Abeer M A Bayounis

Hamad A Alzoman

DDS John A Jansen
email: j.jansen@dent.umcn.nl

Nadir Babay

HEALING OF PERI-IMPLANT TISSUES AFTER FLAPLESS AND FLAPPED IMPLANT INSTALLATION

Keywords:

published or not. The documents may come

INTRODUCTION

Currently, the installation of dental implants can be considered as a routine method in the rehabilitation of partially and completely edentulous patients. Initially, dental implants were installed using a surgical protocol, which involved the elevation of a mucoperiosteal flap.

The rationale for this approach was to prevent infection and ingrowth of gingival tissue in between the implant and the bone margins of the implant bed.

However, it is known from periodontal surgery that any flap reflection always results in bone resorption and changes of the crestal bone level [START_REF] Nobuto | Microvascular response in the periosteum following mucoperiosteal flap surgery in dogs: Angiogenesis and bone resorption and formation[END_REF], Wood et al. 1972). In view of this problem, a flapless surgical approach was already introduced in the late 1970s by [START_REF] Ledermann | Vollprothetische versorgung des atrophierten problem -unterkiefers mit hilfe von CBS implantaten[END_REF]. In this procedure a motor-driven circular tissue punch, or a circumferential incision utilizing a surgical blade was used to remove the soft tissue at the implant site without any surgical flap elevation (Sclar 2007). Another approach of flapless implant surgery is penetrating with a round bur directly through the mucosa into the alveolar bone.

Besides the suggested reduced crestal bone resorption, flapless surgery is associated with several other advantages, like: (1) a reduced surgical time and less traumatic surgery, which results in minimal bleeding and an accelerated postsurgical healing as well as allows the patient to resume normal oral hygiene procedures immediately after surgery [START_REF] Becker | Minimally invasive flapless implant surgery: A prospective multicenter study[END_REF], Fortin et al. 2006), and (2) better maintenance of the soft tissue profiles, including the gingival margins of adjacent teeth and the interdental papilla [START_REF] Ramfjord | Healing after exposure of periosteum on the alveolar process[END_REF], Wood et al. 1972[START_REF] Jeong | Flapless implant surgery: an experimental study[END_REF][START_REF] Cairo | Soft tissue management at implant sites[END_REF].

Despite these evident advantages, the major drawback of flapless implant surgery is that it is a F o r P e e r R e v i e w 5 for external irrigation during the implant bed preparation. Also, the surgeon cannot manipulate the soft tissues to achieve a proper adaptation of keratinized gingiva (Sclar, 2007).

In addition to these advantages and disadvantages, there are also some strict anatomical requirements formulated to allow the performance of a flapless procedure. These include the availability of: (1) a sufficient bone width and height because the lack of a direct view of the bone topography, (2) adequate keratinized tissue due to the inevitable sacrifice of some keratinized tissue, and (3) the absence of significant tissue undercuts to prevent dehiscence and fenestrations [START_REF] Hahn | Single-stage, immediate loading and flapless surgery[END_REF][START_REF] Campelo | Flapless implant surgery: A 10-year clinical retrospective analysis[END_REF][START_REF] Kan | Flapless anterior implant surgery: A surgical and prosthodontic rationale[END_REF].

A lot of literature is already available to support the reliability and safety of flapless implant surgery [START_REF] Campelo | Flapless implant surgery: A 10-year clinical retrospective analysis[END_REF][START_REF] Rocci | Immediate loading in the maxilla using flapless surgery, implants placed in predetermined positions, and prefabricated provisional restorations: A retrospective 3-year clinical study[END_REF][START_REF] Blanco | Ridge alterations following immediate implant placement in the dog: flap versus flapless surgery[END_REF][START_REF] Becker | Minimally invasive flapless implant placement: follow-up results from a multicenter study[END_REF], de Bruyn et al. 2009). Most of these studies deal with retrospective and prospective studies, in which frequently image-guided templates are used for the installation of the implants [START_REF] Azari | Flapless implant surgery: Review of the literature and report of 2 cases with computer-guided surgical approach[END_REF], D'haese et al. 2009[START_REF] Komiyama | Soft tissue conditions and marginal bone changes around immediately loaded implants inserted in edentate jaws following computer guided treatment planning and flapless surgery: A ≥ 1-year clinical follow-up study[END_REF][START_REF] Lindeboom | A comparison of two implant techniques on patient-based outcome measures: a report of flapless vs. conventional flapped implant placement[END_REF].

Evaluation of the efficacy of the procedure is based on pocket depth measurements as well as marginal bone loss using radiographs. However, it has been reported before that the use of these clinical parameters overrate the histological marginal bone level [START_REF] Caulier | The relationship of some histologic parameters, radiographic evaluations, and periotest measurements of oral implants: An experimental animal study[END_REF].

Besides the limited diagnostic value of these evaluation techniques, only two studies are available which provide histological data about the effect of flapless surgery on peri-implant bone loss [START_REF] Becker | Histologic evaluation of implants following flapless and flapped surgery: A study in canines[END_REF][START_REF] Lee | Effects of soft tissue punch size on the healing of peri-implant tissue in flapless implant surgery[END_REF]. Unfortunately, the data, as presented in these studies, do not corroborate with each other. [START_REF] Becker | Histologic evaluation of implants following flapless and flapped surgery: A study in canines[END_REF], who installed the implants by direct drilling to the mucosa, found favorable histological data. In contrast, [START_REF] Lee | Effects of soft tissue punch size on the healing of peri-implant tissue in flapless implant surgery[END_REF], who used tissue punches with various sizes in their flapless approach, reported a possible negative effect on the junctional epithelial as well as crestal bone response, which was found to be depending on the diameter of the used tissue punch. In view of the apparent lack of histological data dealing with the effect of flapless surgery on implant healing, the present dog study was done to investigate the consequences of different flapless surgical procedures (i.e., tissue punch and direct approach) on peri-implant response tissue changes.

The null hypothesis was that there are no significant differences in the histological outcome of bone-to-implant response, crestal bone level, barrier epithelium and connective tissue thickness when utilizing different surgical approaches.

The alternative hypothesis was that the used surgical approach in dental implant placement will affect bone-to-implant response, crestal bone level alterations, barrier epithelium and connective tissue thickness.

MATERIALS AND METHODS

Animals

The study was performed at the Animal Research Center at the affiliated King Khalid University Hospital. Ten healthy adult Beagle dogs, 1-2 years of age, with an average weight of 8 to 15 kg, were used. All dogs were housed in single cages. After tooth extractions and dental implant installation, the dogs were fed with soft food. The study protocol was approved by the Animals Ethical Committee of King Saud University and performed according institutional regulations.

Surgical Procedures

Anesthesia

Before surgery, the dogs were premedicated using atropine (Neozine, Rhodia, Brazil) 0.5mg/kg intramuscularly (IM) to prevent against salivation and vomiting. Anesthesia was induced by injection of Ketamine HCl® (ketamine 10%: 8-10 mg/kg; Tekam Al Hikam Pharmaceuticals, Amman, Jordan) and Rompun® xylazine (Seton 2%: 1-3 mg/kg; Laboratorios Calier, Barcelona, Spain) IM. Local anesthesia with xylocaine® (30 mg of 2% lidocaine with 1:80,000 epinephrine; Astra, Sodertalje, Sweden) was used at the site of tooth extraction to control bleeding. Also, Duphapen strep B.P® (injectable preparation of streptomycin, 2 ml/kg; Solvay, Massa, Italy) was given during surgery and post-operatively.

Tooth Extraction

Bilateral mandibular second and third premolars (PM2 and PM3) were extracted in each dog.

