Journal of Cranio-Maxillo-Facial Surgery 43 (2015) 559e566

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Extra-oral defect augmentation using autologous, bovine and equine bone blocks: A preclinical histomorphometrical comparative study Tobias Moest*, Falk Wehrhan, Rainer Lutz, Christian Martin Schmitt, Friedrich Wilhelm Neukam, Karl Andreas Schlegel Department of Oral and Maxillofacial Surgery, University Erlangen e Nuremberg, Glückstraße 11, D-91054 Erlangen, Germany

a r t i c l e i n f o

a b s t r a c t

Article history: Paper received 19 December 2014 Accepted 16 February 2015 Available online 24 February 2015

Objectives: This study aimed to compare autologous bone (AB), bovine bone (BB), and equine bone (EB) blocks with regard to de novo bone formation, connective tissue, and residual bone substitute material portions in a standardized defect animal model. Material and methods: In the frontal skull of 20 pigs, 106 standardized cylindrical “critical size defects” were prepared. Defects were randomly filled with AB, BB, and EB blocks. After a healing period of 30 and 60 days, de novo bone formation, residual bone substitute material, and connective tissue portion was assessed by means of histomorphometry (Toluidine blue O staining). ManneWhitney U-tests were used to evaluate differences between the groups. Results: The de novo bone formation was significantly higher in the AB group in comparison to the xenogeneic groups (p < 0.05). After 30 days, EB showed significantly (p < 0.05) more newly formed bone compared to the BB group. The soft tissue formation was significantly higher in the BB and EB group. Defects augmented with BB showed significantly (p < 0.05) higher portions of bone substitute materials compared to sides augmented with EB after 30 days. Conclusion: In the extra-oral model, AB blocks were superior concerning de novo bone formation. No clinical advantages of EB blocks could be observed. © 2015 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

Keywords: Alveolar bone grafting Bone substitute Replacement material Bone

1. Introduction Lack of bone quantity due to alveolar atrophy, periodontal infection, trauma, or missing ridge preservation in the context of tooth extraction may compromise the insertion of dental implants and hinder rehabilitation with implant-supported dentures. To obtain adequate bone quantity and quality, regenerative therapies are now established and integrated into the clinical daily routine. Several techniques with various grafting materials have been proposed for bone regeneration (Chiapasco et al., 2006). Autologous bone is still considered to be the gold standard, due to osteogenetic, osteoconductive, and osteoinductive properties (Dragoo and Sullivan, 1973). Since autogenous bone is endogenous, the risk of rejection does not exist. However, the availability, the tendency to undergo partial resorption, the need for an additional

* Corresponding author. Tel.: þ49 9131 8543728; fax: þ49 9131 8534219. E-mail address: [email protected] (T. Moest).

surgery, the associated surgical risks, and the higher patient morbidity (limping, anaesthesia, paraesthesia, residual defects, pain), hospitalization, higher costs, and longer treatment time represent significant limitations in contrast to the use of bonesubstitute materials (Chiapasco et al., 2006; Clavero and Lundgren, 2003; Herford and Dean, 2011; Kessler et al., 2005; Nkenke et al., 2001; Noia et al., 2011). To avoid these limitations, the application of allogeneic (Acocella et al., 2012; Damlar et al., 2015; Schlee and Rothamel, 2013), alloplastic (Alfotawei et al., 2014; Jodia et al., 2014; Yun et al., 2014), or xenogeneic (Jensen et al., 2006) bone substitute materials has been discussed as an alternative. For the application of natural bone substitute materials biocompatibility, nonantigenic and non-infectious properties are mandatory. Moreover, grafting materials should not inhibit normal cell activity/invasion on its surface and disturb the natural bone remodelling process. To allow consolidation of the augmented side, the surface of implanted material should be conductive for osteoblasts/osteoclasts and should be resorbable.

http://dx.doi.org/10.1016/j.jcms.2015.02.012 1010-5182/© 2015 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

