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Annals of Anatomy 204 (2016) 63–70

Contents lists available at ScienceDirect

Annals of Anatomy

jou rn al hom ep age: www.elsev ier .com/ locate /aanat

esearch Article

ultiple gingival recession coverage with an allogeneic biostaticascia lata graft using the tunnel technique—A histologicalssessment�

acek Zureka, Marzena Dominiakb, Krzysztof Tomaszekc, Ute Botzenhartd,∗,omasz Gedrangeb,d, Wojciech Bednarze

Special Medical Practis Stomatologia, Srebrna 48 Street, Pl-42-612 Tarnowskie Góry, PolandDental Surgery Department, Silesian Piast Medical University of Wroclaw, Krakowska 26 Street, Pl-50-425 Wrocław, PolandSpecial Medical Practice, Pawia 12 Street, Pl-42-612 Tarnowskie Góry, PolandDepartment of Orthodontics, Carl Gustav Carus Campus, Technische Universität Dresden, Fetscherstr. 74, D-01309 Dresden, GermanySpecialistic Outpatient Medical Clinic MEDIDENT, Okulickiego 19 Street, Pl-38-300 Gorlice, Poland

r t i c l e i n f o

rticle history:eceived 5 August 2015eceived in revised form 14 October 2015ccepted 10 November 2015

eywords:llograftascia lataonnective tissueingival recessionibroblasts

a b s t r a c t

Background: Autogenous connective tissue graft (CTG) that can be safely harvested from the palatalmucosa is limited. Often a multi-stage surgical procedure is needed to cover multiple gingival reces-sions (MGR). To address this problem, efforts are being made to explore substitutes suitable in size toensure surgical treatment in a single visit.The objective of the present study was the histological evalua-tion of tissue in the recipient site after augmentation with a hydrated biostatic Fascia Lata Allograft (FLA)in conjunction with MGR coverage at different healing stages.Material and methods: Twelve patients needing bilateral multiple gingival recession coverage participatedin this study. On the test side, the tunnel technique with FLA was used, while CTG, harvested from thepalatal mucosa, was used to cover MGR on the control side. Histological assessment was performed 3, 6,9 and 12 months after augmentation.Results: FLA was well tolerated by the host tissue. During all investigation periods histological images ofall patients in the test side revealed a slow process of incorporation of the material grafted in the host

connective tissue, showing a colonization of the graft with host fibroblasts and formation of new bloodvessels. After 12 months, the graft had fully remodeled into connective tissue of the host gingiva.Conclusion: Apart from the limitations of the present study, we conclude that the FLA may serve as asubstitute for autogenous CTG harvested from the palatal mucosa and can be applied as a technique forcovering MGR in a single visit.

© 2015 Elsevier GmbH. All rights reserved.

. Introduction

Gingival recession is a problematic issue in modern periodontol-

gy and also an important topic for orthodontic treatment planningonsidering critical values of bone-soft tissue morphology andirection of tooth movement (Warmuz et al., 2014, 2015). It is

� The work was performed in the Department of Periodontal Disease and Oralucosal, the Department of Conservative Dentistry with Endodontics University of

ilesia, the Periodontal Disease Clinic and Oral Mucosal in Zabrze and the Depart-ent of Oral Surgery Wroclaw, Medical University. Own founding was source of

nancial support.∗ Corresponding author. Tel.: +0049 351 4582718; fax: +0049 351 4585318.

E-mail address: ute.botzenhart@uniklinikum-dresden.de (U. Botzenhart).

ttp://dx.doi.org/10.1016/j.aanat.2015.11.002940-9602/© 2015 Elsevier GmbH. All rights reserved.

characterized by partially exposed root surfaces of one or moreteeth in a clear form without accompanying features of inflamma-tion (Dominiak and Gedrange, 2014). The pathogenesis of gingivalrecession is complex and the effectiveness of therapeutic proce-dures largely depends on the identification of those etiologicalfactors. However, the most significant causative factors appear tobe traumatic tooth brushing techniques and an accumulation ofplaque as a result of inadequate dental hygiene (Dominiak andGedrange, 2014), but also the direction of orthodontic tooth move-ment seems to have a significant influence on the development andprogression of gingival recession, especially in the front section of

the mandible. Warmuz et al. (2015), for example, could demon-strate that, in patients with skeletal class III malocclusion, gingivalrecession of the lower incisor teeth occurred significantly more fre-quently in cases in which a camouflage treatment with retrusion of

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he front teeth instead of a surgical correction, with more verticalositioning of the front teeth in the alveolar ridge, was chosen.

