Sculean_et_al-2014-Journal_of_Clinical_Periodontology (1).pdf

Soft tissue wound healingaround teeth and dental implantsSculean A, Gruber R, Bosshardt DD. Soft tissue wound healing around teeth anddental implants. J Clin Periodontol 2014; 41 (Suppl. 15): S6S22. doi: 10.1111/jcpe.12206.

AbstractAim: To provide an overview on the biology and soft tissue wound healingaround teeth and dental implants.Material and Methods: This narrative review focuses on cell biology and histol-ogy of soft tissue wounds around natural teeth and dental implants.Results and conclusions: The available data indicate that:

(a) Oral wounds follow a similar pattern.(b) The tissue specificities of the gingival, alveolar and palatal mucosa appear

to be innately and not necessarily functionally determined.(c) The granulation tissue originating from the periodontal ligament or from

connective tissue originally covered by keratinized epithelium has thepotential to induce keratinization. However, it also appears that deep pal-atal connective tissue may not have the same potential to induce keratini-zation as the palatal connective tissue originating from an immediatelysubepithelial area.

(d) Epithelial healing following non-surgical and surgical periodontal therapyappears to be completed after a period of 714 days. Structural integrityof a maturing wound between a denuded root surface and a soft tissueflap is achieved at approximately 14-days post-surgery.

(e) The formation of the biological width and maturation of the barrierfunction around transmucosal implants requires 68 weeks of healing.

(f) The established peri-implant soft connective tissue resembles a scar tissuein composition, fibre orientation, and vasculature.

(g) The peri-implant junctional epithelium may reach a greater final lengthunder certain conditions such as implants placed into fresh extractionsockets versus conventional implant procedures in healed sites.

Anton Sculean1, Reinhard Gruber1,2

and Dieter D. Bosshardt1,3

1Department of Periodontology, School of

Dental Medicine, University of Bern, Bern,

Switzerland; 2Laboratory of Oral Cell Biology,

School of Dental Medicine, University of

Bern, Bern, Switzerland; 3Robert K. Schenk

Laboratory of Oral Histology, School of

Dental Medicine, University of Bern, Bern,

Switzerland

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Key words: alveolar mucosa; dental

implants; gingiva; non-surgical periodontal

therapy; periodontal surgery; soft tissue

grafting; wound healing

Accepted for publication 12 November 2013

The last decades have improved ourunderstanding of the mechanisms oforal wound healing and how thisknowledge translates into todays

clinical treatment concepts. This nar-rative review aims to provide anoverview on the cellular aspects ofsoft tissue healing including the clas-

sical stages of wound repair and theimplication for oral wound healing,emphasizing the role of TGF-b.

Wound healing in the oral cavityis not only restricted to healing fol-lowing accidental trauma or surgery,but it also encompasses the biologicalevents following a variety of patho-logical conditions such as cancer andinfections (Gurtner et al. 2008).

Wound healing does not alwaysresult in a restitutio ad integrum, butit may end up with a scar tissue.This is not only true for the classical

Conflict of interest and source of funding statement

Anton Sculean has received grants from: Straumann, ITI, Geistlich and OsteologyFoundation and speakers fees from: Straumann, Geistlich and Osteology Founda-tion.Dieter Bosshardt has received grants from: Straumann, ITI, Geistlich and Osteol-ogy Foundation and speakers fees from Osteology Foundation.Reinhard Gruber has received grants from: Straumann, ITI, Geistlich and Osteol-ogy Foundation and speakers fees from Osteology Foundation.

2014 John Wiley & Sons A/S. Published by John Wiley & Sons LtdS6

J Clin Periodontol 2014; 41 (Suppl. 15): S6S22 doi: 10.1111/jcpe.12206

skin injury, but also relevant for thehealing following periodontal sur-gery. Embryonic and foetal wounds,however, have a much higher poten-tial to regenerate.

The aim of this review was toprovide an overview on the mostimportant biologic events duringhealing of soft tissue wounds in theoral cavity as related to teeth anddental implants. This review will,however, not address the biologicalevents during healing of hard tissuesassociated with periodontitis or peri-implantitis.

Cell biology of soft tissue healing

Periodontal repair can be achieved,periodontal regeneration remains achallenge (Bosshardt & Sculean2009). The overall goal is to providepatients with a less invasive, fast,save and predictable therapy that re-establishes a healthy periodontal sit-uation to maintain the teeth. Toachieve this goal, surgical techniqueshave been refined and biomaterialsand growth factors are applied tosupport the natural process ofwound healing and repair/regenera-tion (Susin & Wikesjo 2013, Boss-hardt 2008, Sculean et al. 2008,Kaigler et al. 2011, Stavropoulos &Wikesjo 2012). Advances in peri-odontal therapy were usually basedon the deep understanding of thefundamental cellular processes ofperiodontal regeneration and repair basically, to provide optimal localconditions, thus enhancing theregeneration process. In the follow-ing a brief summary of the basic bio-logical aspects of soft tissue healingis provided.

Most information on soft tissuehealing comes from studies with epi-dermal also termed cutaneous orskin wound healing. Much less isknown about the cellular aspects ofsoft tissue healing in the oral mucosa(see below). Therefore, it is currentlyassumed that wound healing repre-sents a conserved process while thecellular processes of periodontal andperi-implant soft tissue repair resem-ble, at least partly, the healing ofskin wounds (Wikesjo & Selvig 1999,Polimeni et al. 2006). This assump-tion might, however, provoke criti-cism, for example, because scarformation occurs less in the oral cav-ity than in skin wounds (see below).

On the other hand, the examplesprovided here may offer the scientificbasis to determine, if the respectivemechanisms also account responsiblefor periodontal repair or even regen-eration. Today, sophisticated pre-clinical models are available, forexample allowing to understandingthe involvement of a particular celltype in wound healing, or trackingone particular cell type in the ongo-ing process of wound healing. Noveldevelopments in analytical method-ology such as genomics and proteo-mics have opened the door for abetter understanding of the molecu-lar regulation of the complex woundhealing process.

Summarizing the current knowl-edge on wound healing is beyondthe scope of this review, here refer-ring to the existing excellent litera-ture (Martin 1997, Singer & Clark1999, Gurtner et al. 2008, Shaw &Martin 2009, Nauta et al. 2011).Instead, we highlight some aspectsthat appear to be relevant for peri-odontal and peri-implant soft tissuehealing as it should help to under-stand the clinical consequences ofcurrent periodontal treatment con-cepts. Moreover, the cellular basicsof general wound healing shouldprovide inspiration to furtherimprove or develop treatment strate-gies for soft tissue repair and regen-eration as relates to teeth and dentalimplants. The next sections willfocus on the following aspects:

Provide a brief summary of theclassical stages of wound repair

Compare healing of oral soft tis-sues with classical skin woundhealing

Summarize the role of innate andadaptive immunity in soft tissuewound healing

Provide our current understand-ing on the genetic basis of woundhealing exemplified by TGF-band associated genes.

Brief summary of the classical stages of

wound repair

The haemostatic phase is initiated bytissue injury including defects afterperiodontal surgery (Dickinson et al.2013). The defect site is sealed rapidlyoff by the forming blood clot thatbasically originated from blood coag-

ulation. Extravasated platelets areactivated and aggregate together withother blood-derived cells such as neu-trophils and red blood cells in theblood clot, also termed blood coagu-lum. The main component of theextracellular matrix is the newlyformed fibrin meshwork that alsoincludes other proteins for cell adhe-sion such as fibronection and vitrone-tin (Clark et al. 2004, Reheman et al.2005). This conglomerate of cells andthe fibrin-rich matrix is frequentlytermed provisional extracellularmatrix as it will be later replaced bythe granulation tissue. The formationof the blood clot is also the kick-startfor the recruiting of inflammatorycells into the defect site.