Therefore, the premolars were hemisectioned using a high speed hand piece and extracted

Oral implant installation

After a healing period of three months, dental implants were installed. Bone fill of the extraction sockets was confirmed by radiography. The same protocol for anesthesia and antibiotics was used as for tooth extraction. In total 30 dental implants were installed with a length of 8 mm, a diameter of 3.3 mm, a 2.8 mm high smooth neck), and a 3.5 mm shoulder diameter (Straumann® Dental Implant System, SLActive® surface, Basel, Switzerland).

The implants were placed following one of three surgical approaches:

A.

Flapped surgical approach; A full thickness flap was reflected by using a crestal incision connected to two vertical incisions. After exposure of the alveolar ridge, implants sites were prepared using a low-speed drill with copious external cooling with saline solution. A graded series of drills was used for the implant bed preparation. The final drill had a diameter of 2.8 mm, which was followed by implant installation. After implant placement, the flaps were sutured back with 4/0 Vicryl® resorbable sutures.

B.

Flapless surgical approach;

1. Circular soft tissue punch; The soft tissue preparation of the implant site was done using a motor-driven 5-mm-wide circular tissue punch at the center of the implant placement site. Subsequently, the implant bed was prepared following the same sequence as used for the flapped surgical approach.

2. Direct round bur; No soft tissue preparation was done at all, but the same implant bed preparation steps were followed, starting with a round bur (diameter 1.4 mm), which was used to penetrate the soft tissue directly into the bone . The implants in all of the groups were installed in a non-submerged position with their "smooth" permucosal part penetrating through the mucosa. Care was taken to place all implants at the same height and to avoid perforation of the buccal or lingual cortical plates. It was attempted to place the implants in such a way that the marginal level of the sand-blasted and acid-etched (SLA) -coated surface was leveled with the alveolar bone crest. In order to achieve this in the flapless group, bone probing was performed immediately before implant installation, taking into consideration that the smooth surface of the implant had a height of 2.8 mm.

Each dog received a total of three oral implants, two at one side of the mandible and one at the other side.Implant installation and randomization was done according the schedule as listed in Table 1.

Specimen preparation

Three months after implant installation, all dogs were euthanized by an overdose of ketamine 10% (8-10mg/kg) and xylazine 2% (1-3 mg/kg) IM. Subsequently, the mandibles were harvested and IsoMet precision saw (Buehler, Düsseldorf, Germany) was used to cut the specimens in separate bone blocks containing one implant each. Each specimen was kept individually in 5% formalin and the containers were labeled according to the dog number, the quadrant and the surgical technique used for each implant.

Micro CT

After fixation in phosphate-buffered formaldehyde solution (pH=7.4) and dehydration in In addition, calibration rods with standardized bone mineral density were scanned as reference. Cone-Beam reconstruction (version 2.15, Skyscan ® ) was performed. All scan and reconstruction parameters applied were identical for all specimens and calibration rods.

The data were analyzed by CT Analyser (version 1.4, Skyscan ® ). The region of interest (ROI)

was specified as an annular area with a diameter of 1.5 mm surrounding the implants over an area from the first thread to the last thread. In this area bone volume (BV) was determined.

Bone volume (mm 3 ) was expressed as a percentage of the total ROI volume, using the equation:

Bone volume/Total ROI tissue volume x100%

Histological procedures and histomorphometrical evaluation

After micro-CT analysis, the specimens were prepared for histological and histomorphometrical evaluation. First, the specimens were dehydrated in ethanol and embedded in methylmethacrylate (MMA). After polymerization in MMA, three thin (10 µm)

non-decalcified sections in bucco-lingual direction were prepared parallel to the long axis of the implant with a modified diamond blade sawing microtome technique (Leica, SP1600, Nussboch, Germany). All sections were stained with basic fuchsin and methylene blue and were examined with a light microscope (Zeiss -Axio Imager Z1 automated microscope with AxioCam MRc5 digital camera and AxioVision V6.3.2. acquisition software, Göttingen, Germany). In addition, digital image analysis software (Leica Qwin Pro, Leica Microsystems Imaging Solutions, Cambridge, UK) was used for histomorphometrical measurements.

The following parameters were assessed (Figure 1): All histomorphometric procedures were performed on three representative sections of each implant and done blindly by two different experienced operators (VC and LH).

Statistical analysis

All measurements were statistically evaluated using commercial available software program (SPSS 16.0, SPSS Inc., Chicago, USA) . Data were analyzed using paired t-testing. A Shapiro-Wilk test was done to determine that all data were from a normal distribution.

Differences were considered statistically significant when the P-value was less than 0.05.

RESULTS

Clinical observations

A total of 30 dental implants were placed, which showed all primary stability at the moment of installation. All implants were inserted in an undersized mode in the high density bone of the dog mandible. The implants were always surrounded by an attached gingiva. At the time of euthanasia, clinical examination showed uneventful healing for 26 implants, without any sign of clinical mobility. No complications, as swelling, inflammation or exudation of the pergingival tissues was observed. Three dental implants, as placed with the tissue punch technique, appeared clinically to be mobile (dog 6 -right/distal implant, dog 7 -right implant, dog 10 -right implant) and one implant (dog 7 -left/mesial implant) placed, using the direct round bur technique, was found to be lost.

Micro-CT Measurements

Analysis of the micro-CT images indicated a bone volume percentage of 55 ± 9 for the flapped surgical approach group, while the tissue punch and direct approach groups had a bone volume percentage of 51 ± 4 and 54 ± 5, respectively. Statistical testing revealed that no significant differences existed in bone volume percentages between the groups (P = 0.4475).

Histological analysis

Flapped approach

The bone tissue, as present around the implants installed using the flapped approach, appeared to be mature and was characterized by the presence of osteocytes and Haversian systems (Figure 2). High remodeling activity was observed only occasionally. Such areas of high remodeling activity were always seen at some distance of the implant interface. The bone was always in close contact with the implant surface and no intervening fibrous tissue layer was 13 present (Figure 2B). Remodeling lacunae were observed at the implant-bone interface. Only one implant showed significant loss of crestal bone. The two most coronal screw-threads of this implant became exposed. Further around three implants, crestal bone loss was seen till the first coronal screw thread. For all the other implants, the bone made its first contact with the implant surface above the first screw-thread.

Junctional epithelium was in contact with the implant surface (Figure 2A). Some inflammatory response was always present in the connective tissue. This inflammatory response was characterized by the presence of plasma cells.

Direct flapless approach

The bone as well as gingival (junctional epithelium and connective tissue) response to the implants, as installed with the direct flapless approach, was very similar to the flapped installed implants. The bone was again mature with very limited remodeling activity and in tight contact with the implant surface (Figure 3). Four implants showed crestal bone loss till the first coronal screw-thread, while the first implant-bone contact for the other implants was always above the first screw-thread (Figure 3).

A junctional epithelium with some inflammatory response in the connective tissue was seen around all implants (Figure 3).

Punch flapless approach

The peri-implant tissue response around the implants installed with the punch flapless approach was not always consistent and differed from the implants installed with the two other approaches. The three implants, which were found to be clinically mobile, showed a very significant bone loss and the four most coronal screw-threads became exposed (Figure 5). The apical region of these implants (including the remaining 2 screw-threads) were F o r P e e r R e v i e w 14 covered with bone, which was in intimate contact with the implant surface (Figure 4). Three other implants showed crestal bone loss till the second coronal screw-thread. For the other four implants, the first implant-bone contact was above the first screw-thread. The implants that showed crestal bone loss were also surrounded by a thick sub-epithelial connective tissue layer, which was slightly inflamed. Around these implants a long junctional epithelium with a deeper sulcus was seen compared with the implants, which showed no crestal bone loss.

