Ui-Won Jung* In-Kyeong Lee* Jin-Young Park Daniel S. Thoma €mmerle Christoph H. F. Ha Ronald E. Jung
The efficacy of BMP-2 preloaded on bone substitute or hydrogel for bone regeneration at peri-implant defects in dogs
Authors’ affiliations: Ui-Won Jung, In-Kyeong Lee, Jin-Young Park, Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University, Seoul, South Korea Daniel S. Thoma, Christoph H. F. H€ ammerle, Ronald E. Jung, Department of Fixed and Removable Prosthodontics and Dental Material Science, Dental School, University of Zurich, Zurich, Switzerland
Key words: Bone morphogenetic protein-2, bone substitutes, dental implants, drug delivery
Corresponding author: Prof. Ui-Won Jung Department of Periodontology, Research Institute for Periodontal Regeneration College of Dentistry, Yonsei University, 50-1, Yonsei-ro, Seodaemun-gu Seoul 120-752, South Korea Tel.: +82-2-2228 3185 Fax: +82-2-392 0398 E-mail: [email protected]
apicocoronally, and 8 mm mesiodistally) were surgically prepared on buccal sides of the left and
system, hydrogel, polyethylene glycol Abstract Objectives: The objective of this experiment was to test whether or not a synthetic bone substitute (SBS) was more effective than a polyethylene glycol hydrogel as a carrier material for bone morphogenetic protein-2 (BMP-2) when attempting to regenerate bone. Material and methods: Two identical, box-type dehiscence defects (4 9 4 mm buccolingually and right edentulous ridge in five beagle dogs. Following implant placement, the defects either received (i) no graft, (ii) SBS+hydrogel, (iii) SBS+BMP-2 loaded hydrogel, and (iv) BMP-2-loaded SBS+hydrogel. The animals were euthanized at 8 weeks postsurgery. Radiographic and histomorphometric analyses were performed. Results: The hydrogel alone was not able to stabilize the grafted bone particles at 8 weeks, and SBS+hydrogel group did not significantly differ from the control group in all volumetric measurements. On the other hand, extensively regenerated new bone was connected with most of the remaining SBS particles in the BMP-2 groups. The BMP-2 groups exhibited significantly greater new bone formation (10.65 mm3 and 1.47 mm2 in the SBS+BMP-2-loaded hydrogel group; 14.17 mm3 and 0.93 mm2 in the BMP-2-loaded SBS+hydrogel) than non-BMP-2 groups (1.27 mm3 and 0.00 mm2 in the control group; 2.01 mm3 and 0.19 mm2 in the SBS+hydrogel group) in volumetric and histomorphometric analyses (P < 0.001). However, there were no significant differences between both BMP-2 groups. Conclusion: BMP-2 could yield enhanced bone regeneration in the critical-size peri-implant defects regardless of whether SBS or hydrogel is used for preloading, although the outcomes seem to be more reproducible with BMP-2 preloaded on SBS.
*These authors contributed equally to this study. Date: Accepted 27 August 2014 To cite this article: Park J-Y, Thoma DS, H€ammerle CHF, Jung RE. The efficacy of BMP-2 preloaded on bone substitute or hydrogel for bone regeneration at peri-implant defects in dogs. Clin. Oral Impl. Res. 26, 2015, 1456–1465. doi: 10.1111/clr.12491
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Complete embedment of dental implants in bone tissue is a prerequisite for long-term success. However, clinical situations with sufficient width of alveolar bone for the placement of an implant with the adequate diameter are hard to encounter. It is well documented in the literature that after tooth extraction variety of soft and hard tissue alterations are initiated and lead to a reduced alveolar ridge contour (Amler et al. 1960; Pietrokovski & Massler 1967; Jung et al. 2013a). As a consequence, coronal dehiscence-type defect often occurs around implant placed in the atrophied alveolar ridge. To cover the entire surface of the implant by bone tissue, various types of regenerative procedures have been attempted (H€ammerle
et al. 1996, 2002). Due to invasiveness of harvesting autogenous bone, various bone substitutes have been developed and demonstrated successful clinical outcomes (Springer et al. 2004; Hallman & Thor 2008; Jensen & Terheyden 2009). Nevertheless, as osteoconductive bone substitute has limited bone regenerative property, it is short of being the perfect alternative of autogenous bone. Recombinant human bone morphogenetic protein-2 (rhBMP-2) has emerged as a potent osteoinductive solution. The osteogenic effect of rhBMP-2 has been widely investigated and demonstrated successful bone regeneration (Choi et al. 2002; Kim et al. 2002; Wikesj€ o et al. 2003; Huang et al. 2008; Jung et al. 2008, 2009c). rhBMP-2 in a colla-
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Jung et al Hydrogel and bone substitute as a BMP-2 carrier
gen carrier has been commercially available for maxillofacial reconstruction since 2007. However, high dose of rhBMP-2 (1.5 mg/ml) is needed to exhibit biological activity when used in a collagen carrier. High cost and safety issues might be barriers to common clinical use of rhBMP-2. The development of an optimal carrier system could solve these problems, as it has been shown that the regenerative potential of growth and differentiation factors depend upon the carrier material (Hunt et al. 2001; Kim et al. 2005; Hong et al. 2006; Jung et al. 2006b). Such a carrier material should be biocompatible, mechanically stable within defect spaces, have sufficient plasticity for easy defect filling and manipulation, and eventually be replaced by newly formed bone. In addition to these requirements, the release kinetics of rhBMP2 from the carrier material is an important criterion for optimum bone regeneration and also for reducing the dose of rhBMP-2. Hydrogels made of polyethylene glycol (PEG) can serve as an in situ forming matrix for optimal cell ingrowth and retention of bioactive proteins (Lutolf et al. 2003). In recent in vitro and in vivo studies, it was demonstrated that beneficial release kinetics of rhBMP-2 can be achieved by entrapping rhBMP-2 as well as a parathyroid hormone peptide in this synthetic matrix (Weber et al. 2002; Jung et al. 2006a, 2007a,b, 2008; H€ anseler et al. 2012). In regard to mechanical properties, the hydrogel alone may be insufficient to maintain space against the pressure of soft tissue. To reinforce the mechanical strength, a combination of particulated bone substitutes with the PEG hydrogel enabled highly efficient and localized bone regeneration (Jung et al. 2007a, 2009a,b; Herten et al. 2009; Thoma et al. 2009). Calcium phosphates have been of great interest as a synthetic bone substitute (SBS) material due to its chemical similarity with inorganic component composing human bone (Daculsi et al. 1989). In addition, its formulation and configuration are easily modifiable, and various composites of hydroxyapatite and b-tricalcium phosphate are available in dental field. Hydroxyapatite is known to be scantily bioresorbable while b-tricalcium phosphate more readily undergoes bioresorption. The resorption rate can be controlled by the proportion of hydroxyapatite and b-tricalcium phosphate. The objective of this experiment was to test whether or not the SBS was more effective than the PEG hydrogel as a carrier material for BMP-2 when attempting to regenerate bone.
Materials and methods Preparation of the rhBMP-2 and the carrier system
The rhBMP-2 expressed in Escherichia coli was produced at the research institute, Cowellmedi Co. LTD, South Korea. Briefly, the RNAs extracted from the human osteosarcoma cells were reverse-transcribed into cDNA encoding the mature peptide of BMP-2 protein, which was amplified by polymerase chain reaction (PCR). The PCR product was cloned into a plasmid vector. Following highcell-density cultivation, the cultured biomass was collected via repeated centrifugation from which the inclusion bodies were harvested. The inclusion bodies were solubilized and diluted in a renaturation buffer, and then, the active rhBMP-2 proteins were purified and eluted.
Experimental design
A total of 20 implants (SLActiveâ Bone Level Implant, Straumann AG, Basel, Switzerland; a diameter of 3.3 mm and a length of 8 mm) were bilaterally placed in the edentulous mandible. In four peri-implant dehiscence defects, the following four treatment modalities were randomly applied to achieve even number of distribution into each defect location within the mandible.