Prior to periodontal surgery, conservative treatment should bendertaken in order to eliminate potential causative factors. Theositive long-term effect of treatment depends on close and effec-ive cooperation between the patient and the attending physician,ho should monitor the course of tissue healing and prevent the

ecurrence or formation of any new gingival defects.Surgical management can involve a number of techniques. The

ost commonly applied methods involve the use of coronal and lat-ral repositioned flaps, combined with free gingival or connectiveissue grafts in tandem with GTR procedures (Harris, 2000; McGuirend Cochran, 2003; Nelson, 1987; Nickles et al., 2010; Zucchellit al., 2014). In regenerative periodontal therapy in severe cases,utogenous transplantation is considered the gold standard (Zietekt al., 2008) and subepithelial connective tissue has been proveno have the highest clinical effectiveness and the best aestheticesults (Nickles et al., 2010; Zucchelli et al., 2014). However, autoge-ous connective tissue procedures entail a second operating field,

longer operating time, patient discomfort, and a larger amountf analgesics (Fickl et al., 2014; Fletcher et al., 2011; Zucchellit al., 2010). To eliminate these inconveniences, attempts are beingade to utilize substitutes of autogenous connective tissue; i.e.

enogenous or allogeneic grafts (Barker et al., 2010; Hodde et al.,007; McGuire and Cochran, 2003; Wang et al., 2014).

The only allogeneic material used for surgical covering of gin-ival recessions, broadening the keratinized gingiva zone, and foringival augmentation, is the acellular human dermal matrix allo-raft. Although a number of papers discuss the use of human fasciaata fermoris allografts in dental surgery, there are no studies thatescribe its role in gingival recession coverage (Callan, 1993; Sezert al., 2004). The histological advantage of autogenous connectiveissue compared to acellular dermal allografts (Alloderm), is theresence of cells and a network of blood vessels that considerablyromotes the incorporation in the recipient site. As a result, Allo-erm is used in combination with keratinocytes, fibroblasts androwth factors to further accelerate the healing and to improvelinical effects (Novaes et al., 2007; Zurek et al., 2015). According tohaussain Miller et al. (2002), gingival fibroblasts are more effec-ive in remodeling connective tissue and ensuring faster healinghan dermal fibroblasts. Fibroblasts are the main cellular compo-ent of fibrous connective tissue. They are spindle-shaped with anval nucleus and with one or several nucleoli. The cytoplasm con-ains numerous ribosomes and polysomes, extended mitochondria,n endoplasmic reticulum and single vacuoles. Light microscopylso revealed evenly distributed bundles of microfibers in the cyto-lasm. The fibroblasts are arranged parallel to one another in weakoncentrations inside the tissue (Poggi et al., 2000). Allograft-typeaterials in the recipient site act on the basis of a barrier mem-

rane that forms a place for gingival fibroblasts, which are mainlyesponsible for the production and construction of the connectiveissue of the gingiva and the root cementum (acellular, extrinsicber cementum). In addition, there is no need to re-perform therocedure to remove material due to the resorption of the collagen,ontained in the implanted allogeneic material (Sezer et al., 2004).emoral Fascia lata is biocompatible and well tolerated by the tissuen donor sites. Immune system cells directed against foreign bodies

ere not shown to be present. Choe and Bell (2001) demonstratedhe presence of intact DNA in freeze-dried gamma irradiated cadav-ric fascia lata and acellular cadaveric dermis in comparison to freshuman rectus fascia. Hathaway and Choe (2002) demonstratedhe presence of intact DNA that had not been completely elim-

nated when preparing 4 different commercial human allograftsssessed in their study. Fitzgerald et al. (2000) did not observe theresence of any HLA donor antigens 1 year after the grafts hadeen implanted, using both freeze-dried fascia lata allografts and

omy 204 (2016) 63–70

Tutoplast fascia lata allografts, and additionally stressed that theyhad been replaced by host antigens.