Histomorphometrical Measurements

The results of the histomorphometrical measurements for the crestal bone level, bone-toimplant contact%, connective tissue thickness and barrier epithelium length are listed in Table 2. Further analysis of the data revealed that the crestal bone level with the implant surface for the punch method was at a significantly lower level than for the implants installed with the two other procedures (Table 3).

Statistical testing of the bone-to-implant contact measurements indicated that the bone-toimplant contact varied significantly between the three surgical approaches. The implants inserted with the punch technique revealed a significantly lower amount of bone-implant contact compared with the direct and flap technique, while no significant difference existed between the flap vs. direct technique (Table 3).

While the soft tissue measurements also seem to imply that the punch technique results in a thicker connective tissue layer as well as deeper gingival sulcus, this was not completely confirmed by the statistical analysis. Statistical analysis of the connective tissue thickness data showed that the barrier epithelium length varied significantly between the three groups (Table 3). A deeper sulcus was found around the dental implants installed with the punch vs. direct as well as flap vs. punch approach.

Deleted: Testing

Deleted:

DISCUSSION

The aim of the current study was to investigate the consequences of different flapless (i.e., tissue punch and direct approach) as well as flapped surgical procedures on bone and gingiva response. It has to be noticed that in our study design a tissue punch with a diameter of 5 mm was used in the flapless surgical approach. The use of this punch created a gingival defect, which was wider than the implant shoulder diameter (3.5 mm). This was done to avoid any contact between the implant surface and gingival tissues during implant installation in order to prevent the displacement of epithelial cells and/or fibroblasts into the bone bed. On the other hand, it cannot be excluded that the mismatch in diameter has supported the enhanced ingrowth of the gingival epithelium. Also, no oral hygiene regime was done after implant installation. Although, an effect of this omission on the final study results cannot be completely excluded, it has to be emphasized that such a hygiene procedure was excluded for all surgical approaches. Therefore, we assume that the wound healing conditions were similar for all dogs and did not interfere with the outcome.

At the end of the three month implantation period, three of the implants installed with the punch method appeared to be clinically mobile, one of the implants inserted with the direct approach was found to be lost, while no complications were observed for the flapped placed implants. This makes the survival rate for the three surgical methods: 7/10 (punch), 9/10 (direct) and 10/10 (flapped) respectively. These data corroborate with a study as performed by [START_REF] Rocci | Immediate loading in the maxilla using flapless surgery, implants placed in predetermined positions, and prefabricated provisional restorations: A retrospective 3-year clinical study[END_REF], who used flapless surgery to place implants in the maxilla of 46 patients.

On the other hand, in the studies of e.g. [START_REF] Becker | Minimally invasive flapless implant surgery: A prospective multicenter study[END_REF] the initial use and care for their implants can be given to patients in order to prevent overloading during the initial healing stage, while in dogs overloading can only be prevented by feeding them with soft dog chow.

The parameters as selected for the evaluation of the tissue response were: bone volume (by micro-CT), crestal bone level (BL), bone-to-implant (BIC) contact%, connective tissue thickness and barrier epithelium length (all measured by light microscopy). The light microscopical assessment was done to obtain more information about the hard and soft tissue response in the very close vicinity of the implant, while micro-CT was used to obtain more information about bone density and quality of the implant site and its effect on the final interfacial bone response. Both the BIC and marginal bone loss affect the overall success of a dental implant. BIC is providing information about the bone integrative capacity of an implant material and is considered as a major determinant of implant stability after initial healing, since a higher BIC results in a higher resistance to shear strength [START_REF] Buser | Effect of various titanium surface configurations on osseointegration and clinical implant stability[END_REF]. The BIC% measurement in the histomorphometrical procedure, as used in the current experimental design, was done form the first implant-to-bone contact till the apex of the implant. In addition, the bone level distance from the top of the implant till the first implant-to-bone contact was determined, which represented the marginal bone loss. This was done, because BIC% data are assuming an uniform distribution of bone contact over the complete length of the implant surface, while this has not to be the case. Marginal bone level changes, as characterized by first implant bone-contact will influence the BIC, but can also affect the soft tissue level [START_REF] Chang | Implant supported singletooth replacements compared to contralateral natural teeth, crown and soft tissue dimensions[END_REF] as well as the overall success of the implant. As a consequence, it has to be recommended that always several different histomorphometrical parameters are assessed, which describe the interfacial tissue response as accurate as possible.

Subsequently, these parameters can be related to each other as was done in the current study for BIC and marginal bone loss. Further, to allow the crestal bone level measurements, it was attempted to install all implants with the marginal level of roughened surface leveled with the alveolar bone crest. Despite the bone probing, it has to be emphasized that it cannot be excluded that in the flapless group not a completely perfect fit was achieved. Still, it is supposed that the obtained data are valid, as the final goal was not to observe significant changes in crestal bone level, which is still feasible in the current approach. The degree of bone trabecularity, also known as bone density or bone quality (and histomorphometrically expressed as bone volume), represents the amount of bone matrix as present in a particular area of the jaws. It is supposed that a relation exists between the amount of bone trabeculae and their thickness, i.e. the bone volume, and the BIC as obtained at the end of the implant healing period. More numerous and thicker trabeculae will result in a higher BIC [START_REF] Ichikawa | Three-dimensional bone response to commercially pure titanium, hydroxyapatite, and calcium-ion-mixing titanium in rabbits[END_REF], Trisi et al. 2002). An appropriate technique for determining bone volume around implants is by making use of micro-CT [START_REF] Schouten | The quantitative assessment of peri-implant bone responses using histomorphometry and microcomputed tomography[END_REF]). The micro-CT measurements in the current study showed that there was no significant difference in bone volume in an area of 1.5 mm surrounding the dental implant for the three surgical techniques. This indicates that there was no difference in the degree of bone trabecularity or bone quality between the various implant sites and that all observed effects are due to the used surgical conditions.

The histological evaluation and histomorphometrical measurements indicated that the punched surgical approach resulted in more crestal bone loss, less BIC and increased barrier epithelium length. of the occurrence of statistical significant difference can be enhanced due to the fact that the three "punched" implants, which appeared to be mobile after three months of implantation, were included in the histomorphometrical analysis. Although clinical testing before harvesting suggested the presence of mobility, these implants showed still a close bone contact at their apical part with a BIC of resp. 10%, 20% and 27%. This limited apical BIC also influenced the first bone contact and barrier epithelium length. If these implants had been removed from the analysis, only a significant difference in BIC between the punch vs. direct 18 approach would have been found. Nevertheless, it was decided to maintain these data, as the implants were still present at the end of the experimental time and an outlier test did not support the exclusion of these data.

Our histological and histomorphometrical evaluation corroborates with the studies of [START_REF] Becker | Histologic evaluation of implants following flapless and flapped surgery: A study in canines[END_REF] as well as [START_REF] Lee | Effects of soft tissue punch size on the healing of peri-implant tissue in flapless implant surgery[END_REF]. Becker et al. used the direct and flapped approach and found no significant differences in BIC% and crestal bone level between these two techniques. Lee et al. compared the effect of three different punch diameters on the bone and gingival healing. The diameter of the installed implants was 4 mm and they observed that an increased junctional epithelium length, probing dept as well as marginal bone level occurred when the mucosa was punched with a 5 mm punch compared with the use of a 3 and 4 mm punch. Lee suggested that for the 5 mm tissue punch a too wide gap was created between the implant neck and mucosa, which delayed the healing of the peri-implant mucosa. This explanation is based on the interrupted vascularization theory, which hypothesizes that separation and detachment of the periosteum from the underlying bone surface causes vascular damage and an acute inflammatory response. This will result in resorption of the exposed bone surface [START_REF] Brägger | Remodeling of interdental alveolar bone after periodontal flap procedure assessed by means of computer-assisted densitometer image analysis[END_REF]. The periosteum will be removed when the tissue punch is used. In our study design, such an effect will even be enhanced due the larger discrepancy in diameter between tissue punch and implant (5 mm vs. 3.3 mm). This can explain why three of our "punched" implants became mobile, while mobility or implant loss was not reported by Lee et al. Recently, also de Sanctis et al (2010) The comparable histomorphometrical data between the direct flapless and flapped approach suggests that the direct drilling of the implant bed through the mucosa does not force soft tissue into the bone, as the BL, BIC% and implant stability were not found to be jeopardized.

