Evaluation of the tissue reaction to a new bilayered collagen matrix in vivo and its translation

to the clinic

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Biomed. Mater. 6 (2011) 015010 (12pp) doi:10.1088/1748-6041/6/1/015010

Evaluation of the tissue reaction to a newbilayered collagen matrix in vivo and itstranslation to the clinicShahram Ghanaati1,2,8, Markus Schlee3, Matthew J Webber4,Ines Willershausen5, Mike Barbeck1, Ela Balic6, Christoph Gorlach6,Samuel I Stupp7, Robert A Sader2 and C James Kirkpatrick11 REPAIR-Lab, Institute of Pathology, Johannes Gutenberg University, Mainz, Germany2 Clinic for Maxillofacial and Plastic Surgery, Johann Wolfgang Goethe University, Frankfurt Am Main,Germany3 Bayreuther Strasse 39, D-91301, Forchheim, Germany4 Department of Biomedical Engineering, Northwestern University, Evanston, IL 60208, USA5 Institute for Dental Material Sciences and Technology, University Medical Center of the JohannesGutenberg University, Mainz, Germany6 Geistlich Pharma AG, Wolhusen, Switzerland7 Department of Materials Science and Engineering, Chemistry, and Medicine, Northwestern University,Evanston, IL 60208, USA

E-mail: ghanaati@uni-mainz.de

Received 24 August 2010Accepted for publication 9 December 2010Published 17 January 2011Online at stacks.iop.org/BMM/6/015010

AbstractThis study evaluates a new collagen matrix that is designed with a bilayered structure in orderto promote guided tissue regeneration and integration within the host tissue. This materialinduced a mild tissue reaction when assessed in a murine model and was well integrated withinthe host tissue, persisting in the implantation bed throughout the in vivo study. A more porouslayer was rapidly infiltrated by host mesenchymal cells, while a layer designed to be a barrierallowed cell attachment and host tissue integration, but at the same time remainedimpermeable to invading cells for the first 30 days of the study. The tissue reaction wasfavorable, and unlike a typical foreign body response, did not include the presence ofmultinucleated giant cells, lymphocytes, or granulation tissue. In the context of translation, weshow preliminary results from the clinical use of this biomaterial applied to soft tissueregeneration in the treatment of gingival tissue recession and exposed roots of human teeth.Such a condition would greatly benefit from guided tissue regeneration strategies. Ourfindings demonstrate that this material successfully promoted the ingrowth of gingival tissueand reversed gingival tissue recession. Of particular importance is the fact that the histologicalevidence from these human studies corroborates our findings in the murine model, with thebarrier layer preventing unspecific tissue ingrowth, as the scaffold becomes infiltrated bymesenchymal cells from adjacent tissue into the porous layer. Also in the clinical situation nomultinucleated giant cells, no granulation tissue and no evidence of a marked inflammatoryresponse were observed. In conclusion, this bilayered matrix elicits a favorable tissue reaction,demonstrates potential as a barrier for preferential tissue ingrowth, and achieves a desirabletherapeutic result when applied in humans for soft tissue regeneration.

(Some figures in this article are in colour only in the electronic version)

8 Author to whom any correspondence should be addressed.

1748-6041/11/015010+12$33.00 1 2011 IOP Publishing Ltd Printed in the UK

Biomed. Mater. 6 (2011) 015010 S Ghanaati et al

1. Introduction

The loss of soft tissue in the oral cavity caused by eitherreceding gingival tissue or scar tissue, in addition to beingunaesthetic, can be a tremendous source of pain and suffering.Current gold-standard tissue transfer procedures involvetransplanting an oral autograft, but these can cause additionaldiscomfort and are accompanied by a risk of fatality whentissue is harvested from other parts of the oral cavity suchas the palate due to the proximity of major blood vessels.Synthetic strategies, including the application of biomaterialmembranes, have been developed to combat this withinoral and maxillofacial surgery over the past two decadesusing concepts from the so-called guided tissue regeneration(GTR). In GTR, the goal is to promote specifically healthyconnective tissue ingrowth, while inhibiting that of fibroticscar tissue. This is akin to guided bone regeneration (GBR),where a defect in bone is treated by similar means inorder to facilitate bone ingrowth within this defect, whileminimizing ingrowth of reactive fibrotic tissue. Membranousbiomaterials have been applied to promote preferentially theingrowth of cells involved in the tissue regeneration process,while preventing ingrowth from undesired tissue into thedefect [15]. For a successful outcome, barrier membranesused in GTR must possess certain properties, includingbiocompatibility, preferential tissue integration, place-holdercharacteristics, and physiochemical stability [2, 6]. Thefirst generation of such membranes were primarily made ofpoly(tetraflouroethylene) (ePTFE) [7], a polymer with highstability in biological systems. However, this non-resorbablemembrane requires a second intervention for its retrieval,accompanied by further risk of infection, potential damageto the newly regenerated tissue, and an increased likelihoodof further intervention [8, 9]. Thus, the drawbacks associatedwith retrieval have led to the development of bio-resorbablebarrier materials. Bio-resorbable polyesters with differentbiodegradation properties have been developed for variousapplications in reconstructive surgery including GTR [1012]. However, no satisfying outcome was achieved with thesemembranes, as they exhibited a fast degradation related to aforeign body reaction, which did not permit the desired controlof tissue regeneration.