• • • •
Control group: empty SBS+hydrogel group: SBS mixed with hydrogel SBS+BMP/hydrogel group: SBS mixed with BMP-2 (0.1 mg)-loaded hydrogel BMP/SBS+hydrogel group: BMP-2 (0.1 mg)-loaded SBS mixed with hydrogel
Surgical procedures
Synthetic matrix and bioactive peptides
Anesthesia
This study used a PEG-based hydrogel as a synthetic matrix and carrier system for rhBMP-2. Preparation of the gel was performed in a manner similar to previously published protocols (Elbert & Hubbell 2001; Elbert et al. 2001; Jung et al. 2007a). Shortly, a base catalyzed Michael type addition of a thiol to an acrylate group each linked to a PEG molecule which are mixed in an automatic mixing syringe constitutes the ground mechanism for the gelation. The network conformation obtained after a gelation time of about 4 min degrades in puffer by hydrolysis in vitro within 10 days. The SBS (Straumann Bone Ceramicâ, Straumann AG, Basel, Switzerland) used in this study is fully synthetic biphasic calcium phosphate consisting a mixture of 60% hydroxyapatite and 40% of b-tricalcium phosphate. The bone substitute demonstrates partial resorption and substitution with vital bone (Cordaro et al. 2008; Froum et al. 2008).
Surgical procedures were performed under general anesthesia induced by intravenous injection of atropine (0.04 mg/kg; Kwangmyung Pharmaceutical Ind. Co. Ltd., Seoul, Korea) and intramuscular injection of a combination of xylazine (Rompun, Bayer Korea Co., Seoul, Korea) and ketamine (Ketara, Yuhan Co., Seoul, Korea) followed by inhalation anesthesia (Gerolan, Choongwae Pharmaceutical Co., Seoul, Korea). Routine dental infiltration anesthesia was used at the surgical sites. The mandibular forth premolar and first molar (P4 and M1) were bilaterally extracted. The flap was replaced and sutured. Oral prophylaxis was performed on the remaining dentition in conjunction with the extractions. The stitches were removed 10 days postextraction. The defect sites were allowed to heal for 8 weeks. A horizontal midcrestal incision was made on the edentulous experimental site, and vertical incisions were made through the mucogingival junction extending into the alveolar mucosa at the mesial and distal end of the defect. The mucoperiosteal flaps were elevated to expose the edentulous alveolar ridge. Two identical, box-type dehiscence defects (4 9 4 mm buccolingually and apicocoronally, and 8 mm mesiodistally) were surgically prepared on buccal sides of the left and right edentulous ridge. In each of the four defects, an implant was placed following serial drilling according to the standard protocol. The implant’s shoulder was located at the level of the alveolar crest (Fig. 1a and 1b). Of SBS, 0.175 g was weighed into a bowl. Each component of the PEG hydrogel matrix
Animals
Five male beagle dogs with approximate mean age 18 months and weight 15 kg were used. All animals were free of heart worms and were quarantined. The animals were also examined to have intact dentition with healthy periodontium. All experiments were performed at the animal laboratory accredited by Association for Assessment and Accreditation of Laboratory Animal (AAALAC) international, after the ethical approval of the institutional Animal Care and Use Committee regarding animal selection, management, and surgery protocol.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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Jung et al Hydrogel and bone substitute as a BMP-2 carrier
(a)
•
(b)
Any augmented volume on either side of the defect in its mesiodistal aspect was excluded.
The following parameters were calculated within the defect:
• • • • (c)
(d)
Total augmented volume (TAV; mm3) New bone volume (NBV; mm3) Remaining bone substitute volume (RBV; mm3) Non-mineralized tissue volume (NMV; mm3)
Histomorphometric analysis
Fig. 1. Clinical photographs representing surgical procedures. Lateral view (a) and occlusal view (b) following preparation of box-type defects with buccal dehiscence on the edentulous ridge. Lateral view (c) and occlusal view (d) following implant placement and simultaneous bone grafting with the mixture of synthetic bone substitute and hydrogel. M = mesial. D = distal.
(74 ll PEG A, 74 ll PEG B, 57 ll activator) were placed into the bowl next to the SBS granules. For the SBS+BMP/hydrogel group, 23 ll of rhBMP-2 (4.4 mg/ml) was added to the mixture. Using a spatula, the solutions were mixed with the SBC granules and applied to the defect (Fig. 1c and 1d). For the BMP/SBS+hydrogel group, the SBS granules were soaked in 150 ll of rhBMP-2 (0.67 mg/ ml) for 4 h in a refrigerator (4°C) prior to the experiment, and moisture was allowed to evaporate. For the rhBMP-2 groups, gelation took place within 10–11 min, and for positive control group, 5–6 min. Groups were assigned so that they can be distributed into each defect site the same number of times. The buccal flaps were carefully released without tension and sutured. Each dog was maintained on a soft diet throughout the study. Postsurgical management included intramuscular administration of antibiotics (cefazolin sodium 20 mg/kg; Yuhan Corporation, Seoul, Korea) and daily topical application of a 0.2% chlorhexidine solution (Hexamedinâ, Bukwang Pharmaceutical Co., Seoul, Korea) for infection control. Two weeks postoperatively, the sutures were removed. The animals were euthanized at 8 weeks using an overdose of pentobarbital (90– 120 mg/kg; IV). Block sections including
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grafted sites and surrounding alveolar bone and soft tissues were collected and fixed in 10% formalin for 10 days. A micro-CT (SkyScan 1072, SkyScan, Aartselaar, Belgium) was taken at a resolution of 35 lm (100 kV, 100 lA). The scanned data set was processed in DICOM format, and the area of interest was reconstructed with three-dimensional (3D) software (OnDemand3Dâ, Cybermed, Seoul, Korea). Then, the samples were dehydrated in a graded series of ethanol. Thereafter, they were embedded in methyl methacrylate without being decalcified according to standard procedures. The specimens were vertically sectioned in the center of the implants in a buccolingual direction and were stained with hematoxylin–eosin. Analysis Micro-CT analysis
The overall augmented contour was evaluated at the 3D reconstructed view. Boundaries were set as follows to standardize the region of augmented volume to be analyzed.
• •
Any augmented volume directly superior or lingual to the implant in its axial plane was excluded. Any augmented volume inferior to the buccal extension of inferior border of the defect was excluded.
The sections of each defect site were observed using a light microscopy (Olympus Multi-view microscope BH2, Tokyo, Japan). Histomorphometric analyses were performed by one masked experienced examiner, using image analysis software (Image-Pro PlusTM, Media Cybernetic, Silver Springs, MD, USA). The area of interest (AOI) was made of 1 mm width and 4 mm height from the implant platform. The following measurements were analyzed and recorded within the AOI (Fig. 2):
• • • • •
New bone area (NBA; mm2) Remaining bone substitute area (RBA; mm2) Bone-to-implant contact (BIC; %) BC-S (mm): distance from the bone crest to the implant shoulder CO-S (mm): distance from the most coronal point of osseointegration to the implant shoulder.
Statistical analysis
All statistical analyses were performed with software R, version 2.15.2 (http://www.r-project.org). The nonparametric mixed model was used to compare radiographic and histomorphometric parameters between four groups (Brunner & Langer 2000) with treatment group (Control, SBS+hydrogel, SBS+BMP/hydrogel, and BMP/SBS+hydrogel) as an independent factor and dog number as a random factor. Statistical significance level was 5%. Post hoc analyses were performed by pairwise comparison with modified p-value of 0.0083 rather than 0.05 (0.0167 for control value was zero).
Results Clinical findings
The addition of rhBMP-2 solution into both PEG hydrogel and bone substitute resulted in an extended gelation time, which caused
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Jung et al Hydrogel and bone substitute as a BMP-2 carrier
Fig. 2. Illustration of measuring parameters for histomorphometric analysis. Red box = area of interest (1 9 4 mm). Blue arrowhead = the shoulder of implant. Yellow arrowhead = the most coronal point of newly formed bone. White arrowhead = the most coronal point of osseointegration. I = implant.
delay during surgery. All implants and grafted materials remained submerged, and no complications were observed throughout the entire experimental period. Volumetric analysis: micro-CT
In the two BMP-2 groups, the overbuilt materials on the buccal aspect of the implants preserved their grafted dome shape and maintained their coverage of the implant surfaces. In the control group, however, the implant surface remained exposed, and only partial coverage was achieved and grafted materials were spread out in the SBS+hydrogel group (Fig. 3). In terms of NBV, SBS+BMP/hydrogel group and BMP/SBS+hydrogel group exhibited a significantly larger volume than the control and SBS+hydrogel group (P < 0.001; Table 1 and Fig. 4). RBV of both BMP-2 groups showed a statistically significant difference to that of the SBS+hydrogel group (P < 0.001, Table 1). Histological observation
No inflammatory reaction or failure of osseointegration was found in any of the samples. In the control group, minimal bone formation was observed, and the buccal defect was mostly occupied by fibrous tissue (Fig. 5). A periosteum-like dense connective tissue layer lined the implant surface at the defect.