Although, the soft tissue height was measured using a periodontal probe prior to the installation of the implants of the direct flapless group to assure the position of the implant, one of the implants as installed by the direct technique was still lost. This can be due to the inevitable lack of visibility in relation to the anatomy of the alveolar ridge as well as a reduced access for external irrigation during the drilling of the implant bed (Sclar 2007).

Perhaps, a solution as observed for the currently observed failures with the flapless techniques, is the use of a recently suggested mini-incision approach [START_REF] Jeong | Flapless implant surgery using a miniincision[END_REF]). This technique allows a submerged positioning of the dental implant, which can support the bone healing response.

CONCLUSIONS

The results, as obtained in the current dog study, indicate that a flapless surgical technique can be used for the installation of oral implants. However, but caution should be exercised, because in our study design the healing of both bone and gingival tissue around implants installed with a punched flapless technique was hampered. Therefore, the use of a tissue punch, which is much wider than the implant diameter, has to be avoided as it endangers the outcome of the implantation procedure. Histological section of an implant installed with the punch approach. Significant crestal bone resorption did occur resulting in exposure of the first three coronal screw-threads. The gingival tissue was loosely adhering to the implant surface and detachment (arrow) occurred during retrieval of the implant (original magnification obj. 5x, bar = 500µm).

ACKNOWLEDGEMENT

  sized dental elevator as well as forceps. Following tooth extraction, the extraction wounds were approximated and closed with 4/0 Vicryl resorbable sutures.

  ethanol 70%, three-dimensional micro-computed tomography (µCT) images were made to analyze the bone mineral density and bone volume of the implant surrounding bone mass. The specimens were wrapped in Parafilm M ® (Pechiney Plastic Packaging, Chicago, USA) to scanning. Then, all samples were scanned at an energy of 101 kV and an intensity of 96 µA with a resolution of 37.41 µm pixel using an aluminum filter (1mm) (Skyscan-1072 X-ray microtomograph, TomoNT version 3N.5, Skyscan ® , Kontich Belgium).

  epithelium length (BEL): The barrier epithelium length was measured by drawing a line over the top of the gingival epithelium perpendicular on the implant surface. Subsequently, the length of the barrier epithelium was measured from this line to the boundary of the junctional epithelium. B. Connective tissue thickness (CTT): The thickness of the connective tissue from the apical limitation of the gingival epithelium to the first bone-to-implant contact. C. Bone level (BL): The distance from the top of the implant to the bone crest in contact with the implant surface. D. Percentage of bone contact at the interface (BIC): The amount of bone contact was defined as the percentage of implant length at which there was direct bone-to-implant contact without intervening soft tissue layers. Measurements for bone-to-implant contact were performed along the implant interface from the most coronal bone contact till the apex of the implant.

Figure 1 :

 1 Figure 1: Schematic drawing of the histomorphometric parameters.

  Light micrograph showing the bone as well as gingival tissues around an implant installed using the flapped approach. No crestal bone resorption is present and the bone is in direct contact with the implant surface. (A: original magnification obj. 5x, bar = 1000µm; B: original magnification obj. 10x, bar = 100µm). R = remodelling lacuna, JE = junctional epithelium 386x191mm (150 x 150 DPI) Light micrograph of an implant installed using a direct approach. The bone is in close contact with the implant surface (A and C). The first implant-bone contact (1st) occurred coronal of the first screw thread (A). The junctional epithelium (JE) contacts the implant surface and the subepithelial connective tissue (CT) layer is slightly inflamed (B) (A: original magnification obj. 5x, bar = 1000µm; B: original magnification obj. 10x, bar = 500µm; C: original magnification obj. 5x, bar = 200µmLight micrograph of an implant installed using a direct approach. The bone is in close contact with the implant surface (A and C). The first implant-bone contact (1st) occurred coronal of the first screw thread (A). The junctional epithelium (JE) contacts the implant surface and the subepithelial connective tissue (CT) layer is slightly inflamed (B) (A: original magnification obj. 5x, bar = 1000µm; B: original magnification obj. 10x, bar = 500µm; C: original magnification obj. 5x, bar = 200µm). 612x162mm (150 x 150 DPI) Histological section of an implant installed with the punch approach. Significant crestal bone resorption did occur resulting in exposure of the first three coronal screw-threads. The gingival tissue was loosely adhering to the implant surface and detachment (arrow) occurred during retrieval of the implant (original magnification obj. 5x, bar = 500µm). 691x89mm (150 x 150 DPI)

Table 3

 3

	),
	Deleted: but

Table 1 :

 1 Natasja van Dijk for her help in the histological preparation of the specimen as well as Vincent Cuijpers and Liao Hongbing for their assistance in the micro-CT and histomorphometrical analysis. Further, we thank dr.Ewald Bronkhorst for his assistance in the statistical analysis of the data. The implants were kindly provided by Straumann company, Switzerland. Oral implant location for the different surgical approaches.

	The authors thank
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	20

Table 2 :

 2 Histomorphometrical measurements.

	Surgical	1 st Bone	Bone-implant-	Connective	Barrier
	technique	Contact	contact (%)	tissue thickness	epithelium
		(in µm)		(in µm)	length
					(in µm)
	Flap	3420 ± 762	70 ± 13	1498 ± 559	1383 ± 350
	Punch	5358 ± 1681	48 ± 24	2047 ± 1227	2278 ± 1217
	Direct	3843 ± 433	73 ± 13	1476 ± 375	1107 ± 318

Table 3 :

 3 Statistical comparisons for the various implant installation techniques (P-value and 95% Confidence Interval).

	Surgical	1 st Bone Contact	Bone-implant-	Connective tissue	Barrier epithelium
	technique			contact		thickness		length
		P-value 95% CI	P-value 95% CI	P-value 95% CI	P-value 95% CI
	Flap vs.	0.003	[-3038…-	0.002	[10…34] 0.258	[-	0.037	[-1723…-
	Punch		836]				1579…481]		68]
	Flap vs.	0.114	[-	0.775	[-	0.86	-577…493] 0.128	[-633…96]
	Direct		132…1006]		11…14]
	Punch vs.	0.045	[39…2641] 0.008	[-36…-7] 0.361	[-	0.032	[128…2167]
	Direct						464…1134]

Journal of Clinical Periodontology -PROOFJournal of Clinical Periodontology -PROOF

Journal of Clinical Periodontology -PROOF

The study was self-funded by the authors and their institution.

Journal of Clinical Periodontology -PROOF

Journal of Clinical Periodontology -PROOF


Prof. Dr Anton ; Friedmann
email: anton.friedmann@uni-wh.de

Kirsten Gissel

Mouaz Soudan

Bernd-Michael ; Kleber

Sandu Pitaru

Thomas Dietrich

Charitécentrum

Operative Dentistry Periodontology Gissel

Kirsten ; Charitécentrum

Dr M Noff

Morphometric results on mineralized tissue compound

Keywords: GBR, collagen membrane, implants, bone augmentation, morphometry lateral augmentation, non-cross linked collagen, ribose cross linked collagen, morphometry, randomized trial

come

Randomized controlled trial on lateral augmentation using two collagen membranes. Morphometric results on mineralized tissue compound.

