J Mater Sci: Mater Med (2015)26:194 DOI 10.1007/s10856-015-5526-z
BIOMATERIALS SYNTHESIS AND CHARACTERIZATION
Original Research
Influence of synthetic polyethylene glycol hydrogels on new bone formation during mandibular augmentation procedures in Goettingen minipigs Phillipp Brockmeyer1 • Katharina Kramer2 • Sebastian Krohn3 • Philipp Kauffmann1 • Corinna Mauth4 • Michel Dard5 • Henning Schliephake1 Rudolf Matthias Gruber1
•
Received: 18 March 2015 / Accepted: 28 May 2015 Ó Springer Science+Business Media New York 2015
Abstract Polyethylene glycol hydrogels (PEG) have been used as slow release carrier for osteoinductive growth factors in order to achieve a retarded delivery. However, there have been concerns about negative effects on bone regeneration. This study aims to test whether PEG hydrogels themselves affect new bone formation (NBF), when used as a carrier during mandibular augmentation procedures. In a randomized split-mouth design, bilateral mandibular bone defects were surgically created in 12 Goettingen minipigs, and subsequently augmented, using PEG hydrogel on one side of the mandible. The contralateral sides, without PEG, served as controls. After 4 and 12 weeks, bone formation was evaluated in six animals each. A comparison of the data, using a three-way analysis of variance (ANOVA), revealed a significant effect of the healing time and the region of the graft on the distribution and enhancement of NBF (P \ .0001, respectively). Although a 0.3 % (95 %-CI [-5.5; 4.8]) lower volume density of newly formed bone could be observed over all PEG hydrogel sections, in contrast to the contralateral controls, the analysis revealed no
& Phillipp Brockmeyer [email protected] 1
Department of Oral and Maxillofacial Surgery, University Medical Centre Goettingen, Robert-Koch-Str. 40, 37075 Go¯ttingen, Germany
2
Department of Medical Statistics, University Medical Centre Goettingen, Go¯ttingen, Germany
3
Department of Prosthodontics, University Medical Centre Goettingen, Go¯ttingen, Germany
4
Private Practice, Basel, Switzerland
5
Department of Periodontology and Implant Dentistry, College of Dentistry, New York University, New York, NY, USA
clinically significant effects of the PEG hydrogel treatment on the total level (P = 0.90), and the distribution of NBF (P = 0.54). In conclusion, PEG hydrogels do not affect NBF when used as a carrier for osteoinductive growth factors during mandibular augmentation procedures.
1 Introduction Combinations of synthetic carriers, along with osteoinductive acting growth factors, have been discussed as an alternative to autogenous bone grafts, to avoid donor site morbidity [1], and to improve new bone formation (NBF) [2], in reconstructive surgery. Among the known growth factors, bone morphogenetic proteins (BMPs) [2–5], and, in particular, BMP-2 [6–8], showed their osteoinductive character in many investigations. The Food and Drug Administration (FDA) approved a combination of recombinant human BMP-2 (rhBMP-2), with an absorbable collagen sponge (ACS) as a bone substitute material (BSM), for the treatment of degenerative disc disease (DDD) [9], open tibia shaft fractures [10], and sinus floor [3] and localized alveolar ridge augmentations [11]. In the use of osteoinductive growth factors, the carrier is crucial [12]. Ideal carrier materials should be malleable (in the anatomically desired form), possess sufficient mechanical resistance, be absorbable while regenerating bone, and provide the controlled release of active osteoinductive growth factors [12]. While superficially adsorptive-coated materials release factors by a burst release with a loss of up to 80 per cent of activity within the first 48 h after implantation [13], bioactive proteins can also be incorporated into mechanically stable, resorbable carriers [14], which slow the release. Polyethylene glycol hydrogels (PEG) were studied as a carrier for BMP-2 [15], with the advantage of suppressing a burst release of the
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factor. Liquid or solid PEG hydrogels are formed out of ethylene glycol monomers in a polymerization reaction [16]. Preclinically, PEG hydrogels formed a relatively inert matrix, which retained bioactive proteins [17–19]. Because of its entrapment in the hydrogel, rhBMP-2 release could be retarded in these investigations [17–19]. However, it is not yet clear whether PEG hydrogels may influence NBF due to their structure. Hydrogels have specific physical and biological properties which include mechanisms and dynamics of gel formation, mechanical properties and degradation behaviour [20] which may affect surrounding tissue. Moreover, the pH-value seems to be crucial for a seamless integration of the hydrogel into the surrounding tissue [21]. It is described that most cells of the extracellular matrix (ECM) have no receptors to bind to the hydrogel-forming polymers [20], leading to inhibition of cell–matrix adhesion [22]. For this reason, it is questionable whether the PEG hydrogel matrix could adversely affect the integration of new bone formation into the scaffold. Towards this end, we aim to investigate whether PEG hydrogels may affect NBF during the repair of mandibular bone defects in a split mouth design in 12 Goettingen minipigs.