Fig. 3. 3D reconstructed micro-CT views and cross-sectional views of control, SBS+hydrogel, SBS+BMP/hydrogel, and BMP/SBS+hydrogel groups.
Table 1. Volumetric analysis of micro-computed tomography (n = 5; mm3) NBV Control Mean SD Median SBS+hydrogel Mean SD Median SBS+BMP/hydrogel Mean SD Median BMP/SBS+hydrogel Mean SD Median
1.75 2.23 1.27
RBV
NMV
TAV
NA NA
510.48 219.39 552.28
512.23 218.25 552.39
3.47 4.19 2.01
4.46 4.51 2.54
588.83 359.25 504.75
596.76 353.18 508.73
10.75 5.61 10.65*†
12.44 8.48 12.16†
643.25 194.12 583.27
666.44 187.53 611.95
13.80 5.63 14.17*†
15.77 6.34 16.75†
628.43 190.61 550.06
657.99 180.07 591.14
NBV, new bone volume; RBV, remaining bone substitute volume; NMV, non-mineralized tissue volume; TAV, total augmented volume; SBS, Synthetic bone substitute. *Significantly different from the control group (P < 0.001). †Significantly different from the SBS+hydrogel group (P < 0.001).
Following 8 weeks of healing, most of the grafted materials at the defect site had disappeared in the SBS+hydrogel group (Fig. 6). Only one histologic sample (#5 dog) showed a cluster of remaining materials. The SBS particles on the outer layer of grafted mass were surrounded by connective tissue without connection with bone. SBS particles at the inner region of the defect were in contact with newly formed bone sprouting from the floor of the defects. Compared to the non-BMP-2 groups, the BMP-2 groups including the SBS+BMP/hydro-
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
gel and the BMP/SBS+hydrogel groups maintained the original grafted dimension with prominent bone regeneration. In some samples, NB extended over the top of the implants. The two BMP-2 groups exhibited similar healing patterns irrespective of the carrier of BMP-2, whether it was the graft particle or the hydrogel. A concentric lamellation process began forming primary osteons at the NB surrounding the RBC. In the SBS+BMP/hydrogel group (Fig. 7), most of the remaining SBS particles were in close contact with the projection of NB. A few particles
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Jung et al Hydrogel and bone substitute as a BMP-2 carrier
Fig. 4. Scatter plot and the median (the small bar) representing the new bone volume (mm3). *Significantly different (P < 0.001).
(a)
(b)
presented contact with bone even at the outer connective tissue layer. Thin layer of new bone lined the majority of the length of the implant surface within the defect. The NB exhibited high cellularity and vascularity demarcating the mature old bone having well-developed lamellae and Haversian system. Highly dense Volkmann’s canals running perpendicularly to the osteons were found at the border of the old bone (Fig. 8 ). In the BMP/SBS+hydrogel group (Fig. 9), the original grafted volume seemed to be not only maintained but rather increased. SBS particles were observed lined up beneath the outer connective tissue layer. Substantial amount of new bone were formed making bony bridges between the particles. A large portion of the remaining SBS particles in the two BMP-2 groups were embedded in NB. Some SBS particles away from the new bone connection were existed outside of the graft bulk and embedded in soft tissue. A periosteum-like layer lined outside of the grafted materials and multinucleated giant cells appeared to be involved in the resorption process of the remaining SBS particles featuring concave surfaces (Fig. 10). In the high magnified view, almost completely resorbed bone particles were observed in the process of resorption in a soap-bubble-like appearance. Histomorphometric analysis
Fig. 5. Low (a) and high (b; the white box of a) magnified histologic views of the control group (H & E). V = vessels. P = periosteum-like connective tissue layer. I = implant.
(a)
(b)
Both BMP-2 groups exhibited significantly higher NBA, CO-S, BC-S, and BIC values than non-BMP-2 groups (P < 0.001, Tables 2 and 3 & Fig. 11). However, there was no difference between SBS+BMP/hydrogel and BMP/SBS+hydrogel groups in all measurements.
(c)
Discussion
Fig. 6. Histologic view of the SBS/hydrogel group (H & E). Three samples [#1 (a), #2 (b), and #5 (c)] out of a total of five with remaining bone substitute particles (RBS). NB = new bone. CT = connective tissue. I = implant.
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Bone regeneration at the standardized periimplant dehiscence defects was compared according to four different treatment modalities, and the addition of rhBMP-2 resulted in significantly greater new bone formation and more BIC than groups treated without rhBMP-2. The control group, which had no treatment, exhibited minimal resolution of the surgically created osseous defects. As the peri-implant box-type dehiscence defect prepared in the present study could not be resolved spontaneously, the size of the defect could be considered as a critical-sized defect. In such a large defect, the use of membrane is especially needed for favorable guided bone regeneration (GBR). Several studies reported
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Jung et al Hydrogel and bone substitute as a BMP-2 carrier
(a)
(b)
Fig. 7. Histologic view of the SBS+BMP/hydrogel group (H & E). (a) Low magnification (b) High magnified view of the top of the defect. NB = new bone. RBS = remaining bone substitute. I = implant.
(a)
(b)
Fig. 8. Histologic view of the SBS+BMP/hydrogel group (H & E). (a) Low magnification (b) Highly dense Volkmann’s canals (VC) running perpendicularly to the osteons found at the border of the old bone. NB = new bone. I = implant.
(a)
(b)
(c)
Fig. 9. Histologic view of the BMP/SBS+hydrogel group (H & E). (a) Low magnification (b) High magnified view of the top half of the defect (c) High magnified view of the bottom half of the defect. NB = new bone, RBS = remaining bone substitute. I = implant.
that the addition of membrane significantly enhanced bone regeneration and prevented fibrous encapsulation compared with sites treated without a membrane in dehiscence defect as well as lateral ridge augmentation
(Park et al. 2008; Urban et al. 2011; Fu et al. 2013; Jung et al. 2013b). Amorphous sol of PEG hydrogel before gelation can be customized to fit into the irregular defect configuration. Gel state of
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
hydrogel after setting can endure the external pressure having an extent of mechanical strength. Compared to the conventional barrier membrane, the injectable hydrogel is easy to use, as a pin or screw for fixation of the membrane is not required. In this study, PEG hydrogel was used as a membrane alternative for GBR procedure. Beside its hydrophilicity and biocompatibility, PEG has cellocclusive characteristic, which is one of the requisites for the barrier membrane. PEG gels consisting of multi-arm PEG molecules could prevent cell penetration, as the distances between the cross-linking points are smaller than the dimension of a cell (Wechsler et al. 2008). PEG hydrogel is a versatile material that can be used as a delivery system for bioactive molecules as well as a barrier membrane for GBR procedure (Jung et al. 2006a, 2008, 2009a,b). The optimal degradation time and physical properties of PEG hydrogel can be modulated depending on the purpose of use (Thoma et al. 2011, 2014). The dense network showed an increased degradation time and maintained the shape, while the loose network showed a relatively decreased degradation time. All PEG hydrogel groups with different degradation times have been shown to be biocompatible, as demonstrated by Dahlin using human gingival fibroblasts (Dahlin et al. 2014). However, too long-lasting nonporous gel mass might retard the initial recruitment and infiltration of the osteoprogenitor cells into the defect and jeopardize proper release of the captured molecules, in particular, when used as a delivery system of the growth factors. Therefore, the appropriate degradation time might be a critical factor to fulfill the requirements of an ideal carrier. Degradation time of the PEG hydrogel used in the present study was controlled to be degraded within 10 days in vitro. In the present in vivo study, it confirmed that no PEG hydrogel remained in histologic observation in all samples at 8 weeks. In SBS+hydrogel group without addition of BMP-2, only one sample exhibited localized SBS particles. Two of five samples showed no bone graft materials and minimal SBS particles were observed at the center of defect in the other two samples. Such observation could be explained that premature degradation of PEG hydrogel cannot stabilize the grafted particles before consolidation, as the osseous defect was large and uncontainable. Similarly, bovine bone particles were scattered in the non-contained type one-wall intrabony defect due to early resorption of the pure collagen matrix (Jung et al. 2011). Therefore, the PEG
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(a)
(b)
Fig. 10. (a) Distant isolated particles featuring concave surfaces of resorption lined with multinucleated giant cells (BMP/SBS+hydrogel group) (b) Almost completely resorbed bone particle displaying a soap-bubble-like appearance (BMP/hydrogel+SBS group). Arrowheads = multinucleated giant cells. RBS = remaining bone substitute.