The purpose of this study has been to provide a histologi-cal assessment of tissue in the recipient site at different healingstages of a hydrated biostatic femoral fascia lata graft (Fascia LataAllograft—FLA) in augmentation and multiple gingival recessioncoverage procedures.

2. Material and methods

A total of 12 generally healthy patients with an average age of27 years, who had given their informed consent prior to treatment,including 7 women, took part in the study. Power analysis was per-formed with the software “R” (version 3.2.2; The R Foundation ofstatistical computing) giving a power of 86% (n = 12; alpha = 0.05).The study was carried out in accordance with the recommendationsof the Helsinki Convention of 1975, updated in 2000, and approvedby the Bioethics Committee of the Medical University of Silesia inKatowice (No. KNW/0022/KB1/107/12) as well as by the BioethicsCommittee of the Wrocław Medical University (No. KB-104/2014).

Patients with bilateral gingival recession over 2 mm in height onthe facial aspect of their maxillary teeth were included in the study.The subjects were registered and prepared for gingival recessioncoverage in accordance with accepted dental practice guidelines,which included an informed consent form. Initial periodontal ther-apy, including recommended oral hygiene measures and adultprophylaxis, was performed prior to surgery.

The test side underwent multiple gingival recession coverageusing a tunnel technique combined with a Fascia Lata Allograft(FLA). FLA is a highly cross-linked, hydrated collagen matrix builtfrom type I and type III collagen, prepared, preserved and stored,respectively, in the Katowice (Poland) Tissue Bank in a waydescribed earlier (Zurek et al., 2015). The same procedure wasobserved for the control-side, although with autogenous connec-tive tissue graft, harvested from the palatal mucosa, being usedinstead. The recipient sites of the test side were histologicallyassessed in all patients. Biopsies were obtained from each patient, at4 observation intervals: 3, 6, 9 and 12 months after the procedures,respectively. By reason of multiple recession coverage, a differentinterdental space was chosen for the biopsies at the different obser-vation periods, respectively, so that a total of 12 biopsies could beobtained from each time point. Due to the widely known literatureabout the histology of autogenous connective tissue graft (Romanet al., 2010), histological samples had only been collected from thetest side.

2.1. Surgical procedure

Prior to treatment professional oral hygiene instruction wasprovided to the patients. In case of dental calculus, it was removedwith ultrasound scaler. The gingival augmentation and recessioncoverage procedures were performed in outpatient conditionsunder local anesthesia of 4% articaine with 1:100000 ephinephrine(Ubistesin® forte 3M ESPE, Seefeld, Germany). The test and controltreatments were performed during the same surgical appoint-ment (split-mouth study). During the treatment, root planningwas carried out with Gracey curettes (American Eagle instrumentsInc., Missoula, Montana USA). Autogenous connective tissue washarvested from the palatal mucosa via single incision techniquedevised by Hurzeler and Weng (1999). A collagen sponge (Antema®,Molteni Corlo, Italy) was applied to the wound in the donor site and

the wound was closed with surgical sutures. In both proceduresthe preparation on the recipient side was identical and consisted ofthe forming of supraperiosteal envelopes at each of the affectedteeth, which were then combined with each other to create a

J. Zurek et al. / Annals of Anatomy 204 (2016) 63–70 65

Table 1Overview of the course of the treatment including pre- and postsurgical applications.

