Carina B. Johansson
Tomas Albrektsson
Mats Hallman
A randomized and controlled clinical
trial of two different compositions of
deproteinized bovine bone and
autogenous bone used for lateral
ridge augmentation
Authors’ affiliations:
Arne Mordenfeld, Mats Hallman,Department of Oral & Maxillofacial Surgery, Public Health Service, G€avle, Sweden
Arne Mordenfeld, Mats Hallman,Centre for Research and Development, Uppsala University/ G€avleborg County Council, G€avleborg, Sweden Arne Mordenfeld, Tomas Albrektsson,Department of Materials Science & Technology, Malm€o University, Malm€o, Sweden
Tomas Albrektsson,Department of Biomaterials, Institute for Clinical Sciences, the Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
Carina B. Johansson,Department of Prosthodontics/Dental Materials Science, the Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
Corresponding author: Dr. Arne Mordenfeld
Clinic for Oral Maxillofacial Surgery Public Health Service
G€avle Hospital SE 80187 G€avle, Sweden Tel.: +46 2615 5645 Fax: +46 2615 5347
e-mail: arne.mordenfeld@lg.se
Key words: autogenous bone graft, bovine hydroxyapatite, clinical study, fibrin glue, histology, maxillary sinus floor augmentation
Abstract
Objective: The aim of the study was to radiologically and histologically evaluate the graft healing and volumetric changes after lateral augmentation with two different compositions of
deproteinized bovine bone (DPBB) and autogenous bone (AB).
Material and methods: Thirteen patients with a mean age of 59.6 12.1 years (six men and seven women) were included in this randomized and controlled trial, designed as a split-mouth study. Ten edentulous and four partially edentulous jaws with an alveolar ridge width of 4 mm were laterally augmented with a graft composition of 60 : 40 (DPBB/AB) on one side and 90 : 10 (DPBB/AB) on the contralateral side. Cone beam computed tomography (CB/CT) was obtained immediately postoperatively and after a healing period of 7.5 months. Width changes were measured on CB/CT scans. After a mean healing period of 8.1 months (range, 7.9–8.3), biopsies were retrieved perpendicular to the crest from each graft by means of a trephine bur.
Histomorphometry was performed, and the following variables were recorded: Ingrowth of new bone (percentage of total graft width), percentage of DPBB, bone and soft tissue, and percentage of DPBB particles in contact with bone.
Results: The mean gained width of the alveolar crest after 7.5 months was significantly more for the 60 : 40 mixture compared with the 90 : 10 mixture, 3.5 (1.3) mm and 2.9 (1.3) mm, respectively. There was a significant difference in graft width reduction between 60 : 40 and 90 : 10 after 7.5 months, 37 (19.9)% and 46.9 (23.5)%, respectively. New bone ingrowth had occurred in 82.1 (23.3)% and 82.3 (26.6)% of the graft, respectively. There were no statistical differences between fractions of different tissues between the 90 : 10 and 60 : 40 compositions. However, there were significantly more soft tissue and less new bone formation closer to the periosteum compared with the graft portion closer to the residual bone in both 60 : 40 and 90 : 10 compositions.
Conclusions: There was significantly less graft width reduction with a mixture of 60 : 40 (DPBB/AB) compared with a mixture of 90 : 10 composition, but the results from the histomorphometry showed no statistical differences comparing the groups.
Horizontal resorption of the alveolar ridge and subsequent lack of adequate width for optimal implant placement are frequent con-ditions following teeth removal (Araujo & Lindhe 2005). A number of surgical proce-dures have been utilized to reconstruct the alveolar crest. These procedures include “split-ridge” osteotomy for lateral expansion, osteodistraction, bone grafting with different grafting materials (autogenous bone, allograft, xenograft, and alloplastic materials), and
guided bone regeneration (GBR) alone or in
combination with grafting materials
(Widmark et al. 1997; Sjostrom et al. 2007; Donos et al. 2008; Hammerle et al. 2008; Chiapasco et al. 2009; Jensen & Terheyden 2009; Gonzalez-Garcia et al. 2011).
When reconstruction of larger edentulous areas (e.g., totally edentulous jaws) is per-formed, the use of bone grafts from the iliac crest has often been necessary due to the large graft volume needed. The disadvantages
Date:
Accepted 6 February 2013
To cite this article:
Mordenfeld A, Johansson CB, Albrektsson T, Hallman M. A randomized and controlled clinical trial of two different compositions of deproteinized bovine bone and autogenous bone used for lateral ridge augmentation.
Clin. Oral Impl. Res.25, 2014, 310–320
with the use of autogenous bone (AB) include the morbidity from the donor site, extended operating time, higher surgical skill demands, limited available bone, and higher costs. In addition, the resorption rate of AB is unpre-dictable and may vary between 12% and 80% (Widmark et al. 1997; Smolka et al. 2006; Sbordone et al. 2009; Dasmah et al. 2011). To overcome these problems, bone substitutes have been widely used for local horizontal and vertical defects (Rocchietta et al. 2008; Jensen & Terheyden 2009).
Deproteinized bovine bone (DPBB) has been frequently used for maxillary sinus floor augmentation and lateral ridge augmentation and is the most well-documented bone sub-stitute on the market (Hellem et al. 2003; Es-posito et al. 2008; Hallman & Thor 2008; Nkenke & Stelzle 2009). It has been claimed that DPBB has an extremely slow resorption rate, if any at all, and would be suitable in areas where minor resorption is pertinent (Skoglund et al. 1997; Schlegel & Donath 1998; Hallman & Thor 2008; Mordenfeld et al. 2010). Moreover, DPBB has excellent osteoconductive properties and possesses the ability to allow revascularization (Galindo-Moreno et al. 2010). AB has been added to DPBB in different ratios, in grafting proce-dures, to add osteoinductive properties and subsequently enhance bone formation (Hall-man et al. 2001; Tadjoedin et al. 2003; Del Fabbro et al. 2004; Simion et al. 2007; Esposi-to et al. 2010; Urban et al. 2011). In a recent animal study, it was concluded that the graft volume was better preserved when DPBB was added to AB grafts, and the volumetric reduc-tion in the graft was significantly influenced by the ratio of DPBB and AB after sinus floor augmentation (Jensen et al. 2011). In addi-tion, it appears that the ratio of DPBB and AB influences bone remodeling patterns (Gal-indo-Moreno et al. 2011). On the other hand, it has been reported in a randomized con-trolled trial, in which bone formation was scintigraphically and histologically evaluated, that addition of AB to DPBB in a ratio 1 : 4 did not lead to enhanced new bone formation in comparison with 100% DPBB 4 months after sinus floor augmentation (Pikdoken
et al. 2011). Lateral augmentation with
DPBB has been performed in both animal studies (Stavropoulos et al. 2001; Araujo et al. 2002; De Santis et al. 2012) and human studies (Simion et al. 2007; Urban et al. 2011).