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Xenografts carry only a small risk of contamination from infectious diseases, do not compromise the patient's remaining tissue, and have chemicalephysical characteristics that are similar to those of human bone. Bone substitute materials are offered as granules (Aloy-Prosper et al., 2011; Jensen et al., 2007), gels (Bosshardt et al., 2014; Xu et al., 2011) or blocks (Laino et al., 2014; Schwarz et al., 2010). Mechanical characteristics of granules and gels do not allow the grafting material to be fixed stably on the alveolar ridge, but require a membrane to stay in place. Gels and granules are appropriate for reconstructing small space-making defects with good regenerative potential, but are less useful for nonespace-providing defects, such as vertical, horizontal, or combined defects. In these cases, the application of stable grafts such as bone blocks have been tested (Felice et al., 2008; Li et al., 2013; Nissan et al., 2011). However, the success rate of grafting procedures using bone blocks are not described uniformly in the literature. Autologous bone blocks show reliable clinical applicability, whereas osseous consolidation of block-configured screwable xenogenous bone blocks are limited because of less osteoconductive properties for either lateral or vertical ridge augmentation procedures (Rothamel et al., 2008, Rothamel et al., 2009). To induce osseous integration of xenogenous bone blocks, an equine hydroxyapatite bone substitute block (Geistlich Pharma AG, Wolhusen, Switzerland) with sufficient mechanical properties to enable fixation and a collagen component for accelerated osseous consolidation has been developed. For this reason, superior osseous defect regeneration in comparison to that of bovine bone blocks was expected. However, the few data concerning the applicability and biocompatibility of equine bone substitute material are contradictory. Preclinical studies have shown significant bone formation and bone growth into the equine bone blocks (Schwarz et al., 2010; Zecha et al., 2011), whereas, in clinical comparative studies, significantly more equine block grafts have failed (Felice et al., 2013). To assess the influence of equine bone substitutes on bone regeneration correctly, a comparison of equine grafts with other bone substitutes in a standardized defect with high regenerative potential is necessary. The aim of the present experimental study was to evaluate and compare the biocompatibility and resorbability of equine- and bovine-derived bone substitutes in comparison to autologous bone blocks in a standardized, extra-oral, “critical size defect” model in pigs. 2. Material and methods 2.1. Animals The domestic pig was the animal of choice. Twenty pigs with an age of 18 ± 4 months were included in this study. Under circadian day and night rhythm, the animals were kept in an open enclosure of 6 m2 at an ambient room temperature of 18  C ± 1  C. The pigs received standardized pig mast fodder (Garant Tiernahrung GmbH, €chlarn, Austria) as well as water ad libitum. During the entire Po experimental period, the animals were under sequential veterinary control. The research project was approved by a state Animal Research Committee (approval no. 22.1/3879/003/2008).

