Clinical and Histological changes after ridge preservation with two xenografts: preliminary results from a multicentre randomized controlled clinical trial

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Article

Reference

Clinical and Histological changes after ridge preservation with two xenografts: preliminary results from a multicentre randomized

controlled clinical trial

BARONE, Antonio, et al.

Abstract

To evaluate and compare clinical and histological changes after ridge preservation procedures with those of spontaneous healing.

BARONE, Antonio, et al. Clinical and Histological changes after ridge preservation with two xenografts: preliminary results from a multicentre randomized controlled clinical trial. Journal of Clinical Periodontology, 2017, vol. 44, no. 2, p. 204-214

DOI : 10.1111/jcpe.12655 PMID : 27883211

Available at:

http://archive-ouverte.unige.ch/unige:93735

Disclaimer: layout of this document may differ from the published version.

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Received Date : 28-Jan-2016 Revised Date : 18-Oct-2016 Accepted Date : 18-Nov-2016

Article type : Randomized Clinical Trial

Tissue Changes after ridge preservation with two xenografts. Preliminary results from a multicenter randomized controlled clinical trial

RUNNING HEAD: Xenografts for ridge preservation

AUTHORS:

⌘†

Antonio Barone, DDS, PhD, MSc, Professor and Chairman

*† Paolo Toti, BSc, DDS, Research Fellow

‡ Alessandro Quaranta, DDS, PhD, Professor and Chairman

*† Fortunato Alfonsi, DDS

§ Alessandro Cucchi, DDS

|| Bruno Negri, DDS

¶ Roberto Di Felice, MD, DDS

*† Saverio Marchionni, DDS, Research Fellow

*† Jose’ Luis Calvo-Guirado, DDS, PhD, MSc, Professor and Chairman

*† Ugo Covani, MD, DDS, Full Professor and Chairman

≠ Ulf Nannmark, DDS, PhD, Associate Professor

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AUTHORS’ AFFILIATIONS:

⌘Unit of Oral Surgery and Implantology, Department of Surgery, University of Geneva,

Switzerland. Formerly: Department of Surgical, Medical, Molecular and Critical Area Pathology, University of Pisa, Italy.

* Department of Surgical, Medical, Molecular and Critical Area Pathology, University of Pisa, Italy.

† Tuscan Dental Institute, Versilia General Hospital, Via Aurelia 335, Lido di Camaiore (LU), Italy.

‡ Associate Professor, Department of Dentistry, University of West Australia, Perth,

Australia. Formerly: Department of Odontology and Specialized Clinical Sciences (DISCO), Marche Polytechnic University, Torrette di Ancona, Italy;

§ Department of Surgery, University of Verona, Verona, Italy

|| Department of Implantology, University of Murcia, Spain

¶ Private Practice, Ascoli Piceno, Italy

≠ Department of Oral & Maxillofacial Surgery, the Sahlgrenska Academy Gothenburg University, Gothenburg, Sweden

CORRESPONDING AUTHOR:

Prof. Antonio Barone (DDS, PhD, MSc)

Professor and Chairman, Unit of Oral Surgery and Implantology, Department of Surgery, University of Geneva, Switzerland.

Centre Universitaire de Médecine Dentaire Rue Barthélémy-Menn 19

1205 Genève, Switzerland Tel: +41 22 3794080

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Fax: +41 22 3727604

E-mail: barosurg@gmail.com

Fax number and e-mail can be published.

KEY WORDS: alveolar bone loss, alveoloplasty, bone remodeling, xenograft, tooth socket

ACKNOWLEDGMENTS: None.

SOURCE OF FUNDING: No external funding was available for this study.

ABSTRACT

Objectives: To evaluate and compare clinical and histological changes after ridge preservation procedures with those of spontaneous healing.

Materials_and_Methods: Ninety patients were enrolled in the present randomized controlled clinical trial and underwent single-tooth extraction in the premolar/molar areas.

Thirty sites were grafted with collagenated cortico-cancellous (coll), 30 sites with cortical (cort) porcine bone and 30 sites underwent natural healing. Primary (vertical and horizontal bone changes after 3-month) and secondary outcomes (histomorphometric after 3 months) were evaluated at implant placement.

Results:The vertical bone changes at the grafted sockets were significantly (Ps< 0.0001) lower (0.30mm for cort- and 0.57mm for coll-group) when compared to non-grafted sockets (2.10mm for nat-group). Moreover, the width reduction of the coll (0.93mm) and cort (1.33mm) groups was significantly lower (P<0.0001) than the non-grafted group (3.60mm).

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The analysis of subgroups attested that when premolar- and molar-sites were compared, the buccal bone loss appeared to be dependent both on tooth position and grafting material employed.

Conclusion: The ridge preservation procedures had significantly better outcomes when compared to natural healing. The biomaterials did not differ for maintenance of bone width;

even though, the bone height seemed to be better preserved with the cortical porcine bone.