To the best of our knowledge, different ratios of DPBB and AB have not been com-pared for lateral augmentation in a clinical, randomized, and controlled study.
The aim of this study was to radiologically and histomorphometrically evaluate the heal-ing and volumetric changes of graft after lat-eral augmentation with 2 different ratios of DPBB and AB.
We hypothesized that there are no histo-morphometric differences in healing or any differences in width and volumetric reduc-tion in two different mixtures of DPBB and AB used for lateral ridge augmentation.
Material and methods
Study design and patientsThis randomized clinical trial (Appendix S1) was designed as a split-mouth study.
Totally or partially, bilaterally edentulous patients in need of lateral augmentation of the alveolar crest prior to implant treat-ment were included in the study according to the following inclusion criteria: adult patients (20–80 years of age), no uncon-trolled systemic disease, no smoking habits (free of smoking habits more than 1 month prior to treatment), no history of radiation to the area of treatment, adequate height of the alveolar crest, but the width of the alveolar crest should be 4 mm measured by means of cone beam computed tomogra-phy (i-CATâ Cone Beam 3-D Imaging Sys-tem; Imaging Sciences International Inc,
Hatfield, PA, USA). All patients were
required to read, understand, and sign an informed consent before they were included in the study.
A total of 13 patients (six men and seven women) with a mean age of 59.6 years (range, 29–75) participated in the study. One patient had both jaws included in the study. Eight totally edentulous maxillae, two totally eden-tulous mandibles, two bilaterally, partially edentulous maxillae, and two bilaterally, par-tially edentulous mandibles were treated.
The study was approved by the Regional Ethical Review Board in Uppsala, Sweden. Ridge augmentation and graft retrieval All procedures were performed by the same surgeon under local anesthesia and oral seda-tion with 5–10 mg midazolam (Dormicum, Roche AB, Stockholm, Sweden). As a prophy-lactic measure, all patients received 2 g of V-penicillin (Kavepenin, Astra AB, So¨dert€alje, Sweden) x 3 for 10 days starting 1 h preopera-tively or clindamycin (Dalacin, Pfizer AB, Stockholm, Sweden) 300 mg9 3 for 10 days in event of penicillin allergy. Anesthesia was induced by lidocaine (2%) with epinephrine (1 : 80,000) (Xylocain/adrenalin, Astra AB).
After a slightly lingual-oriented crestal incision and vertical releasing incisions, a mucoperiosteal flap was elevated bilaterally and reflected buccally to expose the edentu-lous area to be augmented. In the dentated area, the vertical incisions were at least one tooth away from the surgical site and at the edentulous area at least 5 mm beyond the defect to be augmented. Care was taken to free the buccal wall from periosteum, and the cortical bone was perforated with a small round bur (Fig. 1a). The mucoperiosteal flap was elongated through a careful incision of the periosteum from one releasing incision to the other. If further elongation of the flap was necessary to ensure passive closure of the wound, this was performed with blunt dissection using a hemostat.
An incision was performed 5 mm inferior to the attached gingiva from the first premo-lar to the anterior part of the mandibupremo-lar ramus. By means of a thin fissure bur, a
monocortical autogenous bone block of
approximately 2–3 cm 9 1 cm was retrieved, and a bone mill (Roswitha Quetin Dental Produkte, Leimen, Germany) was used to particulate the bone block. In all cases, the harvesting of the bone graft was performed unilaterally.
DPBB particles (particle size 0.25–1 mm, Bio-Ossâ; Geistlich Pharmaceutical, Wolhu-sen, Switzerland) were mixed by weight with the AB particles in a 90 : 10 (mean; 1.2 g Bio-Ossâ: 0.14 g autogenous bone) and 60 : 40 (mean; 1.1 g Bio-Ossâ: 0.7 g AB) mixture, along with autogenous blood from the surgi-cal site. To each of the graft mixtures, 0.25 ml of fibrin glue (Tisseel, Duo Quick Immuno, Vienna, Austria) was added to make the graft moldable and avoid the parti-cles to migrate from the augmented area.
At this time, the sides were randomized to either a 90 : 10 mixture or a 60 : 40 mixture.
The allocation sequence was computer-generated by a statistician at G€avleborg County Hospital, Sweden, and concealed in envelopes until randomization.
The grafts were placed on the buccal aspect of the alveolar ridge, layer by layer, gently pressed to the recipient site, and thrombin was added to catalyze the setting of the fibrin glue (Fig. 1b,c). In totally eden-tulous jaws, the different graft mixtures were fused in the midline. All grafts were covered by collagen membranes (Bio-Gideâ;
Geistlich Pharmaceutical, Wollhausen,
Switzerland) without any further fixation (Fig. 1d). The incisions were carefully closed with horizontal mattress sutures as well as single interrupted and continuous resorbable
(4–0 Vicrylâ; Johnson & Johnson AB,
Sollentuna, Sweden) sutures.
The patients were not allowed to wear den-tures for a minimum of 1 month, and after that, dentures were carefully adjusted and relined with soft material so that no extend-ing lateral parts were in contact with the grafted area. Patients were instructed to rinse with a 0.1% solution of chlorhexidine twice a day until suture removal. After a healing period of 10–14 days, the sutures were removed and the patients were checked for complications monthly for 3 months. In case of membrane exposure, chlorhexidine rinsing was prolonged.
Biopsy retrieval and implant installation Eight months, mean: 8.1 months (range, 7.9–8.3), after the grafting procedure, biopsies from the grafts were retrieved and oral implants were installed (Fig. 2).