2 Materials and methods 2.1 Animals and study design Since the Minipig [23], and the sample size [24, 25] have been proven in a number of preclinical studies in the field of implant dentistry and oral and maxillofacial surgery, the present evaluation was performed on 12 adult female Goettingen minipigs (Ellegard Goettingen Minipigs ApS, Dalmose, Denmark), carried out in accordance with regulations governing animal housing (German Animal Welfare Act and Directive 2010/63/EU). The Lower Saxony State Office for Consumer Protection and Food Safety (registration no.: 3314.42502-04-013/09), and the committee on animal welfare of the University of Goettingen, approved this study. Two veterinarians observed all animals throughout the study period for signs of pain or unacceptable conditions. The animals were randomly assigned to two groups (Group A: 4 weeks of observation; Group B: 12 weeks of observation). In both groups, in a split-mouth design, one side of the mandible was randomly allocated to the experimental treatment (PEG hydrogel), while the contralateral sides served as control (no PEG applied).
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intramuscular administration of azeparone (StresnilÒ, Jansen; Neuss, Germany; 5 mg/kg bw) and intravenous injection of thiopental (TrapanalÒ, Byk-Gulden; Konstanz, Germany; 7 mg/kg bw). Inhalation anaesthesia was maintained with isoflurance (2 %). Dipidolor (Janssen; 110 lg/kg bw per h) and ketamine (WDT; Garbsen, Germany; 2.5 mg/ kg bw per h) were administered for 3 days to alleviate postoperative pain. Perioperative antibiotic protection was provided by penicillin and streptomycin (AnimedicÒ, Animedica; Senden-Boesensell, Germany; 40 mg penicillin/60 mg streptomycin/kg bw per day). Sixteen weeks after extraction, partial thickness alveolar defects (20 L 9 8 W 9 8 H mm) were surgically created in the extracted areas on both sides of the mandibles. On the test sides, PEG hydrogel blocks were applied to the bone defects, whereas in the contralateral controls no PEG was used and the defects were left empty. To prepare the PEG hydrogel blocks, 136.5 ll of PEG A (PEG A, Mn = 15 kDa) and PEG B (PEG B, Mn = 3.5 kDa; Institut Strautmann AG, Basel, Switzerland) solution mix, 672 ll of sterile water and 105 ll of activator solution were assembled together, and immediately added to a block mould. PEG hydrogel blocks were shaped exactly in accordance with the created defects. Wound closure was performed using Vicryl sutures (Vicryl R, 3x0; Ethicon, Norderstedt, Germany). At the end of the observation period (4 and 12 weeks, respectively), the tissue blocks of the bone defects were retrieved and fixed in 10 % phosphate buffered neutral formalin solution. 2.3 Histology The tissue blocks were infiltrated with composite resin (Technovit 9100; Heraeus-Kulzer, Hanau, Germany) and cut to a thickness of 200 lm perpendicular to the axis of the local jawbone (Exakt, Norderstedt, Germany). Specimens were ground and polished to approximately 25 lm of thickness, as described by Donath and Breuner [26]. Up to 15 sections of the defect area were prepared. In order to take into account the expansion of the bone defects, two sections from the lateral region (mesial and distal, respectively), and one section from the central region of the graft were used for the histomorphometric evaluation. Since the Smith–Karagianes staining (methylene blue/alizarin red S) has proven for histological bone preparations [27], and provides good contrast between bone and soft tissues, it was performed for the microscopic examination and the histomorphometric assessment of new bone formation (Zeiss, Oberkochen, Germany).
2.2 Surgery 2.4 Histomorphometry In an initial surgical procedure, all premolars and first molars were extracted from both sides of the mandible under general anaesthesia. General anaesthesia was induced by
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A Zeiss Axiovert 200 microscope in combination with a digital camera (Axiovision 4.4 software, Axiocam; Zeiss)
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was used to generate combined sequential colour images in a 95 magnification. Complete images of the samples were assembled from several individual images with the MosaiX software (Zeiss). For histomorphometric evaluation of the sections, the image analysis software Axiovision Imaging (Zeiss Microlmaging GmbH, Goettingen, Germany) was used. The image analysing systems were calibrated at a predefined distance. In order to evaluate the gradient of NBF, depending on the distance to the local mandibular bone, six L-shaped regions of interest (ROIs, each 1 mm of height) were defined parallel to the border between local bone and augmentation material (Fig. 1). Each ROI was evaluated for their volume density (VD) of newly formed bone tissue. VD was defined as percentage of newly formed bone within the area of analysis.
Inc., Cary, NC, USA). The diagrams presented show the representative mean values with the corresponding 95 % confidence intervals (95 %-CI).