GmbH, Tübingen, Germany). Then, 30 and 60 days after defect preparation, randomly selected animals were killed. 2.3. Anaesthesia protocol Before the surgical procedures, the pigs were fasted overnight and handled according the following anaesthesia protocol. After an intramuscular injection of medetomidine (Domitor, Pfizer, Karlsruhe, Germany), anaesthesia was initiated using an intravenous administration of Ketamine HCl (Ketavet; Ratiopharm, Ulm, Germany). To maintain hydration, animals received a constant infusion of lactated Ringer's solution while being anesthetized. Perioperative antibiosis was administered 1 hour preoperatively and for 2 days post-operatively to reduce the risk of infection (Streptomycin, 0.5 g/day, Grunenthal, Stolberg, Germany). A veterinarian performed the anaesthesia and the peri-operative control of vital signs. Postoperative pain control was achieved by administering analge€ hringer Mannheim GmbH, Mannheim, Germany), sics (Temgesic, Bo 0.05 mg/kg every 12 hours, for 3 days following surgery. 2.4. Surgical procedure After application of a local anaesthetic in the area of the frontal skull (Ultracain DS forte, Hoechst GmbH, Frankfurt a. M., Germany), an incision was performed, and the soft tissue and the periosteum were mobilized. Identical bony defects were created with a trephine burr with a diameter of 10 mm and a depth of 10 mm (Roland Schmid, Fürth, Germany) according to an established critical size defect model (Schlegel et al., 2003, 2009). The defects were positioned at least 1 cm apart from each other to avoid biologic interactions (Fig. 1). Defects were prepared under copious irrigation with sterile 0.9% physiological saline solution. The continuity of the tabula interna remained intact. To guarantee standardized defect preparation, defect depth was controlled with a ruler. A chisel was used for additional defect preparation. After preparation, the bony defects were randomly filled with size-adapted grafting materials: AB, EB containing remnants of equine collagen I/III (Geistlich Bio-Graft block, width: 10 mm, thickness: 5 mm, height: 10 mm; Geistlich Pharma AG, Wolhusen, Switzerland), and BB (Geistlich BioOss spongiosa block, width: 20 mm, thickness: 10 mm, height: 10 mm, Geistlich Pharma AG). Autogenous bone was harvested during the surgical procedures and reused for autogenous bone grafting. Following the grafting procedures, a nonecross-linked native bioresorbable collagen membrane of porcine origin was adapted over the entire defect area. The periosteum and skin over the defects were sutured in two layers (Vicryl 3.0; Vicryl 1.0; Ethicon Co., Norderstedt, Germany). Due to anatomical configurations, 5.3 defects were prepared per animal. 2.5. Animal sacrifice and retrial of specimen After the designated healing period of 30 and 60 days, the animals were sacrificed. The pigs were sedated by an intramuscular injection of Azaperone (1 mg/kg) and Midazolam (1 mg/kg). Euthanasia was performed by an intravascular injection of 20% pentobarbital solution into an ear vein until cardiac arrest. The foreheads were immediately dissected and stored at 80  C.

2.2. Study protocol and randomisation 2.6. Specimen fixation In the frontal skull, 106 standardized critical size defects were prepared and randomly filled with autologous bone (AB), bovine bone (BB), or equine bone (EB) blocks. The randomization procedure was based on a computer-generated list (RandList; DatInf

To identify defect localization, computed tomographic (CT) analyses (Department of Radiology, University of ErlangenNuremberg; Director: Prof. Dr. M. Uder) were performed of all

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Fig. 1. Schematic presentation of defect location, configuration, and block augmentation.

specimens in the frontal and transversal plane. Afterwards, the specimens were dissected from the ossa frontalia and subsequently fixed by immersion in 1.4% paraformaldehyde at room temperature to render the organic matrix insoluble. The specimens were dehydrated in an ascending alcohol series at room temperature (Shandon Citadel 1000, Shandon GmbH, Frankfurt, Germany). Xylol was used as an intermediate fixative. Technovit 9100 (Heraeus Kulzer, Kulzer Division, Wertheim Germany) was used for embedding. To avoid any negative influence of polymerization heat, the polymerization was performed in a cold atmosphere (4  C). After 20 hours, the specimens were completely polymerized. 2.7. Histological preparation For histological preparation, the embedded bone samples were cut in the middle through the defect. The specimens were grinded into thin sections (30 mm) using a precision saw and a special grinding machine (both Exakt Apparatebau GmbH, Norderstedt, Germany). The slides were transferred in 10% H2O2- solution for 5 minutes. Followed by rinsing under cold running water, the specimens were stained for 10 minutes with Toluidine blue O (SigmaeAldrich Chemie GmbH, Munich, Germany). Excess stain was removed by rinsing the specimens under running water. Toluidine blue Oestained specimens were examined under a light microscope (Magnification:  10; Axio Imager. A1; Zeiss, Jena, Germany) by digitizing the specimens with an attached video camera (QICAM FAST 1394, Qimaging, Burnaby, Canada) (Fig. 2). Histological pictures were stored in TIFF format and evaluated with Bioquant Osteo Software 2013 v13.2.6 (Bioquant Image Analysis Corporation, Nashville, TN, USA). 2.8. Evaluation parameters The evaluation of the Toluidine blue Oestained specimens distinctly showed margins of the defect preparation, whereby the proportion of newly formed bone, remaining bone substitute material (BSM), and connective tissue within the defect could be precisely evaluated. Soft and hard tissue proportions were given as percentages of the total defect volume. 2.9. Statistical analysis Statistical analyses were performed using SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). Mean values and standard