The inflammatory phase parallelsthe haemostatic phase. Neutrophilsare attracted by chemokines, thecomplement system, and by peptidesreleased during cleavage of fibrino-gen (Kolaczkowska & Kubes 2013).Extravasation and migration of cellsin the surrounding tissue is con-trolled by endothelial cells (Shi &Pamer 2011, Kolaczkowska & Kubes2013). Neutrophils and monocytesappear at defect sites within one and24 h respectively. Neutrophils cleanthe wound site as they kill invadingbacteria and release proteases beforethey are removed via phagocytosis.Macrophages are a heterogeneouspopulation, as they can be involvedin inflammation (M1-macrophages)but also switch to an anti-inflamma-tory M2a phenotype (Mantovaniet al. 2013, Novak & Koh 2013). Ingeneral, resolution of inflammationis a controlled process involving lipidmediators (Serhan et al. 2008). Theprimarily catabolic inflammatoryprocess is transient, but crucial forthe following steps of the anabolicphase of new tissue formation.

The phase of new tissue formationis initiated by the formation of thegranulation tissue, a morphologicterm that reflects the highly vascular-ized tissue made of fibroblast and anextracellular matrix. The transition ofthe catabolic to the anabolic phaserequires activation of a complex pro-cess involving at least three cell types:endothelial cells, fibroblasts and theepithelial cells. The cellular originsare partially resolved: (i) Endothelialcells, required for the formation ofnew capillaries, can be derived fromendothelial cells of the original blood

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

Oral soft tissue healing S7

vessels but also from the circulatingendothelial progenitors (Potente et al.2011). (ii) Fibroblasts can be derivedfrom the connective tissue in thewound edges, from monocyte-derivedfibrocytes (Grieb et al. 2011, Reilkoffet al. 2011), from vessel-derived peri-cytes (Grieb et al. 2011) and possiblyalso by a process termed epithelial-mesenchymal transition (Weber et al.2012). (iii) Epithelial cells originatefrom the keratinocytes at the woundedges, but at least in the skin, stemcells of the hair follicle can contributeto the re-epithelialization (Blanpain &Fuchs 2009, Cordeiro & Jacinto2013). A proportion of the fibroblastsachieve a phenotype that resemblessmooth muscle cells (Tomasek et al.2013). These myofibroblasts can drawthe wound edges together and arethus critical components of woundhealing (Klingberg et al. 2013).

The long-term remodelling phasethat ends up with scar tissue startswith the resolution phase. Most of themyofibroblasts, fibroblasts, endothe-lial cells and macrophages undergoapoptosis, leaving the collagen-richextracellular matrix containing only afew cells. The signals leading to cellgroup suicide are not clear (Hinz2007). Also, we have to accept thatbesides the aesthetic drawbacks of ascar tissue, also the biomechanicalcapacity is less than it was beforeinjury. Scar tissue formation, alsotermed fibrosis, is the main pathologi-cal factor of a variety of pathologieslinked with inflammation. Fibrosis,seen in the liver, lung, heart, kidneyand skin, is a significant global diseaseburden. The pathophysiology offibrosis, however, remains an enigma(Meneghin & Hogaboam 2007). Thus,great effort is paid on the control ofscarring, basically to avoid scar for-mation (Wynn & Ramalingam 2012).In periodontal wound healing, sub-epithelial connective tissue grafts canend up with a dense tissue, which isconsidered to provide long-term sta-bility of the area (Thoma et al. 2011,Santagata et al. 2012). Therefore, it isreasonable to suggest that a denseand stable soft tissue can bear clinicaladvantage.

Compare oral with classical skin wound

healing

First, it is necessary to summarizestudies comparing oral with skin

wound healing. For example, mouseoral wound healing can be fasterthan skin wounds, at least followingan experimental mucosa incision ortongue excision respectively (Sciubbaet al. 1978, Szpaderska et al. 2003b).Also in pig models, injured oralmucosa showed reduced scar forma-tion compared to skin incisions(Wong et al. 2009). Moreover, pigoral mucosal wounds showed similarmolecular composition and clinicaland histological scar scores tohuman oral mucosal wounds (Wonget al. 2009). However, in mousemodels with punch biopsies in thescalp and palate, oral healing wasslower than dermal repair, likelybecause of persisting inflammatorystimuli (Nooh & Graves 2003). Inthe latter study, epithelial and con-nective tissue bridging of excisionalwounds was delayed compared todermal wounds and also neutrophilswere more abundant in the oral thanin the skin wounds (Nooh & Graves2003). Interestingly, the inflamma-tory cytokine IL-1 plays a role inoral wound healing, but not in der-mal wound healing, likely becauseIL-1 is necessary to control thedefence mechanisms against com-mensal bacteria in the oral cavity(Graves et al. 2001). Taken together,the majority of studies indicate thatoral mucosa likely heals faster andwith less scar tissue than do skinwounds. The underlying mechanismsremain a matter of speculation butmay include aspects unique to theoral cavity such as the presence ofsaliva with its well-recognized bio-logical activities (Zelles et al. 1995,Schapher et al. 2011). For example,the cutaneous and bone healing ofsublingual sialadenectomized micewas slower than that of sham-oper-ated controls (Bodner et al. 1991a,b). Importantly, palatal wound heal-ing is delayed in desalivated rats andlarger wounds are more sensitive todesalivation than smaller wounds(Bodner et al. 1992, 1993). Overall,the search for explanations why oralmucosa heals faster and with lessscar tissue formation than skinwounds continues.

Summarize the role of innate and adaptive

immunity in soft tissue wound healing

Mice lacking neutrophils or macro-phages can efficiently heal skin

wounds, as long as microbial infec-tion is controlled (Martin et al.2003). Thrombocytopenic miceexhibited altered wound inflamma-tion but no delay in dermal woundhealing (Szpaderska et al. 2003a).Mast cell-deficient mice are charac-terized by a decrease in neutrophilsbut no other aspects of wound heal-ing (Egozi et al. 2003, Nauta et al.2013). Taken together, it seems thatneither of the inflammatory cells isessential for wound healing when thedefects are not challenged by micro-bial or other contamination. Thus,the models not necessarily representthe clinical scenario of the woundsite after periodontal surgery. More-over, there may be functional redun-dancies between various cells types.Inflammation usually provokesslower healing and more scarring(Martin & Leibovich 2005). Thus,reducing the inflammatory responsein a defect site might have a benefi-cial impact on wound healing (Mar-tin & Leibovich 2005).

Wound healing was enhanced bythe depletion of T helper and cyto-toxic lymphocytes (Efron et al. 1990)and in athymic nude mice that lacka normally developed T cell (Barbulet al. 1989). T-cells are heteroge-neous, suggesting the existence of apopulation that stimulates woundhealing. For example, mice lacking apopulation of dendritic epidermal Tcells exhibit a delay in wound clo-sure (Jameson et al. 2002, Havran &Jameson 2010). B-cells are alsoinvolved in wound healing (Nishioet al. 2009). For a review on themechanisms by which the immunesystem regulates wound healing seePark & Barbul (2004). Overall, thereis increasing evidence that lympho-cytes are involved in the control ofwound healing clearly more detailsare wanted, in particular with regardto periodontal soft tissue healing.

Genetic basis of wound healing

exemplified by TGF-b and associated

genes

Understanding wound healing at themolecular level provides the scientificbasis to develop targeted strategiesmainly with the intension to over-come delayed wound healing or tocontrol excessive scar formation.For example, TGF-b1 has pleiotro-pic functions; including increased


S8 Sculean et al.

collagen synthesis by fibroblasts andtheir conversion into myofibroblasts,also with gingival cells (Hong et al.1999, Sobral et al. 2011). In TGF-b1-deficient mice, early skin woundhealing proceeded almost normally(Brown et al. 1995). However, micethat lack Smad3, which transducessignals from TGF-b, show acceler-ated cutaneous and palatal woundhealing compared with wild-typemice (Ashcroft et al. 1999, Jinnoet al. 2009). Targeting of Smad3with small interfering RNA alsoaccelerates wound-healing and inhib-its wound contraction in palatal mu-coperiosteal wounds (Yoneda et al.2013). Moreover, IFN-c knockoutmice exhibited an accelerated woundhealing and there seems to be acrosstalk with TGF-b signallingpathways (Ishida et al. 2004). Over-all, these and other (Liaw et al.1998, Padmakumar et al. 2012,Zhang et al. 2012, Guo et al. 2013)genetic models have helped to revealrole of TGF-b in wound healing andto use these pivotal aspects to designtargeted therapies that may also sup-port periodontal wound healing.TGF-b is exemplified as one out ofmultiple factors that are key regula-tors of wound healing. For the othermolecules involved in wound healingsee the recent reviews (Martin 1997,Singer & Clark 1999, Gurtner et al.2008, Shaw & Martin 2009, Nautaet al. 2011). It is not the intention ofthis part to review the clinical appli-cation of the respective moleculessuch as platelet-derived growth fac-tor-BB (Hollinger et al. 2008)and basic fibroblastic growth fac-tors (bFGF or FGF-2) (Murakami2011).

