogenetic protein-7: a pilot radiological and histological study in humans Giuseppe Corinaldesi a , Luigi Piersanti b,∗ , Adriano Piattelli c , Giovanna Iezzi c , Francesco Pieri a , Claudio Marchetti b a b c
University of Bologna, Department of Oral and Dental Sciences, Via San Vitale 59, 40125 Bologna, Italy University of Bologna, Department of Oral and Maxillofacial Surgery, Via Massarenti 9, 40138 Bologna, Italy Dental School University of Chieti-Pescara, Oral Medicine and Pathology, Viale Pindaro 42, 65127 Pescara, Italy
Accepted 10 June 2012 Available online 4 July 2012
Abstract The aim of this study was to evaluate the quantity and quality of bony regeneration after we had used recombinant human bone morphogenetic protein-7 (rhBMP-7 to augment the floor of the maxillary sinus. Nine consecutive patients with bilateral posterior maxillary atrophy who required augmentation of the sinus for interposition of implants were treated simultaneously with rhBMP-7 (Osigraft) with deproteinised bone substitute (0.5 g on the test side) and with deproteinised bone alone (2.0 g on the control side). Computed tomographic images preoperatively, immediately postoperatively, and at 4 months postoperatively showed a mean (SD) postoperative gain of 10.8 (3.0) mm on the test side and of 10.2 (1.8) mm on the control side. Histological and histomorphometric analyses of biopsy specimens showed that there was significantly more new bone on the control side (19.9 (6.8)%) than on the test side (6.6 (4.8)%). In this pilot controlled trial of the use of rhBMP-7, histological analyses showed that it resulted in the formation of less bone than treatment with inorganic bovine hydroxyapatite. © 2012 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Keywords: Human study; Bone regeneration; Sinus lift; Bone morphogenetic protein; BMP-7; rhOP-1; Anorganic bovine bone
Introduction Loss of teeth in the posterior maxilla results in rapid horizontal and vertical resorption of alveolar bone because of the lack of intraosseous stimulation by periodontal ligaments. The absence of maxillary molars leads to increased activity of osteoclasts in Schneider’s membrane, which causes pneumatisation of the sinuses within a few months because of the resorption of bone.1
∗
Corresponding author. Tel.: +39 335 6125333. E-mail addresses: [email protected] (G. Corinaldesi), [email protected] (L. Piersanti), [email protected] (A. Piattelli), [email protected] (G. Iezzi), [email protected] (F. Pieri), [email protected] (C. Marchetti).
Various bone-grafting materials have been evaluated for the augmentation of sinuses,2,3 and alloplasts and xenografts have shown promising results.2 Extensive animal2–6 and human7–9 clinical trials have used bovine bone mineral as a grafting material. All these materials are osteoconductive. In contrast, bone morphogenetic proteins (BMPs) induce mesenchymal stem cells to differentiate into osteoblasts and produce new bone tissue.10 Several recombinant forms of BMP, notably rhBMP2 and rhBMP-7, have been shown to induce bone formation in vivo11,12 and have been tested in clinical trials.13 The use of rhBMP-7, also known as osteogenic protein1 (rhOP-1; INN eptotermin ␣), with type 1 collagen as a carrier (Osigraft; Stryker Biotech, West Lebanon, NH, USA) has been approved in Europe for the treatment of tibial non-union under particular clinical conditions (initial
0266-4356/$ – see front matter © 2012 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bjoms.2012.06.004
248
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authorisation 17/05/01, European Union Registration No. EU/1/01/179/001). This product is the first human recombinant BMP approved for clinical use in Europe. Animal studies have shown that rhOP-1 stimulates bony regeneration and reduces healing time when used alone or in combination with osteoconductive biomaterials in sinus augmentation.14 Given the osteoinductive capacity of rhOP-1, the objective of this pilot prospective human control study was to evaluate bone formation in atrophied maxillas that required sinus lifting radiologically, histologically, and histomorphometrically. We compared rhOP-1 with deproteinised bovine bone, with deproteinised bovine bone alone.