2.5 Statistical analysis
3.2 Histological evaluation
The mean volume density of all three sections was used as the endpoint for the analysis. Values were analysed using a three-way analysis of variance (ANOVA), with the factors ‘Time’ (4 vs. 12 weeks), ‘Treatment’ (PEG hydrogel vs. no BSM), and ‘Region of interest’ (ROI 1–6). Subsequently, pairwise comparisons between ROI 1 and ROIs 2–6 were performed; in order to adjust for multiple comparisons the Dunnett procedure was applied in this case. We assumed that the residuals are normally distributed. This assumption might lead to the fact that CIs are not range preserving. All tests were performed at a significance level of a = 5 %, using the statistical software SAS (SAS 9.3; SAS Institute
After 4 weeks of observation, a clear ingrowth of bone into the augmentation, adjacent to the local mandibular bone, was observed on both sides of the mandible (Fig. 2). On the test side, NBF occurred most prominently in ROIs 1 and 2, where dense new bone grew into the augmentation material by osteoconduction. In ROI 3, the centre of the augmentation, newly formed bone decreased significantly. From this region to the periphery (ROIs 4–6), NBF decreased further. On the control sides of the 4-week group, NBF could mainly be observed in ROIs 1 and 2, which appeared as a dense layer directly adjacent to the local mandibular bone. In ROIs 3–6, bone was rarely found.
Fig. 1 Overview of all regions of interest (ROI 1–6) of the augmentation. Smith–Karagianes staining (magnification factor 92.5)
Fig. 2 Overview of the augmentation (4 weeks, control group). Resident bone (RB), new bone formation (NBF), border between resident bone and new bone formation (white line), appositional bone which was not evaluated (AB). Smith–Karagianes staining (magnification factor 92.5)
3 Results 3.1 Clinical evaluation Between 3 and 5 days, a minor transient postoperative swelling could be observed at the surgery site. All animals showed normal wound healing after teeth extraction and augmentation procedures, without signs of inflammation or infection. During the entire study period, there was no adverse event that affected the health of any animal.
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Overall, newly formed bone appeared in small immature trabeculae after 4 weeks, on both sides of the mandible (Fig. 3). After 12 weeks of observation, the gradient of NBF differed significantly from that observed after 4 weeks. More newly formed bone could be observed overall, which was almost indistinguishable from the local jawbone (Fig. 4). On the test side, in ROIs 1 and 2, newly formed bone appeared dense and mature. In ROIs 3–6, an increase in NBF was observed. On the control side, an increase in NBF was observed in all regions, which was hardly distinguishable from the local jawbone.
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3.3 Histomorphometric evaluation After 4 weeks, in all sections, mean bone density of newly formed bone decreased from ROI 1–ROI 6. On the test side (PEG hydrogel applied) VD decreased from 72.6 % (95 %CI [61.8; 83.4]) in ROI 1–2.5 % (95 %-CI [-0.5; 5.6]) in ROI 5, whereas no NBF could be observed in ROI 6. On the control side (no BSM applied), VD decreased from 67.1 % (95 %-CI [56.4; 77.9) in ROI 1–1.3 % (95 %-CI [-1.8; 4.3]) in ROI 5. Also, in these sections, no newly formed bone could be observed in ROI 6 (Fig. 5). After a total of 12 weeks, more newly formed bone could be observed in each region. On the test side (PEG hydrogel applied), VD ranged from 76.8 % (95 %-CI [68.0; 85.5]) in ROI 1–5.0 % (95 %-CI [-5.2; 15.1]) in ROI 6. On the control side (no BSM applied), VD ranged from 76.1 % (95 %-CI [67.3; 84.9]) in ROI 1–7.4 % (95 %-CI [-2.8; 17.5]) in ROI 6 (Fig. 5). The comparison of the data revealed a significant effect of the healing time on NBF (P \ .0001). After 4 weeks, over all sections, 18.7 % (95 %-CI [12.0; 25.4]) less VD of newly formed bone could be observed, in contrast to the 12-week time point. Moreover, a significant influence of the region of the graft on the distribution and enhancement of NBF could be observed (P \ .0001). The highest VD values of NBF were observed in ROI 1. The pairwise comparison of ROI 1, with all other regions of interest, revealed significant differences in NBF between ROI 1 and ROIs 2–6 (all P values\0.0007). Although a 0.3 % (95 %-CI
Fig. 3 Details of new bone formation in ROI 1 of the augmentation (4 weeks, PEG group). Smith–Karagianes staining (magnification factor 963)
Fig. 4 Overview of the augmentation (12 weeks, PEG group). Resident bone (RB), new bone formation (NBF), border between resident bone and new bone formation (white line), appositional bone which was not evaluated (AB). Smith–Karagianes staining (magnification factor 92.5)
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Fig. 5 Diagram illustrating the volume density of new bone formation after 4 and 12 weeks. Mean volume density and corresponding 95 % confidence intervals. PEG hydrogel treatment after 4 weeks (green), PEG hydrogel treatment after 12 weeks (red), no treatment after 4 weeks (blue), no treatment after 12 weeks (black) (Color figure online)
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[-5.5; 4.8]) lower volume density of newly formed bone could be observed over all PEG hydrogel sections, in contrast to the contralateral controls, the analysis revealed no clinically significant effects of the PEG hydrogel treatment on the total level (P = 0.90), and the distribution of NBF (P = 0.54).