deviations among animals were calculated for each group. To determine distribution, the data rows were examined by using the KolmogoroveSmirnov test. The ManneWhitney U-test was used for between-group comparison. The overall significance threshold (a) was set at 0.05. 3. Results 3.1. Newly formed bone After 30 and 60 days, significant differences concerning de novo bone formation could be observed. At both investigation time points, the application of AB blocks showed significantly higher bone formation rates in comparison to BB (30 days: p ¼ 0.000: 60 days: p ¼ 0.002) and EB (30 days: p ¼ 0.000, 60 days p: ¼ 0.000) blocks. Within the xenogeneic groups, significantly higher de novo bone formation (p ¼ 0.010) could be observed for the EB group after 30 days. By 60 days after defect preparation, no statistically significant difference (p > 0.05) was observed between the bovine and equine group, whereas de novo bone formation was higher in the BB group (Fig. 3, Table 1). 3.2. Connective tissue The consolidation of autologous and xenogeneic bone blocks showed differences concerning connective tissue proportions of the regenerated defects. By 30 days after defect preparation, AB blocks showed significantly less connective tissue proportions (p ¼ 0.000, BB and EB) in comparison to defects augmented with xenogeneic substitutes. The soft tissues percentage in the defects filled with EB were lower but not significant. After 60 days, the defects of BB and EB blocks also showed significantly higher (p ¼ 0.009, BB; p ¼ 0.002, EB) soft tissue portions in comparison to the AB blocks. Differences within the xenogeneic groups were not statistically significant (p > 0.05) (Fig. 4, Table 1). 3.3. Residual bone substitute material Since de novo formed bone could not be distinguished from the autologous bone graft, the proportion of remaining bone substitute materials could not be specified in the AB group. After 30 days, the defects augmented with BB showed significantly higher proportions of bone substitute materials compared to the sides augmented with EB (p ¼ 0.043). After 60 days, no

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Fig. 2. Toluidine blue Oestained samples 30 days (first row) and 60 days (second row) after defect preparation. Pictures 1 and 4 illustrate an autologous bone block (AB), pictures 2 and 5 a bovine bone block (BB), and 3 and 6 an equine bone block (EB) grafted standardized bony defects.

statistically significant differences concerning the remaining BSM in the defects were observed (p > 0.05) (Fig. 5, Table 1). 4. Discussion For the first time, to our knowledge, autogenous and xenogeneic bone blocks of different origins have been investigated and compared with each other with regard to de novo bone formation,

connective tissue proportion, and remaining BSM in a standardized, extra-oral, critical size defect animal model. The aims of the study was to assess the clinical applicability of equine and bovine bone blocks by investigating their regeneration potential and comparing them to the gold standard of care, the autologous bone block. The applied animal model was chosen since bone regeneration rate of 1.2e1.5 mm/d is comparable to that in humans (1.0e1.5 mm/d) (Eitel et al., 1981; Schlegel et al., 2006). For

Fig. 3. Boxplots presenting the rate of newly formed bone (in percent) 30 and 60 days after defect preparation measured by Toluidine Blue Oestained area within the defect zones of each study group (AB ¼ autologous bone block; BB ¼ bovine bone block; EB ¼ equine bone block). The median and the inter-quartile range are given. *p-Values were estimated by the ManneWhitney U test.

