J Clin Periodontol 2014; 41 (Suppl. 15): S23–S35 doi: 10.1111/jcpe.12191

Soft tissue wound healing at teeth, dental implants and the edentulous ridge when using barrier membranes, growth and differentiation factors and soft tissue substitutes

Fabio Vignoletti, Javier Nunez and Mariano Sanz Department of Periodontology, University Complutense of Madrid, Madrid, Spain

Vignoletti F, Nunez J, Sanz M. Soft tissue regeneration in the oral cavity: review of the current literature on scaffolds, cells and biologicals. J Clin Periodontol 2014; 41 (Suppl. 15): S23–S35. doi: 10.1111/jcpe.12191.

Abstract Aim: To review the biological processes of wound healing following periodontal and periimplant plastic surgery when different technologies are used in a) the coverage of root and implant dehiscences, b) the augmentation of keratinized tissue (KT) and c) the augmentation of soft tissue volume. Materials & Methods: An electronic search from The National Library of Medicine (MEDLINE-PubMed) was performed: English articles with research focus in oral soft tissue regeneration, providing histological outcomes, either from animal experimental studies or human biopsy material were included. Results: Barrier membranes, enamel matrix derivatives, growth factors, allogeneic and xenogeneic soft tissue substitutes have been used in soft tissue regeneration demonstrating different degrees of regeneration. In root coverage, these technologies were able to improve new attachment, although none has shown complete regeneration. In KT augmentation, tissue-engineered allogenic products and xenogeneic collagen matrixes demonstrated integration within the host connective tissue and promotion of keratinization. In soft tissue augmentation and periimplant plastic surgery there are no histological data currently available. Conclusions: Soft tissue substitues, growth differentiation factors demonstrated promising histological results in terms of soft tissue regeneration and keratinization, whereas there is a need for further studies to prove their added value in soft tissue augmentation.

Conflict of interest and source of funding statement This workshop was financially supported by an unrestricted educational grant from Geistlich AB, Switzerland to the European Federation of Periodontology. The authors of this manuscript filed detailed conflict of interest related to the topic of the workshop. Disclosures have included having received speaker’s fees, consulting fees and / or research grants from: Geistlich AG, the Osteology Foundation, Straumann AG, Organogenesis, Datum Dental and Nobelbiocare. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Key words: barrier membranes; biologicals; cell therapy; histology; keratinized tissue; mucogingival surgery; recession defect; root coverage; scaffolds; wound healing Accepted for publication 12 November 2013

Different regenerative approaches have been used in the oral cavity with the goal of reconstructing hard and soft tissues. A special focus has been made to regenerate the soft tissues in order to achieve a natural and aesthetically pleasant appearance. In fact, these soft tissue

S23

S24

Vignoletti et al.

regenerative approaches using different surgical techniques and regenerative technologies were collectively termed by the American Academy of Periodontology as Periodontal Plastic Surgery (AAP 1996). Although they were primarily aimed “to correct or eliminate anatomic, developmental or traumatic deformities in morphology, position and/or amount of gingiva surrounding the teeth,” the term was later enlarged to encompass soft tissue augmentation therapies in edentulous ridges and around dental implants. The biology of oral soft tissue wound healing after trauma and flap surgery has been fully reviewed and described in a different review on this issue (Sculean et al. 2014), but in general terms whenever a surgical muco-periosteal flap is raised over a denuded root the tooth – soft tissue interface may lead to different patterns of healing: 1 The most common outcome will be soft tissue repair by formation of a long junctional epithelium characterized by a thin epithelium interposed between the root surface and the gingival connective tissue. 2 In the most apical portion, it is also common the presence of repair by connective tissue attachment where collagen fibres adhere to the root surface. 3 The healing by regeneration will be characterized by de novo formation of cementum and a functionally oriented periodontal ligament, with the establishment of a short epithelial attachment conforming the gingival unit. The different biological outcome will depend not only on the surgical technique, but mostly on the biological environment where this healing takes place (availability of cell types, access to signalling molecules and nutrients, absence of bacterial contamination, etc.). Different technologies have been tested with the goal of improving the soft tissue response after mucogingival surgery, thus seeking for regeneration and reconstruction of the soft tissues. These technologies have varied from using barrier membranes to promote selective cell colonization of the wound, to the use of cells with intrinsic

capability for differentiation into connective tissue, to the use of growth or differentiation factors that may alter the microenvironment enhancing the soft tissue healing process, and more recently the use of scaffolds that favour cell in-growth during wound healing allowing for soft tissue build up. In contrast to periodontal wound healing, the healing of a mucoperiosteal flap over an implant surface will be represented by only one healing pattern (Berglundh et al. 2007): formation of a junctional or barrier epithelium in the marginal part of the peri-implant mucosa and connective tissue repair with the formation of a supracrestal dense connective tissue with collagen fibres oriented parallel to the implant surface in the portion coronal to the bone crest. Research on implant surgery has focused mainly on hard tissue regeneration developing several surgical techniques, technologies and surface treatment modification to enhance osseointegration and bone regeneration around implants, whereas very limited interest has been dedicated to soft tissue regeneration. It is therefore the objective of this review to evaluate the scientific evidence on the available technologies aimed to enhance soft tissue regeneration when used in combination with periodontal plastic surgical procedures. This review was focused on the biological mechanisms and histological outcomes achieved when these technologies are used: a) for root/implant coverage procedures in the treatment of localized gingival recessions (LGR); b) for keratinized tissue augmentation in areas where attached gingiva or mucosa is absent and, c) for soft tissue volume augmentation in edentulous ridges. Search strategy

We have used the electronic database from The National Library of Medicine in Washington, DC (MEDLINE-PubMed) as our main source of scientific information. The search was complemented manually using full-text reviews published between 1995 and 2013 and reference lists from every selected full-text article.

We have used the following search terms: “enamel matrix proteins” OR “enamel matrix derivative proteins” OR “Emdogain” OR “acellular dermal matrix” OR “dermal matrix allograft” OR “AlloDerm” OR “collagen matrix” OR “Mucograft” OR “xenograft” OR “human fibroblast-derived dermal substitute” OR “HF-DDS” OR “platelet” OR “platelet rich plasma (PRP)” OR “soft tissue substitute” OR “membrane” OR “barrier membrane” AND “gingival recession” OR “keratinized tissue” OR “keratinized gingiva” OR “attached gingiva” OR “attached mucosa” OR “keratinized mucosa” OR “recession” OR “mucogingival surgery” OR “root coverage” OR “coronally advanced flap” OR “CAF” OR “soft tissue augmentation” OR “vestibuloplasty” OR “ridge augmentation” OR “soft tissue correction” AND “histology.” Papers were selected when fulfilling the following criteria: Published in peer-reviewed journals and written in English. Main research focus was oral soft tissue regeneration. Providing histological outcomes, either from animal experimental studies or human biopsy material. Papers were excluded when the main focus was periodontal regeneration in angular bony defects or bone regeneration around implants. Results Soft tissue coverage of denuded roots or implant soft tissue defiencies

Localized gingival recession, defined as the exposure of the root surface due to the displacement of the gingival margin apical to the cementoenamel junction (Armitage 1999, Wennstrom 1996) is one of the most frequent indications of periodontal plastic surgery. It may affect single or multiple root surfaces and is frequently associated with aesthetic complaints, root hypersensitivity and difficulties to achieve optimal plaque control (Susin et al. 2004). The ultimate goal of root-coverage procedures is to reconstruct the soft tissues over the recession defect, with good texture and colour integration with the adjacent tissues, shallow