STATEMENT OF CLINICAL RELEVANCE

Scientific_Rationale_for_Study:Ridge preservation procedures could counteract the tissue changes occurring after tooth loss. Few information is available on the potential benefits of different biomaterials when compared with natural healing after tooth extraction.

Principal_Findings:Three months after healing cortical bone was more efficient in preserving the buccal ridge height at molar sites, whilst collagenated cortico-cancellous bone achieved better outcomes at premolar sites.

Practical_Implications:The ridge preservation procedures, regardless of the biomaterial used, showed lower bone dimensional changes when compared to natural healing. The 2 types of biomaterials seemed to show no differences in clinical performance.

INTRODUCTION

The alveolar ridge undergoes unsteady resorption and atrophy following tooth extraction (Devlin_et_al._1991,Lekovic_et_al._1998, Barone_et_al._2008). These changes take place mainly in the first months and continue during the following year, accounting for up to a 50%

loss of the pristine alveolar ridge width (Schropp_et_al._2003). Moreover, 1 year after tooth extraction, the buccal resorption at the midpoint of the extraction sites is higher than that

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registered at the mesial/distal aspects; the alveolar crest tends to shift two-thirds lingually from the original buccal edge (Covani_et_al._2011, Kan_et_al._2011).

Many different biomaterials have been used to counteract the dimensional changes following tooth extraction (Becker_et_al._1998, Brugnami_et_al._1999,Artzi_et_al._2000,

Carmagnola_et_al._2000,Scarano_et_al._2010). Some authors investigated the use of cortical bone allograft versus cancellous bone allograft for ridge preservation procedures (RP); they reported no differences in the percentage of newly formed bone (Eskow_and_Mealay 2014).

Furthermore, several studies suggested that tissue changes after tooth extraction could be limited by ridge preservation techniques performed with collagenated porcine bone and a resorbable membrane (Barone_et_al._2013). The most common xenografts of bovine origin are calcium-deficient carbon apatite without residual collagen; these biomaterials were found to be highly osteoconductive and seemed to reabsorb very slowly or not at all

(Iezzi_et_al._2007,Slotte_et_al._2007). The collagenated porcine bone grafts are constituted by carbonated nanocrystalline hydroxyapatite containing residual collagen. Several studies showed that the collagenated porcine bone grafts had excellent osteoconductive properties without adverse reactions or inflammatory infiltrate (Nannmark_and_Azarmehr_2010, Barone_et_al._2012,Barone_et_al._2013). Moreover, in vivo studies of these biomaterials showed clear signs of reabsorption/remodeling as well as presence of scalloped lacunae (Nannmark_and_Sennerby_2008,Pagliani_et_al._2012).

The surgical procedure might be a possible determinant of ridge preservation outcomes;

according to some studies, the use of a membrane and graft material for ridge preservation procedures requires primary closure with a full-thickness flap (Tan_et_al._2012,

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in an attempt to achieve primary closure have been associated with bone resorption, marginal recession at adjacent teeth, defective papillae and loss of keratinized tissues at the buccal side.

The aim of this randomized controlled trial was to compare and evaluate the clinical and histological outcomes of extraction sockets grafted with cortical porcine bone

(Apatos,_OsteoBiol®;_Tecnoss,_Italy) to those grafted with collagenated cortico-cancellous porcine bone (MP3,_OsteoBiol®;_Tecnoss,_Italy) and fresh extraction sockets which healed naturally.

The two different xenografts were also compared to each other to determine their respective efficacy in preserving the alveolar ridge dimensions following tooth extraction. This study reported preliminary findings at 3 months after ridge preservation; a five-year follow-up was planned to evaluate the procedures over time.

MATERIALS AND METHODS Study design/sample

The present multicenter parallel-group randomized human clinical trial was approved by the Versilia Hospital Research Ethical Committee (ethical approval form 214/2012;

ClinicalTrials.gov Identifier: NCT02644070), and was conducted according to the principles outlined in the Declaration of Helsinki as revised in 2000; this paper is reported according to the CONSORT statement (Schulz_et_al._2010).

Recruitment and enrollment of patients were performed from June 2011 to June 2012 in five centers:

• Universities of Pisa, Ancona, Verona (Italy), Murcia (Spain) and Dr. Di Felice’s private practice.

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Inclusion criteria

All patients -able to sign an informed consent form and 18 years old or older- who required single-tooth extraction and an implant-supported restoration.

Exclusion criteria

• history of systemic diseases that contraindicate oral surgery;

• long-term non-steroidal anti-inflammatory drug therapy;

• oral bisphosphonate therapy;

• pregnancy or lactation;

• unwillingness to return for the follow-up examinations;

• cigarette consumption>10 per day.

Three balanced groups of patients were obtained with the use of a customized software (matlab7.11,_the_MathWorks,_Natick,_MA). The randomization codes were stored in password-protected portable computers and enclosed in sequentially numbered, identical, opaque, sealed envelopes.

The envelopes were opened in numerical order after tooth extraction. A third operator for each center -not involved in enrollment or treatment of patients- performed data collection.

Surgical methods

All patients underwent at least one oral hygiene session in order to provide a more favorable oral environment for wound healing.