4 Discussion The results of various studies have shown that PEG hydrogels due to their physical and biological characteristics may lead to lower cell adhesion [20], which could influence the integration of the new bone formation [19, 22]. Towards this end, the present investigation aims to test the hypothesis that PEG hydrogels influence new bone formation when used as a carrier during mandibular augmentation procedures. To investigate the effect of the healing time on the distribution or enhancement of NBF, two different time points (4 and 12 weeks) were each analysed. After 4 weeks’ observation time, both test and control sides showed a gradient of NBF, which is described in the literature as typical of osteoconductive augmentation materials [28, 29]. The highest rates of newly formed bone were measured in the region close to the locally constant jawbone (ROI 1). From there, the values decreased significantly with increasing distance from the local jawbone (ROI 6). The results are comparable to those described by Busenlechner et al. [30] and Fuerst et al. [31], who investigated the gradient of NBF in different osteoconductive bone substitute materials during sinus floor augmentations in a curve-based manner. After 12 weeks, the gradient of NBF differed from that after 1 month. On both sides of the mandible, a strong increase in NBF could be observed in all regions, still with decreasing gradient. During the ingrowth of bone tissue, osteoblasts and their precursor cells compete with rapidly proliferating and ingrowing cells from the surrounding connective tissue [32]. Probably because of this reason, a sufficient NBF is only possible in the basal regions of the graft near the local jawbone, after this time period [32]. It is described, that most proteins of the extracellular matrix have no receptors to bind to the hydrogel-forming polymers, leading to lower cell adhesion [20]. However, most cell types are able to bind to the amino acid sequence arginine–glycine–aspartic (RGD), and PEG hydrogels have been modified with this peptide to promote cellular adhesion [19, 22, 33–35]. Thoma et al. had investigated the biodegradation and hard and soft tissue integration of various PEG hydrogels in a histomorphometric analysis in rabbits [22], and described an improvement of the bioactive properties upon modification with RGD. Lutolf et al. examined the effect of PEG hydrogels on NBF during repair of critical-sized bone defects in rats’ crania [19]. To
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enhance the cell–matrix attachment of the PEG hydrogels, the authors had used a cell attachment mediating RGD sequence motif, and described an invasion rate of fibroblasts at 7 lm/h, whereas no cell invasion could be observed without the RGD motif. Moreover, critical-sized bone defects in rats’ crania healed with the combination of PEGRGD and BMP-2, but not with the combination of PEG and BMP-2 without RGD [19]. In addition to surface properties, also the pH-value appears to be important for seamless tissue integration [20, 21]. Thoma et al. recently investigated the influence of different pH modifications of PEG hydrogels on the degradation time and bone regeneration in acute and chronic bone defects in 11 minipigs. In the study of thoma and coworkers the authors used hydrogels at a pH of 8.7, 9.0 and 8.4, wherein the hydrogel with a pH of 8.7 showed the highest amount of NBF [21]. Although many studies indicate that PEG hydrogels lead to reduced cell adhesion [20], and may therefore affect new bone formation [19, 22, 36], the PEG hydrogel blocks, used in the present investigation, did not influence the distribution or enhancement of NBF after both time points. The present results indicate that unloaded PEG hydrogels themselves have no positive or negative effect on new bone formation. To use PEG hydrogels as carriers in bone regeneration, further studies are needed in order to determine the ideal material properties to ensure a seamless integration into the surrounding tissue, and to allow the ingrowth of adjacent bone tissue.
5 Conclusion The results of the present study suggest that PEG hydrogels have no enhancing or compromising effect on new bone formation, when used as a carrier for osteoinductive growth factors during mandibular augmentation procedures. Acknowledgments The study has been funded by the Insitute Straumann AG, Basel, Switzerland. The study has been supported by the Commission for Technology and Innovation CTI of Swizerland No. 9201.1 PFLS-LS. Our special thanks are to Ursula Graf-Hausner (Zurich University of Applied Science, ZHAW, Zurich, Switzerland) fort he constructive contributions and expertise. Conflict of interest Authors declare that they have no conflict of interest. Michel M. Dard, DDS, Ph.D is both an employee of Institut Straumann AG (Basel, Switzerland) and a faculty at New York University College of Dentistry (New York, USA). Corinna Mauth was an employee of Institute Straumann AG (Basel, Switzerland).