Table 2. Histometric analysis within the area of interest (n = 5; mm2) Control Mean SD Median SBS+hydrogel Mean SD Median SBS+BMP/hydrogel Mean SD Median BMP/SBS+hydrogel Mean SD Median
NBA
RBA
The others
0.01 0.01 0.00
NA NA
3.99 0.01 4.00
0.23 0.26 0.19*
0.29 0.30 0.30
3.48 0.53 3.63*
1.21 0.49 1.47*†
0.46 0.19 0.47
2.33 0.67 2.06*†
0.96 0.26 0.93*†
0.60 0.22 0.65
2.44 0.32 2.52*†
NBA, new bone area; RBA, remaining bone substitute area. *Significantly different from the control group (P < 0.001). †Significantly different from SBS+hydrogel group (P < 0.001).
Table 3. Histometric linear measurements (n = 5; mm). Control Mean SD Median SBS+hydrogel Mean SD Median SBS+BMP/hydrogel Mean SD Median BMP/SBS+hydrogel Mean SD Median
CO-S
BC-S
4.18 0.35 4.22
4.18 0.35 4.22
3.29 0.76 3.17
2.67 1.10 2.48*
0.86 1.27 0.36*†
0.51 0.94 0.00*†
0.81 0.49 0.90*†
0.25 0.38 0.00*†
CO-S, distance from the most coronal point of osseointegration to the implant shoulder; BC-S, distance from the bone crest to the implant shoulder. *Significantly different from the control group (P < 0.001). †Significantly different from SBS+hydrogel group (P < 0.001).
hydrogel, which has 10 days degradation time in the present study, might not be sufficient to act as a barrier membrane. Jung et al. showed that a different PEG hydrogel with an in vitro degradation time of approximately 4 months resulted in a comparable amount of vertical bone fill and mean dehiscence defect fill with a standard collagen membrane in clinical study (Jung et al. 2009a). In addition, the PEG hydrogel was
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revealed to perform the role of a barrier membrane in non-critical-size defects in rabbits and in dehiscence-type bone defects in dogs (Jung et al. 2006a, 2009b). In these particular studies, the remaining PEG could still be identified after 4 weeks and 6 months of healing period in the histological evaluation, respectively. In situ overbuilt bolus maintained the volume and exhibited excellent bone regen-
eration at the defects in most samples of the BMP-2 groups in spite of early degradation of the PEG hydrogel. Possible explanation would be that BMP-2 might accelerate substitution of PEG hydrogel with dense provisional matrix for future mineralized tissue by stimulation of osteogenic potential at the early stage of healing, which could reinforce the mechanical strength of grafted mass against the external pressure. Therefore, early degradation of the hydrogel might be beneficial for rapid release of BMP-2 molecules. H€anseler et al. compared the differences in release kinetics and biological activity of nonglycosylated rhBMP-2 and glycosylated rhBMP-2 incorporated into the combination of bovine hydroxyapatite with PEG hydrogel in vitro (H€anseler et al. 2012). They confirmed the equivalent cell viability and biological activity between both rhBMP-2 in spite of the difference in release profile. The rhBMP-2 incorporated into PEG hydrogel revealed reduced bioactivity compared to positive control containing rhBMP-2 only. On this phenomenon, they assumed that a chemical interaction between rhBMP-2 and PEG hydrogel during gelation might affect to the reduced bioactivity of the incorporated rhBMP-2 molecule. When the rhBMP-2 was adsorbed to bovine hydroxyapatite, less concentration of active rhBMP-2 was detected compared to incorporation into PEG hydrogel, which might be associated with high affinity of BMP-2 molecules to hydroxyapatite (Boix et al. 2005). Combination of PEG hydrogel for initial burst and hydroxyapatite for sustained release of rhBMP-2 might be optimal to stimulate the progenitor cells (Brown et al. 2011). The release kinetics could be influenced by the degradation of the carrier material itself. As bovine hydroxyapatite is categorized as a non-resorbing material, the effect of the material resorption might be negligible. However, as SBS used in the present study contains high proportion of fast-resorbing tricalcium phosphate, the release by the degradation of the material must be considered. Therefore, further long-term study to evaluate the degradation rate of the SBS itself is needed to fully apprehend BMP-2 release kinetics. When the two BMP-2 groups were compared, greater bone formation was observed when BMP-2 was loaded onto SBS rather than the hydrogel. When hydrogel is used as the carrier, hydrogel is applied to the defect in mixture with BMP-2 at the final step. Therefore, it did not need an additional soaking time and could minimize the loss of BMP-2 due to bleeding or aspiration during surgery.
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Jung et al Hydrogel and bone substitute as a BMP-2 carrier
(a)
(b)
(c)
(d)
Fig. 11. Scatter plot and the median (the small bar) representing (a) the new bone area, (b) bone-to-implant contact within the defect, and (c and d) the linear measurements (n = 5). CO-S = distance from the bone crest to the implant shoulder. BC-S = distance from the most coronal point of osseointegration to the implant shoulder.*Significantly different (P < 0.001).
However, as addition of BMP-2 to both hydrogel and SBS delayed gelation time and prolonged waiting time prior to flap closure, flaps had to be closed before complete gelation took place and substantial amount of BMP-2 might have been adsorbed undesirably to the surrounding soft tissue. In this case, the hydrogel may still be in its sol state, which would not have enough viscosity to maintain the grafted shape and prone to collapse. In the present study, the SBS+BMP/hydrogel group also showed a wider range of BIC compared to the SBS/BMP+hydrogel group. This might be caused by a further delay in gelation because of direct loading on hydrogel. Moreover, graft materials were overbuilt in consideration of the shrinkage during healing, which would have been sub-
jected to pressure from the overlying soft tissues. To obtain a more predictable gelation time, the concentration of the activating solution needs to be closely adjusted in consideration of the dilution of the mixture following the addition of BMP-2 solution as well as contamination with blood and saliva inside the oral cavity. It is concluded that BMP-2 could yield enhanced bone regeneration in the criticalsize peri-implant defects regardless of whether SBS or hydrogel is used for preloading, although the outcomes seem to be more reproducible with BMP-2 preloaded on SBS.
Acknowledgements:
We thank the Institut Straumann AG for providing us
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
with the PEG hydrogel, synthetic bone substitute, and dental implants. We also gratefully thank Dr Minglan Chang and Ms Sora Yoon for helping with the experimental surgeries and histomorphometric analyses.
Conflict of interest The authors declare no conflict of interest.
Source of funding statement This study was financially supported by an ITI research grant from the ITI foundation (821-2012).
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Lutolf, M.P., Weber, F.E., Schmoekel, H.G., Schense, J.C., Kohler, T., Muller, R. & Hubbell, J.A. (2003) Repair of bone defects using synthetic mimetics of collagenous extracellular matrices. Nature Biotechnology 21: 513–518. Park, S.H., Lee, K.W., Oh, T.J., Misch, C.E., Shotwell, J. & Wang, H.L. (2008) Effect of absorbable membranes on sandwich bone augmentation. Clinical Oral Implants Research 19: 32–41. Pietrokovski, J. & Massler, M. (1967) Alveolar ridge resorption following tooth extraction. The Journal of Prosthetic Dentistry 17: 21–27. Springer, I.N., Terheyden, H., Geiss, S., Harle, F., Hedderich, J. & Acil, Y. (2004) Particulated bone grafts–effectiveness of bone cell supply. Clinical Oral Implants Research 15: 205–212. Thoma, D.S., Halg, G.A., Dard, M.M., Seibl, R., H€ammerle, C.H. & Jung, R.E. (2009) Evaluation of a new biodegradable membrane to prevent gingival ingrowth into mandibular bone defects in
minipigs. Clinical Oral Implants Research 20: 7–16. Thoma, D.S., Schneider, D., Mir-Mari, J., H€ammerle, C.H., Gemperli, A.C., Molenberg, A., Dard, M. & Jung, R.E. (2014) Biodegradation and bone formation of various polyethylene glycol hydrogels in acute and chronic sites in mini-pigs. Clinical Oral Implants Research 25: 511–521. Thoma, D.S., Subramani, K., Weber, F.E., Luder, H.U., H€ammerle, C.H. & Jung, R.E. (2011) Biodegradation, soft and hard tissue integration of various polyethylene glycol hydrogels: a histomorphometric study in rabbits. Clinical Oral Implants Research 22: 1247–1254. Urban, I.A., Nagursky, H. & Lozada, J.L. (2011) Horizontal ridge augmentation with a resorbable membrane and particulated autogenous bone with or without anorganic bovine bone-derived mineral: a prospective case series in 22 patients. The International Journal of Oral & Maxillofacial Implants 26: 404–414.
