Fabio Vignoletti Javier Nu~ nez Francesco de Sanctis Monica Lopez Raul Caffesse Mariano Sanz

Authors’ affiliations: Fabio Vignoletti, Javier Nu~ nez, Francesco de Sanctis, Raul Caffesse, Mariano Sanz, Department of Periodontology, Faculty of Dentistry, Complutense University of Madrid, Madrid, Spain Monica Lopez, School of Veterinary, University of Santiago de Compostela, Santiago de Compostela, Spain Corresponding author: Prof. Mariano Sanz Department of Periodontology, Faculty of Dentistry Complutense University of Madrid Plaza Ram on y Cajal 28040 Madrid Spain Tel.: +34 913942021 Fax: +34 913941901 e-mail: [email protected]

Healing of a xenogeneic collagen matrix for keratinized tissue augmentation

Key words: collagen matrix, histology, keratinized tissue, soft tissue augmentation, soft tissue

substitute, xenogeneic graft Abstract Aim: To evaluate histologically the healing of a xenogeneic collagen matrix (CM) used to augment the width of keratinized tissue around teeth. Materials and methods: The gingiva on the buccal aspect of mandibular and maxillary premolars was surgically excised on 12 minipigs. After 1 month of plaque accumulation, the resulting defects were randomly treated by a periosteal retention procedure (control site) or by placing a collagen matrix after an apically repositioned flap (CM) (test site). Clinical and histological outcomes were evaluated at 1, 4 and at 12 weeks. Results: Clinically, no gain of keratinized tissue was noted in either group. Histometrically, the thickness of the gingival unit was significantly higher in the test group at 1 month, although these differences were not significant at 3 months. There was a tendency in the test group towards less bone resorption (0.7 mm) and apical displacement (0.5 mm) of the gingival margin at 3 months. Conclusions: The tested CM demonstrated uneventful healing, being resorbed within the surrounding tissues in absence of significant inflammation. When compared with periosteal retention alone, the CM group attained similar clinical and histological outcomes.

Date: Accepted 3 June 2014 To cite this article: Vignoletti F, Nu~nez J, de Sanctis F, Lopez M, Caffesse R, Sanz M. Healing of a xenogeneic collagen matrix for keratinized tissue augmentation. Clin. Oral Impl. Res. 26, 2015, 545–552 doi: 10.1111/clr.12441

The gingiva is a specialized mucosa covered with keratin or parakeratin that includes the free and the attached gingiva and extends from the gingival margin to the mucogingival junction. The clinical concept of the need of a certain dimension of KG to maintain the stability of the soft tissue lining and to preserve periodontal health has evolved in the last 30 years. While in the 70s, the prevailing idea was that a minimum of 2 mm of KG was needed to maintain gingival health (Lang & Loe 1972), this concept changed when results of clinical and experimental investigations clearly showed that the absence of attached keratinized tissue was compatible with the maintenance of periodontal health and tissue stability, provided there was no significant gingival inflammation as a result of adequate plaque control (Dorfman et al. 1982; Wennstrom & Lindhe 1983). In spite of this evidence, there are clinical conditions where a plaque-free environment cannot be assured, and the presence of chronic inflammation in areas with minimal amount of keratinized tissue may lead to attachment loss and

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

to soft tissue recession (Ericsson & Lindhe 1984) (Hangorsky & Bissada 1980; de Trey & Bernimoulin 1980). A 5-year prospective case series study (Valderhaug & Birkeland 1976) showed that sites lacking keratinized tissue had a significantly higher rate of gingival inflammation, attachment loss and gingival recession. In these clinical situations, a mucogingival surgical procedure to increase the width and thickness of the attached gingiva may be indicated. Similarly, the stability of the tissues around dental implants in sites with lack of keratinized mucosa is a matter of controversy. Although there are prospective studies reporting stable peri-implant marginal bone levels independent of the amount of keratinized mucosa (Adell 1985), there are other studies, mainly retrospective case series (Chung et al. 2007), which have reported significantly higher amounts of plaque, mucosal inflammation and recession in sites with a lack of keratinized mucosa, as well as a significant negative correlation between the width and thickness of the peri-implant mucosa and the