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

Oral soft tissue regeneration probing depths and absence of inflammation. This goal can be predictably achieved by the proper use of surgical techniques, basically pedicle flaps (laterally or coronally positioned) that cover the denuded root surface, with or without the concomitant use of an autograft (AG). Several systematic reviews have evaluated the efficacy of these procedures reporting percentages of complete root coverage ranging from between 35% and 97%, being the subepithelial connective tissue graft (SCTG) harvested from the patient’s palatal mucosa, the surgical approach that provided the best outcomes (Chambrone et al. 2010). To avoid this second surgical site, pedicle flaps, mainly the coronally advanced flap (CAF) has been advocated. Cairo et al. (2008) evaluated systematically the literature on its efficacy when used alone or in combination of other technologies aimed to enhance the soft tissue healing capability of the CAF procedure. They reported that both, the use of enamel matrix derivatives or the placement of a CTG when combined with CAF, enhanced the probability to obtain complete root coverage and keratinized tissue augmentation. The nature of the healing of this root coverage surgical procedures have been investigated in animal experimental studies and in isolated human case reports. Caffesse et al. (1984) surgically created localized gingival defects by removing an area of 5 9 7 mm of the buccal alveolar plate and exposing the root surfaces. These defects were left untreated for 2 months and then mucogingival lateral sliding flaps were performed to cover the recessions. Clinically, the authors reported a 50% of mean root coverage and histologically 40– 50% of this root coverage consisted on direct connective tissue attachment to the root surface, while the remaining 50–60% consisted on a junctional epithelial interphase. There was no evidence of cementum deposition and therefore, no soft tissue regeneration was attained with this flap procedure. There are controversial results on the possible influence of conditioning the root surface with citric acid on the histological outcomes of the CAF procedure. In non-human primates, Woodyard et al. (1984) dem-

onstrated the positive influence by demonstrating new cementum deposition coronal to the notch and connective tissue attachment to the tooth surface, while there was epithelium proliferation apical to the root notch with no evidence of new connective tissue attachment in the controls. Conversely, Gottlow et al. (1986) in a similar experimental model in beagle dogs, reported a similar amount of new attachment in both groups, demonstrating that citric acid conditioning did not enhance additional new attachment, although different from the previous experimental investigations the amount of new attachment extended a varying distance coronal to the presurgical level of the gingival margin in both groups. When evaluating the histological results of the different periodontal plastic procedures using human biopsy material from case reports, the results were very heterogeneous. Pasquinelli et al. (1995) reported the histological outcomes after treating a LGR with an autogenous graft. The histological outcomes demonstrated 2.6 mm of junctional epithelium, 4.4 mm of new connective tissue attachment and 4 mm of new bone formation although authors did not use a reference notch fort the histological evaluation. Bruno & Bowers (2000) reported that most of the clinical attachment achieved after a successful root coverage with CTG was due histologically to connective tissue adhesion, while Goldstein et al. (2001) demonstrated periodontal regeneration after CTG placement on exposed root surfaces. In contrast with these results, Harris (1999) evaluated the healing of a connective tissue AG in combination with a double pedicle flap on mandibular premolars. They reported that epithelial attachment was the most predominant form of interface after 30 months of healing. There was no new connective tissue attachment, new bone, nor cementum. These findings are consistent with a similar human case report (Majzoub et al. 2001) also describing histologically the interface after a root coverage surgical procedure using a CTG in combination with CAF. After 12 months of healing the most predominant healing was a long junctional epithelium very close

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

S25

to the bone crest. Only minimal cementum formation with inserting collagen fibres was observed. In summary, these histological findings indicate that the interface between the soft tissues and a previously exposed root surface following pedicle flaps, with or without the use of autogenous grafts is mostly represented by a long junctional epithelium. In order to enhance soft tissue regeneration and hence these histological outcomes, different technologies have been evaluated. Barrier membranes Their use is based on the knowledge obtained from periodontal regeneration studies and the principles of guided tissue regeneration (GTR), which demonstrated that the placement of a barrier membrane between the surgical flap and the root surface, allowed for selective cell repopulation of the wound and promoted periodontal regeneration with the formation of new cementum, new connective tissue attachment and new bone (Melcher 1976). Several authors (Tinti et al. 1992, Pini Prato et al. 1992) proposed to promote new attachment on denuded root surfaces using the same GTR principles. The results from experimental studies evaluating histological outcomes on the use of bioabsorbable and non-bioabsorbable membranes in conjunction with the CAF procedure for the treatment of LGR are summarized in Table 1. In general, there is a trend towards less apical migration of the junctional epithelium when a barrier membrane was used, although the differences as compared to control treatment (CAF alone) were minimal and never statistically significant. Different barrier membranes have been investigated, although there is only one study comparing bioabsorbable versus non-bioabsorbable barrier membranes. The authors compared two barrier membranes (bioabsorbable and ePTFE) with non-barrier controls (CAF with and without Scaling and Root Planning) reporting minimal differences in terms of epithelial down-growth, although a clear tendency towards a shorter epithelial attachment was observed in the two GTR groups (da Silva Pereira et al. 2000). In one study, two

8 weeks 4 months

Chronic

Acute dehiscense

Chronic

Chronic

3 beagle dogs

6 beagle dogs

4 non-human primates

7 beagle dogs

5 mongrel dogs

6 mongrel dogs

8 mongrel dogs

16 non-human primates 7 mongrel dogs

6 mongrel dogs

Gottlow et al. (1990)

Lundgren et al. (1995)

Weng et al. (1997)

Casati et al. (2000)

Da Silva Pereira (2000)

Lee et al. (2002) Hammarstrom et al. (1997) Sallum et al. (2004) Sallum et al. (2006)

Chronic No APF

Chronic

Chronic

Chronic

Acute dehiscense

Chronic

4, 16 weeks

3 months

3 months

4 months

6 weeks

3 months

3 months

1, 9, 14, 21, 28, 35 days 14, 21, 28, 42 days

Gottlow et al. (1986)

Chronic

Chronic

2 non-human primates 6 non-human primates

Healing period

Caffesse et al. (1984) Woodyard et al. (1984)

Defect

Animal model

Authors

Allogenic Soft tissue substitute

Emdogain

Barrier membrane Emdogain

Barrier membrane

Barrier membrane

Barrier membrane

Barrier membrane

Barrier membrane

–

0.05  00.7 0.04  0.05 0.06  0.08**

0.78  0.50  2.10  (40) 2.29  (40) CAF + ADMG

CAF‡ CAF + BM¶, ‡ CAF + EMD PGA CAF + EMD EMD + GTR CAF

CAF (no SRP)

CAF

CAF + ePTFE

CAF + PLA

CAF

CAF + PLA

CAF + CTG

0.92

0.32 0.44 0.46

2.2  0.8 (39.2) § 0.8  0.6 (13.7) § 6.45  1.84 (NA) 5.48  2.11 (NA) 0.11  0.13 (1.70) 0.87  0.54 (13.10) 0.1  0.11 (2) 0.00  0.00 (0) 0.18  0.09 (2) 0.07  0.08 (1) 1.10  1.38§ 2.45  1.74§ NA

1.3  0.2 (23.2) § 2.3  1.2 (39.6) § 2.97  0.54 (NA) 4.14  1.29 (NA) 1.96  0.81 (27.71) 3.05  0.97 (42.01) 0.96  0.60 (20) 0.88  0.56 (18) 2.52  1.52 (50) 2.20  1.10 (44) 1.08  0.56 1.22  0.70 NA CAF (Test, Guidor) CAF (Ctr, Vicryl) CAF + ePTFE

0.05  0.08**

NA

NA

NA

§

NA

0.85†, NA

4.50  1.70 (NA) 2.87  2.51 (NA) 1.19  0.53 (NA) 1.41  0.28 (NA) 2.73  1.21* (NA) § 0.95  0.60* (NA) § 0.92  1.05* (NA) § 0.90  0.59* (NA) 0.54  1.62 1.68  1.70 NA (65)

5.72  1.59 (NA) 4.98  2.48 (NA) 3.87  1.59 (61.00) 2.45  0.35 (36.80) 3.78  0.56 (76)§ 3.85  0.86 (78) § 1.97  1.02 (38) § 2.85  1.01 (55) 0.30  0.90 0.41  1.22 NA (75)

1.55

0.60  1.36

2.01  0.82 1.40  0.82 0.35  0.82

NA

NA

0.72 1.10 0.96

0.8  0.2§ 1.4  0.2 (33.3) 1.4  0.3 (30.2) NA

3.72  3.78  2.90  (55) 2.35  (42)

NA

0.4  0.5

10.4% of defect

NA NA 2.2% of defect

NA NA NA

0.00  0.00 (0)

0.57  0.50 (10)

0.16  0.24 (4)

0.13  0.28 (2)

0.54  0.50 (8.13)

0.60  1.12 (9.50)

NA

NA

NA

NA

0.40  0.03§

NA NA

NA

NA

Recession (%)

NA 0.4  0.6

NA

NA

Bone mm (%)

NA 2.2  1.2 (36.6) 2.2  1.2 (39.5) 3.4  0.2 (74.3)§ 1.7  0.2 (36.9)§ NA

NA

1.13§

CAF

NA

NA

Cementum formation mm (%)

4.78 (44)

Connective tissue mm (%)

CAF + ePTFE

CAF + Citric acid ‡ CAF ‡ CAF + Citric acid CAF

–

5.41 (56)

Epithelium mm (%)

Histometry

NA NA (29 5  12.2) NA (38.5  14.8) NA

LRF‡

Surgery

–

Device

Table 1. Results from histometric measurements from pre-clinical studies on root coverage in LGR. Mean values  SD in mm (%), negative values mean bone crest is apival to the notch. BM, Barrier Mambrane

S26 Vignoletti et al.