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Weber, F.E., Eyrich, G., Gratz, K.W., Maly, F.E. & Sailer, H.F. (2002) Slow and continuous application of human recombinant bone morphogenetic protein via biodegradable poly(lactide-co-glycolide) foamspheres. International Journal of Oral and Maxillofacial Surgery 31: 60–65. Wechsler, S., Fehr, D., Molenberg, A., Raeber, G., Schense, J.C. & Weber, F.E. (2008) A novel, tissue occlusive poly(ethylene glycol) hydrogel material. Journal of Biomedical Materials Research. Part A 85: 285–292. Wikesj€ o, U.M., Qahash, M., Thomson, R.C., Cook, A.D., Rohrer, M.D., Wozney, J.M. & Hardwick, W.R. (2003) Space-providing expanded polytetrafluoroethylene devices define alveolar augmentation at dental implants induced by recombinant human bone morphogenetic protein 2 in an absorbable collagen sponge carrier. Clinical Implant Dentistry and Related Research 5: 112–123.
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The efficacy of BMP-2 preloaded on bone substitute or hydrogel for bone regeneration at peri-implant defects in dogs
Authors’ affiliations: Ui-Won Jung, In-Kyeong Lee, Jin-Young Park, Department of Periodontology, Research Institute for Periodontal Regeneration, College of Dentistry, Yonsei University, Seoul, South Korea Daniel S. Thoma, Christoph H. F. H€ ammerle, Ronald E. Jung, Department of Fixed and Removable Prosthodontics and Dental Material Science, Dental School, University of Zurich, Zurich, Switzerland
Key words: Bone morphogenetic protein-2, bone substitutes, dental implants, drug delivery
Corresponding author: Prof. Ui-Won Jung Department of Periodontology, Research Institute for Periodontal Regeneration College of Dentistry, Yonsei University, 50-1, Yonsei-ro, Seodaemun-gu Seoul 120-752, South Korea Tel.: +82-2-2228 3185 Fax: +82-2-392 0398 E-mail: [email protected]
apicocoronally, and 8 mm mesiodistally) were surgically prepared on buccal sides of the left and
system, hydrogel, polyethylene glycol Abstract Objectives: The objective of this experiment was to test whether or not a synthetic bone substitute (SBS) was more effective than a polyethylene glycol hydrogel as a carrier material for bone morphogenetic protein-2 (BMP-2) when attempting to regenerate bone. Material and methods: Two identical, box-type dehiscence defects (4 9 4 mm buccolingually and right edentulous ridge in five beagle dogs. Following implant placement, the defects either received (i) no graft, (ii) SBS+hydrogel, (iii) SBS+BMP-2 loaded hydrogel, and (iv) BMP-2-loaded SBS+hydrogel. The animals were euthanized at 8 weeks postsurgery. Radiographic and histomorphometric analyses were performed. Results: The hydrogel alone was not able to stabilize the grafted bone particles at 8 weeks, and SBS+hydrogel group did not significantly differ from the control group in all volumetric measurements. On the other hand, extensively regenerated new bone was connected with most of the remaining SBS particles in the BMP-2 groups. The BMP-2 groups exhibited significantly greater new bone formation (10.65 mm3 and 1.47 mm2 in the SBS+BMP-2-loaded hydrogel group; 14.17 mm3 and 0.93 mm2 in the BMP-2-loaded SBS+hydrogel) than non-BMP-2 groups (1.27 mm3 and 0.00 mm2 in the control group; 2.01 mm3 and 0.19 mm2 in the SBS+hydrogel group) in volumetric and histomorphometric analyses (P < 0.001). However, there were no significant differences between both BMP-2 groups. Conclusion: BMP-2 could yield enhanced bone regeneration in the critical-size peri-implant defects regardless of whether SBS or hydrogel is used for preloading, although the outcomes seem to be more reproducible with BMP-2 preloaded on SBS.
*These authors contributed equally to this study. Date: Accepted 27 August 2014 To cite this article: Park J-Y, Thoma DS, H€ammerle CHF, Jung RE. The efficacy of BMP-2 preloaded on bone substitute or hydrogel for bone regeneration at peri-implant defects in dogs. Clin. Oral Impl. Res. 26, 2015, 1456–1465. doi: 10.1111/clr.12491
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Complete embedment of dental implants in bone tissue is a prerequisite for long-term success. However, clinical situations with sufficient width of alveolar bone for the placement of an implant with the adequate diameter are hard to encounter. It is well documented in the literature that after tooth extraction variety of soft and hard tissue alterations are initiated and lead to a reduced alveolar ridge contour (Amler et al. 1960; Pietrokovski & Massler 1967; Jung et al. 2013a). As a consequence, coronal dehiscence-type defect often occurs around implant placed in the atrophied alveolar ridge. To cover the entire surface of the implant by bone tissue, various types of regenerative procedures have been attempted (H€ammerle
et al. 1996, 2002). Due to invasiveness of harvesting autogenous bone, various bone substitutes have been developed and demonstrated successful clinical outcomes (Springer et al. 2004; Hallman & Thor 2008; Jensen & Terheyden 2009). Nevertheless, as osteoconductive bone substitute has limited bone regenerative property, it is short of being the perfect alternative of autogenous bone. Recombinant human bone morphogenetic protein-2 (rhBMP-2) has emerged as a potent osteoinductive solution. The osteogenic effect of rhBMP-2 has been widely investigated and demonstrated successful bone regeneration (Choi et al. 2002; Kim et al. 2002; Wikesj€ o et al. 2003; Huang et al. 2008; Jung et al. 2008, 2009c). rhBMP-2 in a colla-
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Jung et al Hydrogel and bone substitute as a BMP-2 carrier
gen carrier has been commercially available for maxillofacial reconstruction since 2007. However, high dose of rhBMP-2 (1.5 mg/ml) is needed to exhibit biological activity when used in a collagen carrier. High cost and safety issues might be barriers to common clinical use of rhBMP-2. The development of an optimal carrier system could solve these problems, as it has been shown that the regenerative potential of growth and differentiation factors depend upon the carrier material (Hunt et al. 2001; Kim et al. 2005; Hong et al. 2006; Jung et al. 2006b). Such a carrier material should be biocompatible, mechanically stable within defect spaces, have sufficient plasticity for easy defect filling and manipulation, and eventually be replaced by newly formed bone. In addition to these requirements, the release kinetics of rhBMP2 from the carrier material is an important criterion for optimum bone regeneration and also for reducing the dose of rhBMP-2. Hydrogels made of polyethylene glycol (PEG) can serve as an in situ forming matrix for optimal cell ingrowth and retention of bioactive proteins (Lutolf et al. 2003). In recent in vitro and in vivo studies, it was demonstrated that beneficial release kinetics of rhBMP-2 can be achieved by entrapping rhBMP-2 as well as a parathyroid hormone peptide in this synthetic matrix (Weber et al. 2002; Jung et al. 2006a, 2007a,b, 2008; H€ anseler et al. 2012). In regard to mechanical properties, the hydrogel alone may be insufficient to maintain space against the pressure of soft tissue. To reinforce the mechanical strength, a combination of particulated bone substitutes with the PEG hydrogel enabled highly efficient and localized bone regeneration (Jung et al. 2007a, 2009a,b; Herten et al. 2009; Thoma et al. 2009). Calcium phosphates have been of great interest as a synthetic bone substitute (SBS) material due to its chemical similarity with inorganic component composing human bone (Daculsi et al. 1989). In addition, its formulation and configuration are easily modifiable, and various composites of hydroxyapatite and b-tricalcium phosphate are available in dental field. Hydroxyapatite is known to be scantily bioresorbable while b-tricalcium phosphate more readily undergoes bioresorption. The resorption rate can be controlled by the proportion of hydroxyapatite and b-tricalcium phosphate. The objective of this experiment was to test whether or not the SBS was more effective than the PEG hydrogel as a carrier material for BMP-2 when attempting to regenerate bone.