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occurrence of mucosal recession (Zigdon & Machtei 2008). Moreover, one experimental study in monkeys reported that sites lacking keratinized mucosa around implants developed long-term chronic inflammation and bone loss when left to accumulate plaque (Warrer et al. 1995). Several surgical techniques have been used to augment the gingival tissue dimension. Among these, the free gingival graft (FGG) is the most frequently used and studied (Sullivan & Atkins 1968). The efficacy of these techniques has been recently evaluated in a systematic review (Thoma et al. 2009): from a total of 12 studies included, the use of the apically positioned flap plus the application of an autogenous graft (APF-AG) resulted in a statistically significant weighted mean difference of 4.49 mm compared with contralateral sites given with the APF alone. Experimental animal studies have demonstrated that these transplanted tissues retain their original structure and specificity leading to keratinization (Karring et al. 1971). The use of epithelialized grafts, although highly effective, often results in compromised aesthetic outcomes and hence, free connective grafts have been proposed providing enhanced aesthetics (Edel 1975), although often resulting in a higher percentage of shrinkage during healing (Roccuzzo et al. 2002). In patients with fixed partial prosthesis, where abutments had minimal amount of keratinized tissue (1 mm), the use of connective tissue grafts (CTGs) resulted in significant gingival augmentation, which favoured plaque control, reduced gingival inflammation and attachment loss, when compared with equivalent nongrafted sites (Orsini et al. 2004). Both surgical procedures are, however, associated with patient morbidity due to the need of harvesting an autograft from the palate and hence, the use of soft tissue substitutes of allogenic or xenogeneic origin has been tested (Thoma et al. 2009). A xenogenic collagen matrix (CM) of porcine origin has been recently investigated in the treatment of recession defects around teeth (McGuire & Scheyer 2010; Jepsen et al. 2013). It has also been used to augment keratinized tissue around teeth and implants supporting fixed prosthetic restorations, reporting comparable results to those achieved with autogenous CTGs (Sanz et al. 2009) (Lorenzo et al. 2012). Similar outcomes were also reported when compared with free gingival grafts combined with vestibuloplasty procedures (Schmitt et al. 2013). Although the histological outcomes of this xenogeneic collagen matrix were reported when used in combination with the coronally advanced flap for the treatment of recession

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defects (Vignoletti et al. 2011), there is limited information on the healing of this soft tissue substitute when sutured in a nonsubmerged environment, together with the periosteal retention procedure for enhancing the width of keratinized tissue (Jung et al. 2011). It is therefore, the objective of this preclinical investigation to evaluate histo-morphometrically the healing of this CM when used for keratinized tissue augmentation.

Material and methods Experimental design

This study was performed according to the modified arrive guidelines for preclinical experimental investigations (Vignoletti & Abrahamsson 2012) and designed as a randomized controlled experimental study employing 12, 24-month old, female Gottingen minipigs. Animals

The 35–40 kg minipigs were kept at the Veterinary Teaching Hospital Rof Codina in Lugo, Spain, in a purpose-designed centre for large experimental animals. The Regional Ethics Committee for Animal Research at the University of Lugo approved the protocol. The animals were kept on a soft diet and subject to oral hygiene by mechanical cleaning once every 3 weeks during the experimental study, being always 1 week before each surgical procedure. The animals were divided into three groups to allow the evaluation of three healing periods, at 1, 4 and 12 weeks after treatment. Each group included four minipigs that provided four study sites each, two test and two controls. Study device

The xenogeneic collagen matrix of porcine origin (Mucograftâ, Geistlich Pharma AG [Wolhusen, Switzerland]) is a class III medical device according to the Medical Device Directive 93/42 (Medical Device Directive REF 30773.1, approved by the FDA for use in the US). This three-dimensional matrix consists of two functional structures: a thin compact layer consisting of collagen fibres in a wide dense porous layer where the collagen fibres are loosely arranged. This scaffold, mostly spongy, provides a space that favours the formation of a blood clot and the ingrowth of tissue from adjacent sites.