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

1 week, 1, 3 months Chronic 12 minipigs Vignoletti et al. 2011

*Bone area. †N-JE: distance from the apical edge of the root notch (N) to the most apical level of the junctional epithelium. ‡Values representing last healing period. §Statistically significant difference. ¶Bovine type I collagen membrane (Biomend Regular). **Connective tissue adhesion. Histometric measurement from the most coronal cementum to the most apical JE, i.e. connective tissue fibres not inserted into root cementum. ADMG, acellular dermal matrix graft; APF, apically positioned flap; BM, barrier membrane; CAF, coronally advanced flap; CM, collagen matrix; CTG, subepithelial connective tissue graft; EMD, enamel matrix derivative; e-PTFE, expanded polytetrafluorethylene; GTR, guided tissue regeneration; NA, not available; PGA, propylene glicol alginate; PLA, polylactic acid membrane; SRP, scaling and root planning.

NA NA 0.16  0.54 0.20  0.75 0.75  0.25 1.08  0.41 2.79  0.84 2.26  0.23 CAF‡ CAF + CM‡

0.12  0.16** 0.28  0.32**

2.2% of defect 10.4% of defect 0.35  0.49 0.61  0.55 2.22  0.44 2.27  0.42 0.47  0.44** 0.18  0.08** 1.79  0.46 1.21  0.35

Epithelium mm (%)

CAF + CTG CAF + ADMG

Allogenic Soft tissue substitute Xenogeneic soft tissue substitute 3 months Chronic 3 minipigs Nunez et al. 2009

Animal model Authors

Table 1. (continued)

Defect

Healing period

Device

Surgery

Histometry

Connective tissue mm (%)

Cementum formation mm (%)

Bone mm (%)

Recession (%)

Oral soft tissue regeneration

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

S27

bioabsorbable membranes were compared reporting significant differences in the amount of epithelial down-growth in favour of the test membrane (Guidor) (Lundgren et al. 2005). The results in terms of new cementum and new connective tissue attachment are heterogeneous. When a non-bioabsorbable membrane consisting on expanded polytetrafluoroethylene (e-PTFE) was compared with the CAF procedure to cover surgically created root dehiscences (Gottlow et al. 1990), differences in terms of new cementum formation with inserting collagen fibres were significant when compared with the non-barrier controls (74.3% versus 36.9% respectively). In contrast, Weng et al. (1998) compared the use of a barrier membrane versus the use of a CTG in combination with the CAF and the amount of new connective tissue attachment was very similar between the two groups (5.72 [SD 1.59] mm versus 4.98 [SD 2.48] mm respectively). This latter finding is consistent with results reported by Casati et al. (2000) in a similar investigation reporting 3.87 (SD 1.59) mm for the GTR-group and 2.45 (SD 0.35) mm for the control group (CAF alone). da Silva Pereira et al. (2000) demonstrated a clear tendency towards better histological outcomes for the GTR groups, which reported similar results irrespective of the type of barrier membrane, although differences were significant between the GTR groups and the CAF group. Similarly, Lee et al. (2002) observed statistically significant differences in terms of new connective tissue comparing the use of a bovine collagen membrane versus CAF alone, although in both groups the amount of new cementum formation was minimal. In these experimental studies, results on new bone formation have shown that this event was a common finding in both test and control groups, although there is a consistent tendency towards more new bone formation in the groups using a barrier membrane (see Table 1). Similar histological results have been reported in the few human case reports available in the scientific literature. New connective tissue attachment (3.66 mm) with new cementum (2.48 mm) and new bone (1.48 mm) were reported following

S28

Vignoletti et al.

GTR by Cortellini et al. (1993) on a case report where a LGR was treated with a CAF and a non-bioabsorbable membrane (ePTFE) and histologically evaluated 6 months after the removal of the membrane. In a similar case report, 8 months after the removal of the membrane reported 5.6 mm of new cementum and connective tissue attachment and 6.7 mm of newly formed bone (Parma-Benfenati & Tinti 1998). In summary, the histological results on the use of barrier membranes in conjunction with CAF surgical procedures indicate improved healing with increased amounts of new cementum and connective tissue attachment formation and concomitantly a reduced junctional epithelium, although results are very heterogeneous and differences rarely statistically significant. This may be due to the i) design of these experimental studies with minimal sample sizes, ii) the surgical protocol, that is, chronic versus acute surgically created defects and iii) difficulties in standardizing the histological measurements. These differences in wound healing, however, are most likely not relevant clinically, as demonstrated in different systematic reviews (Roccuzzo et al. 2002, Cairo et al. 2008) and this fact may be due to the clear anatomical limitations, since the available space between the flap/membrane and the root surface once the flap has been coronally repositioned over the exposed root surface is minimal, what limits the organization of the clot and the selective cell repopulation needed for soft tissue regeneration. Growth and differentiation factors Use of enamel matrix derivative than (EMD-Emdogainâ). Similar barrier membranes, the biological potential and clinical uses of EMD have been extensively studied in periodontal regeneration. The source of proteins is porcine, with a high degree of homology with human enamel proteins and their mechanism of action is biomimetic since this group of proteins have an important role in the embryonic root development and cementogenesis. Based on the evidence of its efficacy in periodontal regeneration studies, both experimental and clinical (Bosshardt & Sculean 2009), it seems reasonable

to study the effect of EMD in the treatment of recession defects in conjunction with the CAF procedure (Castellanos et al. 2006). Hammarstr€ om et al. (1997) were the first to apply EMD on a denuded root surface in surgically created dehiscence defects of 6 mm in monkeys. After 8 weeks of healing, the root surface showed regeneration with new acellular cementum, periodontal ligament and alveolar bone. This cementum was firmly attached to the underlying dentin surface and new collagen fibres were perpendicularly oriented and attached between the cementum and the newly formed alveolar bone. Histometrically, 60–80% of the apicocoronal extension of the cementum defect was regenerated due to EMD, with the apical extension of the epithelium usually ending at the coronal end of the new cementum. Based on these promising results, several experimental studies in animals and humans have evaluated the biological potential and the histological outcomes after using EMD to treat LGR defects. Sallum et al. (2004) evaluated the possible synergistic effect of EMD and GTR in root dehiscencetype defect in dogs. After 4 months of healing the histological outcomes in both groups were very similar in regards to the apical extension of the junctional epithelium, new cementum formation and the amount of new bone. The authors concluded that EMD in combination with GTR barriers did not provide additional benefits to the use of EMD alone. The first histological demonstration of true periodontal regeneration in humans was presented by Heijl (1997). A surgically created buccal dehiscence defect in a mandibular incisor was treated with EMD. After 4 months of healing the microscopic examination revealed a new periodontal ligament with inserting and functionally-oriented collagen fibres and an associated alveolar bone was also present. The new cementum covered 73% of the original defect. Regain of bone was 65% of the presurgical bone height. The possible added value of EMD to the CTG in the treatment of recession defects was studied histologically in humans by Carnio et al. (2002). They used

four teeth (two with Miller class II recession and the other two with Miller class III recession) that received CTG plus EMD. After 6 and 12 months of healing, the teeth and adjacent soft tissues were extracted and studied histologically. There were no differences between both recession defects (Miller class II and class III), nor between the 6 month and the 1 year specimens, all demonstrating a short junctional epithelium with the majority of the area covered by the CTG demonstrating connective tissue adhesion by connective tissue fibres running parallel to the root surface. No formation of new cementum and bone was observed. This study therefore demonstrated that the combination of EMD and CTG did not have a beneficial effect on the nature of the attachment achieved and did not promote regeneration. Contrastly, Rasperini et al. 2000 (HYPERLINK \l “_ENREF_55” \o “Rasperini, 2000 #5849” Rasperini et al.) in one tooth with Miller class III treated with CTG plus EMD observed new connective tissue attachment, new cementum and new bone in the notch area. McGuire & Cochran (2003) after 6 month of healing demonstrated new cellular cementum lining the treated root surface with islands of bone although periodontal dental ligament fibres were not inserting between the new cementum and the bone. These studies therefore demonstrated that the combination of EMD and CTG did not have a beneficial effect on the nature of the attachment achieved and did not promote regeneration. The biological potential of EMD as a differentiation and proliferation factor for mesenchymal cells and fibroblasts derived from the periodontal ligament has been demonstrated by in vitro studies (Hoang et al. 2000). EMD promotes the transformation of gingival fibroblasts to actively participate in new connective tissue attachment to root surfaces (Cattaneo et al. 2003). These effects of EMD on the connective tissue cells have been described in humans by Lafzi et al. (2007) who treated two contra-lateral recession defects, one with EMD application and the other without, together with the laterally positioned flap. After 10 days of healing biopsy specimens