Collagen-based materials have also been explored forapplications to GTR strategies. Collagen is the mostabundant family of proteins in the human body andis physiologically ubiquitous. Moreover, neutrophils,monocytes, and fibroblasts recruited during wound healingrelease matrix metalloproteases (MMP), which results inenzymatic biodegradation of collagen [13]. Its naturalorigin combined with its relative ease of biodegradationmake collagen a broad candidate for biomaterial applications.Collagen type I is also known to have angiogenic potential[6, 14, 15], a characteristic that makes it further desirable topromote the ingrowth of healthy tissue. In applications asmembranes, however, the biodegradation of collagen provedto be a major disadvantage, since it limited the time scaleover which the membrane exhibited a barrier function. Inorder to decrease the rate of collagen degradation and enhance

membrane stability, several cross-linking techniques havebeen applied [11, 1619]. While this leads to increasedmechanical stability of the membrane [11, 16, 19], it alsoinhibits the attachment and proliferation of human periodontalligament fibroblasts and osteoblasts compared to nativecollagen [20]. Cross-linked collagen has also been reportedto induce a severe foreign body reaction in vivo [21]. As aconsequence, alternative processing techniques for collagenhave been developed. One such technique involves thecombination of both non-cross-linked native collagen III,which undergoes a relatively fast degradation, and collagen I,which is more resistant, in order to regulate the biodegradationof membranes applied in GTR. Recently, a bilayered non-cross-linked collagen-I/III membrane (BioGide R) implantedsubcutaneously in rats was demonstrated to undergo ahomogeneous and transmembranous vascularization after 2weeks and persisted in the tissue for up to 4 weeks [21,22]. It was also shown that membrane thickness continuouslydecreased, with a breakdown 4 weeks after implantation. Afast transmembranous vascularization, while enhancing thetissue integration of the scaffold, at the same time results in aloss of the barrier function of the material. This could provedetrimental to applications in GTR.

The aim of this study was to assess the tissue reaction toMucograft R (MG), a new non-cross-linked collagen matrix. Itis composed of collagen type I and type III without furthercross-linking or chemical treatment. The MG matrix isbilayered with one side being thin, smooth and of low-porosity,while the other is a more porous three-dimensional network(figure 1). In this histological study, special emphasis wasplaced on the comparative tissue integration on both sides ofthe matrix, focusing on host tissue reaction to the materialat each of its different surfaces and the cells involved in thisreaction. Additionally, the material thickness was measuredhistomorphometrically throughout the time points of the study.Finally, we compare our observations developed in a murinemodel to those from a pilot human application of MG to treatsoft tissue defects within the oral cavity, so as to evaluate howthe information gained from our small animal study translateddirectly into the observations from the use of MG as a matrix totherapeutically reconstruct human gingival soft tissue. Thus,it was hoped to test the validity of the animal model for clinicaltranslation purposes.

2. Materials and methods

2.1. Production of Mucograft R

Mucograft R (MG) is a pure collagen matrix obtained by aproprietary standardized manufacturing process and sterilizedby gamma irradiation. The device has been CE-markedand 510(k) FDA-approved. The matrix is made of collagentype I and type III without further cross-linking or chemicaltreatment. The collagen is extracted from a veterinary certifiedporcine source and is carefully purified to avoid antigenicreactions. The collagen is processed into a bilayered matrixwith one side being a thin, smooth and low-porosity compactlayer (CL) and the other a more porous three-dimensional

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(A)

(B )

(C )

(D)

(E)

Figure 1. Scanning electron microscopy of the MG scaffold, showing a low magnification cross section (A). In addition, the face (B, left)and cross-section (C) of the low-porosity compact layer of this scaffold is shown, along with the face (D) and cross-section of the moreporous spongy layer (E). CL = compact layer and SL = spongy layer.

spongy layer (SL) (figure 1). The low-porosity surface, madefrom porcine peritoneum, has elastic properties that permitsuturing to the host mucosal margins. The porous surface,derived from porcine skin, allows tissue adherence favoringwound healing and promoting cell integration. The porosityis obtained through defined parameters and a controlledlyophilization process. This side is turned toward the bonedefect and/or soft tissue to encourage bone-forming cells andtissue growth and to stabilize the blood clot. Both layers arecombined through a biophysical interweaving process withoutany chemical manipulation. The volume fraction of pores inthe matrix is 90% and the size distribution for these poresranges from 5 to 200 m, with smaller pores being primarilylocated on the compact layer and larger pores found in thespongy layer.