After a single dose of 2 g amoxicillin (Imacillinâ, Meda AB, Solna, Sweden) and local anesthesia induced by lidocaine (2%) with epinephrine (1 : 80,000), a crestal inci-sion with releasing inciinci-sions was performed. Buccal and lingual flaps were carefully reflected. After planning of the implant sites, biopsies were retrieved from the grafts with a trephine bur (3.1 mm in diameter) in a hori-zontal direction 5–7 mm apical to the top of the alveolar crest and at least 1 cm away from the midline in edentulous cases. One
core sample was retrieved from each graft (i.e., one biopsy from each side). The biopsies were immediately immersed in formalin fixative and sent to the laboratory for further processing.
Seventy-one surface-modified oral implants with titanium dioxide (OsseospeedTM; Astra
Tech AB, Mo¨lndal, Sweden) were installed in a two-stage procedure according to the manu-facturer’s protocol. After placement of cover screws, the flaps were adapted with resorb-able sutures (Vicrylâ).
Radiographic examination and evaluation
Cone beam computed tomography (i-CATâ
Cone Beam 3-D Imaging System; Imaging Sciences International Inc) (CB/CT) scans
were obtained preoperatively, immediately after grafting, and 7.5 months (approximately 2 weeks before implant installation for treat-ment planning) after the grafting procedure. Volumetric and width measurements were performed to compare differences between the graft mixtures (90 : 10 and 60 : 40) after a healing period of 7.5 months. Volumetric measurements of lateral ridge grafts were per-formed from all CB/CT scans in the ImageJ software (version 1.45, downloaded from http://rsb.info.nih.gov/ij/download.html) by a single radiologist. Grafted bone could easily be distinguished from residual bone by den-sity and structure on the scans immediately after grafting. However, after 7.5 months, the distinction between graft and cortical bone was more difficult, especially in the mandi-ble. Each 0.4- or 0.3-mm slice was displayed on a computer monitor, and a drawing func-tion was used to manually outline the mar-gins of the graft to calculate the graft area for each coronal slice. The graft volumes were estimated according to the method described by Uchida (Uchida et al. 1998), using the following formula:
Vn
i¼1¼
X dS Dh
The volume of each section was dV= dS x Δh, where dS is the area of the graft in a given section, andΔh is the slice thickness of the section. Hence, the volume (V) of the graft to a height of n mm was calculated as the sum of the volumes of each section (dV).
All width measurements were made on
tomographic slices perpendicular to the
longitudinal axis of the alveolar crest, using i-Cat Vision software, 3 mm and 6 mm from the top of the crest, at the anticipated implant sites. In the midline of the jaws, on the axial view, an anatomic reproducible landmark (e.g., the nasal spine or foramen incisivum) was defined and a straight line was drawn through it. The distance from the measuring point to this line, at the middle of the alveolar crest, was obtained using the software ruler. The same anatomic land-marks and distances were used for measure-ments on CB/CT scans immediately after grafting and 7.5 months after the grafting procedure (Fig. 3). The width of the residual bone and the total width (residual bone + bone graft) after grafting were obtained from the CB/CT scans immediately after grafting, as the graft could easily be distinguished from the residual bone, using the software ruler. On the CB/CT scans 7.5 months after grafting, only the total width was measured at the anticipated implant sites (Fig. 4), as it
Fig. 2. After 8 months of graft healing, Astra Osseo-speedTM
implants were installed and biopsies were retrieved from both graft compositions (90 : 10 and 60 : 40) perpendicular to the alveolar crest (black arrows).
(a) (b)
(c) (d)
Fig. 1. (a) Buccal view of an exposed and perforated (after drilling with a small round bur), extremely thin, alveolar crest of a totally edentulous mandible prior to lateral augmentation.(b) Lateral ridge augmentation of a totally eden-tulous mandible. After randomization, a mixture of DPBB/AB (60 : 40) was placed on the buccal aspect of the alveo-lar ridge from the midline and posterior on the left side. (c) A mixture of DPBB/AB (90 : 10) was placed on the right side of this patient. All grafts were placed, layer by layer, and thrombin was added to catalyze the setting of the fibrin glue. (d) All grafts were covered by collagen membranes, Bio-Gideâ, without any further fixation.
was difficult to distinguish between cortical bone and graft in a few cases. The graft width after 7.5 months was calculated by subtrac-tion of the total width with the width of the
residual bone obtained from the scans
immediately after grafting.
Histologic preparation and assessment In brief, the specimens were immersed in 4% neutral-buffered formaldehyde solution, rinsed in water followed by dehydration in a graded series of ethanol, pre-infiltrated in diluted resin followed by pure resin, and embedded in light-curing resin (Technoviteâ 7200 VLC; Kulzer & Co GmbH, Wehrheim, Germany). The samples were divided in the long axis of the biopsy and mounted on a sup-porting plexi-glass slide. One thick section was taken from the center of each biopsy, ground to a thickness of about 10–15 lm, according to the technique described by Donath and Breuner (Donath & Breuner 1982)
using the Exaktâ system (ExaktApparatebau, Norderstedt, Germany), and stained with toluidine blue mixed with pyronin-G. The undecalcified, histologically stained sections were analyzed histologically and histomorho-metrically in a LeitzMetallux 3 light micro-scope equipped with a Microvid (Ernst Leitz GmbH, Wetzlar, Germany) morphometric system connected to a personal computer. All
measurements were performed manually,
directly through the eyepiece of the micro-scope with a mouse connected to the com-puter. The 2.59 objective was used, resulting in a 25 9 magnification revealing the entire biopsy in the field of view for quantitative analyses. The same person performed all mea-surements. The grafts were identified in the specimens and divided into two equally long segments, one facing the recipient bone and one facing the recipient periosteum (Fig. 5). The following histomorphometric measure-ments were made in all specimens:
1. Total length of the graft (mm),
2. Ingrowth of newly formed bone into the graft (percentage of the total length of the graft, measured from the recipient bone),
3. Total bone area in percentage of the graft facing bone and periosteum, respectively, 4. Total area of the DPBB in percentage of
the graft facing bone and periosteum, respectively,
5. Total area of soft tissue in percentage of the graft facing bone and periosteum, respectively,
6. The degree of DPBB–bone contact (per-centage of total surface length for each particle) in the graft facing bone and peri-osteum, respectively, and