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BIOMATERIALS SYNTHESIS AND CHARACTERIZATION
Original Research
Influence of synthetic polyethylene glycol hydrogels on new bone formation during mandibular augmentation procedures in Goettingen minipigs Phillipp Brockmeyer1 • Katharina Kramer2 • Sebastian Krohn3 • Philipp Kauffmann1 • Corinna Mauth4 • Michel Dard5 • Henning Schliephake1 Rudolf Matthias Gruber1
•
Received: 18 March 2015 / Accepted: 28 May 2015 Ó Springer Science+Business Media New York 2015
Abstract Polyethylene glycol hydrogels (PEG) have been used as slow release carrier for osteoinductive growth factors in order to achieve a retarded delivery. However, there have been concerns about negative effects on bone regeneration. This study aims to test whether PEG hydrogels themselves affect new bone formation (NBF), when used as a carrier during mandibular augmentation procedures. In a randomized split-mouth design, bilateral mandibular bone defects were surgically created in 12 Goettingen minipigs, and subsequently augmented, using PEG hydrogel on one side of the mandible. The contralateral sides, without PEG, served as controls. After 4 and 12 weeks, bone formation was evaluated in six animals each. A comparison of the data, using a three-way analysis of variance (ANOVA), revealed a significant effect of the healing time and the region of the graft on the distribution and enhancement of NBF (P \ .0001, respectively). Although a 0.3 % (95 %-CI [-5.5; 4.8]) lower volume density of newly formed bone could be observed over all PEG hydrogel sections, in contrast to the contralateral controls, the analysis revealed no
& Phillipp Brockmeyer [email protected] 1
Department of Oral and Maxillofacial Surgery, University Medical Centre Goettingen, Robert-Koch-Str. 40, 37075 Go¯ttingen, Germany
2
Department of Medical Statistics, University Medical Centre Goettingen, Go¯ttingen, Germany
3
Department of Prosthodontics, University Medical Centre Goettingen, Go¯ttingen, Germany
4
Private Practice, Basel, Switzerland
5
Department of Periodontology and Implant Dentistry, College of Dentistry, New York University, New York, NY, USA
clinically significant effects of the PEG hydrogel treatment on the total level (P = 0.90), and the distribution of NBF (P = 0.54). In conclusion, PEG hydrogels do not affect NBF when used as a carrier for osteoinductive growth factors during mandibular augmentation procedures.
1 Introduction Combinations of synthetic carriers, along with osteoinductive acting growth factors, have been discussed as an alternative to autogenous bone grafts, to avoid donor site morbidity [1], and to improve new bone formation (NBF) [2], in reconstructive surgery. Among the known growth factors, bone morphogenetic proteins (BMPs) [2–5], and, in particular, BMP-2 [6–8], showed their osteoinductive character in many investigations. The Food and Drug Administration (FDA) approved a combination of recombinant human BMP-2 (rhBMP-2), with an absorbable collagen sponge (ACS) as a bone substitute material (BSM), for the treatment of degenerative disc disease (DDD) [9], open tibia shaft fractures [10], and sinus floor [3] and localized alveolar ridge augmentations [11]. In the use of osteoinductive growth factors, the carrier is crucial [12]. Ideal carrier materials should be malleable (in the anatomically desired form), possess sufficient mechanical resistance, be absorbable while regenerating bone, and provide the controlled release of active osteoinductive growth factors [12]. While superficially adsorptive-coated materials release factors by a burst release with a loss of up to 80 per cent of activity within the first 48 h after implantation [13], bioactive proteins can also be incorporated into mechanically stable, resorbable carriers [14], which slow the release. Polyethylene glycol hydrogels (PEG) were studied as a carrier for BMP-2 [15], with the advantage of suppressing a burst release of the
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factor. Liquid or solid PEG hydrogels are formed out of ethylene glycol monomers in a polymerization reaction [16]. Preclinically, PEG hydrogels formed a relatively inert matrix, which retained bioactive proteins [17–19]. Because of its entrapment in the hydrogel, rhBMP-2 release could be retarded in these investigations [17–19]. However, it is not yet clear whether PEG hydrogels may influence NBF due to their structure. Hydrogels have specific physical and biological properties which include mechanisms and dynamics of gel formation, mechanical properties and degradation behaviour [20] which may affect surrounding tissue. Moreover, the pH-value seems to be crucial for a seamless integration of the hydrogel into the surrounding tissue [21]. It is described that most cells of the extracellular matrix (ECM) have no receptors to bind to the hydrogel-forming polymers [20], leading to inhibition of cell–matrix adhesion [22]. For this reason, it is questionable whether the PEG hydrogel matrix could adversely affect the integration of new bone formation into the scaffold. Towards this end, we aim to investigate whether PEG hydrogels may affect NBF during the repair of mandibular bone defects in a split mouth design in 12 Goettingen minipigs.