T. Moest et al. / Journal of Cranio-Maxillo-Facial Surgery 43 (2015) 559e566 Table 1 Means and standard deviations (SD) for newly formed bone (NF), connective tissue (CT), and residual bone substitute material (BSM) 30 and 60 days after defect preparation for extra-oral defects filled with autologous bone (AB), bovine bone (BB), and equine bone (EB) blocks. Observation period

Group

Parameter

Extra-Oral defect Mean

± SD

30 days

AB

NF CT BSM NF CT BSM NF CT BSM NF CT BSM NF CT BSM NF CT BSM

61.50 38.50 0.00 14.60 66.40 19.00 25.30 61.90 12.90 57.20 42.80 0.00 16.90 66.40 18.20 15.70 65.30 19.00

5.50 5.50 0.00 5.60 9.00 7.50 13.10 11.50 9.70 10.50 10.50 0.00 5.90 11.70 13.30 8.00 10.80 11.80

BB

EB

60 days

AB

BB

EB

this reason, the generated results can reliably be extrapolated to human beings. The animal model is well investigated and cited to test the regeneration potential of various bone substitute materials (Schlegel et al., 2004, Schlegel et al., 2003, Schlegel et al., 2006, Schlegel et al., 2009; Stockmann et al., 2012; Wehrhan et al., 2012). The forehead region is simple to prepare, and offers space to prepare defects with enough distance to avoid influence among the different groups (Von Wilmowsky et al., 2013). The defect localization in the forehead region offers ideal conditions for osseous regeneration, since wound healing is not affected by mastification or the colonization of oral bacteria. Wound closure

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with two layers represents a stable closure. To protect the defects from infection, the surgical side was additionally covered with a bioresorbable membrane. Furthermore, the membrane shields soft tissue and periostum proliferation, which influences osseous regeneration. Studies show that the periosteum contains a high number of osteogenic precursor cells (Squier et al., 1990; Zhu et al., 2006), although greater bone volumes and accelerated defect regeneration can be observed (Agata et al., 2007). By using membranes, the influence of applied BSM on osseous regeneration starting from the defect walls can be precisely investigated. As appropriate investigation time points, 30 and 60 days post-grafting were selected, since the biocompatibility of the applied equine blocks and the immunological host reaction was unknown. Because the avoidance of micro-movement represents a prerequisite to investigate osseous regeneration and consolidation of BSM, the bone blocks were fixed with a vertical osteosynthesis screw to guarantee stable fixation. A stable block fixation allows the formation of a blood clot, ingrowth of mesenchymal stem, and progenitor cells as well as neovascularization, which is crucial in the early healing phase. However, the results of our study show that 12.8% of the bone blocks after 30 days and 25.7% of applied blocks after 60 days were affected by granulomatous inflammation. Postoperative care and restrictions are of great concern after bone grafting and can make a difference, leading to either success or failure after grafting. In this respect, animal studies in particular are challenging. The compliance for correct post-operative behavior such as the avoidance of mechanical irritation cannot be ensured. The resulting micro-movements due to mechanical forces lead to loss of the blood clot and decrease vessel formation, wound dehiscence, membrane, and defect exposure whereby defect regeneration can be negatively influenced. Clinical data confirm the limited success rate of xenogeneic bone blocks (Pistilli et al., 2014). To examine the applicability of the used bone blocks, we measured the proportion of newly formed bone, connective tissue,

Fig. 4. Boxplots presenting the rate of connective tissue (in percent) 30 and 60 days after defect preparation, measured by the Toluidine Blue Oestained area within the defect zones of each study group (AB ¼ autologous bone block; BB ¼ bovine bone block; EB ¼ equine bone block). The median and the inter-quartile range are given. *p Values were estimated by the ManneWhitney U test.