general anaesthesia using a mixture of isoflurane (IsobaVet, Intervet, Madrid, Spain) 2 l/h and oxygen with a mechanical respirator. The animals were previously sedated with a cocktail containing 80 lg/kg of Medetomidine (Domtor, Pfizer, Madrid, Spain), 20 mg/kg of Butorphanol (Torbugesics, Fort Dodge, Gerona, Spain) and 1 mg/kg of Atropine Sulphate (Atropinas, Instituto Farmaceutico FAS, Burgos, Spain), then intubated, anaesthetized and monitored with continuous electrocardiography throughout the surgical procedure. The experimental and control defects were created by eliminating the buccal gingiva on the mandibular and maxillary PI or PIII (baseline one), as previously reported (Wennstrom et al. 1981). In brief, two vertical releasing incisions separated 10 mm from one another extending beyond the mucogingival junction, were performed at the mesio-buccal and disto-buccal aspects of the experimental sites and muco-periosteal full-thickness flaps were increased exposing the buccal alveolar bone, which was left to heal spontaneously once the flap was excised (Fig. 1a,b). One month after creating the defect (baseline 2), the treatment surgeries were carried out. Under similar anaesthesia, an intrasulcular incision was made to raise a partial-thickness flap creating a periosteal bed free of any muscle attachment, measuring 12 9 10 mm, approximately. The resulting flap was excised at the base of the newly created vestibule (Fig. 1c). A notch was then prepared with a round diamond bur in the root surface at the level of the bone crest (Fig. 1c). At this time, randomization was disclosed using a computer-generated list. Defects were assigned to either control sites, having the periosteal retention vestibulopasty alone (V) or test sites with the same surgical procedure combined with the placement of the CM (V + CM). The CM was trimmed and secured to the recipient bed with a combination of t-mattress and loop resorbable sutures (Vicryl, 4.0) (Fig. 1d). Clinical evaluation

Before the experimental surgeries and prior to euthanasia, the width of keratinized tissue was measured from the gingival margin to the mucogingival line with a NC15 periodontal probe (PCP-UNC 15; Hu-Friedy Manufacturing Co., Chicago, IL, USA). Histological processing

Interventions

All surgical procedures were performed under sterile conditions in an operating room, and

At the assigned times, the animals were sedated and euthanized through an overdose of sodium pentobarbital (Dolethal©, Vetoquinol,

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Vignoletti et al  Histological healing of a xenogeneic collagen matrix

(a)

(b)

(c)

(d)

Fig. 2. Cross section of the buccal dento-gingival region representing test and control groups after 1 week of healing. (a). Test. Very thin discontinuous epithelium, composed of just a few layers of cells. (b) Detail of (a). The connective tissue presented a dense inflammatory infiltrate, rich in polymorphonuclear leucocytes and abundant blood vessels. Dense network of collagen fibres identifying the structure of the implanted matrix could be observed upon the bone crest (dotted box). (c). Control group. Very thin but continuous epithelium starting to migrate apically. (d) detail of (c). Inflammatory infiltrate and abundant angiogenesis. Except for the detection of the implanted collagen matrix, test and control specimens showed similar characteristics. Haematoxylin and eosin (HE) stain.

(a)

(b)

(c)

(d)

Fig. 3. Cross section of the buccal dento-gingival region representing test and control groups after 1 month of healing. Both structures show improved tissue healing conditions (a). Test group. Mature oral epithelium with thin and deep rete pegs and a dense connective tissue. (b) Detail of (a). Infiltrated connective tissue underneath the sulcular epithelium. (c) Control group. Oral epithelium with wide and shallow rete pegs formation and healthy connective tissue structure. (d) Detail of (c). Dense inflammatory infiltrate underneath a thin junctional epithelium within the notch area. Haematoxylin and eosin (HE) stain.

abundant blood vessels could be detected. The alveolar crest showed evident osteoclastic resorption (Fig. 2a,c). Except for the

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presence of the implanted collagen matrix, test and control specimens showed similar histological characteristics.

At the end of 1 month, the improved tissue healing was evident in all specimens (Fig. 3a–d). In the test sites, the oral

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Vignoletti et al  Histological healing of a xenogeneic collagen matrix

(a)

(b)

(c)

(d)

Fig. 2. Cross section of the buccal dento-gingival region representing test and control groups after 1 week of healing. (a). Test. Very thin discontinuous epithelium, composed of just a few layers of cells. (b) Detail of (a). The connective tissue presented a dense inflammatory infiltrate, rich in polymorphonuclear leucocytes and abundant blood vessels. Dense network of collagen fibres identifying the structure of the implanted matrix could be observed upon the bone crest (dotted box). (c). Control group. Very thin but continuous epithelium starting to migrate apically. (d) detail of (c). Inflammatory infiltrate and abundant angiogenesis. Except for the detection of the implanted collagen matrix, test and control specimens showed similar characteristics. Haematoxylin and eosin (HE) stain.