2 Materials and methods 2.1 Animals and study design Since the Minipig [23], and the sample size [24, 25] have been proven in a number of preclinical studies in the field of implant dentistry and oral and maxillofacial surgery, the present evaluation was performed on 12 adult female Goettingen minipigs (Ellegard Goettingen Minipigs ApS, Dalmose, Denmark), carried out in accordance with regulations governing animal housing (German Animal Welfare Act and Directive 2010/63/EU). The Lower Saxony State Office for Consumer Protection and Food Safety (registration no.: 3314.42502-04-013/09), and the committee on animal welfare of the University of Goettingen, approved this study. Two veterinarians observed all animals throughout the study period for signs of pain or unacceptable conditions. The animals were randomly assigned to two groups (Group A: 4 weeks of observation; Group B: 12 weeks of observation). In both groups, in a split-mouth design, one side of the mandible was randomly allocated to the experimental treatment (PEG hydrogel), while the contralateral sides served as control (no PEG applied).
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intramuscular administration of azeparone (StresnilÒ, Jansen; Neuss, Germany; 5 mg/kg bw) and intravenous injection of thiopental (TrapanalÒ, Byk-Gulden; Konstanz, Germany; 7 mg/kg bw). Inhalation anaesthesia was maintained with isoflurance (2 %). Dipidolor (Janssen; 110 lg/kg bw per h) and ketamine (WDT; Garbsen, Germany; 2.5 mg/ kg bw per h) were administered for 3 days to alleviate postoperative pain. Perioperative antibiotic protection was provided by penicillin and streptomycin (AnimedicÒ, Animedica; Senden-Boesensell, Germany; 40 mg penicillin/60 mg streptomycin/kg bw per day). Sixteen weeks after extraction, partial thickness alveolar defects (20 L 9 8 W 9 8 H mm) were surgically created in the extracted areas on both sides of the mandibles. On the test sides, PEG hydrogel blocks were applied to the bone defects, whereas in the contralateral controls no PEG was used and the defects were left empty. To prepare the PEG hydrogel blocks, 136.5 ll of PEG A (PEG A, Mn = 15 kDa) and PEG B (PEG B, Mn = 3.5 kDa; Institut Strautmann AG, Basel, Switzerland) solution mix, 672 ll of sterile water and 105 ll of activator solution were assembled together, and immediately added to a block mould. PEG hydrogel blocks were shaped exactly in accordance with the created defects. Wound closure was performed using Vicryl sutures (Vicryl R, 3x0; Ethicon, Norderstedt, Germany). At the end of the observation period (4 and 12 weeks, respectively), the tissue blocks of the bone defects were retrieved and fixed in 10 % phosphate buffered neutral formalin solution. 2.3 Histology The tissue blocks were infiltrated with composite resin (Technovit 9100; Heraeus-Kulzer, Hanau, Germany) and cut to a thickness of 200 lm perpendicular to the axis of the local jawbone (Exakt, Norderstedt, Germany). Specimens were ground and polished to approximately 25 lm of thickness, as described by Donath and Breuner [26]. Up to 15 sections of the defect area were prepared. In order to take into account the expansion of the bone defects, two sections from the lateral region (mesial and distal, respectively), and one section from the central region of the graft were used for the histomorphometric evaluation. Since the Smith–Karagianes staining (methylene blue/alizarin red S) has proven for histological bone preparations [27], and provides good contrast between bone and soft tissues, it was performed for the microscopic examination and the histomorphometric assessment of new bone formation (Zeiss, Oberkochen, Germany).
2.2 Surgery 2.4 Histomorphometry In an initial surgical procedure, all premolars and first molars were extracted from both sides of the mandible under general anaesthesia. General anaesthesia was induced by
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A Zeiss Axiovert 200 microscope in combination with a digital camera (Axiovision 4.4 software, Axiocam; Zeiss)
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was used to generate combined sequential colour images in a 95 magnification. Complete images of the samples were assembled from several individual images with the MosaiX software (Zeiss). For histomorphometric evaluation of the sections, the image analysis software Axiovision Imaging (Zeiss Microlmaging GmbH, Goettingen, Germany) was used. The image analysing systems were calibrated at a predefined distance. In order to evaluate the gradient of NBF, depending on the distance to the local mandibular bone, six L-shaped regions of interest (ROIs, each 1 mm of height) were defined parallel to the border between local bone and augmentation material (Fig. 1). Each ROI was evaluated for their volume density (VD) of newly formed bone tissue. VD was defined as percentage of newly formed bone within the area of analysis.
Inc., Cary, NC, USA). The diagrams presented show the representative mean values with the corresponding 95 % confidence intervals (95 %-CI).