Clinical relevance

Scientific rationale for the study: Limited data are available regarding morphometric outcome in terms of mineralized tissue gain if either cross linked or non-cross linked collagen membranes were used in lateral augmentation.

Principial findings:

Results indicate both membranes having potential to effectively support GBR in dehiscence type of defect. Dehiscence of the soft tissue occurred in 70.5% of test and in 55% of control sites. However, significantly higher amount of newly mineralized tissue in a most critical zone argues for the use of cross linked collagen membranes although more exposures occurred in test sites.

Practical implications: Cross linked membranes combined with biphasic calcium phosphate are suitable for GBR and likely to support formation of significant amount of newly mineralized tissue under unlikely clinical conditions.

Introduction

Guided bone regeneration (GBR) is one of several well documented, evidence based augmentation techniques [START_REF] Hammerle | Guided bone regeneration at oral implant sites[END_REF][START_REF] Mcallister | Bone augmentation techniques[END_REF].

Several animal and clinical studies showed gain in marginal bone, using resorbable membranes in combination with an underlying, osteoconductive membranesupporting material [START_REF] Hammerle | Single stage surgery combining transmucosal implant placement with guided bone regeneration and bioresorbable materials[END_REF][START_REF] Hockers | The combined use of bioresorbable membranes and xenografts or autografts in the treatment of bone defects around implants. A study in beagle dogs[END_REF][START_REF] Strietzel | Healing pattern of bone defects covered by different membrane types--a histologic study in the porcine mandible[END_REF][START_REF] Zitzmann | Long-term results of implants treated with guided bone regeneration: a 5-year prospective study[END_REF]. Collagen membranes, as the most frequently used type of degradable membranes, lack enough stiffness for space maintenance and tend to collapse [START_REF] Strietzel | Healing pattern of bone defects covered by different membrane types--a histologic study in the porcine mandible[END_REF]. Therefore, titanium re-inforced non-degradable e-PTFE membranes are still favored by some clinicians. Early exposure of barrier membranes to the oral environment jeopardizes the outcome due to infection, mostly manifested around non-resorbable membranes, or due to rapid disintegration in case of resorbable membrane [START_REF] De Sanctis | Bacterial colonization of barrier material and periodontal regeneration[END_REF][START_REF] Mayrand | Detection of collagenase activity in oral bacteria[END_REF][START_REF] Nowzari | Microbiologic and clinical study of polytetrafluoroethylene membranes for guided bone regeneration around implants[END_REF][START_REF] Sela | Enzymatic degradation of collagen-guided tissue regeneration membranes by periodontal bacteria[END_REF][START_REF] Simion | A comparative study of the effectiveness of e-PTFE membranes with and without early exposure during the healing period[END_REF][START_REF] Tempro | Colonization of retrieved polytetrafluoroethylene membranes: morphological and microbiological observations[END_REF].

Collagen cross-linking contributes to prolonged membrane barrier function [START_REF] Bornstein | Effect of two different bioabsorbable collagen membranes on guided bone regeneration: a comparative histomorphometric study in the dog mandible[END_REF][START_REF] Von Arx | Membrane durability and tissue response of different bioresorbable barrier membranes: a histologic study in the rabbit calvarium[END_REF]. Barriers with high degradation rates could have a shorter-than-indicated effect [START_REF] Moses | Biodegradation of three different collagen membranes in the rat calvarium: a comparative study[END_REF]. Membrane degradation starts shortly after implantation [START_REF] Von Arx | Membrane durability and tissue response of different bioresorbable barrier membranes: a histologic study in the rabbit calvarium[END_REF]. The integration pattern of various collagen membranes into soft tissues were analyzed in recently published animal studies [START_REF] Rothamel | Biodegradation of differently cross-linked collagen membranes: an experimental study in the rat[END_REF][START_REF] Schwarz | Angiogenesis pattern of native and cross-linked collagen membranes: an immunohistochemical study in the rat[END_REF]. A larger membrane porosity (less collagen contents per area) would allow for cell ingrowths within the membrane, resulting in better tissue [START_REF] Rothamel | Biodegradation of differently cross-linked collagen membranes: an experimental study in the rat[END_REF][START_REF] Schwarz | Angiogenesis pattern of native and cross-linked collagen membranes: an immunohistochemical study in the rat[END_REF], but may result in reduced barrier function [START_REF] Rothamel | Biocompatibility of various collagen membranes in cultures of human PDL fibroblasts and human osteoblast-like cells[END_REF]. Importantly, nutrient diffusion for cell proliferation and differentiation was not affected by collagen cross linking vs. noncross linking in an in-vitro study [START_REF] Friedmann | Cytobiocompatibility of collagen and ePTFE membranes on osteoblast-like cells in vitro[END_REF]. Significantly more new together with either a non-degradable (e-PTFE) or a newly introduced slowly degrading polyethylene glycol membranes vs. controls grafted without membranes was recently confirmed by [START_REF] Jung | Evaluation of an in situ formed synthetic hydrogel as a biodegradable membrane for guided bone regeneration[END_REF]. Nevertheless, the discussion upon the benefits and disadvantages of cross-linked collagen material as slowly degradable vs. membranes from native collagen, a rapidly degrading type is still ongoing. In this respect, there is no data regarding the efficiency of resorbable membranes to support bone gain in cases in which bone augmentation is performed with simultaneous installation of implants with transgingival surgical elements.

Morphometric assessment of outcomes in bone augmentation in terms of volume gain has been the topic for in vitro and clinical studies [START_REF] Kohles | A morphometric evaluation of allograft matrix combinations in the treatment of osseous defects in a baboon model[END_REF][START_REF] Llambes | Vertical guided bone regeneration with bioabsorbable barriers[END_REF][START_REF] Proussaefs | The use of intraorally harvested autogenous block grafts for vertical alveolar ridge augmentation: a human study[END_REF][START_REF] Proussaefs | A method to assess the clinical outcome of ridge augmentation procedures[END_REF][START_REF] Studer | Quantitative measurement of volume changes induced by oral plastic surgery: validation of an optical method using different geometrically-formed specimens[END_REF][START_REF] Tai | Prospective analysis of secondary alveolar bone grafting using computed tomography[END_REF][START_REF] Windisch | A new optical method to evaluate three-dimensional volume changes of alveolar contours: a methodological in vitro study[END_REF]. Our group introduced a method to perform morphometric measurements on casts obtained from impressions of the residual alveolar ridge taken during surgery and of the post-augmented ridge taken during re-entry [START_REF] Pitaru | Ribose-crosslinked collagen membranes are more effective than ePTFE membranes in supporting bone augmentation in humans: a morphometric study[END_REF].