Healing of soft tissue wounds atnatural teeth

This section will attempt to providethe biological background of soft tis-sue healing around natural teeth andto give an overview on the mostimportant histological events follow-ing non-surgical and various peri-odontal surgical procedures.

Role of connective tissue and sulcular

environment in determining epithelial

differentiation

The question whether the specificityof the epithelium is determined by

hereditary mechanisms rather than byfunctional adaptation has been ele-gantly investigated in monkeys byKarring et al. (1971). Following exci-sion of the buccal crevicular epithe-lium adjacent to the maxillary and/ormandibular premolars, mucoperio-steal flaps were raised, separate pedi-cles of gingiva and alveolar mucosaprepared and the flaps were trans-posed and sutured. Furthermore, freepalatal grafts including epitheliumand lamina propria, were transplantedto the maxillary and/or mandibularalveolar mucosa. The experimentalprocedures were designed to yieldobservation periods of 5 and 14 days,18, 10 and 12 months.

The histological evaluation indi-cated that during the early woundhealing period (e.g. 5-day grafts) thesuperficial layers of the epitheliumwere desquamated and an abun-dance of mitoses was present in thebasal layers of gingival and alveolarmucosal transplants. Epithelial pro-liferation started from the edges ofthe grafts and the adjacent tissues,while regeneration of the supraalveo-lar connective tissue appeared to beinitiated within the periodontal liga-ment. At 14 days the entire surfaceof the grafts was already coveredwith a thin layer of epithelium. At30 days, the transplanted grafts werecompletely covered by a thin epithe-lium layer but displayed identical tis-sue characteristics to those of thecorresponding control tissues. After2 months, the grafted tissue demon-strated clinical and histological fea-tures identical to those of therespective donor tissues. Moreover,both the clinical and the histologicalfindings have indicated that the tis-sue specificities of the gingival, alve-olar and palatal mucosa wereconserved after heterotopic trans-plantation, thus suggesting that theclinical and structural features ofthese tissues are rather geneticallythan functionally determined.

The finding that after completeexcision of the keratinized tissuessurrounding the teeth (e.g. free andattached gingiva) a zone of gingivawill always reform, was later corrob-orated in animals and humans (Wen-nstrom et al. 1981, Wennstrom 1983,Wennstrom & Lindhe 1983). In allthese studies a narrow zone of gin-giva was always observed to regener-ate following complete excision (e.g.

gingivectomy). Interestingly, at9 months following surgery, com-plete excision of the gingival unitappeared to lead to a wider zone ofattached gingiva compared to aflap-excision approach (Wen-nstrom 1983). This finding wasexplained by the more pronouncedformation of granulation tissue origi-nating from the periodontal ligamentspace following gingivectomy thanfollowing the flap-excisionapproach.

Further evidence for the pivotalrole of gingival connective tissue indetermining epithelial differentiationhas been provided by a subsequentanimal study where free connectivetissue grafts were transplanted fromeither gingiva (test) or non- kerati-nized alveolar mucosa (controls),into areas of the alveolar mucosa(Karring et al. 1975b). The graftswere implanted into pouches createdin the connective tissue of the alveo-lar mucosa as close as possible tothe overlying epithelium. Followinga period of 34 weeks, the trans-plants were exposed by removal ofthe overlying epithelium, thus allow-ing epithelialization from the sur-rounding non-keratinized alveolarmucosa. The clinical and histologicalevaluation after a healing periodbetween 1 and 12 months has shownthat the gingival connective tissuegrafts became covered with kerati-nized epithelium, displaying the samecharacteristics as those of normalgingival epithelium, while the alveo-lar mucosa transplants were coveredwith non-keratinized epithelium.These findings indicate that the spec-ificity of these epithelia is geneticallydetermined and their differentiationis mainly dependent on stimuli fromthe underlying connective tissues.They also suggest that the granula-tion tissue proliferating from thealveolar mucosa will produce a non-keratinized epithelium, whereas thatoriginating from the supra-alveolarconnective tissue or from theperiodontal ligament will lead to akeratinized epithelium. These obser-vations in animals were later alsoconfirmed in humans (Edel 1974,Edel & Faccini 1977).

In a first study, 14 free palatalconnective tissue grafts without epi-thelium were transplanted to partial-thickness sites prepared in patientswith an inadequate width of



attached gingiva (Edel 1974). It wasreported that clinically, the graft sur-faces appeared keratinized afteralready 2 weeks and an increase inthe width of keratinized tissueoccurred. A subsequent study hasevaluated histologically 10 connec-tive tissue grafts transplanted to par-tial-thickness sites (Edel & Faccini1977). Eight grafts were completelyfree of epithelium, while two graftshad one thin layer of retained epi-thelium oriented at the apical edgeof the graft. The grafts were placedin a way to be in contact with bothkeratinized and non-keratinizedmucosa. The histological evaluationhas demonstrated that at 24 weeks,the epithelium covering the gingivalconnective tissue grafts displayedkeratinization with a normal archi-tecture. Interestingly, in the twocases where a keratinized epithelialborder was left on the grafts, an epi-thelial down-growth between thegraft and the recipient site wasobserved indicating no apparentadvantage of leaving a border ofkeratinized epithelium on a grafttransplanted in an area where alveo-lar mucosa was originally present.

An important question, whichstill needs to be definitely clarified,is related to the possible differencesinherent in deep and superficial con-nective tissue in determining epithe-lial keratinization. In a nicelydesigned experiment, a thick palatalepithelial-connective tissue graft wasexcised and split into two thinnergrafts (e.g. one epithelial-connectivetissue graft and one connective tis-sue graft) (Ouhayoun et al. 1988).The grafts were transplanted intocontra-lateral areas lacking kerati-nized mucosa. Following a healingperiod of 3 months, biopsies wereexcised and examined by means ofroutine histology, immunofluores-cence and gel electrophoresis. Theresults have shown that while theepithelial-connective tissue graftsdisplayed histological and biochemi-cal characteristics of keratinizedmucosa (e.g. gingiva) the deep con-nective tissue grafts expressed fea-tures belonging to both keratinizedand non-keratinized mucosa. Theseobservation appear to suggest thatdeep palatal connective tissue, maynot have the same potential toinduce keratinization of non-kerati-nized epithelial cell as the palatal

connective tissue originating froman immediately subepithelial area.Comparable findings in humanswere also reported by others indi-cating that palatal connective tissuegrafts or free gingival grafts trans-planted into areas of non-kerati-nized mucosa may not alwaysdevelop the characteristics of kerati-nized mucosa (Bernimoulin & Sch-roeder 1980, Bernimoulin & Lange1973, Lange & Bernimoulin 1974).On the other hand, it is interestingto note that despite the fact that theconnective tissue underlying the epi-thelium appears to determine thecharacteristics of the overlying epi-thelium, the sulcular epithelium isnot keratinized. The questionwhether the sulcular environmentmay influence keratinization of sul-cular epithelium has been evaluatedin two animal experiments (Caffesseet al. 1977, Caffesse et al. 1979b).