Patients were recruited and treated by clinicians who had received training and calibration for the accuracy of measurements during a 1-week session (April 2011). To test the reliability, five model casts were selected and measured twice by each operator on separate days.

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All patients received prophylactic antibiotic therapy of 2g of amoxicillin (or clindamycin 600mg if allergic to penicillins) 1 h before the extraction procedure and continued to take the antibiotic postoperatively, 1g amoxicillin (or 300mg clindamycin) twice a day for 5 days. All patients rinsed for 1 min with chlorhexidine mouthwash 0.2% prior to the surgery (and twice a day for the following 3 weeks) and were treated under local anesthesia using lidocaine with adrenaline 1:50,000. Tooth extraction was performed without raising a full thickness flap, and if necessary the tooth was sectioned to make the extraction the least traumatic possible (Figure 1).

The randomization envelope was opened and the surgeon followed the indication to treat the extraction socket as follows: (1) extraction sockets with spontaneous healing; (2) extraction sockets grafted with collagenated cortico-cancellous porcine bone, with a particle size between 600 and 1000 m (MP3, OsteoBiol®,_Tecnoss®,_Coazze,_Italy); (3) extraction sockets grafted with cortical porcine bone, with a particle size between 600 and 1000 m (Apatos,_OsteoBiol®,_Tecnoss®,_Coazze,_Italy).

In the test groups, the extraction sockets were grafted up to the buccal and palatal alveolar bone walls and, subsequently, a collagen membrane (Evolution,_OsteoBiol®,_Tecnoss®, Coazze,_Italy) was gently pushed under the interdental papilla with the use of periotomes (PT1,3,4,5_Hu-Friedy_Mfg.B.V._P.O._Box_29025_NL-3001_GA_Rotterdam) without raising a flap; the soft tissues at the level of interdental papilla were prepared with a pouch procedure. The collagen membrane was used in all test sites to cover the fresh extraction socket entrance without covering the bone walls. Sutures were used to stabilize the membrane in order to prevent loss of graft particles; therefore the collagen membrane was left exposed

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to the oral cavity. In the control group, sutures were used to stabilize the blood clot. No releasing incisions or mucoperiosteal flaps were performed in any of the groups.

Patients were instructed to continue with prophylactic antibiotic therapy and naproxen sodium 550mg tablets were prescribed as an anti-inflammatory to be taken twice a day.

Three months after healing, all the experimental sites were re-entered to harvest bone

biopsies (Trepan_bur_227A.204.0232,_Komet_Italia_S.r.l._Milan,_Italy) and to insert dental implants (BT_Evo,_Biotec,_Vicenza,_Italy) according to the occlusal hole of the surgical stent (Figure 2), which was used for all measurements. The bone samples were sent to the Institute of Biomedicine, the Sahlgrenska Academy Gothenburg University, Sweden for histological examination.

Variables

Descriptive variables were registered: age, gender, smoking habits and location.

Primary predictor variable

• Preservation: cort-group, cortical porcine graft group; coll-group, pre-hydrated collagenated cortico-cancellous porcine graft group; nat-group: non-grafted group.

Secondary predictor variables

• Tooth site: premolar or molar.

• Buccal Bone Thickness (BBT): it was measured at baseline, at the mid-facial level of the buccal bone plate using a surgical caliper exactly at the buccal hole of the stent; low-BBT group with a thickness of<1.5mm, and high-BBT group with a thickness of1.5mm;

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• Socket type: extraction socket evaluation was made according to the classification suggested by Juodzbalys (Table 1) (Juodzbalys_et_al._2008) Socket type I: adequate extraction socket; Socket type II: compromised extraction socket; Socket type III: deficient extraction socket.

Primary outcome variables

All anatomical measurements were taken at baseline and at 3 months after tooth extraction.

The following variables were registered to the nearest millimeter:

• Vertical bone changes were evaluated with the use of a custom made stent at mesial, distal, buccal and lingual/palatal sites; it was set as the distance between the reference point and the most apical point of the marginal bone level. The stent was placed on the adjacent teeth to allow reproducible vertical measurements, as shown in Figure 2A. Changes at the vertical bone level were evaluated by subtracting the postoperative value from the respective baseline value (VBL)

• Buccal-Lingual Width was measured at the most coronal level of the socket as the distance between buccal and lingual/palatal plate with a periodontal probe aligned from lingual/palatal to buccal insertion hole, as shown in Figure 2B. Changes of bone width were calculated by subtracting the baseline value from the post-operative value (BLW)

Secondary outcome variable

• Histomorphometric parameters such as newly formed bone (NFB), non-mineralized tissues (NMT) and residual graft particles percentages (RGP) were calculated.

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Histological Processing

The bone biopsies were decalcified in ethylenediaminetetraacetic acid (10%) for a period of 2 weeks, afterwards X-rayed in order to verify the decalcification procedure. After

dehydratation in graded series of ethanol, the specimens were embedded in paraffin and sectioned by a wafering high speed rotating blade microtome; the sections were subsequently ground down to about 5-10 µm by a grinding machine and stained with hematoxylin-eosine and modified Mallory aniline blue.