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

Oral soft tissue regeneration were obtained from the dento-gingival region immediately above the alveolar crest. The non-EMD specimen contained non-active fibroblasts (flattened spindle-shaped form with peripherally located and crescent-like heterochromatin) and signs of apoptosis were frequent. In contrast, the fibroblasts on the EMD site shown clear signs of activity (rounded morphology with plump cytoplasm and euchromatic nuclei, containing numerous mitochondria and welldeveloped rough endoplasmic reticulum) and signs of apoptosis were rarely detected. Also the extracellular matrix was very different. In the EMD site, it was well organized and rich of collagen bundles, while in the non-EMD site the collagen fibres were sparse and not completely formed. The scarce human histological information on the use of EMD and CAF to treat recession defects is derived from case reports in hopeless teeth and Miller class IIIIV defects. These extreme cases are probably far from the expected clinical use and therefore are not good examples to demonstrate the biological potential of this biomimetic approach. Use of platelet rich plasma and other growth factors. Platelet rich plasma contains a plethora of substances, including growth factors and components that may have the potential to enhance soft tissue healing, since these factors, mainly the plateletderived growth factor (PDGF) are involved in the wound healing process by promoting angiogenesis, stimulating granulation tissue formation, enhancing initial epithelial migration or improving hemostasis (for review see Bashutski & Wang 2008). This potential biological activity has been advocated to promote regeneration when used together with root-coverage procedures, but unfortunately is limited histological data demonstrating this potential. One pre-clinical histological study compared CAF+CTG+PRP (test) versus the standard of care CAF+CTG after treating of surgically created and Miller Class I recession defects. After 45 days of healing, microscopic evaluation demonstrated that the test group showed more new attachment forma-

tion than the control group. When comparing the two procedures the residual recession was similar in this pre-clinical study (Suaid et al. 2008). Unfortunately, no human histological datum is available to corroborate these findings in humans. The use of recombinant plateletderived growth factors (rhPDGFBB) has been investigated clinically and histologically for its use in periodontal regeneration (Kaigler et al. 2011). McGuire et al. (2009a) reported histological outcomes of using beta-tricalcium phosphate (bTCP) saturated with recombinant human platelet-derived growth factor-BB (rhPDGF-BB) together with CAF, in comparison to CAF + CTG, 9 months after treating surgically created recessions. As part of a study segment from a randomized controlled clinical trial (RCT) comparing beta-tricalcium phosphate (bTCP) + 0.3 mg/ml recombinant human platelet-derived growth factor-BB (rhPDGF-BB) with a bioabsorbable collagen wound-healing dressing and a CAF to a subepithelial CTG in combination with a CAF (McGuire et al. 2009a,b), recession defects were created in six teeth, each requiring extraction for orthodontic therapy. These defects were treated with a CTG (control) or rhPDGF-BB + b-TCP + wound-healing dressing (test), plus CAF. Nine months after surgical correction, en bloc resections were obtained and examined histologically and with micro-computed tomography (CT). In the RCT, test and control treatments demonstrated clinically significant improvements from baseline through month 6, although statistically significant results favoured the CTG for recession depth reduction and root coverage. Conversely, in the histologic/micro-CT portion, all four sites treated with rhPDGFBB + b-TCP showed evidence of regeneration of cementum, periodontal ligament with inserting connective tissue fibres, and supporting alveolar bone, whereas neither CTGtreated site exhibited any signs of periodontal regeneration. In summary, even though the biological plausibility of using recombinant growth factors was demonstrated with histological outcomes, there is still insufficient evidence to support its clinical use.

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

S29

Use of soft tissue substitutes. Even though the use of autografts, mainly the CTG has shown the best efficacy in clinical trials for attaining complete root coverage (Chambrone et al. 2011), the main disadvantage of using autogenous tissue are mainly due to the harvesting procedure with its inherent increased patient’s morbidity (Farnoush 1978, Griffin et al. 2006). Furthermore, anatomical limitations may limit the quantity and quality of available grafting tissue. In order to overcome these limitations, alternative techniques and materials have been developed as an alternative to CTG. In 1980s, allogenic dermal substitutes were developed for the treatment of extensive burn wounds and a similar product, the acellular dermal matrix graft (ADMG) was developed for its use in periodontal plastic surgery. It is composed of extracellular matrix with collagen bundles and elastic fibres as the main components. Its intended mechanism of action is to serve as a three dimensional scaffold that allows the in-growth and repopulation of fibroblasts, blood vessels, and epithelium from surrounding tissues to develop into fully functional gingiva or keratinized mucosa. When clinically compared with CTG in the treatment of gingival recessions no statistically significant differences were observed in terms of percentage of root coverage and amount of keratinized tissue (KT) (Gapski et al. 2005). More recently, a similar concept using a xenogeneic collagen matrix (CM) of porcine origin has been introduced for the treatment of gingival recessions (Mucograftâ). The clinical results when compared to CTG are still preliminary and controversial, with similar outcomes in one study (Cardaropoli et al. 2012) and inferior in another (McGuire & Scheyer 2010), but it seems to provide an added value when combined to CAF alone (Jepsen et al. 2013). In contrast to ADMG and CM which merely consist of a connective tissue matrix, two therapies, the human fibroblast-derived dermal substitute (HF-DDS) and a human skin equivalent (BCT) have been introduced for soft tissue regeneration and both include a cellular component, aiming not only to serve as a scaffold, but to actively promote new connective

S30

Vignoletti et al.

tissue formation. The HF-DDS has been compared clinically to the CTG in combination with CAF with preliminary, although promising results (Wilson et al. 2005). There are few studies reporting histological outcomes when these technologies have been used for the treatment of recession defects. ADMG with the CAF procedure was investigated in dogs at 4, 8 and 12 weeks of healing demonstrating close integration and similar density of the grafted ADMG area with the host connective tissue. Indeed, in some areas, the borders between the allograft and native collagen were difficult to identify with standard H&E (Luczyszyn et al. 2007). Furthermore, ADMG with the CAF procedure, was compared with CAF + CTG in the treatment of surgically created Miller Class I reces~ez et al. 2009). sion defects (N un After 3 months of healing, the histological outcomes were similar in both groups, with a short epithelial attachment and newly formed cementum (2.22  0.44 mm for the CTG and 2.13  0.48 mm for the ADM) and supracrestal connective tissue fibres running perpendicularly to the root surface and inserted in the newly formed cementum. The main difference between both treatments was the gingival thickness, which was significantly higher in the CTG group (2.54  0.93 mm versus 1.45  0.15 mm) in spite of the fact that both grafts had a similar thickness (1 mm) when surgically placed. Similar results were observed by Sallum et al. (2006), although differences were not statistically significant. These experimental studies indicated that regeneration, consisting on the formation of new cementum, new bone and connective tissue attachment to a previously exposed root surface can occur with both CTG or ADMG. In humans, the reported ADMG histological outcomes when used for root coverage are very heterogeneous, mainly since these histological case reports are performed in teeth with hopeless prognosis and very deep recessions, which are clearly not the ideal model to test wound healing (Cummings et al. 2005). These human histological studies have reported in some studies areas of new cementum deposition on the apical portion of

the defect (Cummings et al. 2005), while in others no new cementum was identified nor connective tissue attachment occurred, with the connective tissue collagen fibres running mainly parallel to the root surface (Richardson & Maynard 2002). With the xenogeneic CM there is only one experimental study reporting histological outcomes. In the minipig model, CAF + CM was compared with CAF alone (control) for the treatment of surgically created Miller class I single recession defects (Vignoletti et al. 2011). The xenogeneic matrix was biocompatible with the surrounding connective tissue with presence of inflammatory infiltrate only limited to the supracrestal tissues in the vicinity of the root surface. After 1 month of healing, the CM could not be differentiated from the rest of the supracrestal connective tissue. At 3 months, the histometric results demonstrated a deeper extension of the junctional epithelium in the control group (CAF only), while the test group (CM + CAF) demonstrated an increased length of newly formed cementum. These differences were however not statistically significant. The histological outcomes of the use of CAF + CM in the treatment of recession defects were recently evaluated in a human case report (Camelo et al. 2012). In contrast with the observation from the pre-clinical study, the interface consisted mainly of a long junctional epithelial attachment and connective tissue adhesion without evidence of new cementum or bone formation. Nevertheless, clinical outcomes assessed on two posteriorly extracted premolars demonstrated in between 83% and 100% root coverage. In one pre-clinical study with dental implants (Schwartz et al. 2012), single Miller Class I-like recession-type defects were surgically created at each implant site. Subsequent to a chronification period of 8 weeks, all recession defects were randomly allocated to CAF+CM, CAF+CTG or CAF alone. The histological results for CAF+CM revealed a tissue implant interface similar to CAF+CTG or CAF alone consisting of epithelial attachment and connective tissue adhesion. The amount of coverage of the soft tissue deficiencies was similar for the

three procedures, however both experimental groups improved tissue thickness when compared with CAF alone. No human histological datum is presently available on the treatment of soft tissue deficiencies at implant sites. There is no histological data available on the use of human fibroblast-derived dermal substitute (HFDDS) or human skin equivalent (BCT) to determine the healing that such soft tissue substitutes may promote in combination with the CAF for the treatment of recession defects. Soft tissue augmentation procedures to increase the band of keratinized tissue