The aim of the present randomized clinical trial was to test the efficiency of supporting bone gain and promoting soft tissue healing of a ribose cross-linked collagen membrane (RCLM) vs. a non-cross-linked collagen membrane (NCLM) during augmentation of bone dehiscences and fenestrations with biphasic calcium phosphate and concomitant implant placement using a new morphometric approach for human clinical trials. out under local aneasthesia (Ultracain DS forte, Aventis, Germany) by a single surgeon (AF). All patients received a one-stage procedure including implant placement, concomitant augmentation, and primary wound closure. Defect extensions were recorded using periodontal probe (PCP 15, Hu-Friedy, Leimen, Germany) during surgery. While in vivo measurements were performed by the surgeon, morphometric analyses on the casts obtained were carried out by the Clinical Investigator (KG), who was blinded to the randomized patient assignment. In addition, impressions were taken for ex-vivo measurements by a blinded investigator (KG) using individualised plastic trays and a sterile A-silicone material (Elite® Implant, Zhermack, Germany). These impressions were taken before and after reflection of a muco-periosteal flap exposing the defect area. Straumann Soft Tissue Level implants with 4.1 mm in diameter and varying from 8 to 12mm in length (Institute Straumann AG, Basel, Switzerland) were used in accordance with established guidelines. In brief, recipient site was prepared for placing implant with it rough SLA surface at the crestal level of alveolar bone leaving the machined part of the implant neck above the bone level. Standard Plus (SP) implants with a machined neck of 1.8 mm were used. Biphasic calcium phosphate (BoneCeramic, Institute Straumann AG, Basel, Switzerland) was used for grafting dehiscences and fenestrations in alveolar bone in exposed implant areas. No autogenous bone was used. For easier application, a coagulum was formed by the calcium phosphate grafting material with 1 to 2 ml of the patients' own blood collected in the wound area after the incisions were carried out [START_REF] Artzi | The amount of newly formed bone in sinus grafting procedures depends on tissue depth as well as the type and residual amount of the grafted material[END_REF][START_REF] Friedmann | Histological assessment of augmented jaw bone utilizing a new collagen barrier membrane compared to a standard barrier membrane to protect a granular bone substitute material[END_REF]Friedman, et al., 2009). The composite grafting material was placed in the defect up to the level of the machined surface of the implant, in the vertical direction and up to the completion of the bony envelop in the lateral direction. After application of the augmentation material, envelopes containing the randomization code were opened and membrane placement was performed as assigned. Each membrane was applied in a monolayer. All flaps were coronally advanced by periosteal release to achieve a tension free adaptation of soft tissue margins for complete closure of the wound. The post-operative regimen included twice daily mouth rinses with Chlorhexidine 0.2% (Chlorhexamed, Glaxo Smith Kline, Bühl, Germany). Systemic antibiotics, either amoxicillin 750mg 3xdaily (Amoxicillin-Ratiopharm, Ratiopharm, Germany) or clindamycine 300 mg 4xdaily (Clindamycin-Ratiopharm, Ratiopharm, Germany) were administered for 1 week; and ibuprofen (Ibuprofen-Ratiopharm 600mg, Ratiopharm, Germany) as necessary for postoperative analgesia. Sutures were removed after 14 days. Fixed partial dentures or semi-permanent splinting were used as temporary prosthetic devices only. In edentulous posterior regions, no dentures were applied during the study.

At re-entry after 6 months, an impression was taken to document tissue dimensions and full thickness flaps were then raised using similar incision extensions as during the first surgery, with the exception for lingual flaps which were not raised if the area augmented was sufficiently exposed (Fig. 2a-f). Following thorough removal of any remaining non-mineralized tissue with sterile curettes another impression was taken to document hard-tissue dimensions. In nine cases requiring additional augmentation, BCP and RCLM were used. All implants received gingival formers (Straumann AG, Basel, Switzerland) and the flap was sutured back by single sutures using Monocryl 6.0 suture (Ethicon, Hamburg, Germany).

Morphometric procedure

The primary objective of the study was to quantify the effect of the type of collagen membrane on the level of bone augmentation at the crestal level of endosseous implants. To do this the following morphometric procedure was undertaken. All impressions were cast in stone rock plaster (Octa Stone CN, Hereus -Kulzer, Bensheim, Germany). Casts of impressions obtained at initial surgery and at re-entry (post-augmentation) are designated as casts-0 and casts-1, respectively. Negative templates of casts-1 were obtained by taking silicon (C-silicon, Silaplast®, Detax, Germany) impressions. Using landmarks as teeth adjacent to the augmented site and the implant neck the negative templates were adjusted upon the casts-1 (Fig. 3a+b).

To determine reference points and to ascertain reproducibility in positioning one and the same template on casts obtained at both surgeries, the top aspects of the templates were trimmed parallel with the basis of cast 1; furthermore the side walls of templates were trimmed perpendicular to top and bottom aspect of the templates (Fig. 4a-d). While kept together, the casts-1 and their negative silicon templates were sectioned through bucco-lingual plans that mid-crossed in mesio-distal dimension of implants around which augmentation was performed. A perpendicular was dropped from the edge of template to the middle of the cover screw of a cross-sectioned implant to label central implant axis (Fig. 4a). To assess vertical dimension a parallel perpendicular was marked on the template at the crossing point of alveolar crest and implant surface and the distance between this point and the top plan of the silicon template was measured with a digitized calliper and termed -H1/1 (Fig. 3a; 4a). A perpendicular to H1/1 passing through the crossing point of alveolar crest and implant surface was drawn. The distance between the implant middle axis and the buccal aspect of the alveolar crest was measured on this perpendicular and termed -W1/1 (Fig. 3a; 4a). A second perpendicular, parallel to H1/1 was drawn at the level of most pronounced buccal aspect of augmented area and the distance from the middle of implant to this aspect was recorded and termed -W2/1 (Fig. 4a;4b). The distance from this point to the top plan of the silicon template was measured with a digitized caliper and termed -H2/1 (Fig. 4a). Then, the cross sectioned silicon templates were adapted to the casts-0 obtained during the augmentation procedure using the reference marks mentioned above (Fig. 4c+d). Using the templates as landmarks the casts-0 were sectioned in the same plan as described above for the casts-1. Using the landmark lines drawn on the silicon templates, now adapted to casts-0, the distances measured on casts-1 were measured for casts-0 and termed W1/0; H1/0; W2/0; and H2/0. This methodology ensured that casts-0 and casts-1 were sectioned in the same plans and exactly at the same site, that is a bucco-lingual plan crossing the central axis of the implants (Fig. 4b). Bone gain in width and height was calculated by:

• Crestal (coronal) width gain: ∆W1 = W1/1 -W1/0

• Crestal (coronal) vertical gain: ∆H1 = H1/0 -H1/1

• Apical width gain:

∆W2 = W2/1 -W2/0
• Apical vertical gain: ∆H2 = H2/0 -H2/1

Statistical analysis

Changes in hard and soft-tissue dimensions between baseline and re-entry were outcomes of interest. Data were analyzed in two different ways. Firstly, data of all 37 patients were analyzed using one randomly selected site of those patients with two surgical sites. Comparisons between groups and between baseline and re-entry within groups were made using t-tests and paired t-tests as appropriate. Further analyses used multiple linear regression adjusting for maxillary vs. mandibular defects, anterior and posterior sites, smoking, membrane exposure. Secondly, comparisons between groups were made limited to data of those patients who had two surgical sites (split-mouth comparison) using non-parametric Wilxocon sign rank

Results

Patient recruitment involved 40 patients; three of them were excluded due to the extensive loss of alveolar bone making therefore the implant placement at one stage with augmentation not feasible (Fig. 1). All patients included completed the study, resulting in a total of 37 patients with 46 defect sites. All implants inserted (in total:

73) were well integrated after 6 months of healing. All implants received gingival formers for 4 to 6 weeks being loaded subsequently by single crowns or fixed partial dentures. There were 37 implants placed in 17 defect sites in the test group and 36 implants in 20 sites in the control group (Table 1). The overall survival rate at time point of loading was 100%.

Albeit patient assignment to the test and control groups was performed randomly, groups matched well in regard to patient's age, proportions between females and males and distribution of sites between maxilla and mandible (Table 1). In both groups not-smoking patients and smoking <10 cigarettes/day were represented in similar frequency. A group of nine patients formed a split mouth group and were treated at 2 sites each site with a different membrane. Analysis of the morphometric results within this group indicated not-statistically significant differences between the sites treated with the RCLM and those treated with NCLM. Therefore, only one site per patient has been chosen by secondary randomization assigning one study site and implant per patient, either to tests or to controls. Thus, No adverse events were recorded but one participant in the control group showed hypersensitive reaction to Amoxicillin, which then was exchanged by Zithromax (Azithromycine, Pfizer, Germany).