Examinations were performed in a Nikon Eclipse 80i microscope (teknooptikAB,_Huddinge, Sweden) using X10 to X40 objectives for descriptive evaluation and morphometrical

measurements. All measurements were determined by using an Easy image 2000 system (teknooptikAB,_Huddinge,_Sweden) for area measurements. For each bone biopsy, 3 different sections, measuring about 10mm² each, were analyzed. Finally, the results were expressed as a percentage (i.e., area fraction) of mineralized bone, residual graft materials, and non-mineralized tissue (i.e., connective tissue and/or bone marrow).

Statistical Analysis

Sample size was calculated by using a power of 0.9 (Statistics_Toolbox,_MatLab_7.0.1, The_MathWorks,_Natick,_MA) and compared to outcome data reported in a previous study (Barone_et_al._2008). According to the mean and SD values for horizontal ridge changes and vertical height changes (with a mean difference and a pooled standard deviation of

21.4279mm and 2.92.0512mm, respectively), the sample size was close to 12 for a two- group analysis. The required sample for a three-group analysis was about 18 patients: the final sample size (18 patients for each center) was 90 subjects.

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Each center contributed according to the predetermined 1:1:1 ratio; the fresh extraction socket was considered the unit of randomization. Corrections for balancing the three

experimental groups for confounding factors were not applied. Normal distribution for each outcome variable was checked and confirmed by the Shapiro-Wilk test. A matrix elaborator performed all the analyses (matlab7.11,_The_MathWorks,_Natick,_MA): analysis of variance with two-fixed factor interaction and analysis of covariance were applied; then appropriate post hoc comparison tests were run. The level of statistical significance was set at 0.05 for all analyses but investigators demanded a more stringent level of significance

comparing adjusted p-values to =0.01.

RESULTS

The intra- and inter-reliability coefficients ranges were 0.905-0.980, and 0.468-1, respectively.

One hundred patients were screened for this study; 5 patients were excluded because they refused to be included in a randomized control trial, 3 patients were excluded because during tooth extraction a mucoperiosteal flap had been raised and 2 patients were excluded at tooth extraction because they were affected by an acute infection involving the soft tissues. A total of 90 patients were allocated to the study groups of the trial. Patients' demographic data are reported in Table 2. At the end of the survey, 90 dental implants (BT_EVO;Biotec,_Povolaro of_Dueville,_Vi,_Italy) were placed.

Outcome variables and preservation

Regression analysis suggested that, when the natural group was the reference category, all outcome variables were significantly affected by the ridge preservation procedure employed (Table 3).

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The vertical and horizontal bone changes were reported in Table 4. The grafted sites showed a significant (P<0.0001) lower vertical bone loss at buccal (from -0.301.28mm to -

0.571.54mm) and lingual/palatal (from +0.672.54mm to -1.001.17mm) aspects than that registered at the no-grafting sites. (Table 4). Additionally, the grafted groups behaved significantly better than the non-grafted group in terms of horizontal bone resorption. The cort- and coll-groups had a horizontal bone loss of 1.33±0.71mm and 0.93±1.25mm, respectively, while the nat-group had a horizontal bone loss of 3.60±0.72mm.

No statistically significant differences were registered between the grafted groups for any of the variables except for vertical bone loss at the lingual/palatal aspect (P=0.0039).

Outcome variables and tooth sites

Data analysis showed that tooth site could affect the vertical bone resorption (Table 4).

Indeed the vertical bone loss at buccal aspect was significantly lower (P=0.0013) when a premolar site was grafted with collagenated cortico-cancellous bone (apparent gain of 0.80±1.23mm), on the other hand the vertical bone loss at buccal aspect was significantly lower (P=0.0004) when a molar site was grafted with cortical bone (apparent gain of 0.44±1.14mm).

These differences were observed comparing premolar and molar sites as well, regardless of the biomaterial used (Ps=0.0007).

Outcome variables and buccal bone thickness or socket types

The study groups were further divided into four subgroups according to the Buccal Bone Thickness (low-BBT<1.5mm and high-BBT≥1.5mm) and post-extraction socket morphology (socket type I and socket type II/III). All the grafted subgroups, irrespective of the grafting

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(ranging between 0.17±1.46mm and 1.29±1.07mm) than that of naturally healed sites (ranging between 1.94±0.66mm and 2.31±0.63mm). No statistically significant differences were observed between the two grafted groups (Table 4).

Histomorphometric analysis

Histological data analysis (Table 4, Figure 3) showed a percentage of newly formed bone of 36.8±19.1% and 44.0±14.7% for cort-group and nat-group respectively, whereas coll-group had 41.4±20.6% of newly formed bone; these differences were not statistically significant.

The residual graft particles were 15.5±8.4% in the cort-group and 14.9±7.3% in the coll- group, and the difference was not significant. Results attested that non-mineralized tissues showed a statistically significant difference (P=0.0077) between coll-group (41.415.9%) and nat-group (56.014.7%), respectively.