The gingiva is a specialized mucosa that includes the free and the attached gingiva and extends from the gingival margin to the mucogingival junction. When teeth are extracted, the gingival tissue is lost and the crestal ridge is covered with decreasing amounts of keratinized mucosa, which will serve as lining mucosa when dental implants are placed to restore the lost dentition. It is well demonstrated that the absence of keratinized gingiva may be compatible with the maintenance of periodontal health (Dorfman et al. 1980, Kennedy et al. 1985). There are clinical situations, however, that in presence of chronic inflammation or other traumatic events on a gingival margin without attached keratinized gingiva may lead to loss of attachment and increased recession (Hall 1982). Similar outcomes have been reported in relation to the presence of non-keratinized mucosa around dental implants (Chung et al. 2007, Zigdon & Machtei 2008) and therefore in these clinical situations a soft tissue augmentation surgical procedure may be recommended. The standard surgical technique is the use of an AG to augment the gingival dimensions and to create a band of keratinized tissue around teeth or implants. The efficacy of these techniques has been recently evaluated in a systematic review (Thoma et al. 2009a,b). From a total of 12 studies, 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

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

Oral soft tissue regeneration compared with no treatment. Both free gingival graft (FGG) and CTG have been used for this indication, with the FGG attaining less shrinkage, but poorer aesthetic results. Both techniques are, however, associated with significant patient morbidity due to the need to create a wound at the palatal donor site and mainly for this reason the use of soft tissue substitutes has also been advocated for this clinical indication. Several clinical studies evaluated the ADMG for its capability to increase the width of keratinized tissue around dental implants (Park & Wang 2006) reporting minimal increases of the band of keratinized tissue, mainly due to a significant contraction of the grafted area (58%). When ADMG has been compared with CTG the gain in keratinized mucosa was significantly higher in the CTG when compared with ADM (5.5 mm versus 2.5 mm) (Wei et al. 2000). The porcine CM has also been clinically tested for augmenting keratinized tissue around teeth and implants supporting fixed prosthetic restorations reporting a limited but consistent band of keratinized gingival/mucosal tissue. When compared with the CTG, CM attained comparable results (Sanz et al. 2009, Lorenzo et al. 2012). Tissue engineered cellular dermal grafts (HF-DDS) have also been investigated in clinical trials in comparison with autogenous soft tissue to increase the width of keratinized tissue reporting similar but limited amount of keratinized tissue gain, mainly due to high percentage of shrinkage (McGuire & Nunn 2005). Recently, an extracellular matrix membrane (Dynamatrix) has been processed from the submucosa of the small intestine of pigs retaining the natural composition of extracellular matrix molecules such as collagens (types I, III, IV and VI), glycosaminoglycans, glycoproteins, proteoglycans and growth factors and used as soft tissue substitute to enhance the keratinized tissue around teeth (Nevins et al. 2010a,b). A randomized, controlled split-mouth study was designed to evaluate the safety, feasibility and efficacy of this extracellular matrix membrane for gingival augmentation by comparing the tested membrane in combination

with the apically positioned flap (APF) as compared to the FGG. The FGG obtained approximately two times greater amount of KT at the end of the study. A tissue-engineered skin product (bilayered cell therapy, BCT) using allogenic cell therapy has been investigated to enhance keratinized tissue and wound healing around teeth. BCT is a living product, constructed of type I bovine collagen (extracted from bovine tendons and subsequently purified) and viable allogenic human fibroblasts and keratinocytes isolated from human foreskin. This device should not be considered a tissue-replacement graft, but a celldelivery therapy that encourages healing with the subject’s own tissues. McGuire et al. (2008) evaluated the safety and effectiveness of this tissue-engineered skin product and compared it to a FGG. Similarly to the HF-DDS, the test device demonstrated a significant increase of KT although results were inferior to the FGG. Indeed, the FGG generated statistically significantly (p < 0.001) more KT than the test device (BCT) (4.5 [SD 0.80] mm versus 2.4 [SD 1.02] mm). These results were further corroborated in a larger multicentre randomized controlled study on 96 patients comparing the BCT with the standard of care treatment FGG. Similarly, the FGG generated more keratinized gingiva than the BCT (4.57  1.0 mm versus 3.2  1.1 mm, respectively) (Mc Guire et al. 2011). There are only few studies reporting histological outcomes when these technologies have been used for augmentation of the keratinized gingiva/ mucosa. The healing sequence of ADMG when used in combination with the APF for increasing the amount of keratinized has been described histologically by Scarano et al. (2009). This material was filled with new vessels and fibroblasts in the first 2 weeks of healing. At 6 weeks the area was epithelialized and at 10 weeks the area was fully healed. When evaluated at 6 months the use of ADMG resulted in a healing similar to “scar” tissue with dense connective tissue, rich in elastic fibres (Wei et al. 2002). In attempt to improve the healing, autogenous gingival fibroblast were seeded into

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

S31

ADM and tested for this clinical indication in dogs by comparing ADMG alone and ADMG with seeded fibroblasts. The healing was similar in both groups and at 8 weeks ADMG in both groups was well vascularized and incorporated into the adjacent connective tissue. In both groups, the epithelium was para-keratinized and the connective tissue contained large amounts of elastin fibres. The addition of fibroblasts did not improve the histological outcomes (Novaes et al. 2007). A case report describing the histological outcomes after 3 months of healing of ADMG placed around implants reported minimal amounts of keratinization and presence of inflammatory cells suggesting that the soft tissue substitute did not participate in the healing, but rather sloughed away (Harris 2001). Two studies have evaluated histologically the healing of the CM Mucograft in combination with the APF (Test) in the minipig model in comparison with the APF alone (Control). One study evaluated at teeth the early healing phases reporting histological outcomes at 1 week, 1 month and 3 months (Vignoletti et al., submitted for publication), whereas a second experiment investigated at edentulous ridges the 6-month healing comparing two different prototypes of CM (Jung et al. 2011). The CM showed in the early healing phases that was well-tolerated and promoted epithelialization and new vessel formation. At 3 months, both areas, test and control, have completed epithelialization with a stratified keratinized oral epithelium. Similarly, Jung et al. (2011) observed active connective tissue formation overlaid by a normal epithelial keratinized layer. At 6 months all sites were completely healed, with signs of mature submucosal and epithelial tissues. Muscular fibres, new vessels and signs of innervation were observed. Neither inflammatory reactions nor residues of matrix material was found in sites treated with the two matrices at this later time-point. Histometrically when CM and a periosteal retention procedure was compared with CAF alone, the test group showed an increased thickness of the gingival unit and less crestal bone loss (Vignoletti et al., submitted for publication).

S32

Vignoletti et al.