Morphometric results

Statistical outcomes for all 37 sites including randomly selected one site per patient out of former split mouth randomization are given by Table 3. At reference point 1 (W1 / H1), indicating the most crestal bone-to-implant contact the median gain of mineralized tissue in the vertical dimension (vertical gain; ∆H1) was 1.1mm in the test sites compared to 0.2 mm in the control sites (p = .0463). The median gain in the horizontal dimension (width gain; ∆W1) was 1.8 mm in the test sites vs. 0.7 mm in the control sites (p = .0359). There were no statistically significant associations between the primary outcome and membrane exposure (p = .6845 for ∆H1 and p = .2809 for ∆W1, respectively).

At the second reference point (∆W2 / ∆H2), the most apically accessible point for measurement, the median vertical gains were 2.5 mm and 2.7 mm for the test and control sites, respectively (p = 0.5674). The median width gains at this reference point were 3.0 mm and 2.1 mm for test and control sites, respectively (p = .1189).

In

Discussion

The aim of the present randomized clinical trial was to test the effectivity of ribosecross linked (RCLM; test) membrane vs. a non-cross linked (NCLM; control) membrane in GBR treatment using a new method for morphometric analysis.

Generally, the results indicate that both membranes improved the bone volume and predictably supported GBR procedures at dehiscence-type and fenestration-type defects.

The rate of soft tissue dehiscences during healing was almost equally distributed for both groups. Over 50% of sites were exposed and underwent secondary epithelization. The figures are high compared to the data presented for the group of dehisence-type defects within the review by Jensen & Terheyden. They found rates of soft tissue dehiscences up to 14.5% for sites with resorbable and of 26.3% for those with non-resorbable mebranes [START_REF] Jensen | Bone augmentation procedures in localized defects in the alveolar ridge: clinical results with different bone grafts and bone-substitute materials[END_REF]. Similar observations in regard to RCLM samples were reported previousely by our group [START_REF] Friedmann | Observations on a new collagen barrier membrane in 16 consecutively treated patients. Clinical and histological findings[END_REF]. Moses et al. found the RCLM membrane demonstrating a higher reduction of the bony defect area in cases of premature membrane exposure [START_REF] Moses | Healing of dehiscencetype defects in implants placed together with different barrier membranes: a comparative clinical study[END_REF]. The history of premature membrane exposure may have a negative effect on new bone formation even if soft tissue dehiscence recovers by secondary epithelization. Although ribose-cross linked membranes might be associated with a higher incidence rate of soft tissue dehiscences [START_REF] Moses | Healing of dehiscencetype defects in implants placed together with different barrier membranes: a comparative clinical study[END_REF][START_REF] Tal | Long-term bio-degradation of cross-linked and non-cross-linked collagen barriers in human guided bone regeneration[END_REF], the frequency of concomitant inflammatory reactions reported is almost zero [START_REF] Friedmann | Observations on a new collagen barrier membrane in 16 consecutively treated patients. Clinical and histological findings[END_REF]. On the opposite, the histological report by membranes is beneficial for mineralization process [START_REF] Zubery | Ossification of a novel crosslinked porcine collagen barrier in guided bone regeneration in dogs[END_REF][START_REF] Zubery | Ossification of a collagen membrane crosslinked by sugar: a human case series[END_REF].

In our study Soft Tissue Level Straumann implants were inserted in accordance with the ITI surgical protocol resulting in a position of the machined part of implant neck supracrestally. However, all implants and augmentation sites were planned to heal in the submerged mode according to the study protocol. Therefore complete flap closure required mobilization and coronal advancing of the flap to achieve tension free primary wound closure over the implant neck exceeding almost 2mm coronally to the crest. No grafting material was applied in the supracrestal area of bone dehiscencies laterally and therefore it is conceivable that flap stabilization was under optimal in this study. This lack of membrane support by grafting material might explain the unexpected high frequency of soft tissue dehiscence and premature membrane exposure. In a recent study by Burkhard and Lang tensions exceeding 0.1N determined before suturering the flap resulted in a wound dehiscence rate over 40% (Burkhardt & Lang, 2010). The high prevalence of dehiscences in our study may be a result of mechanical challenges due to masticatory movements as a complementary factor to membrane properties. According to the implant design secondary epithelialisation in this study did not always result in complete soft tissue closure over the cover screw. However, the difference in frequency of dehiscence onset following augmentation and the need of additional augmentation at re-entry was obvious.

The method used to assess bone gain was originally introduced by Pitaru et al.

2006 at Europerio in Madrid, Spain [START_REF] Pitaru | Ribose-crosslinked collagen membranes are more effective than ePTFE membranes in supporting bone augmentation in humans: a morphometric study[END_REF]. Customizing each defect site before and after augmentation in plaster of paris allowed for determining identically positioned reference points on boths. A silikon template transfered the with BCP [START_REF] Friedmann | Ridge augmentation and maxillary sinus grafting with a biphasic calcium phosphate: histologic and histomorphometric observations[END_REF]. Since in this study biopsies for the histomorphometric analysis were harvested during re-entry surgeries from sites augmented in the present study, histologic outcome indicates an effective denudation of bony crest [START_REF] Friedmann | Ridge augmentation and maxillary sinus grafting with a biphasic calcium phosphate: histologic and histomorphometric observations[END_REF]. Taking these observations into account and furthermore by removing all non-mineralized tissue from underneath the flap prior to impression we payed maximum attention to establish accuracy of the impressions. Since all surgeries were performed by one co-worker (AF) we consider the approach standardized for all sites. Our study presents for first time an accurate method for quantifying vertical and horizontal changes of the alveolar ridge in GBR. Furthermore this is first prospective report on efficacy of collagen membranes in one stage GBR with one stage Soft Tissue Level Straumann implants. Lang et al. reported successful implementation of Gore Tex membranes placing them around necks of Straumann implants which, however, healed in transmucosal mode (1994) [START_REF] Lang | Immediate transmucosal implants using the principle of guided tissue regeneration. I. Rationale, clinical procedures and 30-month results[END_REF]. The methods for tracking defect changes introduced in periodontal regeneration as standardized probing from the cemento-enamel junction (CEJ) to the deepest defect extension at baseline and at re-entry surgery [START_REF] Yukna | Multi-center clinical comparison of combination anorganic bovine-derived hydroxyapatite matrix (ABM)/cell binding peptide (P-15) and ABM in human periodontal osseous defects. 6-month results[END_REF][START_REF] Lekovic | Comparison of platelet-rich plasma, bovine porous bone mineral, and guided tissue regeneration versus platelet-rich plasma and bovine porous bone mineral in the treatment of intrabony defects: a reentry study[END_REF] are not applicable for purposes outlined in this study. Even in a situation illustrated by Fig. 2a Retrospective analyses presented by Jensen and Terhyeden at ITI consensus conference 2008 revealed an average fill of 85.5% together with 68.5% completely regenerated defects, respectively, calculated for the group of dehiscence-type of defects if a resorbable membrane was used. The rate of infectious complications was 13.75%. These data are based on a review of 20 studies [START_REF] Jensen | Bone augmentation procedures in localized defects in the alveolar ridge: clinical results with different bone grafts and bone-substitute materials[END_REF].

Our results did not evaluate the level of defect fill, but rather the change in the bone dimensions in the defect. Nevertheless, sites requiring additional augmentation can be considered showing incomplete regeneration. Implants exposed in the area to be grafted are generally considered seeking new bone formation. A failure in apposition of new bone results therefore in an unlikely contact of the implant surface with soft tissues bearing odds for onsetting peri-implantitis. Therefore, the gain in mineralized tissue at the crestal level of implant roughened surface is the crucial parameter in testing the efficacy of GBR procedure. The statistically significant differences in gain of clinically hard mineralized tissues at the crestal border of previously exposed SLAsurface (H1/W1) favors the use of the ribose-cross linked collagen test membrane.