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Fig. 5. Boxplots presenting the rate of residual bone substitute material 30 and 60 days after defect preparation, measured by the Toluidine Blue Oestained area within the defect zones of each study group (AB ¼ autologous bone block; BB ¼ bovine bone block; EB ¼ equine bone block). The median and the inter-quartile range are given. *p Values were estimated by the ManneWhitney U test.

and remaining BSM in standardized defects 30 and 60 days after defect preparation. At both investigation time points, the groups of AB blocks showed superior bone formation rates compared to EB and BB blocks. The healing outcome of autologous grafts in our study is in agreement with several preclinical (De Santis et al., 2014; Yeo et al., 2012) and clinical (Acocella et al., 2010; Buser et al., 1996; Spin-Neto et al., 2014; Von Arx and Buser, 2006) studies. Because of its healing properties, autologous bone transplantation is still used as standard of care. Furthermore, autologous bone grafting seems reasonable for large and nonespace-providing defects. Compared to xenogeneic bone grafts, autologous bone grafts have higher regenerative potential, since mesenchymal stem cells, vital osteoblasts, and their precursors are transplanted in the augmented side (Blokhuis and Arts, 2011; Cypher and Grossman, 1996). These osteogenic properties are responsible for the high regenerative potential of autologous bone grafts in comparison to osteoconductive xenografts. Compared to the defects in the other applied bone blocks, the defects in the AB group showed the highest regeneration rate and the best bone quality within the augmented defect area. Defects filled with autologous bone showed the lowest rate of soft tissue formation. Since soft tissue formation has no function with respect to osseous defect regeneration for, for example, subsequent dental implant insertion, ABs were significantly superior in comparison to the other xenogeneic BSMs. However, autologous bone grafting has some disadvantages that limit its applicability. Autologous bone harvesting is associated with risks, and patients experience additional stress (Nkenke et al., 2001; Schaaf et al., 2010; Weibull et al., 2009). Autologous bone grafts undergo continuous resorption, which makes predictable results more difficult, since long-term stability is not predictable (Aghaloo and Moy, 2007; Tonetti et al., 2008). To minimize risks and the burden for patients, research is still ongoing to generate and evaluate the clinical applicability of a comparable bioactive material with long-term stability.

The applied BSM (Bio-Oss) has been extensively investigated and has shown good bone regeneration potential in various indications (Felice et al., 2010; Li et al., 2013; Rothamel et al., 2009; Schmitt et al., 2013; Steigmann, 2008). Bio-Oss represents a wide interconnecting pore system that is characterized by a macro- and micro-porous structure. The pore structure serves as physical scaffold for the immigration of bone-forming cells (Tapety et al., 2004). The evaluation of the bovine bone blocks at both observations time points showed significantly less newly formed bone in comparison to autologous grafted defects. However, the proportion of connective tissue was significantly higher. Since we observed similar results for the equine bone blocks, one possible reason for the inferior bone formation but superior connective tissue proportion is the structure of both xenogeneic BSMs. Biological healing depends on the direct contact of the bone substitute material to local bone, since higher surfaces areas allow larger areas for biological interaction. Histological observations clearly show the dense structure of autologous bone blocks in comparison to loose equine and bovine bone blocks, whereby osseous regeneration can be significantly influenced. Our findings of EB invasion and organization by fibrous connective tissue are in agreement with previous experimental animal studies reporting on either lateral (Araujo et al., 2002; Schwarz et al., 2008) or vertical ridge augmentation (Simion et al. 2009). With regard to de novo bone formation within the xenogeneic defect groups, after 30 days in the EB group, significantly more newly formed bone could be observed in comparison to that in the BB group. Nevertheless, after 60 days, no significant difference within the xenogeneic groups can be detected. However, in summary, newly formed bone quantity after 30 and 60 days was low for both materials and inferior to ABs. Missing bone regeneration after EB application has also been reported in the literature (Pistilli et al., 2014; Simion et al., 2009). The application of equine-derived bone grafts shows an inferior ability in regard to bone formation and an increased rate of