In the study designed to evaluate the safety, feasibility and efficacy of the extracellular matrix membrane (Dynamatrix) the histological analysis of five pairs of test and control specimens reported very similar keratinized epithelium in absence of significant subjacent inflammatory infiltrate and with presence of rete pegs of similar shape and dimensions (Nevins et al. 2010a,b). No histological data are available on the use of human fibroblastderived dermal substitute (HF-DDS) to augment the keratinized gingiva/ mucosa. McGuire et al. (2008) evaluated the safety and effectiveness of the tissue-engineered skin product (BCM) in comparison to a FGG and took 6-month biopsies (test and control), which demonstrated a normal epithelial architecture by showing an ortho-keratinized stratified squamous epithelium typical of the attached gingiva. Test and control sites demonstrated tissues characteristic of alveolar and gingival mucosal phenotypes and connective tissue with normal architecture. Soft tissue augmentation procedures for ridge augmentation

Autogenous soft tissue grafting procedures, either the FGG or the subepithelial CTG, have been proposed to surgically correct localized alveolar defects, as pre-prosthetic site development and as ridge preservation procedures (Seibert 1983, Studer et al. 2000, Jung et al. 2004). The systematic review on soft tissue volume augmentation by Thoma et al. (2009a,b), reported only one comparative cohort study, in localized alveolar defects, where CTG provided greater volume gain than FGG. Most recently, research has focused on the use of soft tissue substitutes for this indication. ADMG grafts have been used and published in case series for this clinical indication. Authors reported minimal amounts of vertical gain and a significant amount of shrinkage (41.4%) (Batista et al. 2001), whereas no histological data is available on on the use of this soft tissue substitute for this clinical indication. Modified xenogeneic CM have been developed for this indication. The main modification is that these

matrixes contain collage tissue that is chemically cross-linked to offer mechanical stability with a favourable biological behaviour. In a pre-clinical experiment in mice, two prototype collagen matrix 1 (CM1) and 2 (CM2) composed of native porcine collagen I and III, being different by the degree of additional chemical cross-linking, were tested to study their tissue integration, biodegradation and new blood vessel formation (Thoma et al. 2011). CM1 had a denser network structure, whereas CM2 had a looser network structure. Results from the histological analysis demonstrated that the level of crosslinking had a significant influence on the amount of new blood vessel and connective tissue formation, and the degradation of the collagen network. Indeed, the less dense CM2 offered improved angiogenic patterns and enhanced connective tissue formation compared to the more dense network of CM1. This latter CM2 prototype was further tested in a pre-clinical investigation for soft tissue volume augmentation in alveolar ridge defects and compared to the CTG. The volumetric analysis demonstrated no significant differences between the two groups of treatment (Thoma et al. 2010). A subsequent study with this prototype investigated its histological soft tissue integration demonstrating at 84 days a good integration of the CM into the surrounding hard and soft tissues, with presence of blood vessels and a limited number of inflammatory cells (Thoma et al. 2011). These prototypes, however, have not yet been tested clinically. Conclusions

Different barrier membranes, growth and differentiation factors and soft tissue substitutes have been used to promote the healing and soft tissue regeneration. When used in the treatment of localized gingival recessions, the use of barrier membranes provided improved histological outcomes in terms of reduced epithelial attachment and increased new cementum, connective tissue attachment and bone. These improved histological outcomes were however of limited clinical significance and not predictably present in all studies. The use of EMD has also shown hetero-

geneous histological outcomes when used to treat LGR, but there is good evidence on its biological activity promoting new cementum formation and fibroblast proliferation. Similarly, the use of growth factors, such as PRP and RH-PDGF has shown improved histological outcomes, when compared with CTG, although the clinical efficacy seems to be inferior. The use of soft tissue substitutes, either allogenic or xenogeneic, has been tested histologically. ADM showed similar outcomes when compared with CTG. CM when compared with CAF alone, showed improved histological outcomes with a reduced epithelial attachment and increased new cementum. These preliminary histological results with the use of xenogeneic matrixes need to be supported with improved clinical outcomes from clinical trials. For the clinical indication of gingival/mucosal augmentation of keratinized tissue, the most promising results have come from the xenogeneic collagen matrixes and from tissue-engineered allogenic products. Both have resulted in good biocompatibility and a histological picture with absence of inflammation and promotion of keratinization. The use of these soft tissue substitutes must be supported with clinical outcomes from clinical trials, which are still limited, although promising. For the indication of soft tissue augmentation in edentulous ridges we do not have any biomaterial, scaffold or biological providing positive histological outcomes, being the CTG the only available tissue. Some xenogeneic prototypes have been tested in pre-clinical trials, but results are too preliminary. As of today, research on implant surgery has focused mainly on hard tissue regeneration and surface treatment modifications to enhance osseointegration and bone regeneration, whereas very limited interest has been dedicated to soft tissue regeneration around dental implants thus providing only few clinical trials and experimental studies.

References American Academy of Periodontology (1996) Consensus report on mucogingival therapy.Proceedings of the World Workshop in Periodontics. Annals of Periodontology 1, 702–706.

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

Oral soft tissue regeneration Armitage, G. C. (1999) Development of a classification system for periodontal diseases and conditions. Annals of Periodontology 4, 1–6. Bashutski, J. D. & Wang, H. L. (2008) Role of platelet-rich plasma in soft tissue root-coverage procedures: a review. Quintessence International 39, 473–483. Batista, E. L. Jr, Batista, F. C. & Novaes, A. B. Jr (2001) Management of soft tissue ridge deformities with acellular dermal matrix. Clinical approach and outcome after 6 months of treatment. Journal of Periodontology 72, 265–273. Berglundh, T., Abrahamsson, I., Welander, M., Lange, N. P. & Lindhe, J. (2007) Morphogenesis of the peri-implant mucosa: an experimental study in dogs. Clinical Oral Implants Research 18 (1), 1–8. Bosshardt, D. D. & Sculean, A. (2009) Does periodontal tissue regeneration really work? Periodontology 2000 51, 208–219. Bruno, J. F. & Bowers, G. M. (2000) Histology of a human biopsy section following the placement of a subepithelial connective tissue graft. International Journal of Periodontics & Restorative Dentistry 20, 225–231. Caffesse, R. G., Kon, S., Castelli, W. A. & Nasjleti, C. E. (1984) Revascularization following the lateral sliding flap procedure. Journal of Periodontology 55, 352–358. Cairo, F., Pagliaro, U. & Nieri, M. (2008) Treatment of gingival recession with coronally advanced flap procedures: a systematic review. Journal of Clinical Periodontology 35, 136–162. Camelo, M., Nevins, M., Nevins, M. L., Schupbach, P. & Kim, D. M. (2012) Treatment of gingival recession defects with xenogenic collagen matrix: a histologic report. International Journal of Periodontics & Restorative Dentistry 32, 167–173. Cardaropoli, D., Tamagnone, L., Roffredo, A. & Gaveglio, L. (2012) Treatment of gingival recession defects using coronally advanced flap with a porcine collagen matrix compared to coronally advanced flap with connective tissue graft: a randomized controlled clinical trial. Journal of Periodontology 83, 321–328. Carnio, J., Camargo, P. M., Kenney, E. B. & Schenk, R. K. (2002) Histological evaluation of 4 cases of root coverage following a connective tissue graft combined with an enamel matrix derivative preparation. Journal of Periodontology 73, 1534–1543. Casati, M. Z., Sallum, E. A., Caffesse, R. G., Nociti, F. H. Jr, Sallum, A. W. & Pereira, S. L. (2000) Guided tissue regeneration with a bioabsorbable polylactic acid membrane in gingival recessions. A histometric study in dogs. Journal of Periodontology 71, 238–248. Castellanos, A., de la Rosa, M., de la Garza, M. & Caffesse, R. G. (2006) Enamel matrix derivative and coronal flaps to cover marginal tissue recessions. Journal of Periodontology 77, 7–14. Cattaneo, V., Rota, C., Silvestri, M., Piacentini, C., Forlino, A., Gallanti, A., Rasperini, G. & Cetta, G. (2003) Effect of enamel matrix derivative on human periodontal fibroblasts: proliferation, morphology and root surface colonization. An in vitro study. Journal of Periodontal Research 38, 568–574. Chambrone, L., Sukekava, F., Araujo, M. G., Pustiglioni, F. E., Chambrone, L. A. & Lima, L. A. (2010) Root-coverage procedures for the treatment of localized recession-type defects: a Cochrane systematic review. Journal of Periodontology 81, 452–478. Chung, D. M., Oh, T. J., Lee, J., Misch, C. E. & Wang, H. L. (2007) Factors affecting late