7. Number of DPBB particles in the graft facing bone and periosteum, respectively.
Statistics
Results were reported as mean values and standard deviations. Histomorphometric and volumetric differences between 90 : 10 and 60 : 40 (DPBB/AB compositions) were tested using Wilcoxon signed-rank test, and non-parametric confidence intervals were esti-mated using bias-corrected and accelerated (BCa) bootstrapping. Differences in width
changes between the two compositions
adjusted for initial graft width were tested using mixed models, with DPBB/AB compo-sition as a fixed factor and jaw as random fac-tor. Model assumptions, that is, normally distributed random effects with constant var-iance, equal across compositions, were
veri-fied by visual inspection of graphs of
residuals and predicted values, in combina-tion with Shapiro–Wilks test of normality. All statistics were performed using SPSS ver-sion 17 (IBMâ; SPSS Inc., Chicago, IL, USA). A statistical significant difference was con-sidered at P< 0.05.
Results
Clinical data of subjects and surgical sites are presented in Table 1.
Twenty-one of 28 grafts healed unevent-fully, whereas the rest presented with dehi-scences (4 in the 90 : 10 group and 3 in the 60 : 40 group). Of the seven sites with dehi-scences (in two edentulous mandibles, two edentulous maxillae, and two with bilateral posterior edentulous mandibles), five sites healed within 4–6 weeks. In two sites (one totally edentulous mandible and one totally edentulous maxilla), the soft tissue healed after 4 months.
(a) (b)
Fig. 3. In the midline of the jaws, on the axial view of the CB/CT scan, an anatomic reproducible landmark (e.g., the nasal spine or foramen incisivum) was defined and a straight line was drawn through it (black vertical lines). The distance from the measuring point to this line was obtained using the software ruler (black horizontal line). The same anatomic landmarks and distances were used for measurements on CB/CT scans immediately after graft-ing (a) and 7.5 months after the graftgraft-ing procedure (b).
(a) (b)
Fig. 4. (a) The width of the residual bone (black arrow) and the total width (residual bone+ bone graft) (white arrow) after grafting were obtained from the CB/CT scans immediately after grafting, 3 and 6 mm from the top of the crest, as the graft could easily be distinguished from the residual bone. (b) On the CB/CT scans 7.5 months after grafting, only the total width (black arrow) was measured, 3 and 6 mm from the top of the crest, at the anticipated implant sites. It is difficult to distinguish the graft from the residual cortical bone plate.
When the flaps were raised at reentry, 8.1 months (range, 7.9–8.3) after the grafting procedure, some of the particles were found in the soft tissue, especially in the maxilla. In the mandible, it was, in general, easier to separate the flap from the graft, and the regenerated bone appeared more dense and mineralized compared with the graft in the maxilla.
It was possible to install implants in all planned sites, but one (dehiscence healed after 4 months in a totally edentulous max-illa), where the ridge was still too narrow and the graft was very soft at the area of the pre-vious dehiscence.
Clinically, no differences were experienced between the two mixtures (90 : 10 and
60 : 40) regarding graft healing, width of the alveolar crest, or density of the graft during drilling for implants.
Radiographic results
There was a wide individual range in both volumetric and width changes.
The volumetric changes of the grafts are presented in Table 2. There was no signifi-cant difference regarding graft reduction between the 90 : 10 and 60 : 40 mixtures, 55.3% and 53.8%, respectively (P= 0.88).
The width changes are presented in
Tables 3 and 4. There was a significant differ-ence in width reduction between the 90 : 10 and 60 : 40 mixtures when measured 3 mm
from the top of crest, 46.9% (2.7 mm) and 37.0% (2.0 mm), respectively (P= 0.029). There was no significant difference in width reduction between the 90 : 10 and 60 : 40 mixtures when measured 6 mm from the top
of crest, 34.7% (2.3 mm) and 27.2%
(1.8 mm), respectively (P= 0.07). Qualitative observations on histologically stained sections
The bone biopsies differed in size (length and diameter) and structure that one side had a cortical appearance, whereas the other was more spongeous. While DPBB particles were easily recognized and frequently pres-ent, the AB particles were not. Irrespective of mixture, no major qualitative differences were observed. All biopsies demonstrated bone and soft tissue integration of DPBB
particles. Inflammatory cells were
com-monly observed irrespective of sample.
Multinucleated giant cells were more
frequently observed in close relation to light-stained DPBB surfaces.
In most samples and irrespective of mix-ture of DPBB: AB, that is, 90 : 10 or 60 : 40, the transplanted region in the biopsy could easily be judged by the naked eye. The DPBB particles appeared to be quite large and often encapsulated by bone.
Survey pictures illustrated more “bone-integrated particles” in the graft facing the bone side, whereas the other side, the one facing the periosteal side, showed less inte-grated particles (Fig. 6). In higher magnifica-tion, the DPBB particles demonstrated a rather sharp, straight line when in contact to bone. When the DPBB particles were fac-ing soft tissue areas, the surface was com-monly lighter stained. Multinucleated giant cells were often observed in close relation to this area. In some sections, demarcated and quite large encapsulated regions, formed in close vicinity to DPBB particles, could be observed in the soft tissue areas. These for-mations seemed to contain small fragments of DPBB particles. Macrophages and plasma cells were observed in these soft tissue regions outside the DPBB particles. The
amount of inflammatory cells seemed
similar irrespective of biopsy type. In a few samples, eosinophils could be observed in soft tissue areas.