Observation time Treatment

Before treatment • Professional oral hygiene instruction of the patients and how to behave after surgery• Removement of calculus (in case) with ultrasound scaler and polishing of the tooth surfaces with rubber polisher• 500 mg Amoxicillin orally 24 h before the treatment, three times a day

Surgical process and post-surgicaltreatment

• Local anaesthesia of 4% articaine with 1:100,000 ephinephrine• Root planning with Gracey curettes• Surgical procedure with CTG and FLA, respectively, using the tunnel technique and mattress sutures• Instructions how to behave in the operation sides:Prohibition of tooth brushing for 7 days and interdental floss for 2 month, chemical plaque control instead using 0.1% CHXmouth rinse 3 times a day• Dietary instructions:Liquid diet for the first day, followed by a semi-liquid diet for the following 3 days, and a soft diet up to the 14th day aftersurgery

Up to 7 days after surgery • 500 mg Amoxicillin orally three times a day up to 7 days after surgery and oral pain killers if necessary• Prohibition of tooth brushing in the operation sides• CHX mouth rinse (chemical plaque control)

7 Days after surgery • Tooth brushing with a ultra-soft postoperative tooth brush and fluoride toothpaste

2 weeks after surgery • Removal of the sutures (CTG) control-side (with scissors and tweezers)• Professional tooth cleaning with professional toothbrush, rubber polisher and clinic paste

3 weeks after surgery • Removal of the sutures (FLA) test-side (with scissors and tweezers)• Professional tooth cleaning with professional toothbrush, rubber polisher and clinic paste• Normal dental care with a soft toothbrush and fluoride toothpaste

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5 and 7 weeks after surgery • Professional tooth cleaning with profe

ingle space–a tunnel. Thereafter, autogenous or suitably trimmedllogeneic connective tissue was inserted in the tunnel, under theartial thickness flap. After positioning, the grafts were fixed inlace with mattress sutures (Seralene® 6/0 Dss-13 Serag, Wiess-er Naila, Germany). Then the flap together with the grafts wasdvanced coronally and stabilized with suspension sutures on eachooth, with the aim of totally covering the denuded surfaces of theiroots. A dose of 500 mg amoxicillin orally was applied three times aay for 7 days, starting 24 h before surgery. After surgery, oral painillers were applied if necessary. The patients were instructed noto clean the teeth with customary toothbrushes on the operationides over a period of 7 days and to refrain from dental floss for 2onths. Instead, chemical plaque control using a mouth rinse con-

aining 0.1% chlorhexidine 3 times a day was prescribed. Dietarynstructions included a liquid diet on the first day, followed by

semi-liquid diet for the following three days and a soft diet upo the 14th day after surgery. After 7 days of healing time, dentalare with an ultra-soft post-operative brush was performed and,fter 3 weeks, normal dental care was performed with a soft tooth-rush and fluoride toothpaste. According to the literature (Allen,010; Alves et al., 2012; Ayub et al., 2012; Barros et al., 2005; Felipet al., 2007; Shepherd et al., 2009), the sutures were removed 14ays post-surgery in the CTG-side and 3 weeks post-surgery in theLA-side. Initially, every 7 days up to the third week and every 14ays between the 4th and 8th weeks after surgery, a professionalooth cleaning was also performed (W&HR Proxeo Set for Prophy-axis; W&H Dentalwerk Bürmoos GmbH, Bürmoos, Austria). Table 1llustrates the treatment at each time point, respectively.

.2. Biopsy method

After local anesthesia with 4% articaine solution plus andrenaline dilution of 1:100,000 (Ubistesin Forte® 3M ESPE, Neuss,ermany), the biopsy material, 2 mm2 in size, was harvested from

he interdental space lying between the covered gingival reces-ions. Two wedge-shaped incisions were made with scalpel bladeo. 15c (Swann Morton, UK). After harvesting, the material waslaced in a sterile test tube filled with a 5% formalin solution. A

l toothbrush, rubber polisher and clinic paste

hemostatic sponge (Antema®, Molteni Corlo, Italy) was applied tothe wound, which was stabilized with a knot suture (Seralon®, 5/0Ds-12 Serag, Wiessner Naila, Germany).