In a first experiment, 24 intrasul-cular mucoperiosteal flaps wereraised by blunt dissection on the buc-cal aspect of individual teeth includ-ing also the approximal papillae ofthe tooth (Caffesse et al. 1977). Sub-sequently, a split thickness flap wasprepared in the alveolar mucosa api-cal to the flap, by removing the epi-thelium and a thin layer ofconnective tissue. The flaps were thenfolded and sutured in a way that thesulcular epithelium became exposedto the oral cavity. Following surgery,biopsies were taken to allow forobservation periods of 1 h to8 weeks. The findings have indicatedthat the sulcular epithelium has thepotential for keratinization and thatthe contact to the tooth appears todetermine the lack of keratinizationof the sulcular epithelium. In anotherexperiment, the influence of the sul-cular environment on the keratiniza-tion of the outer surface gingivalepithelium was evaluated (Caffesseet al. 1979b). Mucoperiosteal flapswere raised on the buccal aspect ofexperimental teeth, without includingthe approximal papillae and invertedin order to place the outer surfaceepithelium in contact with the tooth.The experimental time intervals var-ied from 1 h to 60 days. The resultshave shown that the outer surfaceepithelium may change its morphol-ogy to a non-keratinized epitheliumdevoid of deep rete pegs when placedin close contact with the tooth and

displayed anatomical characteristicsof sulcular epithelium. It thusappears that the sulcular environ-ment has the capability of controllingthe keratinizing potential of the outersurface gingival epithelium.

The question whether an inflam-matory process may influence epi-thelial keratinization is stillcontroversially discussed. Resultsfrom experimental studies in ani-mals have failed to show that anexperimentally induced acute orchronic inflammation of gingivalconnective tissue may modify theinduced-keratinized sulcular epithe-lium, or the normally keratinizedoral gingival epithelium, if bacterialplaque is removed regularly (Nasj-leti et al. 1984, Caffesse et al.1985). On the other hand, thereduction of gingival inflammationby means of systemic antibiotics,local plaque control and scalingand subgingival scaling may facili-tate keratinization of sulcular epi-thelium (Bye et al. 1980, Caffesseet al. 1980, Caffesse et al. 1982).Furthermore, histological, immuno-fluorescence, electron microscopicobservations in humans have alsoindicated that, in the presence of aninflammatory process, alterations ofgingival epithelia may occur (Levyet al. 1969, Ouhayoun et al. 1990,Tonetti et al. 1994).

Taken together, the availabledata indicate that the granulationtissue originating from the periodon-tal ligament or from connective tis-sue originally covered by keratinizedepithelium has the potential toinduce keratinization. The questionwhether a deep palatal connectivetissue has the same potential toinduce keratinization of non-kerati-nized epithelial cell as the palatalconnective tissue originating from animmediately subepithelial area maybear clinical relevance and should beclarified in further studies.

Soft tissue healing following non-surgical

periodontal therapy

Several histological studies in animalsand humans have evaluated the heal-ing after root planing and soft tissuecurettage. Novaes et al. (1969) haveperformed gingival curettage on thelabial side of upper incisors in dogsand observed that the epithelialattachment was re-established after


S10 Sculean et al.

6 days and remained at the cemen-tum-enamel junction until the end ofthe experiment. Stahl et al. (1971)have treated 80 suprabony pockets in60 adult patients suffering from peri-odontitis by means of curettage androot planing. The histological analy-sis has indicated that at one weekafter curettage, an epithelial liningwas already present in all investi-gated specimens. The early stage aftercurettage was characterized by anincrease in an acute inflammatoryinfiltrate. However, after a healingperiod of 8 weeks, the inflammatoryinfiltrate appeared to be similar in dis-tribution and degree to that observedin non-treated control samples.

Waerhaug (1978) has studied thehealing of the dento-epithelial junc-tion following subgingival plaquecontrol in 39 biopsies from 21patients. Following removal of sub-gingival calculus and plaque and ahealing period varying from 2 weeksto 7 months, block biopsies wereharvested and analysed histologi-cally. The histological analysisrevealed that a normal dento-epithe-lial junction has been routinelyreformed in areas from which sub-gingival calculus and plaque hasbeen removed. The new dento-epi-thelial junction appeared to be com-pleted within a period of 2 weeks.

The healing following periodicroot planing and soft tissue curettagehas been evaluated in a non-humanprimate model (Caton & Zander1979). The soft tissue curettage ofthe pocket wall and marginal gingivawas extended apical to the bottomof the clinical pocket to remove theentire pocket epithelium. The proce-dure was repeated at 3, 6 and9 months after the initial root plan-ing and curettage. The histologicalevaluation has demonstrated that inall cases the healing occurredthrough formation of a long junc-tional epithelium along with no con-nective tissue attachment.

Similar findings have beenrecently reported in humans follow-ing non-surgical therapy performedwith the aid of a dental endoscope.Besides the observation that at6 months following therapy thehealing was characterized by a longjunctional epithelium, completeremoval of calculus and plaque wasassociated with a lack of histologi-cal signs of inflammation (Wilson

et al. 2008). Taken together, theavailable histological evidence indi-cates that the healing followingnon-surgical periodontal therapy ischaracterized by epithelial prolifera-tion, which appears to be completedafter a period of 714 days aftertreatment. Complete removal of cal-culus and plaque was associatedwith a limited or complete lack ofinflammation.

Soft tissue healing following periodontal

surgical procedures

Gingivectomy

The sequential healing events follow-ing gingivectomy have been evaluatedby Novaes et al. (1969). Immediatelyafter surgery, a haemorrhage is pres-ent. At 2 days, a thick clot coveredthe entire wound and a slight epithe-lial migration at the apical margin ofthe wound was observed. At 4 days,the blood clot still covered the majorpart of the wound surfaces, but theepithelial proliferation was clearlyvisible from the oral epitheliumand epithelial attachment cells. At1 week, the wound surface was usu-ally completely epithelized and thesulcus reformed but keratinizationand the reformation of rete pegs wasonly detected at 16 days. Woundmaturation was still detectable until38 days, when no differences betweenthe treated areas and the pristine siteswere detected.

Flap surgery

Several studies have evaluated thehealing following full thickness andpartial thickness flaps. Overall, thehealing follows the same pattern andis characterized by the formation ofa blood clot between the soft andhard tissues. The adherence medi-ated by the blood clot between thesoft and hard tissues appears to beweak and does not seem to be ableto hold them together (Kon et al.1969). At 6 and 7 days, an inflam-matory reaction and an increase invascularization of the remaining con-nective tissue and flap can usually beobserved. At this stage, the flap isstill more prone to separation fromthe subjacent tissues when tension isapplied. At 12 days, the flap is reat-tached to the bone and tooth, whilethe oral gingival epithelium appearsto be keratinized. The rete pegs areof a normal shape. At around

4 weeks, the flap is re-attached tothe tooth by dense, organized, con-nective tissue. At 5 weeks, the tissuesappear to be completely regeneratedand do not show differences com-pared to pristine sites. Comparablefindings have been also reported byCaffesse et al. (1984) and Kon et al.(1984). Bone resorption alwaysoccurs following elevation of fullthickness and partial-thickness flaps.Despite the observation that partialthickness flaps may result in lessbone loss compared to the elevationof fullthickness flaps, they do notseem to completely prevent bone loss(Fickl et al. 2011).

The strength of the flap attach-ment to the tooth during healing fol-lowing periodontal surgery wasevaluated by Hiatt et al. (1968). At 2and 3 days following surgery, 225 gof tension on the silk suture placedthrough the margin of the flap wasneeded to separate the flap from thetooth and the bone and increased to340 g at 1 week. Once the epithelialattachment was severed, the fibrinbeneath the connective tissue surfaceappeared to offer very limitedmechanical resistance. At 2 weeks,the suture was pulled through thegingival margin with a force of1700 g, which separated the flaponly partially from the tooth. At1 month, the flap could not bemechanically separated from thetooth, but a split within the epithe-lium at the point of stress wasobserved microscopically. Thesefindings indicate that the initialattachment of the flap to the toothwas through the epithelium while thefibrin layer did not appear to signifi-cantly contribute to the retention ofthe flap. Moreover, a proper re-adaptation of the mucoperiostealflap to the root surface appeared toinhibit epithelial proliferation anddown-growth. The findings have alsosuggested that the strength of theepithelial attachment to the root isgreater than the attachment betweencells. These findings were later cor-roborated by Wikesjo et al. (1991)suggesting that connective tissueattachment to dentin is mediated byadsorption of plasma proteins to thesurface and subsequent developmentand maturation of the fibrin clot(Wikesjo et al. 1991).