The few AB particles observed (being lighter stained and with empty osteocyte lacunae) were often surrounded by newly formed bone with large osteocytes. The newly formed bone areas facing the marrow cavity/soft tissue regions demonstrated dar-ker-stained rims. These rims may have been Table 1. Clinical data of subjects and surgical sites
Subject
number Gender Age Region
Healing time (mo)
Number of implants
Lost implants Graft Implant
1 M 57 Totally ed. maxilla 8.2 3.0 6
2 M 66 Totally ed. maxilla 8.1 3.3 6 1 (90 : 10)
3 F 58 Partially ed. mand. 8.2 2.9 4
4 M 66 Totally ed. maxilla 8.3 3.0 6
5 F 42 Partially ed. maxilla 8.2 2.8 6
6 F 66 Totally ed. maxilla 8.2 3.0 6 1 (90 : 10)
7 F 68 Totally ed. mand. 8.1 3.0 4
8 M 67 Totally ed. mand. 7.9 5.7 4
9 M 67 Totally ed. maxilla 7.9 3.6 6
10 F 75 Totally ed. maxilla 8.1 3.0 5
11 M 59 Totally ed. maxilla 8.2 3.1 6
12 F 64 Totally ed. maxilla 7.9 3.8 6
13 F 29 Partially ed. maxilla 8.1 3.1 2
14 M 50 Partially ed. mand. 8.2 3.0 4
Total 71 2
Mean (SD) 59.6
(12.1)
8.1(0.1) 3.3 (0.7)
Range 29–75 7.9–8.3 2.8–5.7
mo, months; SD, standard deviation.
Fig. 5. This cut and ground section is from the posterior mandible with a mixture of 60 : 40 (DPBB/AB). The graft was easily identified on the left hand side of the specimen and, for histomorphometry, divided into two equally long segments, one facing the recipient bone and one facing the recipient periosteum. Scale bar, 500lm.
osteoid, although it was not easy to confirm the presence of osteoblasts if on some occa-sions osteoblasts were found entrapped in the osteoid. The AB particles that were sur-rounded by new bone demonstrated irregular-shaped and darker-stained surfaces (cement lines). Osteons with concentric lamella were seen close to AB regions. These boarder sur-faces, between osteons and AB, reminded of Howship’s lacunae but without osteoclasts. When these AB surfaces were facing the soft
tissue regions, macrophages could be
observed close to the bone.
Histomorphometric results
All 28 specimens, 14 from each mixture and grafted site, could be used for histomorphom-etry, and the grafted tissue was easily distin-guished from the residual bone. The AB particles were sparse and mostly resorbed, and no effort was made to differentiate bone from AB in the histomorphometric analyses.
The results from the histomorphometric analyses are presented in Tables 5–7. There was a wide individual range in all histomor-phometric results. There were no significant differences between the 90 : 10 mixture and
the 60 : 40 mixture in the fraction of differ-ent tissues (i.e., bone, DPBB, and soft tissue) (Fig. 7) or in any of the histomorphometric variables analyzed.
In the graft with a mixture of 90 : 10, there was no significant difference in fraction of DPBB between the graft facing residual bone and the graft facing the periosteum (30% and 31%, respectively, P= 0.73), but there was a significant difference in fraction of soft tissue (42% and 53%, respectively, P= 0.003) and fraction of bone formation (29% and 16%, respectively, P= 0.002) (Fig. 8).
In the graft with a mixture of 60 : 40, there was no significant difference in fraction of DPBB between the graft facing residual bone and the graft facing the periosteum (29% and 28%, respectively, P= 0.75), but there was a significant difference in fraction of soft tissue (46% and 59% respectively, P= 0.008) and fraction of bone formation (26% and 13%, respectively, P= 0.001) (Fig. 8).
There was a significant difference in the degree of DPBB–bone contact between the graft facing residual bone and the graft facing the periosteum, both in the 90 : 10 mixture (56% and 34%, respectively, P= 0.003) and in the 60 : 40 mixture (57% and 30%, respec-tively, P= 0.002) (Table 7).
Table 2. Volume measurements of the different grafts at the time of augmentation and after a healing period of 7.5 months
Mixture
After Augmentation After 7.5 months
Reduction in the graft Mean (SD) mm3 (Range) Mean (SD) mm3 (Range) Mean (SD) mm3 (Range) Mean (SD)% (Range) 90 : 10 1979 (732) (554–3079) 876 (415) (14–1681) 1103 (595) (171–2407) 55.3 (20.9) (15.8–97.5) 60 : 40 1823 (607) (693–2670) 786 (326) (380–1377) 1037 (546) (179–2060) 53,8 (16,9) (25.9–83.3) Mean diff 95% CI 156 59 to 373 90 71 to 249 66 148 to 302 1.5 6.8 to 10.8 P-value 0.16 0.30 0.78 0.88
Values are expressed as mean, standard deviation (SD), range and 95% confidence interval (95% CI). The reduction of the grafts is calculated both in mm3and in percentage (%). Differences in volume
between a mixture of DPBB/AB of 90 : 10 and 60 : 40 were tested by Wilcoxon signed-rank test. A statistically significant difference was considered atP < 0.05.
Table 3. Width of the alveolar crest at different time points, measured on CB/CT, 3 mm from the top of the alveolar crest
Mixture
Preoperative After grafting After 7.5 months Gain width at 7.5 months Reduction in the graft Mean (SD) mm (Range) Mean (SD) mm (Range) Mean (SD) mm (Range) Mean (SD) mm (Range) Mean (SD) mm (Range) % 90 : 10 3.1 (1,1) (1.6–6.2) 8.8 (1.3) (6–11.6) 6.0 (1.6) (2.4–9.2) 2.9 (1.3) (0.4–5.2) 2.7 (1.6) (0–7.5) 46.9 (23.5) (0–94.9) 60 : 40 2.7 (0.7) (1.6–4.1) 8.3 (1.1) (6.2–10.8) 6.2 (1.3) (3.2–8.8) 3.5 (1.3) (0.8–6.3) 2.0 (1.3) (0–4.4) 37.0 (19.9) (1.6–83.7) Mean diff 95% CI 0.4 0.2 to 0.7 0.5 0.1 to 1.0 0.20.3 to 0.7 0.61.1 to 0.0 0.7 0.1–1.2 9.9 1.4–18.3 P-value 0.06 0.11 0.60 0.021 0.021 0.029
Values are expressed as mean, standard deviation (SD), range, and 95% confidence interval of the mean difference (95% CI). Differences in width changes between a mixture of DPBB/AB of 90 : 10 and 60 : 40, adjusted for initial graft width (except for preoperative analyses), were tested using mixed models. A statistically significant difference was considered atP < 0.05.