2.2. Scanning electron microscopy

Scanning electron microscopy (SEM) was performed atthe Northwestern University Electron Probe InstrumentationCenter (EPIC, Evanston, IL, USA), using a Hitachi S4800scanning electron microscope with a 5 kV accelerating voltage.To prepare samples for imaging, MG was first hydrated in PBSand subsequently dehydrated in ethanol. It was then dried atthe critical point, cut to reveal a cross section, and coated withroughly 9 nm OsO4 prior to imaging.

2.3. Murine experimental design

The Committee on the Use of Live Animals in Teachingand Research of the State of Rhineland-Palatinate, Germany,approved these studies. In total, 30 female 5-week-oldCD-1 mice were obtained from Charles River Laboratories,

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Germany. The animals were maintained for 1 week beforeuse at the Laboratory Animal Unit, Institute of Pathology,Johannes Gutenberg University of Mainz, Germany, asfollows. They were fed with regular mouse pellets (LaboratoryRodent Chow, Altromin, Germany), water was available adlibitum and there was an artificial lightdark cycle of 12 heach. The mice were randomly distributed into two groups.The first group (n = 4 animals/time point) was implanted withMG while the second group (n = 2 animals per group) servedas a sham-operated control with no biomaterial implantation.The time points evaluated were 3, 10 15, 30 and 60 days.Sterile MG samples of 10 10 cm were implanted with thespongy side facing down into preformed subcutaneous pocketsof animals rostral subscapular region, according to a previouslydescribed method [23]. All animals survived the operation andsubsequent evaluation period without complications.

2.4. Tissue preparation for histology andimmunohistochemistry

After explantation at the specified time points, tissue wasprocessed for histological analysis as has been previouslydescribed [23]. Sections from each region were stained withMayers hematoxylin and eosin (H&E), Azan, Sirius Red,Ladewig, and Movats Pentachrome. Murine vessels andmacrophages were detected immunohistochemically by meansof an anti-rabbit polyclonal CD31 antibody (GeneTex, Inc.,USA) and a monoclonal F4/80 antibody (eBioscience, SanDiego, USA), respectively. The DAKO REALTM EnVisionTMdetection system (DAKO, Glostrup, Denmark) was used forvisualization. Counterstaining of the immunohistologicalsamples was performed with hemalaun.

2.5. Morphological evaluation of the inflammatory responseHistopathological evaluation was carried out using apreviously described method [23, 24]. Briefly, cellularinflammatory component (i.e. neutrophils, lymphocytes,plasma cells, macrophages, giant cells) and biomaterialvascularization and degradation were qualitatively andquantitatively evaluated by light microscopy of histologicalsections and software quantification tools.

2.6. Thickness measurements

Thickness measurements of the matrix were done usingpreviously described histomorphometric methods [23, 24].Briefly, the region of interest containing the MG biomaterialand the corresponding peri-implant tissue was used to generateof a total scan which assembles 100120 images into one largeimage at 100 magnification. For each animal the thicknessof both the compact layer and the spongy layer was measuredat each time point in order to assess the dynamic degradationof these components over time. The material was measuredat up to 15 points along the histological cross-section foreach animal at each time point, and the total thickness wascalculated from the mean of these measurements. This meanwas taken as the total thickness for the layers of the material.

2.7. Pilot human Mucograft R useA clinical application of MG was performed at the privatepractice of one of the co-authors (MS). In this paper, weprovide evidence from this trial only in so far as to confirm ourfindings in vivo and thus support a possible clinical translationof Mucograft R. A future report, detailing the full clinicalmeasures and outcomes of this study, will follow. In thisstudy, a total of 11 patients (7 females, 4 males) who hadat least one buccal recession classified as Miller class I orII were selected and each patient gave written consent foroutcomes of this study to be used anonymously for researchand publication purposes in accordance with local ethicalguidelines. In total, 54 recessions were treated with MG.Patients were otherwise healthy and were verified to haveno allergy to porcine products and no known anaphylacticreaction. After local anesthesia, the exposed root was scaledand planed to the bottom of the pocket with rotating burs,ultrasonic instruments and curettes. Deeper instrumentationwas avoided to prevent fibrous attachment. The root wasflattened, smoothened and decontaminated. No chemicalroot conditioning was performed. After sulcular incision,a coronal displaced split thickness flap without releasingincision was performed, according to the incision methodpreviously described [25]. The flap preparation, in contrastto Zucchelli, was a complete split thickness flap. The flapwas considered to be immobilized enough when it stayedpassively at a level coronal to the cemento-enamel junction.Beside complete root coverage, the aim of this surgery wasto enhance the thickness and stability of gingival tissue. TheMG collagen matrix was placed underneath the prepared flap.After rehydration, the matrix becomes flexible, remains stable,and is readily sutured. The matrix was adapted in sizeand fixed by sling sutures around the recipient teeth. Thematrix was trimmed to reach at least a level 2 mm apicalthe bony margin. The coronal position was 1 mm belowthe cemento-enamel junction. The knots were placed on thelingual aspect, making removal painless and atraumatic. Thecoronal advanced flap completely covered the matrix and wasalso fixed by sling sutures to accomplish a precise adaptationaround teeth. Following surgery, patients were instructed toavoid mechanical trauma to the wound. Sutures were removed2 weeks after surgery. Patients were recalled at time pointsfollowing this for a professional supragingival tooth cleaningand remotivation.