Materials and methods Preparation of the rhBMP-2 and the carrier system
The rhBMP-2 expressed in Escherichia coli was produced at the research institute, Cowellmedi Co. LTD, South Korea. Briefly, the RNAs extracted from the human osteosarcoma cells were reverse-transcribed into cDNA encoding the mature peptide of BMP-2 protein, which was amplified by polymerase chain reaction (PCR). The PCR product was cloned into a plasmid vector. Following highcell-density cultivation, the cultured biomass was collected via repeated centrifugation from which the inclusion bodies were harvested. The inclusion bodies were solubilized and diluted in a renaturation buffer, and then, the active rhBMP-2 proteins were purified and eluted.
Experimental design
A total of 20 implants (SLActiveâ Bone Level Implant, Straumann AG, Basel, Switzerland; a diameter of 3.3 mm and a length of 8 mm) were bilaterally placed in the edentulous mandible. In four peri-implant dehiscence defects, the following four treatment modalities were randomly applied to achieve even number of distribution into each defect location within the mandible.
• • • •
Control group: empty SBS+hydrogel group: SBS mixed with hydrogel SBS+BMP/hydrogel group: SBS mixed with BMP-2 (0.1 mg)-loaded hydrogel BMP/SBS+hydrogel group: BMP-2 (0.1 mg)-loaded SBS mixed with hydrogel
Surgical procedures
Synthetic matrix and bioactive peptides
Anesthesia
This study used a PEG-based hydrogel as a synthetic matrix and carrier system for rhBMP-2. Preparation of the gel was performed in a manner similar to previously published protocols (Elbert & Hubbell 2001; Elbert et al. 2001; Jung et al. 2007a). Shortly, a base catalyzed Michael type addition of a thiol to an acrylate group each linked to a PEG molecule which are mixed in an automatic mixing syringe constitutes the ground mechanism for the gelation. The network conformation obtained after a gelation time of about 4 min degrades in puffer by hydrolysis in vitro within 10 days. The SBS (Straumann Bone Ceramicâ, Straumann AG, Basel, Switzerland) used in this study is fully synthetic biphasic calcium phosphate consisting a mixture of 60% hydroxyapatite and 40% of b-tricalcium phosphate. The bone substitute demonstrates partial resorption and substitution with vital bone (Cordaro et al. 2008; Froum et al. 2008).
Surgical procedures were performed under general anesthesia induced by intravenous injection of atropine (0.04 mg/kg; Kwangmyung Pharmaceutical Ind. Co. Ltd., Seoul, Korea) and intramuscular injection of a combination of xylazine (Rompun, Bayer Korea Co., Seoul, Korea) and ketamine (Ketara, Yuhan Co., Seoul, Korea) followed by inhalation anesthesia (Gerolan, Choongwae Pharmaceutical Co., Seoul, Korea). Routine dental infiltration anesthesia was used at the surgical sites. The mandibular forth premolar and first molar (P4 and M1) were bilaterally extracted. The flap was replaced and sutured. Oral prophylaxis was performed on the remaining dentition in conjunction with the extractions. The stitches were removed 10 days postextraction. The defect sites were allowed to heal for 8 weeks. A horizontal midcrestal incision was made on the edentulous experimental site, and vertical incisions were made through the mucogingival junction extending into the alveolar mucosa at the mesial and distal end of the defect. The mucoperiosteal flaps were elevated to expose the edentulous alveolar ridge. Two identical, box-type dehiscence defects (4 9 4 mm buccolingually and apicocoronally, and 8 mm mesiodistally) were surgically prepared on buccal sides of the left and right edentulous ridge. In each of the four defects, an implant was placed following serial drilling according to the standard protocol. The implant’s shoulder was located at the level of the alveolar crest (Fig. 1a and 1b). Of SBS, 0.175 g was weighed into a bowl. Each component of the PEG hydrogel matrix
Animals
Five male beagle dogs with approximate mean age 18 months and weight 15 kg were used. All animals were free of heart worms and were quarantined. The animals were also examined to have intact dentition with healthy periodontium. All experiments were performed at the animal laboratory accredited by Association for Assessment and Accreditation of Laboratory Animal (AAALAC) international, after the ethical approval of the institutional Animal Care and Use Committee regarding animal selection, management, and surgery protocol.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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(a)
•
(b)
Any augmented volume on either side of the defect in its mesiodistal aspect was excluded.
The following parameters were calculated within the defect:
• • • • (c)
(d)
Total augmented volume (TAV; mm3) New bone volume (NBV; mm3) Remaining bone substitute volume (RBV; mm3) Non-mineralized tissue volume (NMV; mm3)
Histomorphometric analysis
Fig. 1. Clinical photographs representing surgical procedures. Lateral view (a) and occlusal view (b) following preparation of box-type defects with buccal dehiscence on the edentulous ridge. Lateral view (c) and occlusal view (d) following implant placement and simultaneous bone grafting with the mixture of synthetic bone substitute and hydrogel. M = mesial. D = distal.
(74 ll PEG A, 74 ll PEG B, 57 ll activator) were placed into the bowl next to the SBS granules. For the SBS+BMP/hydrogel group, 23 ll of rhBMP-2 (4.4 mg/ml) was added to the mixture. Using a spatula, the solutions were mixed with the SBC granules and applied to the defect (Fig. 1c and 1d). For the BMP/SBS+hydrogel group, the SBS granules were soaked in 150 ll of rhBMP-2 (0.67 mg/ ml) for 4 h in a refrigerator (4°C) prior to the experiment, and moisture was allowed to evaporate. For the rhBMP-2 groups, gelation took place within 10–11 min, and for positive control group, 5–6 min. Groups were assigned so that they can be distributed into each defect site the same number of times. The buccal flaps were carefully released without tension and sutured. Each dog was maintained on a soft diet throughout the study. Postsurgical management included intramuscular administration of antibiotics (cefazolin sodium 20 mg/kg; Yuhan Corporation, Seoul, Korea) and daily topical application of a 0.2% chlorhexidine solution (Hexamedinâ, Bukwang Pharmaceutical Co., Seoul, Korea) for infection control. Two weeks postoperatively, the sutures were removed. The animals were euthanized at 8 weeks using an overdose of pentobarbital (90– 120 mg/kg; IV). Block sections including
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grafted sites and surrounding alveolar bone and soft tissues were collected and fixed in 10% formalin for 10 days. A micro-CT (SkyScan 1072, SkyScan, Aartselaar, Belgium) was taken at a resolution of 35 lm (100 kV, 100 lA). The scanned data set was processed in DICOM format, and the area of interest was reconstructed with three-dimensional (3D) software (OnDemand3Dâ, Cybermed, Seoul, Korea). Then, the samples were dehydrated in a graded series of ethanol. Thereafter, they were embedded in methyl methacrylate without being decalcified according to standard procedures. The specimens were vertically sectioned in the center of the implants in a buccolingual direction and were stained with hematoxylin–eosin. Analysis Micro-CT analysis
The overall augmented contour was evaluated at the 3D reconstructed view. Boundaries were set as follows to standardize the region of augmented volume to be analyzed.
• •
Any augmented volume directly superior or lingual to the implant in its axial plane was excluded. Any augmented volume inferior to the buccal extension of inferior border of the defect was excluded.
The sections of each defect site were observed using a light microscopy (Olympus Multi-view microscope BH2, Tokyo, Japan). Histomorphometric analyses were performed by one masked experienced examiner, using image analysis software (Image-Pro PlusTM, Media Cybernetic, Silver Springs, MD, USA). The area of interest (AOI) was made of 1 mm width and 4 mm height from the implant platform. The following measurements were analyzed and recorded within the AOI (Fig. 2):
• • • • •
New bone area (NBA; mm2) Remaining bone substitute area (RBA; mm2) Bone-to-implant contact (BIC; %) BC-S (mm): distance from the bone crest to the implant shoulder CO-S (mm): distance from the most coronal point of osseointegration to the implant shoulder.
Statistical analysis
All statistical analyses were performed with software R, version 2.15.2 (http://www.r-project.org). The nonparametric mixed model was used to compare radiographic and histomorphometric parameters between four groups (Brunner & Langer 2000) with treatment group (Control, SBS+hydrogel, SBS+BMP/hydrogel, and BMP/SBS+hydrogel) as an independent factor and dog number as a random factor. Statistical significance level was 5%. Post hoc analyses were performed by pairwise comparison with modified p-value of 0.0083 rather than 0.05 (0.0167 for control value was zero).