Table 4. Width of the alveolar crest at different time points, measured on CB/CT, 6 mm from the top of the alveolar crest
Mixture
Preoperative After grafting After 7.5 months Gain width at 7.5 months Reduction in the graft Mean (SD) mm (Range) Mean (SD) mm (Range) Mean (SD) mm (Range) Mean (SD) mm (Range) Mean (SD) mm (Range) % 90 : 10 4.3 (1.2) (1.4–6.5) 10.5 (1.7) (8.4–13.4) 8.2 (1.6) (5.3–11.4) 4.0 (1.4) (0.4–6.3) 2.3 (1.7) (0.3 to 6.3) 34.7 (23.5) (7.5 to 89.7) 60 : 40 3.8 (1.1) (1.7–6) 10.1 (1.8) (6.4–13.7) 8.3 (1.8) (4.2–11.4) 4.5 (1.3) (2.3–7.6) 1.8 (1.4) (0.6 to 5.2) 27.2 (18.7) (8.6 to 69.3) Mean diff 95% CI 0.5 0.03–0.9 0.4 0.2 to 0.9 0.10.7 to 0.5 0.51.2 to 0.1 0.5 0.1 to 1.1 7.4 0.6 to 15.4 P-value 0.034 0.18 0.73 0.07 0.11 0.07
Values are expressed as mean, standard deviation (SD), range, and 95% confidence interval of the mean difference (95% CI). Differences in width changes between a mixture of DPBB/AB of 90 : 10 and 60 : 40, adjusted for initial graft width (except for preoperative analyses), were tested using mixed models. A statistically significant difference was considered atP < 0.05. SD = standard deviation.
Discussion
In this clinical and randomized study, volu-metric and width changes were analyzed and histomorphometric analyses were performed of grafts with two different ratios of DPBB and AB after lateral augmentation of the hori-zontally resorbed alveolar crest. Despite sig-nificantly less width reduction, 3 mm from the top of the alveolar crest, in favor of
60 : 40 (DPBB/AB) ratio compared with
90 : 10 ratio, there were no other significant differences regarding volumetric changes, ingrowth of newly formed bone, and fractions of different tissues in the grafts (i.e., percent-age of DPBB, bone, and soft tissue).
Even though the majority of augmented areas were large defects (10 totally edentulous jaws), it was possible to install implants in all cases.
As lateral ridge augmentation may be quite technique- and operator-sensitive (Aghaloo & Moy 2007), we decided to use one single surgeon for all clinical procedures. It was concluded in a meta-analysis that membrane exposure seemed to have a major negative effect on guided bone regeneration (Machtei 2001). Factors that may impede wound heal-ing include foreign materials, compromised blood supply, and wound tension, which in combination with bacterial contamination may explain the reduced bone formation after
membrane exposures (Wang & Boyapati
2006). In the present study, the membrane exposures were evenly distributed between 90 : 10 (four exposures) and 60 : 40 (three exposures) mixtures and should therefore not have any impact on the results. In larger edentulous areas in need of lateral augmenta-tion, autogenous onlay bone blocks are fre-quently used (Nystrom et al. 1995; Becktor et al. 2004; Thor et al. 2005; Sjostrom et al. 2007; Esposito et al. 2008; Chiapasco et al. 2009). AB possesses both osteoinductive and osteoconductive properties, and bone blocks may be considered more reluctant to bone resorption compared with particulated bone particles. However, the need for donor sites, general anesthesia, high costs and morbidity to the patients have motivated the use of bone substitutes. Alveolar ridge augmenta-tion with the use of membranes in combina-tion with supporting AB or bone substitutes, guided bone regeneration (GBR), is well docu-mented, and several different materials have been used as membranes or bone substitutes (Simion et al. 2007; Donos et al. 2008; Roc-chietta et al. 2008; Beitlitum et al. 2010; Dahlin et al. 2010).
When DPBB has been used in GBR, the addition of AB varies between 0% and 50% (Mayfield et al. 2001; Hellem et al. 2003; Simion et al. 2007; Rocchietta et al. 2008; Urban et al. 2011). From a clinical point of view, it is of great interest to gain knowledge about how the amount of added AB affects the reduction and healing of the DPBB graft, to possibly avoid problems from a donor site. In an animal study, the addition of 25% AB to DPBB accelerated de novo bone formation in osseous defects (Thorwarth et al. 2006). Ten percent AB, used in one of the compositions in the present study, can easily be collected with a bone scraper at the same or neighboring surgical site; however, 40% AB would require another surgical donor site in larger defects.
The width changes after lateral augmenta-tion are often measured clinically by the means of a caliper or a periodontal probe (von Arx & Buser 2006; Hammerle et al. 2008; Beitlitum et al. 2010; Urban et al. 2011). However, if the graft is not totally integrated, parts of the superficial layer might be ele-vated with the flap depending on the surgical handling. This may lead to inconsistent mea-surements depending on the surgical tech-nique at flap elevation rather than the reduction in the graft per se. On the other hand, if the graft is not fully integrated, the
radiographic appearance may not be in
accordance with the clinical situation after flap elevation, which will complicate the
(a) (b)
Fig. 6. These cut and ground sections are from the same maxillary graft with a mixture of 90 : 10 (DPBB/AB). The graft closest to the residual bone seemed to have more particles being surrounded by bone (a) compared with the graft closest to the periosteum (b) demonstrating more particles surrounded by soft tissue. DPBB– deproteinized bovine bone, NB– newly formed bone, ST – soft tissue Scale bars, 200 lm.