One patient in this study was recalled at 6 weeks forcomplications unrelated to the Mucograft R scaffold itself, butto make adjustments to the surgical procedure. In the courseof this procedure, a biopsy was obtained which allowed ahistological evaluation of the performance of the MG scaffoldat this point. Again, written consent was received from thepatient to use the results of this biopsy for research publicationpurposes. The results from this biopsy, and their connectionto the results we have seen in the subcutaneous implantationmodel, will be the primary focus for this report. Tissue wasprocessed for histology by similar means to that describedfor murine tissue and the tissue was stained using H&E andAlizarin Red.

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3. Results

3.1. SEM of the scaffoldSEM of a cross-section of the MG scaffold revealed themorphology of the bilayered matrix (figure 1(A)). Thecompact layer (CL), designed to be less cell permeable, wasfound to have a very low-porosity surface (figure 1(B)), whilethe cross-section of the matrix revealed that this layer consistedof large parallel sheets of densely compressed collagen fibers(figure 1(C)). The spongy layer (SL), designed to facilitatetissue ingrowth, had a surface that was highly porous andhad more randomly aligned and diffusely packed collagenfibers (figure 1(D)), with a cross-section of this region havingvery similar structural characteristics (figure 1(E)). Overall, thestructural characteristics of the matrix suggest it has potentialfor use as a GTR scaffold, with two very structurally differentlayers that would, presumably, promote preferential tissueinfiltration. The cross-sectional thickness of the membranein SEM was of the order of 1.61.8 mm, with a compact layerof roughly 0.4 mm and a spongy layer of roughly 1.3 mm.

3.2. Murine histological results

Animals implanted with MG showed no signs of hemorrhageor necrosis at the implantation site. At 3 days followingimplantation, there was no evidence of capsule formationaround the material. The cells in the peri-implant tissue oneither side of the matrix were mainly fibroblasts with a fewmacrophages, while there was almost no observed neutrophilinfiltration (figure 2(A)). Histologically, both the compactand spongy layers of the MG scaffold were easily detectable.Both components were well integrated within the implantationbed (figure 2(A)). At this early time point, tissue ingrowthwas already observed from the spongy surface of the matrix,indicated by evidence of several infiltrating cells (figure 1(B)).However, on the compact surface of the matrix, a cell-richconnective tissue was observed adjacent to the matrix whichenabled cell attachment but appeared to simultaneously inhibitthe ingrowth of tissue into the matrix, as few cells could beseen infiltrating this side (figure 1(C)).

At day 10, the two components of the matrix were stilldistinctly visible by histology (figure 3(A)). The compact layerstill appeared to inhibit the penetration of the peri-implantcells into the core of the matrix, but the beginnings of aningrowth of peri-implant cells were evident within the layersof the dense component at this time (figure 2(B)). The tissueingrowth into the spongy layer that was originally observed atday 3 had progressed, with more cells reaching deeper withinthe scaffold and beginning to secrete their own matrix withinthe collagen fibrils (figure 3(C)). Immunohistological stainingwith F4/80 showed the presence of macrophages within thematrix and suggests their possible role in the degradation ofthe biomaterial (figure 3(D)).

At day 15, the tendency for tissue ingrowth that was seenon day 10 for both sides of the matrix continued (figure 4(A)).The two layers of the matrix also remained clearly visibleby histology. The compact layer still serves as a barrier forcell ingrowth into the core of the matrix, with some cells

(A)

(B)

(C )

Figure 2. Post-implantation day 3 histological images showing across-section of the scaffold with Azan stain at 100 (A) as well asthe interface between the host tissue and the spongy layer with H&Estain at 400 (B) and the corresponding interface for the compactlayer with H&E stain at 400 (C). Cells (arrows) can be seenpenetrating the spongy layer, which the compact layer shows verylittle cell infiltration. SL = spongy layer, CL = compact layer andST = subcutaneous tissue. All scale bars are 100 m.

being diffusely spread through the thick sheets of collagen(figure 4(B)). The spongy layer was invaded by peri-implanttissue, with a further increase in matrix production by thesehost cells (figure 4(C)). Additionally, immunohistochemicalstudies showed an increase in macrophages in this spongylayer compared to day 10 (figure 4(D)).