Patients and methods Between October 2007 and December 2008, three men and six women (mean age 50 years, range 39–61) who required bilateral augmentation of the maxillary sinus floor to allow rehabilitation with osseointegrated implants were recruited to participate in this study that was approved by the Ethics Committee of S. Orsola-Malpighi Hospital, Bologna, Italy (No. 56/2007/U/Sper 15.05.2007). Participants had no clinical or radiological signs of active or chronic sinus conditions, and no clinical history of cardiac, haemorrhagic, haematological, or kidney disease. The following criteria were used to exclude patients from the study: residual maxillary atrophy of more than 5 mm (the distance between the sinus floor and the alveolar ridge); tobacco misuse (smoking more than 15 cigarettes/day); known hypersensitivity to the active component or to collagen; poor oral hygiene (plaque index for dentate patients 25% or less); pregnancy; current infection at the reconstruction site or ongoing systemic infection; uncontrolled diabetes mellitus or other serious metabolic disease; HIV or hepatitis B or C infection; cancer; history of chemotherapy, radiotherapy, or immunosuppression during the past 5 years; diagnosed autoimmune disorders, current or past use of drugs that may affect bone metabolism or tissue regeneration and repair (such as bisphosphonates); and current therapeutic regimen of steroids or non-steroidal anti-inflammatory drugs. Computed tomographic (CT) images were used to evaluate the vertical height of the sinus obtained postoperatively and variation in this value at the time of insertion of the implant. The quality of the bone was evaluated histologically and histomorphometrically 4 months postoperatively. In particular, the percentages of new bone tissue, soft tissue, and residual Bio-Oss particles were measured with respect to the total area of the sample examined. Clinical evaluation was used to monitor treatment-related adverse events. All operations were done by the same surgeon (GC) under general anaesthesia with nasal endotracheal intubation. Standard surgical access to the lateral maxillary wall was achieved through a mucosal crestal incision and release of vestibular incisions anteriorly and posteriorly. A bony
window was designed on the lateral wall of the maxillary sinus using piezosurgery (Mectron S.p.A., Carasco, Italy). After the sinus membrane had been dissected and raised, block randomisation was used to designate a test side and a control side for each patient. On the test side, the maxillary sinus region was filled after the membrane had been raised with one vial of Osigraft (eptotermin ␣ 3.5 mg in collagen 1 g) that had been reconstituted with saline solution 2.5 ml and sodium chloride 9 mg/ml for injection. The dosage of eptotermin ␣ 3.5 mg was chosen because in previous case reports 2.5 mg had been used with inconsistent bony regeneration in the sinus. The lateral wall was filled with inorganic bovine hydroxyapatite 0.5 g (Bio-Oss; Geistlich Biomaterials, Wolhousen, Switzerland) to maintain the graft in the site and completely fill the sinus. The control site was grafted with inorganic bovine hydroxyapatite 2 g (Bio-Oss; Geistlich Biomaterials) reconstituted with saline solution 2.5 ml. Both sinus grafts were covered with resorbable collagen membrane (Bio-Guide; Geistlich Biomaterials), and primary closure of the soft tissue was by non-absorbable sutures. Ceftriaxone 1 g (Rocefin; Roche S.p.A., Milan, Italy) was given twice daily for a week as prophylaxis against infection. CT evaluations were made preoperatively, immediately postoperatively, and 4 months later to facilitate the planned position of the implant. Surgical augmentation was evaluated by comparing preoperative (T0) and immediately postoperative (T1) CT scans. This calculation was based on height, measured from the lowest point of the alveolar ridge to the maxillary sinus floor, using a line parallel to the hypothetical position of the implant as a reference. The extent of resorption was evaluated at the time of insertion of the implant. This was expressed as a percentage: (T1 − T2) × 100: (T1 − T0), where (T1 − T2) indicated the amount of resorbed graft as a percentage of the total size of the initial graft (T1 − T0) (Table 1). Implants were positioned 4 months postoperatively, and at this time a 3-mm trephine-bur biopsy specimen was taken from the buccal side of the bone. Specimens were stored immediately in 10% buffered formalin and processed to obtain thinly ground sections (Precise 1 automated system; Assing, Rome, Italy). Each specimen was dehydrated in an ascending series of alcohol rinses and embedded in a glycol methacrylate resin (Technovit 7200 VLC; Kulzer, Wehrheim, Germany). After polymerisation, the specimen was sectioned longitudinally along the major axis with a high-precision diamond disc at about 150 m and ground down to about 30 m. Three slides were prepared and stained with basic fuchsin and toluidine blue. Histomorphometric examination was made with a light microscope (Laborlux S; Leitz, Wetzlar, Germany) connected to a high-resolution video camera (KY-F55B 3-CCD; JVC Italy S.p.A., Segrate, Italy) and interfaced with a monitor and personal computer (Pentium III 1200 MMX; Intel, Santa Clara, CA, USA). This optical system was associated with a digitising pad (Matrix Vision GmbH, Oppenweiler, Germany) and a histometry software package
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Table 1 Radiological evaluation with computed tomography of the sinus. Test side (Osigraft, rh-OP-1), control side (Bio-Oss).