DISCUSSION

Several studies have evaluated the effects of tooth extraction on alveolar tissues, which had a volume reduction especially on the buccal side; this resulted in a palatal/lingual shift of the residual crest (Pietrokovski_and_Massler_1967,Schropp_et_al._2003, Barone_et_ al._2008, Martuscelli_et_al._2014). These changes could compromise the aesthetic outcome of final restorations either with implant placement or traditional prosthetic rehabilitations. Although different techniques have been proposed for maintaining original alveolar ridge dimensions, a complete preservation seems to be a non-achievable outcome.

The flapless tooth extraction is generally performed to obtain a reduction of healing time and discomfort. Furthermore, a recent RCT showed that the flapless approach for ridge

preservation was more successful than the flap approach in preserving horizontal ridge dimension and width of keratinized tissues (Barone_et_al._2014). Moreover, a recent

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systematic review (Jambhekar_et_al._2015) on ridge preservation with a flapless approach observed that the amount of bone dimensional changes were lower than that reported with other reviews, which combined data with and without flap approach.

The grafting techniques achieved better outcomes when compared to tooth extraction alone (Iasella_et_al._2003). The present study showed that porcine bone, resorbable membrane and a flapless approach were more effective in controlling the bone changes after tooth extraction when compared to no grafting. Moreover, the present data confirmed findings from other studies in which the horizontal bone resorption in extraction sockets grafted with

collagenated cortico-cancellous porcine bone was significantly lower (1.6mm) than in extraction sockets that had healed naturally (3.6mm) (Barone_et_al._2013). Data from our study confirm the Cosyn & co-workers' hypothesis, which identified tooth location as a predictive factor of remodeling after ridge preservation procedures, though it should be considered that in Cosyn’s study only canine and premolars were evaluated

(Cosyn_et_al._2016). Indeed, the present study showed that the buccal bone plate was better preserved at premolar sites with collagenated porcine bone and at molar sites with cortical bone. The findings from this study as well as from Cosyn’s study (2016) suggested that tooth site and/or thickness of buccal bone plate could affect the outcomes of ridge preservation procedures. Additionally, the present study found that the thinnest buccal bone walls were associated with higher horizontal bone resorption (BLW) in the grafted sites irrespective of the xenograft used. Spinato & co-workers (Spinato_et_al._2014), found greater horizontal bone loss at the spontaneous healing sites when compared to grafted sockets sites when buccal bone thickness was lower than 1mm. The ridge preservation procedures could

counteract the remodeling pattern observed at the non-grafted sites, thus reducing the risks of

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(<1.5mm) had less horizontal bone resorption than those with thick buccal bone walls

(1.5mm) and the difference was significant. Some authors indicated that a greater reduction in the molar area should be expected since a wider socket requires more time for the bone to bridge over the defect (Engler-Hamm_et_al._2011). Their conclusions were in agreement with the findings from other authors who found that horizontal bone reduction was more pronounced for molar sites with the thicker buccal bone walls than for premolar sites with thin buccal bone walls (Araujo_et_al._2005). On the contrary, the present study attested that, horizontal bone loss was greater in low-BBT than in high-BBT for grafted sites. Furthermore, the present study showed that the use of a ridge preservation procedure could better preserve not only ridge width but also height dimension when compared to tooth extraction alone.

Moreover, cortical graft (cort-group) seemed to maintain more effectively, in sites with thick buccal plate, the ridge height when compared to collagenated cortico-cancellous graft (coll- group), which seemed to be more efficient in maintaining bone width. This trend is in accordance with some other authors who found that the loss of alveolar height is greater in the cancellous group compared to the cortical group (Eskow_and_Mealey_2014).

Regarding the histomorphometric evaluation of this study, the percentages of residual graft particles in the cort-group were higher (15.5%) than those found in the coll-group (14.9%), though the difference was not significant. The Newly Formed Bone in the coll-group (41.4%) seemed to be greater than that in the cort-group (36.8%), but none of test groups showed statistically significant differences when compared to the control group (44%). These data could indicate a variation with regards to resorption rate between the 2 biomaterials;

moreover, this would suggest a more promising healing pattern for the collagenated bone when compared to the cortical porcine bone.

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The limitations of the present study were the very short-term of analysis, which is not enough to predict the possible differences over time, and the limited number of patients per each center. The present study will follow the patients for 5 years.

The external validity of this study suggests that irrespective of the biomaterials used, the ridge preservation technique could preserve the vertical and horizontal tissue dimensions better than the natural healing after tooth extraction. It should be considered that the

observational time for this study is still too short to register the biological and biomechanical complications that could occur over time. On the other hand, the ridge preservation technique can be considered an easy procedure that could be performed by a large variety of clinicians to better preserve tissue dimensions after tooth extraction.

CONCLUSIONS

Alveolar ridge preservation with cortical or collagenated cortico-cancellous porcine bone is an effective way to maintain the ridge dimensions after tooth extraction compared to spontaneous healing, though a complete prevention of remodeling is not achievable irrespective of the biomaterials employed.