By day 30 after implantation, significant changeswere evident in the histology of the matrix (figure 5(A)).Specifically, at this time the compact layer of the matrix nolonger maintained its function as a non-penetrable barrier, ascells could be seen to have traversed this layer and enteredthe deeper regions of the scaffold. At this time, cells hadpenetrated the scaffold from both surfaces of the matrix andbecame more dispersed throughout (figures 5(B) and (C)).

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(A) (B )

(C ) (D)

Figure 3. Post-implantation day 10 histological images showing a cross-section of the scaffold with H&E stain at 100 (A) as well as theinterface between the host tissue and the compact layer with H&E stain at 400 (B) and the corresponding interface for the spongy layerwith Movats pentachrome stain at 400 (C). Cells continue to penetrate the spongy layer and secrete their matrix, while the compact layercontinues to inhibit cell penetration of the scaffold, though cells can be seen between the sheets of collagen in this layer. F4/80 staining formacrophages in the spongy layer (D) reveals some at the surface and beginning to enter the scaffold at 400. SL = spongy layer, CL =compact layer and ST = subcutaneous tissue. All scale bars are 100 m.

At day 60 after implantation, the dense component ofMG was much less visible and what remained of the spongylayer of the matrix was invaded by a vascularized connectivetissue (figure 6(A)) and had become very well integrated withinthe implantation bed (figures 6(B) and (C)). There were nomultinucleated giant cells and only a few lymphocytes wereobserved within the implantation bed throughout the entireobservational period, with the degradation of the materialbeing primarily performed by macrophages. Accordingly, atno point in the histological study did we observe a typicalforeign body response.

3.3. Histomorphometric assessment of thicknessUsing histomorphometric measurements of a total scan of thebiomaterial, we were able to track the total MG thicknessas well as the thickness of each bilayer, as these layerswere distinctly visible through histology due to their verydifferent morphologies (figure 7). These measurementsrevealed that the compact layer was more inclined to bethinned by invasion from host cells than the spongy layer.The total scaffold thickness at day 3 was 980 90 m,consisting of roughly the same thickness of a spongy layer(479 80 m) and a compact layer (501 46 m). Boththe total thickness and the thickness of the spongy layerdiffered from those seen in SEM of the scaffold, possiblyas a result of factors arising from the compression of the

spongy layer on subcutaneous implantation, scaffold wetting,or potential histological artifacts. The thickness of the compactlayer is similar regardless of technique. Over the course ofimplantation, the thickness of the spongy layer decreased to289 68 m, a decrease of 40% from the day 3 thickness.The compact layer, on the other hand decreased considerablyin thickness to 137 33 m by day 60, a decrease of almost73% compared to its value on day 3. The MG scaffold as awhole had a thickness of 426 62 m at day 60, representing adecrease in thickness of 57%. The largest change in thicknesswas in the compact layer between days 10 and 15, whichpreceded the breakdown of this layer as a barrier by day 30.

3.4. Scaffold vascularizationUsing CD31 staining, we evaluated the material for its effecton vascularization of the peri-implantary tissue, particularlyat the tissue interface of the spongy layer of the material. At3 days following implantation, there was some evidence ofvessels in the peri-implant space (figure 8(A)), but this did notappear to be a significant increase from normal physiologicallevels. At 10 days, there were still only a few vessels foundin the peri-implant region (figure 8(B)). By 30 days followingimplantation, a few vessels were observed within the scaffoldperiphery (figure 8(C)) and this did not change considerablyby day 60 (figure 8(D)), with the center of the scaffold being,

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(A) (B)

(C) (D)

Figure 4. Post-implantation day 15 histological images showing a cross-section of the scaffold with Ladewig stain at 100 (A) as well asthe interface between the host tissue and the compact layer with Azan stain at 400 (B) and the corresponding interface for the spongy layerwith Movats Pentachrome stain at 400 (C). Cells continue to penetrate the spongy layer and secrete their matrix, while the compact layercontinues to inhibit cell penetration of the scaffold, though cells can be seen between the sheets of collagen in this layer. F4/80 staining formacrophages in the spongy layer (D) reveals even more at the surface and beginning to enter the spongy layer of the scaffold at 400. SL =spongy layer, CL = compact layer and ST = subcutaneous tissue. All scale bars are 100 m.

at most, sparsely vascularized. Overall, at no point was there adramatic increase in vascularization in the peri-implant tissueand the scaffold itself was minimally vascularized, beginningat the periphery and progressing slowly to the center.