implant bone loss: a retrospective analysis. International Journal of Oral and Maxillofacial Implants 22, 117–126. Cortellini, P., Clauser, C. & Prato, G. P. (1993) Histologic assessment of new attachment following the treatment of a human buccal recession by means of a guided tissue regeneration procedure. Journal of Periodontology 64, 387–391. Cummings, L. C., Kaldahl, W. B. & Allen, E. P. (2005) Histologic evaluation of autogenous connective tissue and acellular dermal matrix grafts in humans. Journal of Periodontology 76, 178–186. Dorfman, H. S., Kennedy, J. E. & Bird, W. C. (1980) Longitudinal evaluation of free autogenous gingival grafts. Journal of Clinical Periodontology 7, 316–324. Farnoush, A. (1978) Techniques for the protection and coverage of the donor sites in free soft tissue grafts. Journal of Periodontology 49, 403– 405. Gapski, R., Parks, C. A. & Wang, H. L. (2005) Acellular dermal matrix for mucogingival surgery: a meta-analysis. Journal of Periodontology 76, 1814–1822. Goldstein, M., Boyan, B. D., Cochran, D. L. & Schwartz, Z. (2001) Human histology of new attachment after root coverage using subepithelial connective tissue graft. Journal of Clinical Periodontology 28, 657–662. Gottlow, J., Karring, T. & Nyman, S. (1990) Guided tissue regeneration following treatment of recession-type defects in the monkey. Journal of Periodontology 61, 680–685. Gottlow, J., Nyman, S., Karring, T. & Lindhe, J. (1986) Treatment of localized gingival recessions with coronally displaced flaps and citric acid. An experimental study in the dog. Journal of Clinical Periodontology 13, 57–63. Griffin, T. J., Cheung, W. S., Zavras, A. I. & Damoulis, P. D. (2006) Postoperative complications following gingival augmentation procedures. Journal of Periodontology 77, 2070–2079. Hammarstrom, L. (1997a) Enamel matrix, cementum development and regeneration. Journal of Clinical Periodontology 24, 658–668. Hammarstrom, L. (1997b) The role of enamel matrix proteins in the development of cementum and periodontal tissues. Ciba Foundation Symposium 205, 246–255; discussion 255–260. Hammarstr€ om, L., Heijl, L. & Gestrelius, S. (1997) Periodontal regeneration in a buccal dehiscence model in monkeys after application of enamel matrix proteins. Journal of Clinical Periodontology 24, 669–677. Harris, R. J. (1999) Human histologic evaluation of root coverage obtained with a connective tissue with partial thickness double pedicle graft. A case report.. Journal of Periodontology 70, 813–821. Harris, R. J. (2001) Gingival augmentation with an acellular dermal matrix: human histologic evaluation of a case–placement of the graft on bone. International Journal of Periodontics & Restorative Dentistry 21, 69–75. Hoang, A. M., Oates, T. W. & Cochran, D. L. (2000) In vitro wound healing responses to enamel matrix derivative. Journal of Periodontology 71, 1270–1277. Jepsen, K., Jepsen, S., Zucchelli, G., Stefanini, M., de Sanctis, M., Baldini, N., Greven, B., Heinz, B., Wennstrom, J., Cassel, B., Vignoletti, F. & Sanz, M. (2013) Treatment of gingival recession defects with a coronally advanced flap and a xenogeneic collagen matrix: a multicenter randomized clinical trial. Journal of Clinical Periodontology 40, 82–89.

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

S33

Jung, R. E., Hurzeler, M. B., Thoma, D. S., Khraisat, A. & Hammerle, C. H. (2011) Local tolerance and efficiency of two prototype collagen matrices to increase the width of keratinized tissue. Journal of Clinical Periodontology 38, 173–179. Jung, R. E., Siegenthaler, D. W. & Hammerle, C. H. (2004) Postextraction tissue management: a soft tissue punch technique. International Journal of Periodontics & Restorative Dentistry 24, 545–553. Kaigler, D., Avila, G., Wisner-Lynch, L., Nevins, M. L., Nevins, M., Rasperini, G., Lynch, S. E. & Giannobile, W. V. (2011) Platelet-derived growth factor applications in periodontal and peri-implant bone regeneration. Expert Opinion on Biological Therapy 11, 375–385. Kennedy, J. E., Bird, W. C., Palcanis, K. G. & Dorfman, H. S. (1985) A longitudinal evaluation of varying widths of attached gingiva. Journal of Clinical Periodontology 12, 667–675. Lafzi, A., Farahani, R. M., Tubbs, R. S., Roushangar, L. & Shoja, M. M. (2007) Enamel matrix derivative Emdogain as an adjuvant for a laterally-positioned flap in the treatment of gingival recession: an electron microscopic appraisal. Folia Morphol (Warsz) 66, 100–103. Lee, E. J., Meraw, S. J., Oh, T. J., Giannobile, W. V. & Wang, H. L. (2002) Comparative histologic analysis of coronally advanced flap with and without collagen membrane for root coverage. Journal of Periodontology 73, 779–788. Lorenzo, R., Garcia, V., Orsini, M., Martin, C. & Sanz, M. (2012) Clinical efficacy of a xenogeneic collagen matrix in augmenting keratinized mucosa around implants: a randomized controlled prospective clinical trial. Clinical Oral Implants Research 23, 316–324. Lundgren, D., Laurell, L., Gottlow, J., Rylander, H., Mathisen, T., Nyman, S. & Rask, M. (1995) The influence of the design of two different bioresorbable barriers on the results of guided tissue regeneration therapy. An intraindividual comparative study in the monkey. Journal of Periodontology 66, 605–612. Majzoub, Z., Landi, L., Grusovin, M. G. & Cordioli, G. (2001) Histology of connective tissue graft. A case report. Journal of Periodontology 72, 1607–1615. McGuire, M. K. & Cochran, D. L. (2003) Evaluation of human recession defects treated with coronally advanced flaps and either enamel matrix derivative or connective tissue. Part 2: histological evaluation. Journal of Periodontology 74, 1126–1135. McGuire, M. K. & Nunn, M. E. (2005) Evaluation of the safety and efficacy of periodontal applications of a living tissue-engineered human fibroblast-derived dermal substitute. I. Comparison to the gingival autograft: a randomized controlled pilot study. Journal of Periodontology 76, 867–880. McGuire, M. K. & Scheyer, E. T. (2010) Xenogeneic collagen matrix with coronally advanced flap compared to connective tissue with coronally advanced flap for the treatment of dehiscence-type recession defects. Journal of Periodontology 81, 1108–1117. McGuire, M. K., Scheyer, E. T., Nunn, M. E. & Lavin, P. T. (2008) A pilot study to evaluate a tissue-engineered bilayered cell therapy as an alternative to tissue from the palate. Journal of Periodontology 79, 1847–1856. McGuire, M. K., Scheyer, E. T. & Schupbach, P. (2009a) Growth factor-mediated treatment of recession defects: a randomized controlled trial and histologic and microcomputed tomography

S34

Vignoletti et al.

examination. Journal of Periodontology 80, 550–564. McGuire, M. K., Scheyer, T., Nevins, M. & Schupbach, P. (2009b) Evaluation of human recession defects treated with coronally advanced flaps and either purified recombinant human platelet-derived growth factor-BB with beta tricalcium phosphate or connective tissue: a histologic and microcomputed tomographic examination. International Journal of Periodontics & Restorative Dentistry 29, 7–21. Melcher, A. H. (1976) On the repair potential of periodontal tissues. Journal of Periodontology 47, 256–260. Nevins, M., Nevins, M. L., Camelo, M., Camelo, J. M. B., Schupbach, P. & Kim, D. M. (2010b) The clinical efficacy of DynaMatrix extracellular membrane in augmenting keratinized tissue. International Journal of Periodontics & Restorative Dentistry 30, 151–161. Nevins, M., Nevins, M. L., Camelo, M., Camelo, J. M., Schupbach, P. & Kim, D. M. (2010a) The clinical efficacy of DynaMatrix extracellular membrane in augmenting keratinized tissue. International Journal of Periodontics & Restorative Dentistry 30, 151–161. Novaes, A. B. Jr, Marchesan, J. T., Macedo, G. O. & Palioto, D. B. (2007) Effect of in vitro gingival fibroblast seeding on the in vivo incorporation of acellular dermal matrix allografts in dogs. Journal of Periodontology 78, 296–303. ~ez, J., Caffesse, R., Vignoletti, F., Guerra, F., N un San Roman, F. & Sanz, M. (2009) Clinical and histological evaluation of an acellular dermal matrix allograft in combination with the coronally advanced flap in the treatment of Miller class I recession defects: an experimental study in the mini-pig. Journal of Clinical Periodontology 36, 523–531. Park, S. H. & Wang, H. L. (2006) Management of localized buccal dehiscence defect with allografts and acellular dermal matrix. International Journal of Periodontics & Restorative Dentistry 26, 589–595. Parma-Benfenati, S. & Tinti, C. (1998) Histologic evaluation of new attachment utilizing a titanium-reinforced barrier membrane in a mucogingival recession defect. A case report. Journal of Periodontology 69, 834–839. Pasquinelli, K. L. (1995) The histology of new attachment utilizing a thick autogenous soft tissue graft in an area of deep recession: a case report. International Journal of Periodontics & Restorative Dentistry 15, 248–257. Pini Prato, G., Tinti, C., Vincenzi, G., Magnani, C., Cortellini, P. & Clauser, C. (1992) Guided tissue regeneration versus mucogingival surgery in the treatment of human buccal gingival recession. Journal of Periodontology 63, 919– 928. Rasperini, G., Silvestri, M., Schenk, R. K. & Nevins, M. L. (2000) Clinical and histologic evaluation of human gingival recession treated with a subepithelial connective tissue graft and enamel matrix derivative (Emdogain): a case report. International Journal of Periodontics & Restorative Dentistry 20, 269–275. Richardson, C. R. & Maynard, J. G. (2002) Acellular dermal graft: a human histologic case report. International Journal of Periodontics & Restorative Dentistry 22, 21–29. Roccuzzo, M., Bunino, M., Needleman, I. & Sanz, M. (2002) Periodontal plastic surgery for treatment of localized gingival recessions: a systematic review. Journal of Clinical Periodontology 29 (Suppl. 3), 178–194; discussion 195–176.