No significant differences were found between the two pertaining to the ridge width.

Furthermore, no significant differences regarding the histomorphometric analysis were registered between the two grafted groups.

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Accepted Article

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Accepted Article

Tan, W.L., Wong, T.L., Wong, M.C. & Lang, N.P. (2012) A systematic review of post- extractional alveolar hard and soft tissue dimensional changes in humans. Clinical Oral Implants Research 23(Suppl. 5), 1-21

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TABLE and FIGURE LEGENDS

Table 1: Extraction socket type according to soft tissue and hard tissue conditions.

* Jemt 1997

Table 2: Demographic data, dependent variables and histmorphometric analysis reported for the three experimental groups.

NFB: newly formed bone RGP: residual graft particle NMT: non-mineralized tissue

cort group: cortical porcine bone group

coll group: collagenated cortico-cancellous porcine bone group nat group: non-grafted group.

Table 3: Regression of outcome variables in function of treated groups in comparison with natural healing group and of the several predictors of dimensional changes: tooth site, buccal

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Accepted Article

VBL: Changes of Vertical Bone Level at buccal (b), lingual-palatal (lp) and mesial-distal (md) aspects; BLW: Changes of Buccal-Lingual Width

Table 4: Tissue Changes for the three experimental groups. VBL: Changes of Vertical Bone Level at buccal (b), lingual-palatal (lp) and mesial-distal (md) aspects

BLW: Buccal-Lingual Width

BLW: Changes of Buccal-Lingual Width

BLW*: Changes of Buccal-Lingual Width resulting by ANCOVA correction

cort group: cortical porcine bone group

coll group: pre-hydrated collagenated cortico-cancellous porcine bone group nat group: non-grafted group.

ANOVA variable: VBL; source: preservation_group*tooth_site; sum of sq:17.15; df:2;

mean sq:8.5753; F:12.56; Prob>F:0

ANOVA variable: BLW; source: preservation_group* tooth_site; sum of sq:0.848; df:2;

mean sq:0.424; F:0.51; Prob>F:0.6034

ANOVA variable: VBL; source: preservation_group* buccal bone thickness _group; sum of sq:3.6302; df:2; mean sq:1.81509; F:2.24; Prob>F:0.1123

ANOVA variable: BLW; source: preservation_group* buccal bone thickness _group; sum of sq:4.663; df:2; mean sq:2.3314; F:2.77; Prob>F:0.0686

ANOVA variable: VBL; source: preservation_group*socket_type; sum of sq:0.3327; df:4;

mean sq:0.08317; F:0.09; Prob>F:0.9844

ANOVA variable: BLW; source: preservation_group* socket_type; sum of sq:11.76; df:4;

mean sq:2.94; F:4.19; Prob>F:0.0039

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Accepted Article

Figure 1: Clinical and radiological images of test post-extractive filled sockets: cortical porcine bone (A) Bicuspid before extraction; (B) fresh extraction socket; (C) socket grafted with bone substitute; (D) cross-mattress suture; (E) implant osteotomy; (F) implant

placement; (G–I) periapical radiographs (G) baseline; (H) after 3 months of healing and (I) after implant placement. Collagenated cortico-cancellous porcine bone (J) Bicuspid before extraction; (K) fresh extraction socket; (L) socket grafted with bone substitute; (M) cross- mattress suture; (N) implant osteotomy; (O) implant placement; (P–R) periapical radiographs (P) baseline; (Q) after 3 months of healing and (R) after implant placement.

Figure 2: Anatomic measurements: A) vertical bone level (VBL). B) View of reference stent related to width measurement.

Figure 3: Histological sections from the cortical porcine graft group (Htx-eosine): (A) original magn x4; (B), (C) and (D) original magn x30-x50; histological sections from the prehydrated collagenated cortico-cancellous porcine graft group (Htx-eosine): (E) original magn x4; (F), (G) and (H) original magn x30-x50. newly formed bone, °, residual graft particles, *, and non-mineralized soft tissues, ^.

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Accepted Article

Table 1

type I type II type III

assessment all adequate at least 1 compromized at least 1 deficient soft tissue contour

variations No < 2mm ≥ 2 mm

vertical soft tissue

deficiency No 1 mm to 2 mm >2 mm

keratinized gingival width

on the mid-buccal side

>2 mm 1 mm to 2 mm < 1 mm

status of both mesial and distal papilla*

V (hyperplastic) IV (fills the entire proximal space)

III (lower than the normal contact point)

II (less than half the normal papilla height is present)

I (no papilla)

gingival tissue biotype

Thick (≥ 2 mm)

Moderate (≥ 1 mm to < 2 mm)

Thin (< 1 mm) soft tissue quality

(color, consistency, and contour)

Pink, firm, and smooth

Slightly red and a soft, spongy, and uneven contour

Red/bluish or red with a soft edematous and boggy or craterlike

appearance facial bone thickness

on the mid-buccal side

≥ 2 mm ≥ 1 mm to < 2 mm < 1 mm

facial bone lesion No

Yes

(> 0 mm to < 2 mm)