3.5. Human Mucograft R pilot studyThe murine in vivo studies demonstrated a very favorable tissuereaction as well as preferential control over tissue ingrowth,with cells penetrating the scaffold from the spongy layerbefore they were able to penetrate the compact layer. Thispoints to the possibility of MG scaffolds as a barrier for softtissue regeneration. As mentioned, one clinical need for suchmaterials is for the treatment of exposed gingival roots inteeth. Thus, MG was evaluated for its ability to serve asa barrier and promote tissue ingrowth in this application.Shown here is an example from a single patient with anexposed root from receding gingiva (figure 9(A)) and whoreceived MG as therapy. The gum was retracted to make aflap and MG was inserted so that the spongy side was facingthe tooth (figure 9(B)). The flap was then sutured in placeover the MG scaffold. A follow-up after treatment revealedthat the gum level has returned to normal height, with thecolor and appearance matching that of healthy gingival tissue(figure 9(C)). These results suggest the potential for MG to besuccessfully translated to the clinic in this application.

At 6 weeks following treatment, a biopsy of the scaffoldand surrounding tissue was collected as part of another

procedure. The histology of this biopsy corroboratedthe findings in the murine evaluations. We observeda distinct difference in the tissue reaction of the twolayers (figure 9(D)), with the spongy layer demonstratinggood tissue integration with the surrounding connectivetissue and substantial cell infiltration, while the compactlayer appeared to be much less infiltrated with cells,though it still remained integrated in the host tissue atits surface. The spongy layer was also well integratedwithin the neighboring connective tissue (figure 9(E)).As was seen in the murine model, there was no evidence ofan adverse tissue reaction, with no multinucleated giant cellsand no lymphocytes present. Also, there was minimal scaffoldvascularization, consistent with our findings of a low level ofvascularization in the murine model. The results from thisbiopsy closely mirrored those from our in vivo evaluation interms of tissue reaction and preferential tissue ingrowth.

4. Discussion

In this in vivo evaluation, we examined the tissue reaction toa bilayered porcine collagen I/III matrix with a compact andspongy side. Special emphasis was placed on the integrationof this material within the implantation bed for each of itstwo morphologically distinct surfaces. In the animal study,no capsule formation was observed around the implantedmatrix and there was no acute accumulation of neutrophils

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(A)

(C)

(B)

Figure 5. Post-implantation day 30 histological images showing a cross-section of the scaffold with Azan stain at 100 (A) as well as ahigher magnification image with H&E stain at 200 (B) and a total scan of a histological slide with H&E stain at 100 magnification(C) showing cells that have progressed to the center of the scaffold from both sides, indicating a loss of barrier function for the compactlayer. SL = spongy layer, CL = compact layer and ST = subcutaneous tissue. All scale bars are 100 m.

and macrophages following 3 days of implantation. Thecellular degradation of the biomaterial was initiated primarilyby macrophages, with no evidence of multinucleated giantcells on either matrix surface at any time point of the study.Our findings suggest that there are no issues pertaining to theporcine collagen source, as there was no sign of any adverseimmunological reaction within the host, nor was there evidenceof a foreign body reaction at any point in the study. It is knownthat giant cell formation is dependent on biomaterial surfacemorphology, requiring the adsorption of a specific spectrumof proteins in order to trigger fusion of adherent mononuclearcells into multinucleated giant cells [2527]. As no giantcells were seen, the study demonstrates that macrophagesare sufficient for biodegradation of the MG matrix. Ourpresent findings are in agreement with observations for similarmaterials in other in vivo studies that have demonstrated goodtissue integration and shown no foreign body reaction for non-cross-linked porcine derived collagen I/III membranes [21].

In this study, the center of the MG matrix did notbecome vascularized until some point between days 30 and60, which contrasts with studies in similar collagen I/IIImaterials that demonstrate vascularization 2 weeks followingimplantation [22]. This is most likely attributable to thedifferences in material processing for the MG matrix comparedto this other collagen I/III membrane. We have previouslyshown that changes in material processing for biomaterialscaffolds can have a significant effect on the resulting tissuereaction [23], so that differences in processing methodologycould certainly account for the variance between our results