2.3. Histological preparation

Following fixation, the material was put through a CytadelThermo tissue (Thermo Scientific Cytadel) processor using astandard processor program (rinsing in 50, 90 and 98% alco-hol series in turns for 1 h each, rinsing three times with xyleneand embedding in paraffin). After being embedded in paraf-fin blocks and cut with a semi-automatic ShandonTM FinesseTM

325 microtome to a thickness of 7 �m, the material was dyedwith Bio-Optica Milano S.p.a. Mayer’s hematoxylin and Bio-OpticaMilano S.p.a. Eosin Y solution using a Thermo ScientificTM GeminiAS Automated Slide Stainer.

Histological examination was performed by descriptive analysisunder light microscopy with Olympus BX43 microscope.

3. Results

The histological images from all tested patients were similar,showing the same histological course of healing in each patient,with slight intersubjective variability (Table 2). Three months afterthe gingival recession tunnel coverage procedure using FLA, abiopsy of keratinized tissue was harvested from one of the inter-dental spaces adjacent to the augmentation side and preparedfor histological examination. Microscopic images revealed theimplanted FLA, which was clearly delimited and separated from thetissue of the patient’s host mucous membrane (Fig. 1). All the histo-logical images included autologous and augmentation connectivetissue. At 300× magnification no foreign body reaction or charac-teristics of graft rejection were observed at the contact between theconnective gingival tissue and the FLA. Only minor blood extrava-

sation was visible. No inflammatory infiltration was observed inthe mucous membrane above the implanted fascia fragment. Onlylymphocytic-plasmocyte infiltration, which was physiological, waspresent under the epithelium (Fig. 2a). The grafted fascia fragment

66 J. Zurek et al. / Annals of Anatomy 204 (2016) 63–70

Table 2Overview of the histological assessment of each healing stage.

Observation period Biopsies Healing stage

3 months First • Weak connection between fascia lata allograft and mucosa• Mild lymphocytic infiltration• Fibroblast colonization visible

6 months Second • Firm connection between allograft and mucosa• Prominent fibroblast colonization and production of new collagen bundles in the allograft• Vascularization of the allograft

9 months Third • Strong connection between allograft and mucosa• Identical fibroblast distribution in both: mucosa and allograft• Numerou

12 months Fourth • Connectio• Total inco

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ig. 1. Section from the gingiva 3 months after the procedure without any contactetween the Fascia Lata Allograft and the host mucous membrane (100×). Hämalaunosin (HE) staining. MM = mucous membrane, FLA = Fascia Lata Allograft.

iffered from the fibrous connective tissue of the host mucousembrane in terms of having thinner collagen fibers with a more

ndulating arrangement and of lesser density, which was reflected

n the weaker staining of the fibers. In turn, visible at 600× magnifi-ation, individual fibroblasts were lying between the collagen fibersnd colonizing the graft (Fig. 2b). Features of angiogenesis, both inhe mucous membrane and in the grafted fascia fragment were also

ig. 2. Section from the gingiva 3 months after the procedure with contact between the Faf the highlighted parts of Fig. 2a with features of angiogenesis, small numbers of lympämalaun eosin (HE) staining. MM = mucous membrane, FLA = Fascia Lata Allograft.

s capillaries in the allograft

n between mucosa and allograft almost indistinguishablerporation of the allograft

noticeable. A small number of lymphocytes, but no inflammatoryinfiltrations could be seen in the implanted tissue area (Fig. 2c).

Six months after the procedure, the border between the allo-graft and the host connective tissue was still visible in form of aline of collagen bundles (Fig. 3a). Histological images revealed asimilar number of fibroblasts in both tissue fragments and sprout-ing vessels linking the host connective tissue with the implantedfascia fragment. Single lymphocytes, but no inflammatory infiltra-tion was also visible. At 700× magnification, two types of collagenfibers could be distinguished in the fascia region—more slightlystained, constricted and degenerated fibers originating from thegrafted fascia, colonized by individual fibroblasts, as well as newlyformed fibers, that were more intensely stained, thicker, with amore regular arrangement and a larger number of fibroblasts.Many capillaries were visible throughout the entire fascia fragment(Fig. 3b).