rh-OP-1 1 2 3 4 5 6 7 8 9
Preoperatively
Postoperatively
At 4 months
Resorption at 4 months
Percentage resorption
3.6 4.7 4.4 1.9 2.8 4.3 3.9 3.3 3.6
14.6 10.6 13.6 12.7 15.1 15.2 13.2 12.9 13.2
12.3 8.9 12.7 11.9 14.6 14.7 12.1 11.8 12.7
−2.3 −1.7 −0.9 −0.8 −0.5 −0.5 −1.1 −1.1 −0.5
−15.8 −16.0 −6.6 −6.3 −3.3 −3.3 −8.3 −8.5 −3.8 −8.0 (4.9)
Mean (SD) Bio-Oss 1 2 3 4 5 6 7 8 9
2.6 4.6 1.2 2.3 3.2 4.1 3.2 2.9 4.1
15.4 10.6 12.9 16.4 18.9 15.8 14.1 14.6 13.3
Mean (SD)
with image-capturing capabilities (Image-Pro Plus 4.5; Media Cybernetics Inc., Immagini & Computer Snc., Milan, Italy). Histomorphometric measurements were made to calculate the percentages (the area fractions) of newly formed bone, residual graft materials, and soft-tissue components (connective tissue, or bone marrow, or both). We used the Statistical Package for the Social Sciences (version 11, SPSS Inc., Chicago, IL) to assess the significance of any differences between the test and control groups in the height of alveolar bone below the sinuses and the radiological and histomorphometric results. Group means (SD) were calculated for each variable measured. We used a splitmouth design to assess the significance of the differences between the experimental groups using Student’s t test for paired samples. Probabilities of less than 0.05 were accepted as significant.
Results Clinical and radiological results CT scans showed that the mean (SD) basal bone height was 3.1 (1.0) mm (range 1.9–4.7) on the right side and 2.8 (1.1) mm (range 1.5–4.5) on the left. The height of residual alveolar bone below the sinuses between the test and control side did not differ significantly (p = 0.3). Radiological examination showed that the mean gain in height after the sinus lift was 12.2 (4.2) mm (range = 18.3–5.4) on the test side and 11.9 (2.8) mm (range = 15.7–5.6) on the control side. The mean resorption
12.6 9.3 11.9 15.7 16.6 15.1 13 13.7 12.4
−2.8 −1.3 −1 −0.7 −2.3 −0.7 −1.1 −0.9 −0.9
−18.2 −12.3 −7.8 −4.3 −12.2 −4.4 −7.8 −6.2 −6.8 −8.9 (4.5)
volume 4 months postoperatively was 1.4 (1.2) mm (range 4.3–0.4) on the test side and 1.7 (1.0) mm (range 3.8–0.7) on the control side (Table 2). There were no major complications. Four patients developed small tears of Schneider’s membrane (test side: case 2; and control side: cases 1, 3, and 4) and were treated with resorbable membrane (Bio-Guide; Geistlich Biomaterials). There was no evidence of adverse local or systemic effects at 10 days, 3 weeks, 6 weeks, and 3 months postoperatively. Histological and histomorphometric observations All histological sections in the test group showed formation of new bone only at the most coronal parts of the biopsy specimen, which corresponded to the buccal portion of the augmented sinus. The new bone was primarily lamellar, and was in close contact with the remaining particles of hydroxyapatite. The apical portions of the biopsy specimens that contained only rhOP-1 differed; all these portions were filled with remnants of collagen carrier embedded in loose connective tissue, with no appreciable formation of bone in or around them (Figs. 1 and 2). In contrast, the biopsy specimens of the control sites showed newly formed lamellar bone surrounding particles of hydroxyapatite along their entire lengths. Similar particles were in contact with biological fluids and spaces in marrow in only a few areas. There were no inflammatorycell infiltrates or foreign-body reactions, no gaps at the bone–hydroxyapatite interface, and the bone was always in close contact with the particles. No particles of hydroxyapatite had resorbed (Figs. 3 and 4).
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Table 2 Mean (SD) histomorphometric data 4 months after sinus augmentation with Osigraft, rh-OP1, combined with bovine bone hydroxyapatite (HA) in the buccal portion of the augmented sinuses, or hydroxyapatite alone. Variable
rhOP-1-HA (test)
HA (control)
p value
Percentage of newly formed bone Percentage of remaining HA particles Percentage of soft tissue components
6.55 (4.75) 27.66 (4.74) 65.77 (6.9)
19.88 (6.79) 43 (4.89) 37.11 (5.03)
0.0003 0.0004 <0.0001
Fig. 1. Test site: a limited amount of newly formed bone was present in only the most lateral portion of the specimen in close proximity to the bovine hydroxyapatite particles (H). There were no signs of bony formation in the remaining internal portion where the collagen particles were completely covered by connective tissue (acid fuchsin and toluidine blue, original magnification 12×). Fig. 4. Control site: high-powered image of a single bovine hydroxyapatite particle (H) in close and tight contact with newly formed bone. There were no epithelial cells, connective tissue, or inflammatory cell infiltrates present at the bone/biomaterial interface (acid fuchsin and toluidine blue, original magnification 200×).