and those published for other collagen-I/III materials. Theslow vascularization of MG is likely a result of the barrier-like function of the compact collagen layer, which did notallow cell infiltration until 30 days following implantation.Vascularization of the spongy layer began at 2 weeks, whichis in accordance with published reports. The degree to whichthe material is vascularized is low, perhaps on account of therelatively small amount of granulation tissue formed. Wehave previously shown that similar subcutaneous implantationof silk fibroin (SF) leads to an active granulation tissuewithin the implantation bed and corresponds to a higherdegree of scaffold vascularization [23]. Although SF isalso a natural-based biomaterial, its implantation evoked theactivation of macrophages and their fusion into multinucleatedgiant cells within the implantation bed. These cells, whichwere found on the surface of the SF fibers, were involved indegradation of the SF scaffold and their number decreased asthe material was degraded. Multinucleated giant cells are aknown participant in the foreign body response and arise frommacrophage fusion. Since SF and collagen are both naturallyderived protein-based materials, but yet have different levelsof vascularization, it is possible that multinucleated giant cellsplay a role in scaffold vascularization as part of the foreignbody response. Our hypothesis is that the host tissue reactionis, in some way, related to the ease with which the material isdegraded. Materials based on collagen, which is ubiquitousin mammalian tissue, may not activate the degree of cellularimmune response that a naturally derived but foreign materialsuch as SF will, perhaps due to the structural similarity ofthe implanted collagen to physiological tissue. However,

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(A)

(B)

(C )

Figure 6. Post-implantation day 60 histological images showing across-section of the scaffold with sirius red stain at 100 (A) aswell as higher magnification images of the material-tissue interfacewith Ladewig stain and H&E stain at 200, (B) and (C). Thematerial is totally invaded by cells and appears more as a connectivetissue, though there is only some evidence of blood vessels (arrow inC). The spongy layer of the material is well integrated within thisnew connective tissue. SL = spongy layer and ST = subcutaneoustissue. Arrow heads in (A) show the materialtissue interface, whilethe double-headed arrow demonstrates the spongious layer of thematrix. All scale bars are 100 m.

even among collagen scaffolds, the tissue reaction may varydepending on such parameters as scaffold thickness, porosity,chemical cross-linking, source, and purification methods. Inthis study very little inflammation was seen and, perhapscorrespondingly, a low level of vascularization. However,the density of vascularization is more in accordance withthe physiological levels for this tissue site as was observedfor sham-operated animals [28], which in some applicationsfor biomaterials may be desirable when there is no need fora vascularization in excess of the particular host tissue site.Moreover, the low level of vascularization also likely resultsin a slower material degradation, allowing the material to serve

Figure 7. Histomorphometric analysis of the total thickness of thescaffold as well as the thickness of each of the two layers of thescaffold over time of implantation.

as a place holder and barrier for a longer period of time, anideal characteristic in GTR applications.

We also focused on the materialtissue interface. Afterimplantation the compact layer of the matrix served a barrierfunction, preventing cellular infiltration from the surroundingtissue. Although peri-implant cells were able to intercalatethese layers of collagen in this part of the matrix by day 10, theywere not able to penetrate this layer completely until 30 daysafter implantation. These results emphasize that the compactlayer promotes cell adhesion and tissue integration, but thatinvading cells need up to 30 days in order to traverse this layer.On the spongy layer of the MG matrix, a slow but continuouscell and tissue ingrowth was observed through the porosity ofthis surface. This spongy side, while allowing cells to infiltratewithout much resistance, maintained its structure through theentirety of the study and became integrated into the host tissueas opposed to undergoing degradation. The porosity allowed itto act as a scaffold on which peri-implantary cells, connectivetissue, and microvessels were able to grow and advance to thecenter of the matrix.

Our results from the preliminary human applicationof MG are exciting, as the matrix demonstrated successin reversing the recession of gingival tissue, resulting inmore healthy tissue with a raised gum line in the patient.The aesthetic results are quite remarkable. Furthermore,histological evidence demonstrates that within human tissue,the compact layer of the matrix again serves a barrier function.This gave the periodontal defect the time needed to regenerateby maintaining isolation from the fibrous tissue and oralmucosa for at least 43 days. Along with this, the materialshowed no adverse tissue reaction, an ideal characteristic ofa material to be applied clinically. These results highlightthe functional performance of the MG matrix in a trulytranslational context and emphasize the suitability of thismaterial for application in GTR.

One impressive feature of the combined animal and thepreliminary clinical study is that the human histology from

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(A) (B)

(C) (D)

Figure 8. Immunohistological staining for CD31 in order to visualize blood vessels (arrows) from scaffolds implanted for 3 days (A),10 days (B), 30 days (C), and 60 days (D), all at 400. At most, a mild vascularization is evident in the peri-implantary tissue and within theMG scaffold itself. All scale bars are 100 m.

(A)

(B)

(C)

(D)

(E )

Figure 9. Results from the clinical study applying the MG scaffold to treat gingival tissue recession (arrows), showing one such recession(A) that has been treated by the implantation of the MG scaffold (B) with the compact layer facing out in the image. Following treatment,the gingival tissue level has been restored to normal height (C). Histological results from a biopsy obtained in the course of the studydemonstrate similar findings as those in the in vivo murine model, with the compact layer serving as a barrier, while the spongy layer isinfiltrated with cells and becomes transformed into a connective tissue, demonstrated with Alzarin red stain (D) and H&E stain (E) at 400.SL = spongy layer, CL = compact layer and CT = connective tissue. All scale bars are 100 m.