After 9 months, the grafted fascia fragment had strongly con-nected with the host mucous membrane. The implantation site wasonly distinguishable due to the slightly different arrangement of thecollagen fibers. At 200× magnification the collagen bundles in the

host connective tissue and the graft became stained with the sameintensity, and the distribution of fibroblasts in fibers was identical(Fig. 4a). At 700× magnification, numerous capillaries, penetratingthe graft, were visible at the border of the two areas (Fig. 4b).

scia Lata Allograft and the host mucous membrane (a; 300×). Higher magnificationhocytes (marked by black arrows) but no inflammatory infiltration (b, c; 600×).

J. Zurek et al. / Annals of Anat

Fig. 3. Section from the gingiva 6 months after the procedure. The border betweenthe allograft and the host connective tissue is seen in from of a line of collagenbundles (a; 100×). Single lymphocytes, but no inflammatory infiltration are visible(marked by black arrows). Newly formed collagen fibers, lined by a large number offibroblasts, are more intensely stained (marked by red arrows) (b; 700×). Hämalauneosin (HE) staining. MM = mucous membrane, FLA = Fascia Lata Allograft. (For inter-pretation of the references to color in this figure legend, the reader is referred to theweb version of this article.)

Fig. 4. Section from the gingiva 9 months after the procedure. Only the slightlydifferent arrangement of collagen fibers still indicates the border between thehost connective tissue and the implantation side, illustrating a strong connec-tion between both tissues (a; 200× and b; 700×). Hämalaun eosin (HE) staining.MM = mucous membrane, FLA = Fascia Lata Allograft.

omy 204 (2016) 63–70 67

Twelve months after the procedure, histological images showeda strong connection between the collagen fibers of the host connec-tive tissue and the fibers in the area of the grafted fascia fragment(Fig. 5a and b). The majority of the collagen fibers were thick andintensely stained (Fig. 5c). Numerous vessels originating from thevascular bundle were also visible. No features that may indicateinflammation and “foreign body” reaction in the grafted fascia frag-ment could be detected. The architecture of the connective tissuefrom the biopsy indicated total incorporation of the FL Allograftafter 12 month of healing (Fig. 5a–c).

4. Discussion

Human Fascia Lata Allograft (FLA) is a biodegradable natural tis-sue with high elasticity and flexibility and therefore exhibits tensilestrength and is easy to fit; furthermore it is biologically compatible,has a minimal risk of infection, immunological response and is safeto use (Detorakis et al., 2005; Dufrane et al., 2003; Sezer et al., 2004).Due to stimulative effects on connective tissue formation, it sup-ports a rapid wound healing and is finally replaced by connectivetissue with no immunological or foreign-body reactions (Burres,1999; Groutz et al., 2001; Sezer et al., 2004).

So far FLA was used for several indications, mainly in humanmedicine, as for example ligament reconstruction in orthopedics(Dong et al., 2012; Yamakado et al., 2001), as dura mater substitute(Dufrane et al., 2003), for reconstruction of the orbital floor (Celikozet al., 1997) as well as in urology (Dong et al., 2012). In dentistry FLAwas used for vestibuloplasty (Sezer et al., 2004), rehabilitation oforal mucosal defects (Papakosta et al., 2007), adjacent to implants(Silverstein et al., 1992) or as natural material for augmentation ofsoft tissue prior to implant placement in the edentulous jaw (Callan,1993).

The use of FLA for gingival recession coverage is a highly newscope of application and has to the best of our knowledge rarelybeen described in the literature. Limited clinical data of the use ofFLA in dentistry are available, which are mainly case series (Callan,1993; Papakosta et al., 2007). Only a few studies have histologicallyassessed the remodeling of gingival tissue following augmentationand gingival recession coverage procedures using both connectivetissue and its substitutes (Cummings et al., 2005; Goldstein et al.,2001; Harris, 1998, 1999, 2000; Majzoub et al., 2001; Richardsonand Maynard, 2002), but reports are highly promising.