2.5 Statistical analysis
3.2 Histological evaluation
The mean volume density of all three sections was used as the endpoint for the analysis. Values were analysed using a three-way analysis of variance (ANOVA), with the factors ‘Time’ (4 vs. 12 weeks), ‘Treatment’ (PEG hydrogel vs. no BSM), and ‘Region of interest’ (ROI 1–6). Subsequently, pairwise comparisons between ROI 1 and ROIs 2–6 were performed; in order to adjust for multiple comparisons the Dunnett procedure was applied in this case. We assumed that the residuals are normally distributed. This assumption might lead to the fact that CIs are not range preserving. All tests were performed at a significance level of a = 5 %, using the statistical software SAS (SAS 9.3; SAS Institute
After 4 weeks of observation, a clear ingrowth of bone into the augmentation, adjacent to the local mandibular bone, was observed on both sides of the mandible (Fig. 2). On the test side, NBF occurred most prominently in ROIs 1 and 2, where dense new bone grew into the augmentation material by osteoconduction. In ROI 3, the centre of the augmentation, newly formed bone decreased significantly. From this region to the periphery (ROIs 4–6), NBF decreased further. On the control sides of the 4-week group, NBF could mainly be observed in ROIs 1 and 2, which appeared as a dense layer directly adjacent to the local mandibular bone. In ROIs 3–6, bone was rarely found.
Fig. 1 Overview of all regions of interest (ROI 1–6) of the augmentation. Smith–Karagianes staining (magnification factor 92.5)
Fig. 2 Overview of the augmentation (4 weeks, control group). Resident bone (RB), new bone formation (NBF), border between resident bone and new bone formation (white line), appositional bone which was not evaluated (AB). Smith–Karagianes staining (magnification factor 92.5)
3 Results 3.1 Clinical evaluation Between 3 and 5 days, a minor transient postoperative swelling could be observed at the surgery site. All animals showed normal wound healing after teeth extraction and augmentation procedures, without signs of inflammation or infection. During the entire study period, there was no adverse event that affected the health of any animal.
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Overall, newly formed bone appeared in small immature trabeculae after 4 weeks, on both sides of the mandible (Fig. 3). After 12 weeks of observation, the gradient of NBF differed significantly from that observed after 4 weeks. More newly formed bone could be observed overall, which was almost indistinguishable from the local jawbone (Fig. 4). On the test side, in ROIs 1 and 2, newly formed bone appeared dense and mature. In ROIs 3–6, an increase in NBF was observed. On the control side, an increase in NBF was observed in all regions, which was hardly distinguishable from the local jawbone.
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3.3 Histomorphometric evaluation After 4 weeks, in all sections, mean bone density of newly formed bone decreased from ROI 1–ROI 6. On the test side (PEG hydrogel applied) VD decreased from 72.6 % (95 %CI [61.8; 83.4]) in ROI 1–2.5 % (95 %-CI [-0.5; 5.6]) in ROI 5, whereas no NBF could be observed in ROI 6. On the control side (no BSM applied), VD decreased from 67.1 % (95 %-CI [56.4; 77.9) in ROI 1–1.3 % (95 %-CI [-1.8; 4.3]) in ROI 5. Also, in these sections, no newly formed bone could be observed in ROI 6 (Fig. 5). After a total of 12 weeks, more newly formed bone could be observed in each region. On the test side (PEG hydrogel applied), VD ranged from 76.8 % (95 %-CI [68.0; 85.5]) in ROI 1–5.0 % (95 %-CI [-5.2; 15.1]) in ROI 6. On the control side (no BSM applied), VD ranged from 76.1 % (95 %-CI [67.3; 84.9]) in ROI 1–7.4 % (95 %-CI [-2.8; 17.5]) in ROI 6 (Fig. 5). The comparison of the data revealed a significant effect of the healing time on NBF (P \ .0001). After 4 weeks, over all sections, 18.7 % (95 %-CI [12.0; 25.4]) less VD of newly formed bone could be observed, in contrast to the 12-week time point. Moreover, a significant influence of the region of the graft on the distribution and enhancement of NBF could be observed (P \ .0001). The highest VD values of NBF were observed in ROI 1. The pairwise comparison of ROI 1, with all other regions of interest, revealed significant differences in NBF between ROI 1 and ROIs 2–6 (all P values\0.0007). Although a 0.3 % (95 %-CI
Fig. 3 Details of new bone formation in ROI 1 of the augmentation (4 weeks, PEG group). Smith–Karagianes staining (magnification factor 963)
Fig. 4 Overview of the augmentation (12 weeks, PEG group). Resident bone (RB), new bone formation (NBF), border between resident bone and new bone formation (white line), appositional bone which was not evaluated (AB). Smith–Karagianes staining (magnification factor 92.5)
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Fig. 5 Diagram illustrating the volume density of new bone formation after 4 and 12 weeks. Mean volume density and corresponding 95 % confidence intervals. PEG hydrogel treatment after 4 weeks (green), PEG hydrogel treatment after 12 weeks (red), no treatment after 4 weeks (blue), no treatment after 12 weeks (black) (Color figure online)
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[-5.5; 4.8]) lower volume density of newly formed bone could be observed over all PEG hydrogel sections, in contrast to the contralateral controls, the analysis revealed no clinically significant effects of the PEG hydrogel treatment on the total level (P = 0.90), and the distribution of NBF (P = 0.54).