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connective tissue formation. The observed properties confirm that EB can represent a potential risk factor for a high complication rate either in the immediate or the later post-operative period (Felice et al., 2013; Pistilli et al., 2014). By choosing a later time point of investigation (e.g., 90 days), no improvement concerning osseous xenografts consolidation can be expected, due to fibrous connective tissue formation between the xenografts and the adjacent bony defect walls. 5. Conclusion In the chosen model, autologous bone blocks were superior with regard to bone formation 30 and 60 days after defect preparation. We observed no advantages of equine bone blocks in comparison to bovine bone blocks. Conflict of interest statement The authors declare that there are no conflicts of interest related to this study. Acknowledgement Geistlich Pharma AG (Wolhusen, Switzerland) funded the project. References Acocella A, Bertolai R, Colafranceschi M, Sacco R: Clinical, histological and histomorphometric evaluation of the healing of mandibular ramus bone block grafts for alveolar ridge augmentation before implant placement. J Craniomaxillofac Surg 38: 222e230, 2010 Acocella A, Bertolai R, Ellis 3rd E, Nissan J, Sacco R: Maxillary alveolar ridge reconstruction with monocortical fresh-frozen bone blocks: a clinical, histological and histomorphometric study. J Craniomaxillofac Surg 40: 525e533, 2012 Agata H, Asahina I, Yamazaki Y, Uchida M, Shinohara Y, Honda MJ, et al: Effective bone engineering with periosteum-derived cells. J Dent Res 86: 79e83, 2007 Aghaloo TL, Moy PK: Which hard tissue augmentation techniques are the most successful in furnishing bony support for implant placement? Int J Oral Maxillofac Implant 22(Suppl.): 49e70, 2007 Alfotawei R, Naudi KB, Lappin D, Barbenel J, Di Silvio L, Hunter K, et al: The use of tricalcium phosphate (TCP) and stem cells for the regeneration of osteoperiosteal critical-size mandibular bony defects, an in vitro and preclinical study. J Craniomaxillofac Surg 42: 863e869, 2014 Aloy-Prosper A, Maestre-Ferrin L, Penarrocha-Oltra D, Penarrocha-Diago M: Bone regeneration using particulate grafts: an update. Med Oral Patol Oral Cir Bucal 16: e210e214, 2011 Araújo MG, Sonohara M, Hayacibara R, Cardaropoli G, Lindhe J: Lateral ridge augmentation by the use of grafts comprised of autologous bone or a biomaterial. An experiment in the dog. J Clin Periodontol 29(12): 1122e1231, 2002 Dec Blokhuis TJ, Arts JJ: Bioactive and osteoinductive bone graft substitutes: definitions, facts and myths. Injury 42(Suppl. 2): S26eS29, 2011 Bosshardt DD, Bornstein MM, Carrel JP, Buser D, Bernard JP: Maxillary sinus grafting with a synthetic, nanocrystalline hydroxyapatite-silica gel in humans: histologic and histomorphometric results. Int J Periodont Restor Dent 34: 259e267, 2014 Buser D, Dula K, Hirt HP, Schenk RK: Lateral ridge augmentation using autografts and barrier membranes: a clinical study with 40 partially edentulous patients. J Oral Maxillofac Surg 54: 420e432, 1996 discussion 432e423 Chiapasco M, Zaniboni M, Boisco M: Augmentation procedures for the rehabilitation of deficient edentulous ridges with oral implants. Clin Oral Implant Res 17(Suppl. 2): 136e159, 2006 Clavero J, Lundgren S: Ramus or chin grafts for maxillary sinus inlay and local onlay augmentation: comparison of donor site morbidity and complications. Clin Implant Dent Relat Res 5: 154e160, 2003 Cypher TJ, Grossman JP: Biological principles of bone graft healing. J Foot Ankle Surg 35: 413e417, 1996 Damlar I, Erdogan O, Tatli U, Arpag OF, Gormez U, Ustun Y: Comparison of osteoconductive properties of three different beta-tricalcium phosphate graft materials: a pilot histomorphometric study in a pig model. J Craniomaxillofac Surg 43: 175e180, 2015 De Santis E, Lang NP, Favero G, Beolchini M, Morelli F, Botticelli D: Healing at mandibular block-grafted sites. An experimental study in dogs. Clin Oral Implant Res 44(10): 614e625, 2014

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