Harris, for example, performed a punch biopsy three monthsafter a gingival recession coverage procedure using an allograftacellular dermal matrix, which revealed the presence of elastinfibers and in turn demonstrated the incorporation of the graft inthe host tissue (Harris, 2000). Luczyszyn et al. (2007) also confirmedthe full incorporation of ADMA (Acellular Dermal Matrix) in con-nective tissue 12 weeks following surgery in dogs and Al Hezaimiet al. (2014) assessed the histological results of periodontal tissueremodeling in baboons 16 weeks after a procedure that involved theuse of an extracellular matrix membrane—ECM (Dynamatrix, CookBiotech) in the coverage of surgically induced gingival recessions.ECM is the submucosa of the small intestine of pigs, containingtype I, III, IV and VI collagen (Hodde et al., 2007). Compared to ourstudy this material comes close to the FLA used in our setting. Theauthors also noted new collagen fibers forming a new periodontalligament as well as ECM remnants (Al Hezaimi et al., 2014). Newcollagen fibers could also be detected in our study, which were thin-ner, of lesser density and had a more undulated arrangement after3 months, but with increasing time became thicker and had a more

regular arrangement (after 6 months) until finally, after 9 and 12months, they were no longer distinguishable from the host tissue.

In a randomized study in mongrel dogs, Novaes et al. (2007)placed alloderm alone on one side of earlier formed supraperiosteal

68 J. Zurek et al. / Annals of Anatomy 204 (2016) 63–70

Fig. 5. Section from the gingiva 12 months after the procedure indicating full integration of the FLA in the host connective tissue (a; 200× and b; 400×). Numerous fibroblasts( Well-n w) (c;A der is

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marked by blue arrows) are indicative of a high activity of new fiber production.umerous vessels are originating from the vascular bundle (marked by green arrollograft. (For interpretation of the references to color in this figure legend, the rea

eds around premolars, applied alloderm with autogenous fibro-lasts on the other side, and then covered and sutured them with

partial thickness flap. Histological assessment was performed 2, and 8 weeks after the procedure. After 2 weeks, light microscopyt 40× magnification revealed a zone of dense collagen fibers thatas arranged similarly to those in the surrounding connective tis-

ue. In both groups (alloderm plus fibroblasts and alloderm alone)hinner blood vessels than in the surrounding connective tissueere observed, although they were significantly larger than in the

amples with fibroblasts. Further incorporation into the surround-ng tissue was noted after 4 weeks. In both groups, after that time,he difference between the quantities of blood vessels faded. Like-ise, inflammatory infiltration decreased. After 8 weeks, alloderm

howed even better vascularization, a large amount of collagenbers and a decreased inflammatory infiltration compared to theistological images after 2 and 4 weeks (Novaes et al., 2007).

Our histological images also showed features of angiogenesisoth in the mucous membrane and in the grafted fascia fragment

month after surgery. With increasing time, the number of bloodessels also increased, linking the host connective tissue with themplanted fascia fragment. No foreign body reaction, inflammatorynfiltration or characteristics of graft rejection could be observed athe contact between the connective gingival tissue and the FLA,hich finally, after 12 month, became indistinguishable from theost tissue.

Dominiak et al. (2012) covered gingival recessions with a culturef primary human fibroblasts on a xenogenous collagen membrane,nd also widened and augmented the gingiva of 34 anterior teeth in0 patients. Saczko et al. (2008) described a process that involvedaking biopsies from masticatory mucosa, mechanically isolatingnd culturing the sections. For final growth, the fibroblasts werelaced in a restorable collagen membrane on which they remainedor 3 days. The total cultivation time was 7–10 days. In the recip-

ent sites a partial thickness flap with an intact periosteum wasormed, a collagen carrier with fibroblasts was inserted, and theap was coronally advanced and stabilized with surgical sutures.he sutures were removed after 14 days. Twelve weeks after the

organized new fibers are thick and intensely stained (marked by red arrows) and 700×). Hämalaun eosin (HE) staining. MM = mucous membrane, FLA = Fascia Latareferred to the web version of this article.)