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Biomed. Mater. 6 (2011) 015010 S Ghanaati et al

our pilot evaluation corroborated our results when the materialwas tested in the subcutaneous murine model. It is one thing toevaluate a material in vivo and speculate based on these resultshow the material would perform in a therapeutic context. Inthis study we have direct evidence that our predictions forthe performance of the material formulated in vivo translatevery well to observations in the clinical setting. The timeframe over which the barrier function of the compact layeris maintained does differ, owing perhaps to the differencesin species or in tissue site. However, regardless of whetherMG was placed subcutaneously within a mouse or withinhuman gingival tissue, the matrix maintained its ability topromote preferential tissue ingrowth. Moreover, both studiesreveal a favorable tissue reaction and a physiological levelof scaffold vascularization. It is evident that more extensiveclinical studies are required, and these are currently beingconducted.

The results from the pilot clinical evaluation supportthe findings from our animal model, indicating that thesubcutaneous implantation model can appropriately predictthe tissue reaction and preferential tissue ingrowth for suchmaterials. This is an exciting finding, as it is especiallysatisfying to know that the sacrifice of animals was not withoutpurpose and that the lessons learned in these animal modelstranslated to clinical findings. In addition, observations in theanimal model would suggest that the application of MG is notlimited to only the treatment of gingival recessions. Althoughsuch an application has been shown here to exemplify theclinical applicability of the material. It could certainly find usein applications to promote the ingrowth of healthy tissue in theother locations within the oral cavity, used as a treatment for there-epilthelialization of chronic or non-healing wounds, or usedin a number of other applications for soft tissue regenerationwhere a preferential ingrowth and a barrier to the formation offibrotic tissue are essential.

The results of this study underline the importanceof material composition and morphology for use as GTRscaffolds. The material evaluated here demonstrates idealfunctionality for this role in vivo. The porous side of theMG matrix permits cells to integrate and grow into the centerregion of the scaffold. On the other hand, the compact layerinhibits connective tissue ingrowth. These features allow forpreferential cell ingrowth, a vital characteristic of scaffoldsdesigned for regeneration of soft tissue. Additionally, theMG matrix demonstrates an excellent tissue reaction, resultingin no multinucleated giant cell formation and becoming wellintegrated into the peri-implant tissue. These studies givepromise for the translation of MG as a biomaterial, a promisethat is realized in the example from a pilot human applicationusing MG to treat receding gingival tissue. In this example, theMG matrix performed its function both as a barrier materialand also as a structural component within which functionaltissue was constructed. The results from this study arecongruous with those of our murine studies, and support ourconclusions from these studies that MG is an excellent optionto promote the preferential regeneration of soft tissue.

5. Conclusion

In this study, we have evaluated a new collagen product,Mucograft R, which is designed with a compact and spongylayer in order to serve a barrier function for applicationsin guided tissue regeneration. This represents the first invivo evaluation of this material. The tissue reaction to thismaterial is quite favorable, with minimal inflammation andno multinucleated giant cells. The material persisted in thetissue throughout the study, and while the spongy layer wasinfiltrated early on, the compact layer remained impermeableto invading cells for the first 30 days of the study. Thisdemonstrates promise by indicating preferential cell ingrowthfrom the spongy surface. However, in order to truly realizethe promise of a biomaterial, it must be evaluated in theclinical setting. For this reason, we have also included resultsfrom a pilot clinical evaluation using Mucograft R to treatgingival tissue recession in humans. Our results from thisstudy demonstrate great potential to reverse tissue recessionand promote more healthy gingival tissue. Additionally,histological evidence corroborates our findings in the murinemodel, with the compact layer preventing tissue ingrowthas the scaffold becomes infiltrated from the spongy layer.Overall, the Mucograft R system demonstrates promise inmurine in vivo studies and realizes this therapeutic promise in ahuman application of the material. In terms of the translationof biomaterials, much can be learned from suitable in vivoexperiments, and in this case these in vivo studies correctlypredicted the clinical outcome of the material, although thelatter must be confirmed by more extensive clinical studies.

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1. Introduction2. Materials and methods2.1. Production of Mucograft 2.2. Scanning electron microscopy2.3. Murine experimental design2.4. Tissue preparation for histology and immunohistochemistry2.5. Morphological evaluation of the inflammatory response2.6. Thickness measurements2.7. Pilot human Mucograft use

3. Results3.1. SEM of the scaffold3.2. Murine histological results3.3. Histomorphometric assessment of thickness3.4. Scaffold vascularization3.5. Human Mucograft pilot study

4. Discussion5. ConclusionReferences