(a)

(b)

(c)

(d)

Fig. 3. Cross section of the buccal dento-gingival region representing test and control groups after 1 month of healing. Both structures show improved tissue healing conditions (a). Test group. Mature oral epithelium with thin and deep rete pegs and a dense connective tissue. (b) Detail of (a). Infiltrated connective tissue underneath the sulcular epithelium. (c) Control group. Oral epithelium with wide and shallow rete pegs formation and healthy connective tissue structure. (d) Detail of (c). Dense inflammatory infiltrate underneath a thin junctional epithelium within the notch area. Haematoxylin and eosin (HE) stain.

abundant blood vessels could be detected. The alveolar crest showed evident osteoclastic resorption (Fig. 2a,c). Except for the

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presence of the implanted collagen matrix, test and control specimens showed similar histological characteristics.

At the end of 1 month, the improved tissue healing was evident in all specimens (Fig. 3a–d). In the test sites, the oral

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Vignoletti et al  Histological healing of a xenogeneic collagen matrix

epithelium showed evident keratinization with rete pegs penetrating deep into the connective tissue (Fig. 3a,b). The supracrestal connective tissues evidenced a fibrous structure, rich in fibroblasts and blood vessels, with presence of sparse inflammatory cells, densely concentrated beneath the sulcular epithelium (Fig. 3b). Remnants of the collagen matrix could still be detected in close vicinity to areas of active crestal bone resorption. In the control specimens, the marginal oral epithelium depicted wide and shallow rete pegs without evident keratinization, although a thin band of parakeratinization could be identified in some isolated areas The connective tissue compartment showed similar characteristics to the test sites, although with a higher degree of inflammation and less vascularization (Fig. 3c,d).

(a)

At 3 months, both test and control sections demonstrated full epithelial tissue maturation (Fig. 4a–d). In the test group, the oral epithelium depicted normal rete peg formation with thin and deep epithelial projections spreading into the connective tissue (Fig. 4a). The band of keratinization was evident throughout the oral epithelium with a clear demarcation with the nonkeratinized epithelium at the muco-gingival junction (Fig. 4b). The connective tissue was rich in vessels and cells, mostly fibroblasts spreading within the collagen structure. In the control areas, full keratinization had not been achieved, and the rete pegs were wider and shallower (Fig. 4c,d). In both treatment groups, the junctional epithelia had migrated downwards almost to the level of the notch, with presence of an evident inflammatory infiltrate

(b)

subjacent to the epithelium. This infiltrated area appeared to be larger in the control in comparison with the test specimens. Histometrical evaluation

The results are depicted in Tables 1 and 2. At 1 week, only partial analysis could be performed due to the lack of integrity on many of the specimens. The vertical dimension of the supracrestal soft tissues (G height Bc) measured from the most coronal bone crest (Bc) and the gingival margin (G) was similar between test and control groups throughout the study. The position of the gingival margin (G height aN), however, was located more coronally in the test specimens when compared with the control (0.79 [SD 0.70] vs. 0.23 [SD 0.46] mm, respectively), although these differences were

(d)

(c)

Fig. 4. Cross section of the buccal dento-gingival region representing test and control groups after 3 month of healing. Both structures show healthy tissues besides a manifest dense inflammatory infiltrate limited to the connective tissue underneath the sulcular and junctional epithelia. (a). Test group. Evident band of keratinization throughout the oral epithelium with a clear delimitation of the muco-gingival junction. (b) Detail of (a). Evident epithelial tissue maturation with normal rete peg formation with thin and deep epithelial projections and a well-represented keratinized tissue layer. (c) Control group. Thickened oral epithelium with wide and shallow rete pegs formation and healthy connective tissue structure. Thin sulcular and junctional epithelia reaching the notch. (d) Detail of (c). Not fully developed keratinization of the oral epithelium. Haematoxylin and eosin (HE) stain. Original magnification 209 (b) Original magnification 2009.

Table 1. Mean (SD) histometric measurements of the buccal bone resorption (bone loss), gingival height from the apical notch (G Heigth aN) and from the bone crest (G Heigth Bc) and gingival thickness at the bone crest level (G Thickness Bc) and at the apical notch level (G Thickness aN) in mm Bone loss

G Heigth aN

G Heigth Bc

G thickness Bc

G thickness aN

Healing period

Test

Control

Test

Control

Test

Control

Test

Control

Test

Control

1 week 1 month 3 months

0.98 (0.23) 0.97 (0.04) 1.16 (0.15)

1.37 (0.91) 1.01 (0.38) 1.79 (0.87)

0.93 (0.29) 1.52 (0.60) 0.79 (0.73)

0.84 (0.58) 0.80 (0.46) 0.23 (0.51)