Yes (≥ 2 mm) palatal/lingual bone

lesion No

Yes

(> 0 mm to < 2 mm)

Yes (≥ 2 mm)

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Accepted Article

Table 2

Demographics and dependent variables cort coll nat

patients (n) 30 30 30

age (yrs) 48.212.8 47.29.7 46.910.8

age range (yrs) 25 – 63 31 – 64 28  70

sex (n; males/females) 14/16 10/20 12/18

premolar-to-molar ratio 14:16 10:20 8:22

smoking habit (n; yeas/no) 4/26 6/24 5/25

reasons for tooth extraction (n; decay/endodontic failure/fracture) 10/16/4 14/6/10 14/3/13

amount of graft (cc) 0.60.3 0.70.3 -

Histomorphometric analysis mean ± sd (mm)

cort coll nat

NFB (%) 36.8±19.1 41.4±20.6 44.0±14.7

RGP (%) 15.5±8.4 14.9±7.3 -

NMT (%) 47.819.2 41.415.9 56.014.7

sig. (p-value)

cort vs coll cort vs nat coll vs nat

NFB (%) 0.3401 0.0496 0.4869

RGP (%) 0.8300 - -

NMT (%) 0.4244 0.1644 0.0077

Table 3

unstandardized coefficients

95.0% confidence interval

standardized coefficients

t sig.

model B std. error lower bound

upper

bound beta buccal VBL

(constant) -1.876 0.295 -2.464 -1.289 -6.349 < 0.001

* preservation:

cort 2.080 0.358 1.368 2.793 0.682 5.805 < 0.001

* preservation:

coll 1.817 0.357 1.108 2.526 0.596 5.094 < 0.001 buccal bone

thickness (low/high)

-0.471 0.311 -1.090 0.149 -0.162 -1.512 0.134

socket type (I/II-

III) -0.122 0.275 -0.668 0.424 -0.042 -0.443 0.659 tooth site

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Accepted Article

lingual-palatal

VBL

(constant) -1.601 0.391 -2.378 -0.823 -4.093 < 0.001

* preservation:

cort 3.360 0.474 2.417 4.303 0.800 7.084 < 0.001

* preservation:

coll 1.641 0.472 0.702 2.580 0.391 3.476 < 0.001

* buccal bone thickness (low/high)

-1.128 0.412 -1.948 -0.309 -0.282 -2.738 0.008

III) -0.180 0.363 -0.903 0.542 -0.045 -0.496 0.621 tooth site

(premolar/molar) -0.252 0.365 -0.978 0.475 -0.063 -0.689 0.492 mesial-distal

VBL

(constant) -0.272 0.266 -0.801 0.256 -1.024 0.309

* preservation:

cort -0.811 0.322 -1.452 -0.170 -0.333 -2.516 0.014

* preservation:

coll -0.962 0.321 -1.600 -0.323 -0.394 -2.996 0.004 buccal bone

0.282 0.280 -0.275 0.839 0.122 1.007 0.317

III) 0.238 0.247 -0.253 0.729 0.103 0.964 0.338

tooth site

(premolar/molar) -0.295 0.248 -0.789 0.199 -0.128 -1.187 0.239

WBL

(constant) -3.348 0.221 -3.788 -2.909 -15.165 < 0.001

* preservation:

cort 2.303 0.268 1.770 2.835 0.729 8.600 < 0.001

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Accepted Article

* preservation:

coll 2.723 0.267 2.193 3.253 0.862 10.216 < 0.001 buccal bone

-0.309 0.233 -0.772 0.154 -0.103 -1.328 0.188

* socket type

(I/II-III) -0.462 0.205 -0.870 -0.054 -0.154 -2.252 0.027 tooth site

(premolar/molar) 0.309 0.206 -0.101 0.719 0.104 1.499 0.138

Table 4

primary predictor: experimental groups

mean ± sd (mm) sig. (p-value)

group cort coll nat cort vs coll cort vs nat coll vs nat

size 30 30 30 30 vs 30 30 vs 30 30 vs 30

bVBL (mm) -0.301.28 -0.571.54 -2.100.66 0.4199 < 0.0001 < 0.0001 l/pVBL (mm) 0.672.54 -1.001.17 -2.030.72 0.0039 < 0.0001 0.0001 mdVBL (mm) -0.901.26 -1.081.37 -0.150.38 0.4977 0.0534 0.0003

BLW_baseline (mm) 10.131.99 9.901.39 9.571.36 0.9344 0.4483 0.5413

BLW (mm) -1.33±0.71 -0.93±1.26 -3.60±0.72 0.3895 < 0.0001 < 0.0001

BLW* (mm) -1.28±0.16 -0.93±0.16 -3.66±0.16 0.3740 < 0.0001 < 0.0001 secondary predictor: premolar

size 14 10 8 14 vs 10 14 vs 8 10 vs 8

bVBL (mm) -1.14±0.86 0.80±1.23 -2.25±0.46 0.0013 0.0049 < 0.0001 l/pVBL (mm) 0±2.66 -0.40±1.71 -2.13±0.84 0.7401 0.0066 0.0251 mdVBL (mm) -1.29±1.41 -0.55±1.09 -0.06±0.42 0.2439 0.0434 0.5439