Taken together these data indi-cate that the tensile strength of the



tooth-soft tissue interface stillappears vulnerable to mechanicaltrauma at 7-day post-surgery. Atapproximately 14 days post-surgery,a structural integrity of a maturingwound between a denuded root sur-face and a mucogingival flap, whichcan sufficiently withstand mechanicaltrauma, is achieved. These observa-tions, in turn point to the criticalrole of passive flap adaptation andof suturing to allow undisturbedwound maturation.

Mormann & Ciancio (1977) haveevaluated the effect of various typesof surgical procedures on the gingivalcapillary blood circulation by meansof fluorescein angiography. The circu-lation changes observed suggestedthat flaps receive their major bloodsupply from their apical aspects. Thefull thickness incision in clinicallyhealthy gingiva revealed that theblood supply had predominantlycaudocranial direction from the vesti-bule to the gingival margin. More-over, the findings have also indicatedthat flaps should be broad enough attheir base to include major gingivalvessels and pointed to the importanceof ensuring a tension-free re-adapta-tion to avoid dehiscence. Further-more, partial thickness flaps shouldnot be too thin in order to includemore blood vessels and avoid necro-sis. Later studies have demonstratedthat during the elevation of a muco-periosteal flap the connection of thegingivo-periosteal plexus with theperiodontal ligament vascular plexusis severed and significant vasculartrauma is induced, especially in theinter-dental areas (Nobuto et al.1989, McLean et al. 1995). Thesefindings have been later confirmed ina series of studies evaluating gingivalblood flow by means of laser Dopplerflowmetry after different periodontalsurgical procedures (Donos et al.2005, Retzepi et al. 2007a,b).

The results have shown that theblood flow decreased immediatelyfollowing anaesthesia and remainedat lower values compared to baselineimmediately following flap surgery.The gingival flow presented an over-all increase in comparison to base-line values until the 7th daysfollowing surgery at the inter-dentaland alveolar mucosa sites. At15 days, however, the blood flowvalues were again similar to baseline.The findings have also indicated that

the location of the incisions and theuse of surgical techniques intendingto preserve the inter-dental tissues(i.e. simplified papilla preservationflap) may be associated with fasterrecovery of the gingival blood flowpost-operatively compared with themodified Widman flap (Donos et al.2005, Retzepi et al. 2007a, b).

Healing following denudation tech-niques

The healing following full thicknessand split thickness flaps used in mu-cogingival surgery to increase thewidth of attached gingiva has beenevaluated in several animal experi-ments (Staffileno et al. 1966, Karringet al. 1975a, Pustiglioni et al. 1975,Kon et al. 1978). The origin anddevelopment of granulation tissuefollowing periosteal retention anddenudation procedures has beenevaluated in monkeys by Karringet al. (1975a). The findings haveshown that following periostealexposure and denudation of the alve-olar bone, the granulation tissueoriginated from the residual perio-steal connective tissue, periodontalligament, bone marrow spaces andthe adjacent gingiva and alveolarmucosa. During the initial stages ofhealing, resorption of the labial andbuccal bone took place and theamount of bone loss was influencedby the thickness and structure of thelabial or buccal bone. Generally, theresorption was most severe followingthe denudation technique, while theloss of crestal bone was generallysmaller with the periosteal retentionprocedure than following the com-plete exposure of bone. Moreover,with the denudation technique, a lar-ger portion of the marginal areaswas filled with granulation tissuederiving from the periodontal liga-ment. Granulation tissue derivedfrom the remaining or adjacent gin-gival connective tissue or the peri-odontal ligament appeared to becovered by keratinized epithelium,whereas that originating from theconnective tissue of the alveolarmucosa was covered by non-kerati-nized epithelium. After 1 month, thefree gingiva was regenerated andexhibited a shallow gingival crevice.In the injured mucosa, delicate elas-tic fibres reappeared in the regener-ated tissues after 12 months anddisplayed similar histological charac-

teristics to those of the pristine alve-olar mucosa and probably originatedfrom the intact alveolar mucosa.Generally, no predictable increase inthe width of the gingiva was foundafter any of the two methods.

Taken together, the results sug-gest that the success or failure toextend the width of keratinized tis-sue by surgical means is unpredict-able and depends on the origin ofthe granulation tissue. It may thusbe suggested that the use of trans-plants is a more predictable methodfor increasing the width of kerati-nized tissue.

Healing following soft tissue grafting

Free gingival grafts or free connec-tive tissue grafts have been intro-duced in periodontal therapy toincrease the width of the attachedgingiva and to prevent or treat gingi-val recessions (Gargiulo & Arrocha1967, Oliver et al. 1968, Sullivan &Atkins 1968, Staffileno & Levy 1969,Sugarman 1969, Edel 1974, Edel &Faccini 1977, Caffesse et al. 1979a).The sequential events of the healingand revascularization of free gingivalgrafts when placed over periosteumhas been evaluated in monkeys byOliver et al. (1968). Following prep-aration of a periosteal recipient bedin the maxillary and mandibularanterior region in monkeys, free gin-gival grafts were obtained from thebuccal attached gingiva in the pre-molar area. The grafts were placedover the periosteum and sutured tothe adjacent interproximal tissue,attached gingiva and interproximaltissue. Animals were sacrificed toallow observation periods at 0, 2 4,5, 7, 8, 11, 14, 17, 21, 28 and42 days. The histological evaluationrevealed that the healing of free gin-gival grafts can be divided into threephases: (i) Initial phase (03 days)characterized by a thin layer offibrin separating the periosteumfrom the graft and degeneration ofepithelium and desquamation of theouter layers. (ii) Revascularizationphase (411 days) characterized byminimal resorption of the alveolarcrest, proliferation of fibroblasts intothe area between the graft and peri-osteum. At 5 days all the graft epi-thelium was degenerated anddesquamated. At the same time, athin layer of new epithelial cells pro-liferated over the graft from the


S12 Sculean et al.

adjacent tissues. At day 11, a densefibrous union was observed betweenthe graft and the periosteum. Granu-lation tissue was gradually replacedby fibroblastic proliferation and atday 11 the graft was completely cov-ered by an epithelial layer, whichwas continuous with the marginalepithelium. Vascularization was evi-dent, and capillary ingrowth wasobserved at the base of the graft.(iii) Tissue maturation phase (1142 days). At 14 days of healing, theconnective fibres within the graftwere comparable in staining qualityand appearance to the fibres in thecontrol specimens. The thickness ofthe epithelium had developed morefully at 14 days but no keratiniza-tion was present. Keratinization wasonly detectable at 28 days. At14 days, the number of vesselsthroughout the connective tissue ofthe graft was decreased but in thesame time the connective tissue den-sity increased. The pattern of vascu-larization did not show majorchanges after day 14.

The observations made by Oliveret al. (1968) are generally in agree-ment with those reported by Caffesseet al. (1979a,b) and Staffileno &Levy (1969) have evaluated the heal-ing of free gingival grafts placed oneither periosteum or on denudedbone in monkeys. In cases when thegrafts were placed on bone, a delayin healing was observed. However,by 28 days, there were no differencesin the rate of healing between graftsplaced on bone or on periosteum.However, the periosteal bedappeared to favour better initialadaptation and nourishment of thegraft. Grafts placed directly on boneshowed initially more degenerativechanges and a delay of epithelialmigration. Furthermore, the epithe-lial coverage was restored in 7 dayswhen the grafts were on periosteumand in 14 days when they wereplaced on bone. Keratinization wasfound in both groups at 28 days,while elastic fibres were onlyobserved in cases where the graftswere placed on periosteum. Oneimportant aspect, which needs to beconsidered when using connectivetissue grafts or free gingival grafts, isthe shrinkage, which occurs duringhealing. It has been reported thatthe greatest amount of shrinkageoccurs in the first postoperative

month but can be followed up to360 days and varies between 25%and 45% (Egli et al. 1975, James &McFall 1978, Rateitschak et al.1979, Mormann et al. 1981, Orsiniet al. 2004). While re-vascularizationappears to be more delayed inthicker grafts, less shrinkage wasobserved with increasing graft thick-ness (Egli et al. 1975, Rateitschaket al. 1979, Mormann et al. 1981).