4 Discussion The results of various studies have shown that PEG hydrogels due to their physical and biological characteristics may lead to lower cell adhesion [20], which could influence the integration of the new bone formation [19, 22]. Towards this end, the present investigation aims to test the hypothesis that PEG hydrogels influence new bone formation when used as a carrier during mandibular augmentation procedures. To investigate the effect of the healing time on the distribution or enhancement of NBF, two different time points (4 and 12 weeks) were each analysed. After 4 weeks’ observation time, both test and control sides showed a gradient of NBF, which is described in the literature as typical of osteoconductive augmentation materials [28, 29]. The highest rates of newly formed bone were measured in the region close to the locally constant jawbone (ROI 1). From there, the values decreased significantly with increasing distance from the local jawbone (ROI 6). The results are comparable to those described by Busenlechner et al. [30] and Fuerst et al. [31], who investigated the gradient of NBF in different osteoconductive bone substitute materials during sinus floor augmentations in a curve-based manner. After 12 weeks, the gradient of NBF differed from that after 1 month. On both sides of the mandible, a strong increase in NBF could be observed in all regions, still with decreasing gradient. During the ingrowth of bone tissue, osteoblasts and their precursor cells compete with rapidly proliferating and ingrowing cells from the surrounding connective tissue [32]. Probably because of this reason, a sufficient NBF is only possible in the basal regions of the graft near the local jawbone, after this time period [32]. It is described, that most proteins of the extracellular matrix have no receptors to bind to the hydrogel-forming polymers, leading to lower cell adhesion [20]. However, most cell types are able to bind to the amino acid sequence arginine–glycine–aspartic (RGD), and PEG hydrogels have been modified with this peptide to promote cellular adhesion [19, 22, 33–35]. Thoma et al. had investigated the biodegradation and hard and soft tissue integration of various PEG hydrogels in a histomorphometric analysis in rabbits [22], and described an improvement of the bioactive properties upon modification with RGD. Lutolf et al. examined the effect of PEG hydrogels on NBF during repair of critical-sized bone defects in rats’ crania [19]. To
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enhance the cell–matrix attachment of the PEG hydrogels, the authors had used a cell attachment mediating RGD sequence motif, and described an invasion rate of fibroblasts at 7 lm/h, whereas no cell invasion could be observed without the RGD motif. Moreover, critical-sized bone defects in rats’ crania healed with the combination of PEGRGD and BMP-2, but not with the combination of PEG and BMP-2 without RGD [19]. In addition to surface properties, also the pH-value appears to be important for seamless tissue integration [20, 21]. Thoma et al. recently investigated the influence of different pH modifications of PEG hydrogels on the degradation time and bone regeneration in acute and chronic bone defects in 11 minipigs. In the study of thoma and coworkers the authors used hydrogels at a pH of 8.7, 9.0 and 8.4, wherein the hydrogel with a pH of 8.7 showed the highest amount of NBF [21]. Although many studies indicate that PEG hydrogels lead to reduced cell adhesion [20], and may therefore affect new bone formation [19, 22, 36], the PEG hydrogel blocks, used in the present investigation, did not influence the distribution or enhancement of NBF after both time points. The present results indicate that unloaded PEG hydrogels themselves have no positive or negative effect on new bone formation. To use PEG hydrogels as carriers in bone regeneration, further studies are needed in order to determine the ideal material properties to ensure a seamless integration into the surrounding tissue, and to allow the ingrowth of adjacent bone tissue.
5 Conclusion The results of the present study suggest that PEG hydrogels have no enhancing or compromising effect on new bone formation, when used as a carrier for osteoinductive growth factors during mandibular augmentation procedures. Acknowledgments The study has been funded by the Insitute Straumann AG, Basel, Switzerland. The study has been supported by the Commission for Technology and Innovation CTI of Swizerland No. 9201.1 PFLS-LS. Our special thanks are to Ursula Graf-Hausner (Zurich University of Applied Science, ZHAW, Zurich, Switzerland) fort he constructive contributions and expertise. Conflict of interest Authors declare that they have no conflict of interest. Michel M. Dard, DDS, Ph.D is both an employee of Institut Straumann AG (Basel, Switzerland) and a faculty at New York University College of Dentistry (New York, USA). Corinna Mauth was an employee of Institute Straumann AG (Basel, Switzerland).
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