Table 2 shows the histomorphometric results for the two groups. Control sites (19.9 (6.8%)) had significantly more newly formed bone than did test sites (6.6 (4.8)%, p = 0.0003).
Discussion
Fig. 2. Test site: high-powered image of residual collagen particles (*) completely encapsulated in dense connective tissue with no inflammatory infiltrate (acid fuchsin and toluidine blue, original magnification 100×).
Fig. 3. Control site: bovine hydroxyapatite particles (H) surrounded and joined by newly formed trabecular bone (red) along the entire length of the specimen. There were no osteoclasts or macrophages present (acid fuchsin and toluidine blue, original magnification 20×).
The purpose of this in vivo pilot study was to evaluate the healing response and bony formation in the maxillary sinus stimulated by rhOP-1 in a collagen carrier with the use of rh-BMP-7 both radiographically and histologically. Reconstituted rhOP-1 allowed adequate dimensions of sinus lift, and the membrane had not collapsed after 4 months. The two sides did not differ significantly in the mean quantity of graft resorption, so rhOP-1 maintained membrane-lifting to a degree similar to that of other grafting materials.15 Although no patient developed an adverse reaction between the time of operation and placement of the implant, histological and histomorphometric analyses showed unfavourable results on the test side. No specimen showed histological signs of an inflammatory reaction, but the high percentage of soft tissue may have restricted osteoconduction. Significantly more soft tissue was present in sinuses augmented with BMP-7 than in sinuses grafted with anorganic bovine bone. Histological analyses showed that the collagen particles were completely covered by, and encapsulated within, dense connective tissue with no inflammatory
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infiltrate. Histological examination showed no initiation of bony formation at any test site, indicating that a longer healing time before the biopsy specimen was taken would not have changed the results. This contrasts with the use of rhOP-1 in cases such as the treatment of non-union of long bones, in which there was a significant progression in the success rate at follow-up intervals of 4–5, 7–8, and 9 months or more.16 Animal studies using rhOP-1 have reported significantly more newly formed bone at test sites than at control sites, with the exception of a single non-human primate model in which such a difference was not found. Two of these studies showed that only higher doses were able to increase the amount of newly formed bone significantly. Dose-dependent effects have been reported with the use of rhOP-1, with the following (highest) doses producing the best results: rhOP-1 1000 g in an inorganic bovine hydroxyapatite block to prefabricate vascularised bone grafts in miniature pigs17 ; rhOP-10.5 mg in inorganic bovine hydroxyapatite to treat calvarial defects in baboons18 ; rhOP-1 2.5 mg for augmentation of the maxillary sinus in chimpanzees19 ; and rhOP-1 2.5 mg in collagen matrix for augmentation of the sinus in non-human primates, which demonstrably increased bone mineral density.20 Human studies of raising and augmenting of the maxillary sinus have produced inconsistent results of formation of bone with the use of rhOP-1 2.5 mg in a collagen carrier. In these case reports the histological analysis of bony formation in 4 patients showed new bone in only one sinus at 6 months postoperatively.21,22 Because the dose is important for the successful use of BMP-2, and dose-dependent results have been published,23,24 we used rhOp-1 3.5 mg (1400 g) in an absorbable collagen carrier in the present study. As described previously for the use of BMP-2, the dose and the selection of carrier also presented challenges in the use of BMP-7. Although human clinical studies have evaluated the use of BMP-2 with a self-containing absorbable collagen sponge carrier in the treatment of bony defects, collagen carriers have been studied more extensively. Animal studies have documented inadequate regeneration of bone with the use of collagen alone as a substitute bone graft.25 Collagen may not have sufficiently effective scaffolding capabilities to support the rhBMP-7-induced formation of bone in sinus defects. Absorbable collagen may not provide the three-dimensional structure needed to support the formation of new bone, and non-resorbable materials with greater stability may be more effective. The observations of this pilot study should be interpreted with caution because of the limited size of the histological sample and the prevalence of female patients, with possible diminished regenerative capabilities as a result of the effects of their hormones. In conclusion, the use of rhOP-1 for augmentation of the sinuses gave poor results, with less new bone formed than with inorganic bovine hydroxyapatite. The correct dose and method of use of rhOP-1 should be assessed before it is used further in the augmentation of human sinuses.
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Conflict of interest statement The authors disclose any financial and personal relationship with other people or organisations that could inappropriately influence their work. The vials of Osigraft (eptotermin 3.5 mg in collagen 1 g) used for this study were kindly donated by Stryker Biotech, West Lebanon, NH, USA.
Ethical approval This work was approved by the Ethical Committee of S. Orsola-Malpighi Hospital, Bologna, Italy (No. 56/2007/U/ Sper 15.05.2007) and all the subjects gave informed consent to the work.
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