Soft tissue healing around implants

Dental implants are anchored in jaw-bone through a direct bondingbetween bone and the implant. Suc-cess and survival of an implant do,however, not depend solely on osseo-integration. A soft tissue, which sur-rounds the transmucosal part of adental implant, separates the peri-implant bone from the oral cavity.This soft tissue collar is called peri-implant mucosa (Lindhe et al.2008). The attachment of the soft tis-sue to the implant serves as a biologi-cal seal that prevents thedevelopment of inflammatory peri-implant diseases (i.e. peri-implantmucositis and peri-implantitis). Thus,the soft tissue seal around implantsensures healthy conditions and stableosseointegration and therefore alsothe long-term survival of an implant.

Around teeth, a sophisticated softtissue collar seals the tissues of toothsupport (i.e. alveolar bone, peri-odontal ligament and cementum)against the oral cavity (Bosshardt &Lang 2005). While the soft tissueseal around teeth develops duringtooth eruption, the peri-implantmucosa forms after the creation of awound in oral soft and hard tissues.The wound healing phase may occurfollowing the closure of a mucope-riosteal flap around the neck portionof an implant placed in a so-calledone-stage procedure or after a sec-ond surgical intervention for abut-ment connections to an alreadyinstalled dental implant (two-stageprocedure). Since wound healingoccurs in the presence of a biomate-rial (i.e. a foreign body) at a criticalregion, interference of wound heal-ing events with this biomaterial andadaptation of the soft tissue to thisbiomaterial have to be taken intoconsideration. The aim of this partwas to review the anatomy and his-tology of the soft tissue seal around

transmucosal biomaterials used toreplace missing teeth and to summa-rize its morphogenesis during woundhealing.

Nature and dimensions of peri-implant

mucosa (quantity)

During the process of wound heal-ing, the features of the peri-implantmucosa are established. Many bio-material and surgical factors mayhave an influence on the outcome ofsoft tissue quantity, i.e. the length ofthe peri-implant mucosa. In an excel-lent pioneer study in dogs, Bergl-undh et al. (1991) examinedanatomical and histological featuresof the peri-implant mucosa, whichformed in a two-stage procedure,and compared these with those ofthe gingiva around teeth. The abut-ment consisted of titanium with amachined surface. Two months afterabutment connection, the animalswere enrolled in a careful and metic-ulous plaque control programmeconsisting of cleaning of the abut-ment once daily. Four months afterabutment connection, clinical inspec-tion and radiographic evaluationrevealed healthy conditions. Histo-logically, the peri-implant mucosaconsisted of a well-keratinized oralepithelium, which was located at theexternal surface and connected to athin barrier epithelium (i.e. theequivalent to the junctional epithe-lium around at teeth, which will bereferred to as the peri-implant junc-tional epithelium) facing the abut-ment. This peri-implant junctionalepithelium terminated 2 mm apicalto the coronal soft tissue margin and1.01.5 mm coronal from the peri-implant bone crest. Thus, the meanbiological width (including the sulcusdepth) was 3.80 mm aroundimplants and 3.17 mm around teeth.While there was no statistically sig-nificant difference in the height ofthe junctional epithelium and sulcusdepth between implants and teeth,the height of the soft connective tis-sue was significantly greater aroundimplants than around teeth. Theperi-implant junctional epitheliumand the soft connective adjacent tothe abutment appeared to be indirect contact with the implant abut-ment surface. However, the precisenature of the epithelial and soft con-nective tissue attachments could not



accurately be analysed, since thefracture technique was applied,which included removal of the tita-nium implant before cutting of thehistological sections. This importantstudy in dogs showed that under theconditions chosen, the peri-implantmucosa has a comparable potentialas the gingiva around teeth to pre-vent subgingival plaque formationand subsequent infection.

Effects of implant system on the peri-implant mucosa dimension

While in the above-mentioned studyby Berglundh et al. (1991) theBranemark system (Nobel Biocare,Gothenburg, Sweden) was used, sub-sequent studies revealed that a simi-lar mucosal attachment formed ontitanium in conjunction with differ-ent implant systems (Buser et al.1992, Abrahamsson et al. 1996) andaround intentionally non-submergedand initially submerged implants(Arvidson et al. 1996, Weber et al.1996, Abrahamsson et al. 1999).However, the peri-implant junctionalepithelium was significantly longer ininitially submerged implants towhich an abutment was connectedlater than in intentionally non-sub-merged implants (Weber et al. 1996).The biological width was revisited ina further dog experiment after abut-ment connection to the implant fix-ture with or without a reducedvertical dimension of the oralmucosa (Berglundh & Lindhe 1996).While the peri-implant junctionalepithelium was about 2 mm long,the supraalveolar soft connectivewas about 1.31.8 mm high. Inter-estingly, sites with a reduced muco-sal thickness consistently revealedmarginal bone resorption so that thebiological width could be adjusted.Evaluating the biological widtharound one- and two-piece titaniumimplants that healed unloaded ineither a non-submerged or a sub-merged fashion in dog mandibles,Hermann et al. (2001) suggested thatthe gingival margin is located morecoronally and the biological widthmore similar to teeth in associationwith one-piece non-submergedimplants compared to either two-piece non-submerged or two-piecesubmerged implants. These datawere confirmed in a comparablydesigned dog study with anotherimplant system (Pontes et al. 2008).

Effects of implant material on theperi-implant mucosa dimension

In a dog study, Abrahamsson et al.(1998) demonstrated that the mate-rial used for the abutment had amajor impact on the location of thesoft connective tissue compartment.Sintered ceramic material made ofaluminium (Al2O3) lead to a peri-implant mucosal attachment compa-rable to that adjacent to titaniumabutments. Gold alloy or dental por-celain, however, resulted in inferiorhistological outcome of the peri-implant mucosa. Kohal et al. (2004)and Welander et al. (2008) havedemonstrated the same peri-implantsoft tissue dimensions around tita-nium and zirconia implants installedin the maxilla of monkeys and dogsrespectively.

Effects of implant surface characteris-tics on the peri-implant mucosadimension

The effects of surface macro design,topography, hydrophilicity and vari-ous coatings on the peri-implantmucosa have been evaluated in numer-ous pre-clinical and clinical studies.Organic implant coatings will not bediscussed in this review. Numerous invitro studies have addressed the issueof surface modifications on mesenchy-mal and epithelial cell responses.However, these are not included, sinceit is beyond the scope of this article toreview in vitro studies.

The impact of surface topogra-phy, often characterized by surfaceroughness measurements, on theperi-implant mucosa has been inves-tigated in numerous studies. Coch-ran et al. (1997) noted no differencesin the dimensions of the sulcusdepth, peri-implant junctional epi-thelium and soft connective tissuecontact to implants with a titaniumplasma-sprayed (TPS) surface or asandblasted acid-etched surface.Abrahamsson et al. (2001, 2002)observed similar epithelial and softconnective tissue components on arough (acid etched) and smooth(turned) titanium surface. The bio-logical width was greater on therough surface, however, without astatistically significant difference tothat around a smooth surface. Intwo studies with human biopsymaterial, less epithelial down-growthand a longer soft connective tissue

were found in conjunction with oxi-dized or acid-etched titanium com-pared to a machined surface(Glauser et al. 2005, Ferreira Borges& Dragoo 2010). In a study inbaboons, Watzak et al. (2006) couldshow that implant surface modifica-tions had no significant effect on thebiological width after 18 months offunctional loading. After 3 monthsof healing in dog mandibles, nano-porous TiO2 coatings of one-piecetitanium implants showed similarlength of peri-implant soft connec-tive tissue and epithelium than theuncoated, smooth neck portion ofthe control titanium implants (Rossiet al. 2008). In a dog study, Schwarzet al. (2007) investigated the effectsof surface hydrophilicity and micro-topography on soft and hard tissuehealing at 1, 4, 7, 14 and 28 days.The authors concluded that soft tis-sue integration was influenced byhydrophilicity rather than by micro-topography. Using a new humanmodel, Schwarz et al. (2013) investi-gated the peri-implant soft tissuedimensions after an 8-week healingperiod on specially designed healingabutments with different surfaceroughness and hydrophilicity. Thelength of the peri-implant junctionalepithelium was in the order of 2 mmfor all abutment types without statis-tically significant differences.