BLW_baseline (mm) 9.00±1.24 9.20±0.79 8.25±0.71 0.5548 0.0383 0.0108

BLW (mm) -1.21±0.55 -0.50±1.89 -3.13±0.35 0.9245 0.0001 < 0.0001

BLW* (mm) -1.16±0.31 -0.40±0.37 -3.34±0.45 0.3490 0.0020 < 0.0001 secondary predictor: molar

size 16 20 22 16 vs 20 16 vs 22 20 vs 22

bVBL (mm) 0.44±1.14 -1.25±1.20 -2.05±0.72 0.0004 < 0.0001 0.0280 l/pVBL (mm) 1.25±2.35 -1.30 ±0.66 -2.00±0.69 0.0001 < 0.0001 0.0014 mdVBL (mm) -0.56±1.04 -1.35±1.44 -0.18±0.36 0.0859 0.6773 0.0001

BLW_baseline (mm) 11.13±2.01 10.25±1.51 10.23±0.81 0.1251 0.2102 0.9073

BLW (mm) -1.44±0.83 -1.15±0.76 -3.77±0.75 0.3301 < 0.0001 < 0.0001

BLW* (mm) -1.33±0.19 -1.19±0.17 -3.82±0.16 1.0000 < 0.0001 < 0.0001 premolar vs molar

sig. (p-value)

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Accepted Article

mdVBL (mm) 0.0804 0.0327 0.5997

BLW_baseline (mm) 0.0020 0.0924 0.0005

BLW (mm) 0.6162 0.8703 0.0172

secondary predictor: low-buccal bone thickness < 1.5mm

size 20 14 7 20 vs 14 20 vs 7 14 vs 7

bVBL (mm) -0.35±1.42 -1.29±1.07 -2.14±0.38 0.0610 0.0025 0.0612

BLWbaseline (mm) 10.25±2.04 9.94±1.17 8.86±1.57 0.9010 0.1210 0.0584

BLW (mm) -1.50±0.73 -1.21±0.38 -3.14±0.38 0.2930 0.0003 0.0001

BLW* (mm) -1.47±0.12 -1.20±0.15 -3.27±0.22 0.4900 < 0.0001 < 0.0001 secondary predictor: high-buccal bone thickness  1.5mm

size 10 16 23 10 vs 16 10 vs 23 16 vs 23

bVBL (mm) -0.20±1.03 0.06±1.64 -2.09±0.73 0.8500 < 0.0001 0.0001

BLW_baseline (mm) 9.90±1.96 9.69±1.57 9.78±1.24 0.8517 0.8249 0.6068

BLW (mm) -1.00±0.58 -0.69±1.67 -3.74±0.75 1.0000 < 0.0001 < 0.0001

BLW* (mm) -0.97±0.34 -0.71±0.27 -3.74±0.22 1.0000 < 0.0001 < 0.0001 low-buccal bone thickness vs high-buccal bone thickness

sig. (p-value)

bVBL (mm) 0.6675 0.0294 0.7691

BLW_baseline (mm) 0.8063 0.2796 0.0772

BLW (mm) 0.0556 0.3902 0.0366

secondary predictor: socket type I

size 12 20 17 12 vs 20 12 vs 17 20 vs 17

bVBL (mm) -0.50±1.00 -0.60±1.39 -1.94±0.66 0.7016 0.0004 0.0023

BLW_baseline (mm) 10.33±1.97 10.10±1.33 9.29±1.45 0.7666 0.1149 0.1644

BLW (mm) -1.50±0.52 -1.30±0.77 -3.53±0.72 0.3233 < 0.0001 < 0.0001

BLW* (mm) -1.50±0.21 -1.30±0.16 3.53±0.18 1.0000 < 0.0001 < 0.0001 secondary predictor: socket type II/III

size 18 10 13 18 vs 10 18 vs 13 10 vs 13

bVBL (mm) -0.17±1.46 -0.50±1.89 -2.31±0.63 0.6445 < 0.0001 0.0058

BLW_baseline (mm) 10.00±2.04 9.50±1.49 9.92±1.19 0.6445 0.6099 0.3260

BLW (mm) -1.22±0.81 -0.20±1.72 -3.69±0.75 0.2847 < 0.0001 < 0.0001

BLW* (mm) -1.17±0.22 -0.32±0.29 -3.67±0.26 0.0790 < 0.0001 < 0.0001 socket type I vs socket type II/III

sig. (p-value)

bVBL (mm) 0.6169 0.8056 0.1760

BLW_baseline (mm) 0.7476 0.2172 0.3323

BLW (mm) 0.1151 0.0655 0.6806

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Accepted Article

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