Results Clinical findings
The addition of rhBMP-2 solution into both PEG hydrogel and bone substitute resulted in an extended gelation time, which caused
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Jung et al Hydrogel and bone substitute as a BMP-2 carrier
Fig. 2. Illustration of measuring parameters for histomorphometric analysis. Red box = area of interest (1 9 4 mm). Blue arrowhead = the shoulder of implant. Yellow arrowhead = the most coronal point of newly formed bone. White arrowhead = the most coronal point of osseointegration. I = implant.
delay during surgery. All implants and grafted materials remained submerged, and no complications were observed throughout the entire experimental period. Volumetric analysis: micro-CT
In the two BMP-2 groups, the overbuilt materials on the buccal aspect of the implants preserved their grafted dome shape and maintained their coverage of the implant surfaces. In the control group, however, the implant surface remained exposed, and only partial coverage was achieved and grafted materials were spread out in the SBS+hydrogel group (Fig. 3). In terms of NBV, SBS+BMP/hydrogel group and BMP/SBS+hydrogel group exhibited a significantly larger volume than the control and SBS+hydrogel group (P < 0.001; Table 1 and Fig. 4). RBV of both BMP-2 groups showed a statistically significant difference to that of the SBS+hydrogel group (P < 0.001, Table 1). Histological observation
No inflammatory reaction or failure of osseointegration was found in any of the samples. In the control group, minimal bone formation was observed, and the buccal defect was mostly occupied by fibrous tissue (Fig. 5). A periosteum-like dense connective tissue layer lined the implant surface at the defect.
Fig. 3. 3D reconstructed micro-CT views and cross-sectional views of control, SBS+hydrogel, SBS+BMP/hydrogel, and BMP/SBS+hydrogel groups.
Table 1. Volumetric analysis of micro-computed tomography (n = 5; mm3) NBV Control Mean SD Median SBS+hydrogel Mean SD Median SBS+BMP/hydrogel Mean SD Median BMP/SBS+hydrogel Mean SD Median
1.75 2.23 1.27
RBV
NMV
TAV
NA NA
510.48 219.39 552.28
512.23 218.25 552.39
3.47 4.19 2.01
4.46 4.51 2.54
588.83 359.25 504.75
596.76 353.18 508.73
10.75 5.61 10.65*†
12.44 8.48 12.16†
643.25 194.12 583.27
666.44 187.53 611.95
13.80 5.63 14.17*†
15.77 6.34 16.75†
628.43 190.61 550.06
657.99 180.07 591.14
NBV, new bone volume; RBV, remaining bone substitute volume; NMV, non-mineralized tissue volume; TAV, total augmented volume; SBS, Synthetic bone substitute. *Significantly different from the control group (P < 0.001). †Significantly different from the SBS+hydrogel group (P < 0.001).
Following 8 weeks of healing, most of the grafted materials at the defect site had disappeared in the SBS+hydrogel group (Fig. 6). Only one histologic sample (#5 dog) showed a cluster of remaining materials. The SBS particles on the outer layer of grafted mass were surrounded by connective tissue without connection with bone. SBS particles at the inner region of the defect were in contact with newly formed bone sprouting from the floor of the defects. Compared to the non-BMP-2 groups, the BMP-2 groups including the SBS+BMP/hydro-
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
gel and the BMP/SBS+hydrogel groups maintained the original grafted dimension with prominent bone regeneration. In some samples, NB extended over the top of the implants. The two BMP-2 groups exhibited similar healing patterns irrespective of the carrier of BMP-2, whether it was the graft particle or the hydrogel. A concentric lamellation process began forming primary osteons at the NB surrounding the RBC. In the SBS+BMP/hydrogel group (Fig. 7), most of the remaining SBS particles were in close contact with the projection of NB. A few particles
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Jung et al Hydrogel and bone substitute as a BMP-2 carrier
Fig. 4. Scatter plot and the median (the small bar) representing the new bone volume (mm3). *Significantly different (P < 0.001).
(a)
(b)
presented contact with bone even at the outer connective tissue layer. Thin layer of new bone lined the majority of the length of the implant surface within the defect. The NB exhibited high cellularity and vascularity demarcating the mature old bone having well-developed lamellae and Haversian system. Highly dense Volkmann’s canals running perpendicularly to the osteons were found at the border of the old bone (Fig. 8 ). In the BMP/SBS+hydrogel group (Fig. 9), the original grafted volume seemed to be not only maintained but rather increased. SBS particles were observed lined up beneath the outer connective tissue layer. Substantial amount of new bone were formed making bony bridges between the particles. A large portion of the remaining SBS particles in the two BMP-2 groups were embedded in NB. Some SBS particles away from the new bone connection were existed outside of the graft bulk and embedded in soft tissue. A periosteum-like layer lined outside of the grafted materials and multinucleated giant cells appeared to be involved in the resorption process of the remaining SBS particles featuring concave surfaces (Fig. 10). In the high magnified view, almost completely resorbed bone particles were observed in the process of resorption in a soap-bubble-like appearance. Histomorphometric analysis
Fig. 5. Low (a) and high (b; the white box of a) magnified histologic views of the control group (H & E). V = vessels. P = periosteum-like connective tissue layer. I = implant.
(a)
(b)
Both BMP-2 groups exhibited significantly higher NBA, CO-S, BC-S, and BIC values than non-BMP-2 groups (P < 0.001, Tables 2 and 3 & Fig. 11). However, there was no difference between SBS+BMP/hydrogel and BMP/SBS+hydrogel groups in all measurements.
(c)
Discussion
Fig. 6. Histologic view of the SBS/hydrogel group (H & E). Three samples [#1 (a), #2 (b), and #5 (c)] out of a total of five with remaining bone substitute particles (RBS). NB = new bone. CT = connective tissue. I = implant.
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Bone regeneration at the standardized periimplant dehiscence defects was compared according to four different treatment modalities, and the addition of rhBMP-2 resulted in significantly greater new bone formation and more BIC than groups treated without rhBMP-2. The control group, which had no treatment, exhibited minimal resolution of the surgically created osseous defects. As the peri-implant box-type dehiscence defect prepared in the present study could not be resolved spontaneously, the size of the defect could be considered as a critical-sized defect. In such a large defect, the use of membrane is especially needed for favorable guided bone regeneration (GBR). Several studies reported
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Jung et al Hydrogel and bone substitute as a BMP-2 carrier
(a)
(b)
Fig. 7. Histologic view of the SBS+BMP/hydrogel group (H & E). (a) Low magnification (b) High magnified view of the top of the defect. NB = new bone. RBS = remaining bone substitute. I = implant.
(a)
(b)
Fig. 8. Histologic view of the SBS+BMP/hydrogel group (H & E). (a) Low magnification (b) Highly dense Volkmann’s canals (VC) running perpendicularly to the osteons found at the border of the old bone. NB = new bone. I = implant.
(a)
(b)
(c)
Fig. 9. Histologic view of the BMP/SBS+hydrogel group (H & E). (a) Low magnification (b) High magnified view of the top half of the defect (c) High magnified view of the bottom half of the defect. NB = new bone, RBS = remaining bone substitute. I = implant.