Clinically viable amounts of mineralized tissue was gained in the test group as indicated by without exception positive values for the difference between baseline and 6 months results in height and width parameters (∆H1; ∆W1) of the bone covering previousely exposed implant areas (Tab. 3). There was none additional gain and even a slight loss of bone occurred obviously in some controls as expressed by negative differences in crestal height and width for this group. Taking into account similar frequencies of dehiscence onset in both groups, we had to assume that cases treated with RCLM (tests) had a greater benefit in regard to primary outcome than those treated with NCLM (controls). This interpretation is supported by results of an animal study, which showed significantly greater membrane stability for the RCLM than for NCLM sutured onto oral mucosa in rats, both materials being exactly same as used in our study [START_REF] Rothamel | Biodegradation of differently cross-linked collagen membranes: an experimental study in the rat[END_REF]. The unpublished in vitro data by Pitaru et al. showed greater resistance of RCLM vs. NCLM against bacterial collagenases (personal communication, October 14, 2010). Evaluating at re-entry the degradation status of both collagen membranes histologically Tal et al. found that among 26 initially applied RCLMs 13 were prematurely exposed (50%). Five out of them appeared interrupted and four were completely degraded 6 months following placement, whereas all devices from none-exposed sites remained intact. On the contrary, none of 18 NCLM devices initially applied was detectible at re-entry histologically. The authors concluded RCLM being more resistant against tissue degradation and retaining its integrity for a longer period of time compared to NCLM [START_REF] Tal | Long-term bio-degradation of cross-linked and non-cross-linked collagen barriers in human guided bone regeneration[END_REF].

Although none significant differences between both groups existed in regard to measurements at the second level addressed as ∆H2 / ∆W2, we conclude that the results are clearly superior for the use of RCLM material in lateral augmentation if compared to native collagen membranes, especially in regard to alterations in soft tissue healing.

  animal defects augmented with biphasic calcium phosphate (BCP)

  This study was a randomised controlled, single-blinded pilot clinical trial with an observation period of six months. All participants signed a written consent prior to the beginning of surgical procedures. The study was conducted in accordance with the guidelines of Good Clinical Practice (GCP-ICH) and the principles of the Declaration of Helsinki. The study was approved by the Ethics Committee of the Universitätsmedizin Charité, Berlin, Germany, under protocol number EA2/054/05 and registered at ClinicalTrials.gov under ID: NCT00835432. Patients were recruited from the pool of recall patients at the Department of Periodontology and Synoptic Dentistry or from referring dentists during the years 2005-2006. Included were partially edentulous patients in good general health with one or more teeth missing and requiring bone augmentation with placement of implants. There were no restrictions with regard to the location of the edentulous area, i.e. anterior and posterior regions of the maxilla and/or mandible were included. The study used a block randomization to randomize patients to either the cross-linked test (OSSIX, 3i, Palm Beach, FL, USA) or the non-cross-linked control (BioGide, Geistlich, Wohlhusen, Switzerland) membranes in a 1:1 ratio. This was a pilot study and no sample size calculation was performed. At the beginning of the study, treatment allocation for each patient was placed in a sealed envelope to be opened during the surgery. In patients who had two surgical sites that met the inclusion criteria, both sites were treated concomitantly and the first site received the membrane as per treatment allocation while the second site received the alternative treatment, facilitating comparisons within patients with two surgical sites.Smokers consuming 10 or more cigarettes per day were excluded. All treatments were performed between November 2005 and May 2007. All surgeries were carried

  Zubery et al. demonstrated nicely the ossification of ribose cross linked collagen membrane remnants facing underlying newly mineralized tissues. These observations support the idea that slow degradation of ribose-cross linked collagen

  one cast to the other. Implants inserted concomitantly with augmentation of dehiscence bone defects served as reference points. Albeit the implant necks were positioned supracrestally the equicrestal level of roughened sand blasted and acid-etched (SLA) surface on casts obtained at re-entry was clearly indicated. Therefore, measurements across alveolar crest perpendicular to the most coronal bony margin performed in this study can be considered standardized. In a previous publication our group demonstrated the degree of mineralization in newly formed tissues exceeding 40% in the area grafted laterally

  ridge would result in significant variation in positioning any measuring device at baseline and the re-entry surgeries.

Fig. 1 :

 1 Fig. 1: Flow diagram of study outline.

Fig. 2c :

 2c Fig. 2c: primary closure by coronally advancing the flap tissue for submerged

Fig. 2d :

 2d Fig.2d: the site 6 months later prior to re-entry shows sufficient gain in width

Fig. 3 :

 3 Fig. 3: Schematic drawing of the principle for assessment of the parameters Width

Fig. 3a :

 3a Fig.3a: mid-crestal section across alveolar ridge casted in plaster together

Fig

  Fig 3b: mid-crestal section of the cast obtained at baseline surgery prior to

Fig. 2a :Fig. 4c :Fig. 4d :

 2a4c4d Fig. 2a: Two Straumann Soft Tissue Level implants are placed displaying a dehiscence and a fenestration bone defects on the buccal aspect of the ridge. The necks of both implants are positioned supracrestally according to the protocol of use. 316x249mm (180 x 180 DPI)

Table 2

 2

	represents 37

  both groups of patients, test and control sites which exhibited soft tissue

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	dehiscence required additional augmentation at exposed implant surface. There were
	4 sites in the test group and 5 sites in the control group (23.5% vs. 20%, respectively)
	requiring additional treatment; implants involved received secondary application of
	BCP plus RCLM membranes at re-entry.

Table 1 :

 1 Comprehensive patient data (n = 37). Patient*** indicates sites excluded from statistical analysis by secondary randomization.

Table 2 :

 2 Baseline characteristics for patients and sites randomly selected for statistial analysis (n = 37).

Table 3 :

 3 Median values and ranges of morphometric outcome (* p < 0.05).

	Fig. 1
	Screening
	examination:
	40 patients recruited
	Baseline examination
	F o r	3 patients excluded:
	P e	Inappropriate width of alveolar ridge for one stage augmentation
	e r
	R 37 randomised
	e
	17 patients Test treatment BCP + RCLM	20 patients v i Control treatment e w BCP + NCLM
	Followed up /re-entry: 6 months: 17 patients	Follow up / re-entry: 6 months: 20 patients
	17 patients analysed	20 patients analysed

Journal of Clinical Periodontology -PROOFJournal of Clinical Periodontology -PROOF

Acknowledgements:

We are grateful for initiation of the study by 3i Implant Innovations and their support with study materials. We also highly appreciate the contribution made by Institute Straumann AG supplying this study with additional materials such as BCP, Tissue Level Implants and gingival formers. We are deeply indepted to Asisa Saidowa for providing our group with schematic drawings of the principle used for assessments.

Our gratitude goes also to Mr. Bodenstein and Mr. Schnitzer, who both supported the laboratory part of study providing clinical investigator with lab equipment and facilities.

The study was funded in parts by Biomet 3i (Palm Beach; FL, USA) as unrestricted research grant to Department of Periodontology and Synoptic Dentistry, Charité, Berlin, Germany and by material donations from Institute Straumann AG (Basel, Switzerland). proposing the initial steps for the protocol and providing the membranes as study material. After the company part of this investigation. The final protocol, as described in the manuscript and approved by the Ethic Committee of the Charité University was designed by the principal investigator (AF). The data processing and preparation of the manuscript were completed by the co-authors all named on the cover page. Dr.