1.85 (0.08) 2.58 (0.80) 1.90 (0.91)

2.11 (0.24) 2.05 (0.39) 2.08 (0.46)

1.87 (0.51) 2.28 (0.71) 1.06 (0.50)

1.69 (0.45) 1.32 (0.11) NA

1.17 (0.61) 1.89 (0.18) 1.16 (0.56)

1.06 (0.29) 1.22 (0.33) 1.19 (0.37)

G, gingival; Bc, bone crest; aN, apical notch. NA, Not available for measurement. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

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0.16 (0.05) 0.23 (0.03) 2.98 (1.29) 1.30 (0.78)

Discussion

K, keratin layer; OE, oral epithelium. 1, 2, 3 represents 0.5, 1, 1.5 mm apical to the gingival margin, respectively.

Min

0.10 (0.03) 0.08 (0.07) 0.17 (0.08) 0.71 (0.07)

Max

not statistically significant. The amount of bone loss was less in the test compared with the control group at 3 months (1.16 [SD 0.15] vs. 1.79 [SD 0.87] mm, respectively), although these differences were not statistically significant. The thickness of the gingiva measured at the level of the bone crest and of the apical notch was greater in the test group when compared with the control group at 1 month although these differences were not statistically significant. These differences disappeared towards the end of the study. A longer extension of the keratin layer was observed in the test group, both at 1 and 3 months although these differences were not statistically significant. Similar results were observed when the thickness of the oral epithelium was measured.

0.09 (0.09) 0.08 (0.04) 0.18 (0.09) 0.33 (0.08) 0.12 (0.09) 0.09 (0.04)

Min Max Min

0.27 (0.07) 0.22 (0.06)

Max Min

0.16 (0.11) 0.07 (0.02) 0.24 (0.20) 0.18 (0.06)

Max Min

0.11 (0.06) 0.08 (0.03) 0.34 (0.14) 0.20 (0.04)

Control

Max Min

0.25 (0.13) 0.08 (0.02)

Test Test

Control Test Healing period

1 month 3 months

2.38 (0.95) 0.86 (0.81)

Control Test Control

OE thickness 3 OE thickness 2 OE thickness 1 K length

Table 2. Mean (SD) histometric measurements of the keratin layer length and thickness of the oral epithelium at 0.5, 1 and 1.5 mm apical from the gingival margin in mm

Max

Vignoletti et al  Histological healing of a xenogeneic collagen matrix

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The present study aimed to investigate the histological healing of a xenogeneic CM used as a soft tissue substitute to augment the width of keratinized gingiva around teeth in the minipig model. The mechanism of action of this highly porous collagen matrix was to serve as a scaffold for soft tissue incorporation from the adjacent gingival tissues, thus promoting the ingrowth of fibroblasts and blood vessels and the epithelialization with keratinized gingiva. When a prototype of this CM was tested invitro, it was reported an in-growth of primary human fibroblasts, resulting in an increased expression of extracellular matrix proteins such as collagen type I and fibronectin (Mathes et al. 2010), the promotion of vascular ingrowth and fibrous connective tissue proliferation in the presence of minimal inflammation. The 3-month clinical results demonstrated similar outcomes in terms of increased width of keratinized tissue in both test and control groups. This KT augmentation was minimal and considered clinically not significant in both treatments groups. This outcome is not consistent with a preclinical investigation comparing the APF alone with the APF in combination with two prototypes of CMs, one of them identical to the commercial product used in this investigation (Jung et al. 2011). Although the authors reported similar outcomes between the two test and control groups, a significant increase in the width of the keratinized tissue amounting to approximately 5 mm, was observed. These differences can be explained by the different