Table 5. Histomorphometric results 7.5 months after augmentation
Mixture Number of DPBB particles (SD) range Graft length mm (SD) range Ingrowth of new bone mm (SD) range Ingrowth of new bone% (SD) range B P 90 : 10 18.6 (7.9) 8–34 15.7 (8.2) 2–28 4.0 (1.4) 1.9–5.9 3.1 (1.6) 0.7–5.9 82.3 (26.6) 16.7–100 60 : 40 17.5 (12.8) 4–53 16.1 (7.6) 8–34 4.0 (1.6) 2.1–7.9 3.3 (1.9) 1.2–7.9 82.1 (23.3) 27.7–100 Mean diff 95% CI 1.1 7.4 to 7.4 0.46.6 to 5.4 – 0.191.6 to 0.96 0.11 18.5 to 20.1 P-value 0.19 0.98 – 0.79 0.88
Values are expressed as mean, standard deviation (SD), and range. B; toward recipient bone, P; toward recipient periosteum. Differences in number of DPBB particles, graft length, and ingrowth of new bone into the graft between a mixture of DPBB/AB of 90 : 10 and 60 : 40 were tested by Wilco-xon signed-rank test. diff: difference, 95% CI: 95% confidence interval. A statistically significant difference was considered atP < 0.05.
preoperative planning. The compliance between volumetric and width measurements on radiographs and the clinical situation would probably be more reliable if the healing time of the graft is prolonged and further inte-gration of the graft is allowed. In the present study, the width was measured by means of CB/CT for consistent measurements immedi-ately after augmentation and prior to the secondary surgical intervention, that is, implant placement. After the healing period of 7.5 months, it was, in some cases, difficult to differentiate between cortical bone and the bone graft. To overcome this problem, the total width, including the alveolar crest and the graft, was measured at 7.5 months, and the initial width of the alveolar crest was subtracted to obtain the graft width.
In the present study, the mean horizontal bone gain was 2.9 mm and 3.5 mm for 90 : 10 and 60 : 40, respectively, 3 mm from the top of the crest. There was a statistically
significant difference in reduction in the width between the two compositions in favor of 60 : 40; however, the difference was 0.7 mm and the precise clinical importance for implant placement being uncertain. Six millimeters from the top of the crest the mean horizontal bone gain was 4.0 mm and 4.5 mm, respectively, and there was no
sig-nificant difference between 90 : 10 and
60 : 40 compositions. Subsequently, it seems to be of importance where the measurements are performed as the particulated graft may be reduced or displaced due to pressure from the soft tissue and removable dentition dur-ing masticatdur-ing.
H€ammerle et al. (Hammerle et al. 2008) reported a mean bone gain of 3.6 mm after
lateral augmentation with 100% DPBB
blocks or granules and a collagenous mem-brane (Bio-Gideâ) in 12 consecutive patients, and Urban et al. (Urban et al. 2011) reported a mean bone gain of 5.6 mm after horizontal
ridge augmentation with a resorbable mem-brane and particulated AB with or without DPBB in a prospective case series in 22 patients. DPBB and collagenous membranes have also been used to cover autogenous bone blocks to reduce graft resorption. In 2 of those studies, the mean bone gain was 3.9 mm (Cordaro et al. 2011) and 4.6 mm (von Arx & Buser 2006), respectively. In the latter study, the horizontal measurements were performed 1 mm from the top of the crest; however, in none of the other studies, the authors defined at which vertical level
the horizontal measurements were
per-formed, and comparisons to the present study is therefore problematic.
The reduction in the grafts during the heal-ing period in the present study was extensive, both regarding volume: 53.8% and 55.3% and width: 37.0% and 46.8% (3 mm from the top of the crest) in 60 : 40 and 90 : 10 DPBB/AB compositions, respectively. It has been sug-gested that DPBB particles are non-resorbable or extremely slowly resorbed (Schlegel & Do-nath 1998; Mordenfeld et al. 2010). Further-more, in an experimental study in rat, a shrinkage to about 69% of the height of the initial fill of bovine bone mineral after aug-mentation of calvarial tissue using non-permeable silicon domes was reported (Slotte & Lundgren 1999). The authors suggested that it was rather due to denser packing of the filler material than resorptive activity. Hence, the cause of the reduction in the graft may be attributed to resorption and remodel-ing of the bone particles, blood cloth, and the fibrin glue in combination with soft tissue pressure. In the present study, the 60 : 40 mixture seemed to be more resistant to this reduction closer to the top of the crest; however, it seems that the graft was rather displaced than reduced as there was no signif-icant difference in volume reduction between the mixtures. We find these results difficult to explain, but one explanation for the resis-tance to reduction may be a faster integration of the graft, due to higher percentage of AB present, as DPBB may decelerate bone regen-eration (Jensen et al. 2006). However, this explanation could not be confirmed with the histomorphometry, presumably due to the fact that the biopsies were retrieved further apically than the sites for the radiologic examination (i.e., 3 mm from the top of the crest).
It was difficult to distinguish AB from vital bone, especially in lower magnifications, whereas DPBB particles were easily
recog-nized. Histomorphometry was performed
manually in a lower magnification (x 25) Table 7. Histomorphometric results 7.5 months after augmentation
Site DPBB/bone contact (%) Mean diff 95% CI P-value 90 : 10 60 : 40 B 55.8 (22) 10.7–80.6 56.6 (21.5) 12.7–92.1 0.87.5 to 7.0 0.43 P 34.3 (25.1) 0–85.9 29.9 (27.8) 0–76.8 4.4 6.9 to 15.1 0.46 Mean diff 95% CI 21.5 13.8–29.0 26.7 16.6–36.6 P-value 0.003 0.002
Values are expressed as mean, standard deviation (SD), range, and 95% confidence interval (95% CI). B: in 50% of the graft toward recipient bone as explained in Materials and Methods, P: in 50 % of the graft toward recipient periosteum as explained in Material and Methods, T: total. Differences in DPBB and new bone contact between a mixture of DPBB/AB of 90 : 10 and 60:40 and between P and B were tested by Wilcoxon signed-rank test. A statistically significant difference was considered at P < 0.05.