that the addition of membrane significantly enhanced bone regeneration and prevented fibrous encapsulation compared with sites treated without a membrane in dehiscence defect as well as lateral ridge augmentation
(Park et al. 2008; Urban et al. 2011; Fu et al. 2013; Jung et al. 2013b). Amorphous sol of PEG hydrogel before gelation can be customized to fit into the irregular defect configuration. Gel state of
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
hydrogel after setting can endure the external pressure having an extent of mechanical strength. Compared to the conventional barrier membrane, the injectable hydrogel is easy to use, as a pin or screw for fixation of the membrane is not required. In this study, PEG hydrogel was used as a membrane alternative for GBR procedure. Beside its hydrophilicity and biocompatibility, PEG has cellocclusive characteristic, which is one of the requisites for the barrier membrane. PEG gels consisting of multi-arm PEG molecules could prevent cell penetration, as the distances between the cross-linking points are smaller than the dimension of a cell (Wechsler et al. 2008). PEG hydrogel is a versatile material that can be used as a delivery system for bioactive molecules as well as a barrier membrane for GBR procedure (Jung et al. 2006a, 2008, 2009a,b). The optimal degradation time and physical properties of PEG hydrogel can be modulated depending on the purpose of use (Thoma et al. 2011, 2014). The dense network showed an increased degradation time and maintained the shape, while the loose network showed a relatively decreased degradation time. All PEG hydrogel groups with different degradation times have been shown to be biocompatible, as demonstrated by Dahlin using human gingival fibroblasts (Dahlin et al. 2014). However, too long-lasting nonporous gel mass might retard the initial recruitment and infiltration of the osteoprogenitor cells into the defect and jeopardize proper release of the captured molecules, in particular, when used as a delivery system of the growth factors. Therefore, the appropriate degradation time might be a critical factor to fulfill the requirements of an ideal carrier. Degradation time of the PEG hydrogel used in the present study was controlled to be degraded within 10 days in vitro. In the present in vivo study, it confirmed that no PEG hydrogel remained in histologic observation in all samples at 8 weeks. In SBS+hydrogel group without addition of BMP-2, only one sample exhibited localized SBS particles. Two of five samples showed no bone graft materials and minimal SBS particles were observed at the center of defect in the other two samples. Such observation could be explained that premature degradation of PEG hydrogel cannot stabilize the grafted particles before consolidation, as the osseous defect was large and uncontainable. Similarly, bovine bone particles were scattered in the non-contained type one-wall intrabony defect due to early resorption of the pure collagen matrix (Jung et al. 2011). Therefore, the PEG
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Jung et al Hydrogel and bone substitute as a BMP-2 carrier
(a)
(b)
Fig. 10. (a) Distant isolated particles featuring concave surfaces of resorption lined with multinucleated giant cells (BMP/SBS+hydrogel group) (b) Almost completely resorbed bone particle displaying a soap-bubble-like appearance (BMP/hydrogel+SBS group). Arrowheads = multinucleated giant cells. RBS = remaining bone substitute.
Table 2. Histometric analysis within the area of interest (n = 5; mm2) Control Mean SD Median SBS+hydrogel Mean SD Median SBS+BMP/hydrogel Mean SD Median BMP/SBS+hydrogel Mean SD Median
NBA
RBA
The others
0.01 0.01 0.00
NA NA
3.99 0.01 4.00
0.23 0.26 0.19*
0.29 0.30 0.30
3.48 0.53 3.63*
1.21 0.49 1.47*†
0.46 0.19 0.47
2.33 0.67 2.06*†
0.96 0.26 0.93*†
0.60 0.22 0.65
2.44 0.32 2.52*†
NBA, new bone area; RBA, remaining bone substitute area. *Significantly different from the control group (P < 0.001). †Significantly different from SBS+hydrogel group (P < 0.001).
Table 3. Histometric linear measurements (n = 5; mm). Control Mean SD Median SBS+hydrogel Mean SD Median SBS+BMP/hydrogel Mean SD Median BMP/SBS+hydrogel Mean SD Median
CO-S
BC-S
4.18 0.35 4.22
4.18 0.35 4.22
3.29 0.76 3.17
2.67 1.10 2.48*
0.86 1.27 0.36*†
0.51 0.94 0.00*†
0.81 0.49 0.90*†
0.25 0.38 0.00*†
CO-S, distance from the most coronal point of osseointegration to the implant shoulder; BC-S, distance from the bone crest to the implant shoulder. *Significantly different from the control group (P < 0.001). †Significantly different from SBS+hydrogel group (P < 0.001).
hydrogel, which has 10 days degradation time in the present study, might not be sufficient to act as a barrier membrane. Jung et al. showed that a different PEG hydrogel with an in vitro degradation time of approximately 4 months resulted in a comparable amount of vertical bone fill and mean dehiscence defect fill with a standard collagen membrane in clinical study (Jung et al. 2009a). In addition, the PEG hydrogel was
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revealed to perform the role of a barrier membrane in non-critical-size defects in rabbits and in dehiscence-type bone defects in dogs (Jung et al. 2006a, 2009b). In these particular studies, the remaining PEG could still be identified after 4 weeks and 6 months of healing period in the histological evaluation, respectively. In situ overbuilt bolus maintained the volume and exhibited excellent bone regen-
eration at the defects in most samples of the BMP-2 groups in spite of early degradation of the PEG hydrogel. Possible explanation would be that BMP-2 might accelerate substitution of PEG hydrogel with dense provisional matrix for future mineralized tissue by stimulation of osteogenic potential at the early stage of healing, which could reinforce the mechanical strength of grafted mass against the external pressure. Therefore, early degradation of the hydrogel might be beneficial for rapid release of BMP-2 molecules. H€anseler et al. compared the differences in release kinetics and biological activity of nonglycosylated rhBMP-2 and glycosylated rhBMP-2 incorporated into the combination of bovine hydroxyapatite with PEG hydrogel in vitro (H€anseler et al. 2012). They confirmed the equivalent cell viability and biological activity between both rhBMP-2 in spite of the difference in release profile. The rhBMP-2 incorporated into PEG hydrogel revealed reduced bioactivity compared to positive control containing rhBMP-2 only. On this phenomenon, they assumed that a chemical interaction between rhBMP-2 and PEG hydrogel during gelation might affect to the reduced bioactivity of the incorporated rhBMP-2 molecule. When the rhBMP-2 was adsorbed to bovine hydroxyapatite, less concentration of active rhBMP-2 was detected compared to incorporation into PEG hydrogel, which might be associated with high affinity of BMP-2 molecules to hydroxyapatite (Boix et al. 2005). Combination of PEG hydrogel for initial burst and hydroxyapatite for sustained release of rhBMP-2 might be optimal to stimulate the progenitor cells (Brown et al. 2011). The release kinetics could be influenced by the degradation of the carrier material itself. As bovine hydroxyapatite is categorized as a non-resorbing material, the effect of the material resorption might be negligible. However, as SBS used in the present study contains high proportion of fast-resorbing tricalcium phosphate, the release by the degradation of the material must be considered. Therefore, further long-term study to evaluate the degradation rate of the SBS itself is needed to fully apprehend BMP-2 release kinetics. When the two BMP-2 groups were compared, greater bone formation was observed when BMP-2 was loaded onto SBS rather than the hydrogel. When hydrogel is used as the carrier, hydrogel is applied to the defect in mixture with BMP-2 at the final step. Therefore, it did not need an additional soaking time and could minimize the loss of BMP-2 due to bleeding or aspiration during surgery.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Jung et al Hydrogel and bone substitute as a BMP-2 carrier
(a)
(b)
(c)
(d)
Fig. 11. Scatter plot and the median (the small bar) representing (a) the new bone area, (b) bone-to-implant contact within the defect, and (c and d) the linear measurements (n = 5). CO-S = distance from the bone crest to the implant shoulder. BC-S = distance from the most coronal point of osseointegration to the implant shoulder.*Significantly different (P < 0.001).
However, as addition of BMP-2 to both hydrogel and SBS delayed gelation time and prolonged waiting time prior to flap closure, flaps had to be closed before complete gelation took place and substantial amount of BMP-2 might have been adsorbed undesirably to the surrounding soft tissue. In this case, the hydrogel may still be in its sol state, which would not have enough viscosity to maintain the grafted shape and prone to collapse. In the present study, the SBS+BMP/hydrogel group also showed a wider range of BIC compared to the SBS/BMP+hydrogel group. This might be caused by a further delay in gelation because of direct loading on hydrogel. Moreover, graft materials were overbuilt in consideration of the shrinkage during healing, which would have been sub-
jected to pressure from the overlying soft tissues. To obtain a more predictable gelation time, the concentration of the activating solution needs to be closely adjusted in consideration of the dilution of the mixture following the addition of BMP-2 solution as well as contamination with blood and saliva inside the oral cavity. It is concluded that BMP-2 could yield enhanced bone regeneration in the criticalsize peri-implant defects regardless of whether SBS or hydrogel is used for preloading, although the outcomes seem to be more reproducible with BMP-2 preloaded on SBS.
Acknowledgements:
We thank the Institut Straumann AG for providing us
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
with the PEG hydrogel, synthetic bone substitute, and dental implants. We also gratefully thank Dr Minglan Chang and Ms Sora Yoon for helping with the experimental surgeries and histomorphometric analyses.
Conflict of interest The authors declare no conflict of interest.
Source of funding statement This study was financially supported by an ITI research grant from the ITI foundation (821-2012).
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