surgical protocols utilized. In the present investigation, the surgical excision of the entire zone of the gingiva healed after 1 month of spontaneous plaque accumulation with a small band of keratinized tissue around the experimental teeth, which was then augmented in the second surgery. In the study by Jung et al. (2011), edentulous ridges sites with a pre-existing wide band of keratinized tissue of approximately 3.5–4 mm were treated with either the APF or the APF plus the CM prototypes. A significant increase in the width of keratinized tissue with the APF alone has also been reported in humans (Fagan 1975), although an earlier experimental study reported that this outcome was unpredictable (Karring et al. 1975). Recently, two controlled prospective clinical trials evaluated this CM when used to increase the band of keratinized tissue around prosthetic abutments, and reported similar results when compared with those obtained when placing a connective tissue graft. Both clinical trials reported similar results with a consistent increase in the width of keratinized tissue ranging between 2.5 and 3 mm (Sanz et al. 2009; Lorenzo et al. 2012). The histological behaviour of this CM when placed to heal in a nonsubmerged environment demonstrated that it is incorporated within the surrounding connective tissue. At 1 week, the matrix could be easily identified in a highly inflamed and vascularized connective tissue. At 1 month, only remnants of the matrix could be identified close to areas of crestal bone resorption whereas inflammation was mainly limited to an area underneath the sulcular and junctional epithelia. At 3 months, the matrix was no longer identifiable and a dense fibre-rich connective tissue was observed. Inflammation was confined to an area underneath the sulcular and junctional epithelia. Such inflammation was present at both test and control and is compatible with the healing after an experimental surgery in this animal model under the plaque control conditions of this study. Similar histological observations were reported after 6 months of nonsubmerged healing of a similar CM, demonstrating full integration into the surrounding tissues without signs of inflammation (Jung et al. 2011). The results from the histometric analysis indicate that there were no significant differences between both treatment modalities. These results showed, however, that the use of the CM resulted in reduced bone loss and less mucosal recession. It may be speculated that the CM promotes early stabilization of

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Vignoletti et al  Histological healing of a xenogeneic collagen matrix

the coagulum, thus enhancing the reparative process during wound healing, what may minimize the bone resorption that results in absence of primary intention healing. This coagulum stabilizer effect was also reported in a similar experimental study conducted by our research group when this soft tissue substitute was used in combination with the coronally positioned flap for the treatment of localized gingival recessions (Vignoletti et al. 2011). In summary, these results suggest that the CM may play a protective role, acting as a surgical dressing or a bandage when connective tissues remain exposed. The difference in thickness of the gingiva when comparing the test and control groups was notable at 1 month, however at 3 months of healing, this difference was no longer observed. This reduction in thickness from 1 to 3 months in the test group may be in part associated with shrinkage of the tissues once the soft tissue substitute fully resorbs when used in a nonsubmerged environment. This finding is consistent with the shrinkage associated with the use of this matrix reported in clinical studies, amounting to approximately 60% of the original dimension (Lorenzo et al. 2012). The evaluation of the oral epithelium demonstrated a tendency towards deeper rete pegs

and larger extensions of the keratin layer in the test group, what may translate a more mature healing condition. It is speculated that besides the coagulum stabilizer effect, the collagen matrix may serve as a scaffold to accelerate the migration of epithelial cells from the surrounding tissues and thus promote a faster healing process. Indeed, results from a recent human study evaluating the healing of 6-mm punch biopsies harvested from the palate and treated with the CM, demonstrated accelerated wound healing during the first week and provided better colour match to surrounding tissue, as compared to the untreated control (Thoma et al. 2012). Although bearing in mind these clinically favourable results, the differences reported were not statistically significant and the clinical value of such small differences has to be questioned. It may be argued that this lack of statistical significance is due to the low sample size of this study; however, when using experimental animals and in accordance with the arrive guidelines, there is a general recommendation to reduce and limit the number of animals to use. In conclusion, within the limitations of this preclinical experimental study, the tested CM when used as a soft tissue substitute in combination with the periosteal

retention procedure attained similar clinical and histological outcomes when compared with the control procedure alone.

Acknowledgements: The authors gratefully acknowledge the help of Dr Fernado Munoz (School of Veterinary, University of Santiago de Compostela, Santiago de Compostela, Spain) for the assistance during all surgical procedures and also wish to express appreciation for the diligent support rendered by Christoph G€ orlach (Director Preclinical and Clinical R&D (PCRD), Geistlich Biomaterials) and Lorenz Uebersax (Group Manager Preclinical and Clinical R&D (PCRD), Geistlich Biomaterials) from Geistlich Pharma AG, in monitoring the study. The authors declare that they have no conflict of interests. This study was partially supported by a grant from Geistlich Pharma AG Geistlich Biomaterials. We highly appreciate the collaboration of Dr Nicola Discepoli as a participating surgeon in the experimental surgeries and to Dr Fernando Mun˜oz for the veterinary services and histological processing and evaluation.

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Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Clinical measurements of Keratinized tissue width measured as the distance from the gingival margin to the mucogingival line (mean [SD] mm).

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Healing of a xenogeneic collagen matrix for keratinized tissue augmentation.

To evaluate histologically the healing of a xenogeneic collagen matrix (CM) used to augment the width of keratinized tissue around teeth...
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