Table 6. Histomorphometric results 7.5 months after augmentation Site
DPBB area (%) Bone area (%) Soft tissue area (%)
90 : 10 60 : 40 90 : 10 60 : 40 90 : 10 60 : 40 B 29.6 (9.2) 16.2–46.8 28.8 (11.1) 4.7–44.7 28.7 (13) 9.8–59.9 25.6 (9.8) 8.9–43.6 41.7 (13.8) 16.8–60.9 45.6 (15.7) 22.6–77 P 30.9 (10.4) 7.8–55.7 28.1 (9.3) 11.2–40.4 15.6 (13.3) 0–43.1 13.3 (10.3) 0–30.3 53.5 (13.2) 24.9–69 58.6 (9.3) 37–71.5 T 30.1 (6.7) 19.8–42.9 28.3 (8.9) 12.5–39.4 22.7 (12.4) 5.8–53.8 20.0 (9.1) 6.2–37 47.3 (12.7) 19.7–63.1 51.6 (10.9) 30.4–71.7 Mean diff A 95% CI 1.8 2.4 to 6.1 2.7 2.4 to 8.4 9.1 to 0.14.3 P-value A 0.55 0.73 0.18 Mean diff B 95% CI 1.3 9.6 to 6.0 0.7 4.8 to 5.8 13.1 8.4–17.7 12.3 9.2–15.6 11.817.0 to 6.6 13.020.0 to 5.7 P-value B 0.73 0.75 0.002 0.001 0.003 0.008
Values are expressed as mean, standard deviation (SD), range, and 95% confidence interval (95% CI). B: in 50% of the graft toward recipient bone as explained in Materials and Methods, P: in 50% of the graft toward recipient periosteum as explained in Material and Methods, T: total. Differences in total area of different tissues between a mixture of DPBB/AB of 90 : 10 and 60 : 40 were tested by Wilcoxon signed-rank test (Mean diff A,P-value A) and between P and B (Mean diff B, P-value B). A statistically significant difference was considered atP < 0.05.
after analyzing the material also in higher magnifications. The software program used in the present study did not allow for histo-morphometry in a high magnification as we
wanted to measure the entire biopsy.
Although the AB particles were sparse and
mostly resorbed, the histomorphometric
results may have been slightly different had it been possible to distinguish between origi-nal bone and AB.
New bone formation into the graft may occur both from the residual bone, if prop-erly perforated, and from the periosteum (Gordh et al. 1997; Ortak et al. 2005). In the present study, there was significantly more bone formation closer to the residual bone compared with the portion of the graft closer to the periosteum in both DPBB/AB compo-sitions. This may be explained by the trauma made to the periosteum to achieve
passive closure of the wound, the use of a membrane to obstruct the mesenchymal cells of the “cambium” layer of the perios-teum, and mechanical trauma to the grafted area from the prosthesis. An extended heal-ing time of the graft may increase the ingrowth of newly formed into the graft closer to the periosteum (Araujo et al. 2002). In a clinical study, DPBB particles were grafted to fresh human extraction sockets (Artzi et al. 2000). After a healing period of 9 months, biopsies were retrieved in a coro-nal–apical direction. They found that there was a consistent increase of newly formed bone with a concomitant decrease in soft tissue area fraction from the most superficial histologic sections to the deeper sections. These findings are in accordance with the results of the current study. The authors assumed that difficulty in controlling the stability of the grafted particles probably pro-duced a relatively high percentage of soft tis-sue area fractions. In the present study, fibrin glue was used to increase the mallea-bility and adhesion of the graft to the recipi-ent bone and to stabilize the graft during the early healing period. There are contro-versial opinions on the effect of fibrin glue in the healing process. In an experimental study in four Labrador dogs, it was shown that the percentage of DPBB to bone contact in the alveolar ridge defects treated with DPBB alone was 40% after 3 months, while in the defects treated with DPBB mixed with Tisseelâ, the corresponding integration was only 8%. It was concluded that the adjunct of Tisseelâ may jeopardize the inte-gration of DPBB particles with bone tissue (Carmagnola et al. 2002). In another animal study on bone healing around implants placed in jaw defect augmented with DPBB mixed with fibrin sealer (Tisseelâ), DPBB failed to integrate with the host bone (Carmagnola et al. 2000). These results are in contrast with the findings in the present study. In a review of bio-ceramics and fibrin sealant, the negative effects have essentially been observed in animal studies in contrast to human studies (Le Guehennec et al. 2004). An alternative to fibrin glue to secure the stability of the graft and membrane could be the use of micro screws.
Conclusions
Even though there was no histomorphomet-ric difference between the DPBB/AB compo-sitions, there was significantly less width reduction 3 mm from the top of the crest
Fig. 7. Results from the histomorphometric analyses 8 months after lateral ridge augmentation showing mean per-centage of bone area, soft tissue area, and Bio-Oss (DPBB) area of the two different compositions: 60% DPBB and 40% AB (60 : 40), and 90% DPBB and 10% AB (90 : 10).There were no significant differences between 90 : 10 and 60 : 40 in the percentage of different tissues (i.e., bone, DPBB, and soft tissue). DPBB: deproteinized bovine bone; AB: autogenous bone.
90:10 0 20 40 60 80 Mean(%)
Bone Soft tissue DPBB P = 0.002
P = 0.003
P = 0.73
60:40
Bone Soft tissue DPBB P = 0.001 P = 0.008 P = 0.75 Location in biopsy Bone side Periost side
Fig. 8. Results from the histomorphometric analyses 8.1 months after lateral ridge augmentation comparing mean percentage of bone area, soft tissue area, and Bio-Oss (DPBB) area of the graft facing residual bone (bone side) and periosteum (periost side), respectively, of the two different compositions: 60% DPBB and 40% AB (60 : 40), and 90% DPBB and 10% AB (90 : 10). There was no significant difference in percentage of DPBB between the graft fac-ing residual bone and the graft facfac-ing the periosteum in neither of the compositions, but there was a significant dif-ference in percentage of soft tissue and percentage of bone formation in both compositions. DPBB: deproteinized bovine bone: AB: autogenous bone.
with a mixture of 60 : 40 compared with 90 : 10.
The DPBB particles had a significantly higher degree of integration in newly formed bone closer to the residual bone compared with particles closer to the periosteum in both DPBB/AB compositions.
Further clinical and histologic human stud-ies addressing graft healing/reduction and implant integration, both short and long
term, are needed to make more precise con-clusions of the optimal ratio of DPBB and AB used for lateral augmentation of the alveolar crest.
Acknowledgement:
The authorthanks Hans Ho¨gberg, PhD, and Karl-Erik Westergren, Tech. Lic., for statistical assistance and research technicians Petra
Johansson, Ann Albrektsson, and Maria Hoffman for sample preparation.
Conflict of interest
GeistlichPharma and Astra Tech have sup-ported the study with Bio-Oss and implants (Astra Osseospeed), free of charge.
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Supporting Information
Additional Supporting Information may be found in the online version of this article: Appendix S1. CONSORT 2010 checklist of information to include when reporting a randomised trial