Immediate versus delayed implantloading

Studying immediate versus delayedloading of titanium implants placedin jawbone of monkeys, Siar et al.(2003) and Quaranta et al. (2008)could not detect any significant dif-ferences in the dimensions of theperi-implant sulcular and junctionalepithelia and connective tissue con-tact to the implants.

Composition and structural organization

of the peri-implant mucosa (quality)

Concerning composition, most stud-ies focused on the soft connective tis-sue compartment of the peri-implantmucosa. In particular, amount andstructural organization of fibroblasts,collagen, and blood vessels weredetermined. Fewer fibroblasts andmore collagen fibres were observedin the bulk of the supra-crestal softconnective tissue around implantswith a smooth abutment surface


S14 Sculean et al.

than around teeth (Moon et al.1999). However, very close to theimplant surface the number of fibro-blasts was high and they were inter-posed between thin collagen fibrilsand oriented parallel to the implantsurface (Moon et al. 1999). Surfaceroughness did not seem to have aninfluence on the number of fibro-blasts (Abrahamsson et al. 2002).

In a zone close to the implant sur-face (i.e. 50100 lm away), no bloodvessels were found (Buser et al. 1992,Listgarten et al. 1992). Further awayfrom the implant surface and adjacentto the barrier (junctional) and sulcu-lar epithelia, blood vessels wereobserved (Buser et al. 1992). Thus,the number of blood vessels increasedwith increasing distance from theimplant surface. Compared to teeth,there were less vascular structures inthe supra-crestal soft connective tis-sue near the implant than at a corre-sponding location around teeth(Moon et al. 1999). Using a clearingtechnique to visualize carbon-stainedblood vessels, Berglundh et al. (1994)showed that the vascular network ofthe peri-implant mucosa originatesfrom one large supra-crestal bloodvessel, which branches towards theimplant abutment surface.

Influence of material on collagen fibreorientation

Comparing one-piece machined tita-nium necks with one-piece smoothzirconia implants, no difference wasobserved concerning collagen fibreorientation, that is, the majority ofcollagen fibres were oriented parallelor parallel-oblique to the implantsurfaces (Tete et al. 2009).

Influence of surface topography oncollagen fibre orientation

Collagen fibre orientation was foundto be primarily parallel to implantswith a smooth titanium surface andthe site of fibre insertion into bone wasat the bone crest in dogs (Berglundhet al. 1991, Buser et al. 1992, Listgar-ten et al. 1992). Parallel and uniformcollagen fibre orientation was alsofound around smooth titanium grade4 implants in a rat model at a veryearly healing phase of 4 and 7 days,whereas collagen fibre orientation onrough (alumina grit blasted) titaniumgrade 4 implants was more irregular(Yamano et al. 2011). While these

findings corroborate with otherreports demonstrating a perpendicularinsertion of collagen fibres to the sur-face of porous plasma-sprayed tita-nium implants (Schroeder et al. 1981,Buser et al. 1989), and suggesting thatthe surface texture might affect the col-lagen fibre orientation (Schupbach &Glauser 2007), others concluded thatsurface roughness and different mate-rials did not appear to influence fibreorientation (Listgarten et al. 1992, Co-mut et al. 2001) and amount of colla-gen (Abrahamsson et al. 2002).

Microgrooves are a sort of surfacemodifications that are different fromtopographic modifications intendedto alter surface roughness characteris-tics. In a dog study, it was shown thatcollagen fibres were oriented perpen-dicularly to the laser ablated, micro-grooved abutment surface, whilecollagen fibres on a smooth (machined)surface were oriented parallel to theabutment surface after a 3-month heal-ing period (Nevins et al. 2010). Humanhistological evidence for this attach-ment of perpendicularly oriented colla-gen fibres to a microgrooved abutmentsurface was provided by four implantsretrieved after a 6-month (Nevins et al.2008) and a 10-week (Nevins et al.2012) healing period.

Influence of surface topography oninflammation and defence

Concerning plaque accumulationand inflammatory cells, no relationwas found to surface roughness after4 weeks in a human model (Wenner-berg et al. 2003). However, TiO2-coated implants with a porous sur-face showed less inflammation andless epithelial detachment thanuncoated implants with a smoothneck portion (Rossi et al. 2008). Incontrast, human soft tissue biopsiesretrieved from titanium healing capsafter a 6-month healing periodrevealed more inflammation, highermicrovessel density, and more prolif-eration epithelial cells around arough (acid-etched) surface thanadjacent to a smooth (machined)surface (Degidi et al. 2012).

Implant-tissue interfaces of the peri-

implant mucosa

The study of tissue interactions withmetallic or ceramic biomaterials ham-pers histological evaluation. If thebiomaterial is not removed, only lim-

ited histological techniques andanalyses are possible. On the otherhand, if the biomaterial is removed,some information may irreversiblyget lost or tissue artefacts may impedethe analysis and consequently also theconclusions. Since in most studiesexamining the peri-implant mucosa,the fracture technique (Thomsen &Ericson 1985) was applied to removethe implant, the true composition andarrangement of the tissue-implantinterface could not be analysed.

The ultrastructure of the interfacebetween a metal implant and theperi-implant junctional epitheliumwas first reported on the basis offreeze-fractured preparations of vital-lium implants (James & Schultz1974). Using transmucosal epoxyresin implants, Listgarten & Lai(1975) noted the ultrastructural simi-larity of the intact epithelium-implant interface between implantsand teeth. Subsequently, these find-ings were confirmed for titanium-coated plastic implants (Gould et al.1984), freeze-fractured specimensfrom ceramic (alpha-alumina oxidein single crystalline form) implants(McKinney et al. 1985), and single-sapphire implants (Hashimoto et al.1989). These studies revealed that theepithelial cells attach to differentimplant materials in a fashion com-parable to that of the junctional epi-thelial cells to the tooth surface viahemidesmosomes and a basal lamina.

Analysing the intact interfacebetween soft connective tissue andtitanium-coated epoxy resinimplants, the parallel orientation ofcollagen fibrils to the titanium layerwas confirmed (Listgarten et al.1992, Listgarten 1996). Implantsnormally lack a cementum layer thatcan invest the peri-implant collagenfibres. Thus, the attachment of thesoft connective tissue to the trans-mucosal portion of an implant isregarded as being weaker than softconnective tissue attachment to thesurface of a tooth root. Therefore,improving the quality of the soft tis-sue-implant interface is considered tobe of paramount importance.

Wound healing and morphogenesis of the

peri-implant mucosa after flap surgery in

healed ridges

While the above studies primarilyshowed the dimensional and histo-



logical outcomes of the establishedepithelial and soft connective tissuecomponents of the peri-implantmucosa under various conditions,the wound healing sequence leadingto this establishment has onlyrecently been evaluated. Berglundhet al. (2007) examined the delicateprocess of wound healing and mor-phogenesis in the mucosa aroundnon-submerged commercially puretitanium implants in dogs. Twoweeks after mucosal adaptation tothe marginal portion of the implants,the sutures were removed and a rigidplaque-control program was initi-ated. Healing periods varied from2 h to 12 weeks. The morphogenesiswas analysed in histological sectionsand by means of histomorphometry.

Immediately after implant place-ment, a coagulum occupied theimplant-mucosa interface. Numerousneutrophils infiltrated the blood clotand at 4 days an initial mucosal sealwas established. In the next fewdays, the area with the leucocytesdecreased and was confined to thecoronal portion, whereas fibroblastsand collagen dominated the apicalpart of the implant-tissue interface.Between 1 and 2 weeks of healing,the peri-implant junctional epithe-lium was about 0.5 mm apical to themucosal margin. At 2 weeks, theperi-implant junctional epitheliumstarted to proliferate in the apicaldirection. After 2 weeks, the peri-implant mucosa was rich in cells andblood vessels. At 4 weeks of healing,the peri-implant junctional epithe-lium migrated further apically andoccupied now 40% of the total softtissue implant interface. The softconnective tissue was rich in collagenand fibroblasts and well-organized.The apical migration of the peri-implant junctional epithelium wascompleted between 6 and 8 weeksand the fibroblasts formed a denselayer over the titanium surface atthat time. From 6 to 12 weeks, mat-uration of the soft connective tissuehad occurred and the peri-implantjunctional epithelium occupied about60% of the entire implant soft tissueinterface. Further away from theimplant surface, the number ofblood vessels was low and fibroblastswere located between thin collagenfibres running mainly parallel to theimplant surface. From this study, itcan be concluded that the soft tissue

attachment to transmucosal (i.e.non-submerged) implants made ofcommercially pure titanium with apolished surface in the neck portionrequires at least 6 weeks in this ani-mal model.