Sallum, E. A., Nogueira-Filho, G. R., Casati, M. Z., Pimentel, S. P., Saldanha, J. B. & Nociti, F. H. Jr (2006) Coronally positioned flap with or without acellular dermal matrix graft in gingival recessions: a histometric study. American Journal of Dentistry 19, 128–132. Sallum, E. A., Pimentel, S. P., Saldanha, J. B., Nogueira-Filho, G. R., Casati, M. Z., Nociti, F. H. & Sallum, A. W. (2004) Enamel matrix derivative and guided tissue regeneration in the treatment of dehiscence-type defects: a histomorphometric study in dogs. Journal of Periodontology 75, 1357–1363. Sanz, M., Lorenzo, R., Aranda, J., Martin, C. & Orsini, M. (2009) Clinical evaluation of a new collagen matrix (Mucograft prototype) to enhance the width of keratinized tissue in patients with fixed prosthetic restorations: a randomized prospective clinical trial. Journal of Clinical Periodontology ???, ???–???. doi:10.1111/ j.1600-051X.2009.01460.x. Scarano, A., Barros, R. R. M., Iezzi, G., Piattelli, A. & Novaes, A. B. (2009) Acellular dermal matrix graft for gingival augmentation: a preliminary clinical, histologic, and ultrastructural evaluation. Journal of Periodontology 80, 253– 259. Schwarz, F., Mihatovic, I., Shirakata, Y., Becker, J., Bosshardt, D. & Sculean, A. (2012) Treatment of soft tissue recessions at titanium implants using a resorbable collagen matrix: a pilot study. Clinical Oral Implants Research. doi: 10.1111/clr.12042 [Epub ahead of print]. Sculean, A., Gruber, R. & Bosshardt, D. D. (2014) Soft tissue wound healing around teeth and dental implants. Journal of Clinical Periodontology 41(Suppl. 15), xxx–xxx. Seibert, J. S. (1983) Reconstruction of deformed, partially edentulous ridges, using full thickness onlay grafts. Part II. Prosthetic/periodontal interrelationships. Compendium of Continuing Education in Dentistry 4, 549–562. Seibert, J. S. & Cohen, D. W. (1987) Periodontal considerations in preparation for fixed and removable prosthodontics. Dental Clinics of North America 31, 529–555. da Silva Pereira, S. L., Sallum, A. W., Casati, M. Z., Caffesse, R. G., Weng, D., Nociti, F. H. Jr & Sallum, E. A. (2000) Comparison of bioabsorbable and non-resorbable membranes in the treatment of dehiscence-type defects. A histomorphometric study in dogs. Journal of Periodontology 71, 1306–1314. Studer, S. P., Lehner, C., Bucher, A. & Scharer, P. (2000) Soft tissue correction of a singletooth pontic space: a comparative quantitative volume assessment. Journal of Prosthetic Dentistry 83, 402–411. Susin, C., Dalla Vecchia, C. F., Oppermann, R. V., Haugejorden, O. & Albandar, J. M. (2004) Periodontal attachment loss in an urban population of Brazilian adults: effect of demographic, behavioral, and environmental risk indicators. Journal of Periodontology 75, 1033– 1041. Thoma, D. S., Benic, G. I., Zwahlen, M., H€ ammerle, C. H. F. & Jung, R. E. (2009b) A systematic review assessing soft tissue augmentation techniques. Clinical Oral Implants Research 20 (Suppl. 4), 146–165. Thoma, D. S., Benic, G. I., Zwahlen, M., Hammerle, C. H. & Jung, R. E. (2009a) A systematic review assessing soft tissue augmentation techniques. Clinical Oral Implants Research 20 (Suppl. 4), 146–165. Thoma, D. S., Hammerle, C. H., Cochran, D. L., Jones, A. A., Gorlach, C., Uebersax, L., Ma-

thes, S., Graf-Hausner, U. & Jung, R. E. (2011) Soft tissue volume augmentation by the use of collagen-based matrices in the dog mandible – a histological analysis. Journal of Clinical Periodontology 38, 1063–1070. Thoma, D. S., Jung, R. E., Schneider, D., Cochran, D. L., Ender, A., Jones, A. A., G€ orlach, C., Uebersax, L., Graf-Hausner, U. & H€ ammerle, C. H. F. (2010) Soft tissue volume augmentation by the use of collagen-based matrices: a volumetric analysis. Journal of Clinical Periodontology 37, 659–666. Thoma, D. S., Villar, C. C., Cochran, D. L., Hammerle, C. H. & Jung, R. E. (2012) Tissue integration of collagen-based matrices: an experimental study in mice. Clinical Oral Implants Research 23, 1333–1339. Tinti, C., Vincenzi, G., Cortellini, P., Pini Prato, G. & Clauser, C. (1992) Guided tissue regeneration in the treatment of human facial recession. A 12-case report. Journal of Periodontology 63, 554–560. Vignoletti, F., Nunez, J., Discepoli, N., De Sanctis, F., Caffesse, R., Munoz, F., Lopez, M. & Sanz, M. (2011) Clinical and histological healing of a new collagen matrix in combination with the coronally advanced flap for the treatment of Miller class-I recession defects: an experimental study in the minipig. Journal of Clinical Periodontology 38, 847–855. Vignoletti, F., Nunez, J., Discepoli, N., Caffesse, R., Munoz, F. & Sanz, M. (2014) Healing of a xenogeneic collagen matrix used to enhance the width of keratinized tissue. A pilot histologic study in the minipig. (Submitted for publication). Wei, P. C., Laurell, L., Geivelis, M., Lingen, M. W. & Maddalozzo, D. (2000) Acellular dermal matrix allografts to achieve increased attached gingiva. Part 1. A clinical study. Journal of Periodontology 71, 1297–1305. Wei, P.-C., Laurell, L., Lingen, M. W. & Geivelis, M. (2002) Acellular dermal matrix allografts to achieve increased attached gingiva. Part 2. A histological comparative study. Journal of Periodontology 73, 257–265. Weng, D., Hurzeler, M. B., Quinones, C. R., Pechstadt, B., Mota, L. & Caffesse, R. G. (1998) Healing patterns in recession defects treated with ePTFE membranes and with free connective tissue grafts. A histologic and histometric study in the beagle dog. Journal of Clinical Periodontology 25, 238–245. Wennstrom, J. L. (1996) Mucogingival therapy. Annals of Periodontology 1, 671–701. Woodyard, S. G., Snyder, A. J., Henley, G. & O’Neal, R. B. (1984) A histometric evaluation of the effect of citric acid preparation upon healing of coronally positioned flaps in nonhuman primates. Journal of Periodontology 55, 203– 212. Zigdon, H. & Machtei, E. E. (2008) The dimensions of keratinized mucosa around implants affect clinical and immunological parameters. Clinical Oral Implants Research 19, 387–392.

Address: Mariano Sanz Department of Periodontology Faculty of Odontology University Complutense of Madrid Plaza Ram on y Cajal s/n 28040 Madrid Spain E-mail: [email protected]

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

Oral soft tissue regeneration

Clinical Relevance

Scientific rationale for the study: The evaluation of the histological outcomes of different technologies used in periodontal and periimplant plastic surgery is a prerequisite for identifying therapeutic protocols that can be tested in clinical research and if proven valid,

become part of our current soft tissue regenerative procedures. Principle findings: Soft tissue substitutes and biological products have shown promising histological outcomes in root coverage and gingival augmentation procedures. Cell therapy is a field of great interest, although there are no data from preclinical studies to confirm its biologi-

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

S35

cal potential. No biomaterial is still available for soft tissue volume augmentation. Practical implications: The use of soft tissue substitutes, growth and differiantiation factors and cell therapy may become in the future an alternative to autogenous tissue grafts when aiming to enhance soft tissue regeneration.