procedure a biopsy was extracted from each patient for histologi-cal assessment. Each section contained mature connective tissuecovered by epithelium with a basal membrane. The amount offibroblasts and collagen matrix located in the connective tissuewas moderate. However, no inflammatory infiltration and also noremnants of collagen membrane were found to be present. In thepresent study, 3 months after multiple gingival recession coverageusing a tunnel technique combined with FL Allograft, biopsies wereextracted. Histological images revealed the graft colonized by thehost fibroblasts, the fascia was clearly visible and had undergonevascularization. Neither inflammatory infiltrates nor any foreignbody reaction was visible. In their study, Dominiak et al. (2012)similarly noted the absence of inflammatory infiltration and foreignbody reaction and Callan (1993) as well as Papakosta et al. (2007)also did not clinically observe any graft rejection or infections afterimplantation of fascia lata femoralis or human FL Allograft as cov-erage after bone grafting or as coverage of oral mucosal defects inhumans, respectively.

On the other hand, Richardson and Maynard (2002) reportedthat 16 days after implantation of an acellular dermal allograft dur-ing flap surgery in humans, the histological specimens revealedincomplete incorporation of the graft in the recipient site. The pro-cedure was performed on a 44-year old woman and concerned acanine with a healthy periodontium, on which a full thickness flapwas formed and an ADMA was implanted in a typical position incontact with the root of the tooth and the bone of the alveolar ridge.Due to extensive dental caries, the tooth was to be extracted. Aftertooth extraction and histological preparation, sections containingsoft tissue, tooth and bone were assessed under light microscopy.The most coronal regions in which the ADMA came into contactwith the root were free of blood vessels and no histological attach-ment was found. Only that part located on the surface of the alveolarridge displayed resorption and had been replaced by host connec-

tive tissue (Richardson and Maynard, 2002). In a histological studyin humans, Cummings et al. (2005) showed that ADMA used forgingival augmentation procedure formed an attachment in formof a combination of long junctional epithelium and connective

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issue adhesion six months after surgery. The ADMA implanted areaas colonized by fibroblasts and possessed new collagen fibers, but

lso sustained its own remaining plastic fibers. The course of theew fibers was regular with the majority running parallel to theoot surface. Compared to a human block section assessed at theame time, that is after connective tissue gingival augmentation,he histological image following ADMA implantation was similarCummings et al., 2005).

In our study an assessment of the structure of the clinicalttachment was not included. After 6 months, the histologicalmages in the FL Allograft area and at the border of the hostoft tissue were similar to those described by Cummings et al.2005). The FL Allograft was also incompletely incorporated 6

onths after the procedure. Angiogenesis and new vessels spread-ng toward the implanted fascia were evident at the interfaceetween the fascia and the host connective tissue. After a furtherealing period, histological images showed an increase of the con-ection between the implanted fascia fragment and the mucousembrane by the production of new collagen fibers originating

rom the grafted fascia. A specimen of gingiva assessed 12 monthsfter the augmentation procedure contained fibrous connectiveissue of typical architecture with correctly formed, cigar-shapedbroblasts, indicating a full integration and remodeling of theraft.

. Conclusions

The Fascia Lata Allograft used in multiple gingival recessionoverage procedures did not trigger any inflammatory reactionr foreign body reaction in the host tissue. Fascia Lata Allo-raft was easily colonized by host fibroblasts, which were slowlyemodeled into gingival connective tissue. Bearing in mind theimitations of the present study, we conclude that Fascia Lata Allo-raft may serve as a substitute for autogenous connective tissue,arvested from the masticatory mucosa, which can be used toover multiple gingival recessions. Histopathological examinationevealed that it is well tolerated by the host tissue in the recipientite.

onflict of interest statement

The authors claim that there are no conflicts of interest.

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