Using a new human model, Tom-asi et al. (2013) investigated the mor-phogenesis of the peri-implantmucosa during the first 12 weeks ofhealing. They observed that a softtissue barrier adjacent to titaniumimplants developed completelywithin 8 weeks, which is in agree-ment with observations made indogs (Berglundh et al. 2007, Schwarzet al. 2007, Vignoletti et al. 2009),but not with those from Glauseret al. in humans. Concerning stabil-ity of soft tissue dimensions overtime, it can be concluded that thedimensions of the soft tissue seal (i.e.the biological width) aroundimplants are stable for at least 12(Cochran et al. 1997, Assenza et al.2003) or 15 months (Hermann et al.2000).

Wound healing and morphogenesis of the

peri-implant mucosa after immediate

implant placement into fresh extraction

sockets

Vignoletti et al. (2009) described his-tologically and histomorphometrical-ly the early phases of soft tissuehealing around implants placed intofresh tooth extraction sockets in dogs.They observed a fast apical down-growth of the peri-implant junctionalepithelium within the first week ofhealing and a final biological width ofapproximately 5 mm with a peri-implant junctional epitheliummeasuring 3.03.5 mm at 8 weeks.Similar dimensional outcomes werereported by Rimondini et al. (2005)in minipigs (i.e. 3 mm epitheliallength after 30 and 60 days) and byde Sanctis et al. (2009) around differ-ent implant systems in dogs (i.e. 2.332.70 mm epithelial length after6 weeks). The above mentioned peri-implant soft tissue dimensions differfrom those reported in other studieswhere implants were placed into freshextraction sockets in dogs (Araujoet al. 2005, 2006) and from thosereported after placement into healedridges (see 3.4.). In summary, it canbe concluded that when implants areplaced into fresh extraction socketsthere are conditions that appear to

favour a fast apical migration of theperi-implant junctional epitheliumand the establishment of a greaterfinal biological width dimension, par-ticularly as regards the epithelial com-ponent. The clinical consequences,the conditions that favour and themeasures that reduce the formationof a longer peri-implant junctionalepithelium on implants need to bedetermined.

Flap versus flapless healing of the peri-

implant mucosa

In a dog experiment, teeth wereremoved either flapless or with flapsurgery and implants were immedi-ately placed (Blanco et al. 2008).After a 3-month healing period, thedistance between the peri-implantmucosal margin and the first bone-implant contact was significantlygreater in the flap group compared tothe flapless group (3.69 mm versus3.02 mm). At the edentulous site, theimplantation region may be exposedusing flap surgery (i.e. the bone crestis exposed by a crestal incision) or aflapless approach (i.e. the bone crestis exposed by a soft tissue punch). Ina study in dogs by You et al. (2009),flat bone crests were created followingtooth extractions. Three months later,implants were placed by either theflap or the flapless approach. Threemonths after implant installation, theheight of the peri-implant mucosaand the length of the peri-implantjunctional epithelium were signifi-cantly greater in the flap than in theflapless group. In other studies indogs, a significantly longer peri-implant junctional epithelium formedat both 3 weeks and 3 months (Leeet al. 2010) and at 3 months (Bayo-unis et al. 2011) after implant place-ment, when the punch diameter wasgreater than that of the implant. Thiswas probably also the reason for theformation of a longer peri-implantjunctional epithelium after soft tissuepunching as opposed to flap surgery(Bayounis et al. 2011). These findingssuggest that the diameter of the softtissue punch should be slightly smal-ler than that of the implant to obtainbetter peri-implant mucosa adapta-tion and subsequent healing.

The vascularity of the peri-implantmucosa was investigated after flap andflapless implant installation (Kimet al. 2009). Morphometric measure-


S16 Sculean et al.

ments revealed that there wasincreased vascularity after the flaplessprocedure than after the flapapproach. Mueller et al. (2010, 2011,2012) evaluated marker molecules forinflammation, re-epithelialization,and the implant-epithelial junction inminipigs at 1, 2, 3 and 12 weeks afterimplant installation using the flaplessapproach or flap surgery. Flaplessimplant insertion resulted in lessinflammation (Mueller et al. 2010),early re-epithelialization (Muelleret al. 2011) and significant higherexpressions of integrin a6b4 chain b4at 2 and 12 weeks and laminin 5c2chain for all healing periods (Muelleret al. 2012). A further conclusion wasthat a smooth neck portion is to bepreferred for the flapless approach(Mueller et al. 2010). Using the flap-less approach with and without imme-diate loading, Blanco et al. (2012)found similar soft tissue dimensions indogs around titanium implants after a3-month healing period.

In summary, it seems that theflapless approach has, at least in theshort-term, some advantages overflap surgery, provided that the diam-eter of the soft tissue punch is belowthat of the transmucosal portion ofthe implant. The disadvantage of theflapless approach is that the bonevolume may not accurately be deter-mined. However, the clinical rele-vance of these histological findingsremains to be determined.

Wound healing after probing around

implants

Probing of the soft tissues arounddental implants is an importantparameter during clinical monitoring.The healing of the disrupted soft tis-sue seal as a result of clinical probinghas been studied in dogs from 1 to7 days post-probing around titaniumplasma-sprayed implants (Etter et al.2002). The probe initiated a separa-tion of the peri-implant junctionalepithelium from the implant surface.Although the probe went past themost apical cell of the peri-implantjunctional epithelium, no tissue sepa-ration was detected in the soft con-nective tissue compartment at anytime point. At day 0, the peri-implantjunctional epithelium of five test sideswas completely separated from theimplant surface, while in one case tis-sue separation ended 0.3 mm apical

to the apical termination of the peri-implant junctional epithelium.Primarily leucocytes filled the separa-tion space. One day after probing, aninitial new epithelial attachment wasobserved at the bottom of the separa-tion space, while leucocytes were stillpresent more coronally. The length ofthe epithelial attachment increasedfrom 0.5 mm at day 1 to 1.92 mm atday 7 after probing. From this study,it was concluded that the healing ofthe epithelial attachment adjacent todental implants was complete 5 daysafter clinical probing. Thus, probingdoes not seem to jeopardize mainte-nance of healthy conditions arounddental implants. From another ani-mal study, it was concluded that fre-quent clinical probing at shortintervals during the healing phasecaused dimensional and structuralchanges of the peri-implant mucosalseal (Schwarz et al. 2010). Surfaceroughness and surface chemistry didnot influence the outcome in this dogstudy.

Based on the available data, itwas concluded that:

(a) Wound healing in skin and oralwounds follows a similar pattern.

(b) The tissue specificities of the gin-gival, alveolar and palatalmucosa appear to be innatelyand not necessarily functionallydetermined.

(c) The granulation tissue originat-ing from the periodontal liga-ment or from connective tissueoriginally covered by keratinizedepithelium has the potential toinduce keratinization. However,it also appears that deep palatalconnective tissue, may not havethe same potential to induce ker-atinization as the palatal connec-tive tissue originating from animmediately subepithelial area.

(d) Epithelial healing following non-surgical and surgical periodontaltherapy appears to be completedafter a period of 714 days. Astructural integrity of a maturingwound between a denuded rootsurface and a soft tissue flap isachieved at approximately14 days post surgery.

(e) The formation of the biologicalwidth and maturation of the bar-rier function around transmuco-sal implants requires 68 weeks ofhealing.

(f) The established peri-implant softconnective tissue resembles a scartissue in composition, fibre orien-tation and vasculature.

(g) The peri-implant junctional epi-thelium may reach a greater finallength under certain conditionssuch as implants placed into freshextraction sockets versus conven-tional implant procedures inhealed sites.

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