Comparison of the Capacity of Enamel Matrix Derivative Gel and Enamel Matrix Derivative in Liquid Formulation to Adsorb to Bone Grafting Materials Richard J. Miron, Dieter D. Bosshardt, Daniel Buser, Yufeng Zhang, Stefano Tugulu, Anja Gemperli, Michel Dard, Oana M. Caluseru, Fatiha Chandad, and Anton Sculean 担当:篠田純
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Comparison of the capacity of enamel matrix derivative gel and enamel matrix derivative in liquid formulation to adsorb to bone grafting materials
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Comparison of the Capacity of Enamel Matrix Derivative Gel and Enamel
Matrix Derivative in Liquid Formulation to Adsorb to Bone Grafting Materials Richard J. Miron, Dieter D. Bosshardt, Daniel Buser, Yufeng Zhang, Stefano Tugulu, Anja Gemperli, Michel Dard, Oana M. Caluseru, Fatiha
• CaP は大きなスケールとnano スケールの表面粗さの特徴を示す (Figs. 1E and 1F)。
demonstrating the effects of the polyglycol alginate(PGA) carrier responsible for transporting enamelmatrix proteins in EMD gel (Fig. 2B). The TEM im-ages revealed that the combination of NBM + EMDliquid demonstrated gold-labeled particles of EMDprincipally directly at the surface of NBM scaffolds(Fig. 2C). It was also noted that some gold particleswere observed in the interior of scaffolds, demon-strating that EMD liquid was able to penetrate thesurface of NBM particles when delivered in a liquidformat and adsorb to the interior region of NBMparticles (Fig. 2C). Interestingly, scaffolds coatedwith EMD gel showed a much thicker pattern of goldlabeling, found extended from the surface of graftingparticles (Fig. 2D). Very little gold-labeling particleswere observed on the interior of grafting particlescoated with EMD gel when compared to coating withEMD liquid.
The coating of DFDBA par-ticles followed a similar pattern(Fig. 3). The surface topogra-phy of DFDBA particles was onlyslightly modified after coatingwith EMD liquid and did not affectthe surface characteristics ofDFDBA particles (Fig. 3A). Sim-ilarly to NBM particles, whenDFDBA particles were coatedwith EMD gel, the coating changedthe surface topography and sur-face properties of bone graftingparticles by demonstrating re-gions of thicker coating, likelya result of the PGA carrier used inEMD gel (Fig. 3B). Analysis ofTEM images revealed once againthat DFDBA particles coatedwith EMD liquid demonstratedsurface coating of gold-labeledanti-EMD antibody at the surfaceof grafting particles (Fig. 3C,arrows). In contrast, DFDBAparticles precoated with EMD geldemonstrated surface coatingwith a wide range of gold labelingextended from the surface asa result of the thickness of coatingvia EMD gel (Fig. 3D, arrows).
When CaP particles were coat-ed with EMD liquid, the surfacecharacteristics of particles dem-onstrated slightly modified sur-face topographies (Fig. 4A). Itwas again noted that when CaPparticles were coated with EMDgel, a thick gel-like pattern was
observed on the surface of grafting materials, likelycaused by the PGA carrier (Fig. 4B). Interestingly, theanalysis by TEM demonstrated that the gold labelingshowed staining at variable distances from the surfacein EMD liquid–coated CaP particles (Fig. 4C, arrows).Similarly, the coating of particles with EMD gel furtherincreased the range and distance from the surface ofthe CaP scaffolds (Fig. 4D, arrows).
TEM samples were then quantified for the numberof gold-labeling particles per image, and the averagedistance was calculated from the surface of bonegrafting materials (Fig. 5) (see supplementary Table 1in online Journal of Periodontology). A highernumber of gold labeling was observed on NBMand DFDBA scaffolds coated with EMD liquid whencompared to EMD gel (see supplementary Table 1 inonline Journal of Periodontology). Interestingly, onCaP scaffolds, only a small, non-significant increase
Figure 1.SEM of the bone grafting materials used for this study. Low (·70) (A) and high (·1,200) (B)magnification of a NBM bone graft derived from bovine origin displays bone chips resembling humanbone with visible large and small nanotopographies present on the surface. C) DFDBA demonstratessmaller-sized bone particles that lack mineralized tissues. (·25.) D) The surface properties showa smooth surface at highmagnification (·1,600). E and F) The CaP bone graft demonstrates a surfacewith many large and small nanotopographies. (·25 and ·1,600, respectively.) Scale bars = A) 500mm;B), D), and F) 20 mm; C) and E) 1 mm.
J Periodontol • April 2015 Miron, Bosshardt, Buser, et al.
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RESULTSSEM and TEM
• さらにすべての骨移植材は EMD liquid または EMD gel でコーティングされ、SEM と TEM で表面状態を分析された (see Figs. 2 through 4)。
was observed. The most notable difference was thedistance from which the labeling was observed fromthe scaffold surface. On NBM + EMD liquid andDFDBA + EMD liquid, most adsorption of proteinswas done either within the scaffold or directly on thescaffold surface. Results from the TEM analysis re-vealed values of 8.45 and 44.85 nm from the scaffoldsurface, respectively (Fig. 5) (see supplementaryTable 1 in online Journal of Periodontology). Whenthese scaffolds were coated with EMD gel, this distanceincreased to 906.45 and 1,133.65 nm, respectively.This same trend was also observed on CaP scaffolds,although to a lesser extent (Fig. 5) (supplementaryTable 1 in online Journal of Periodontology).
Quantification of Amelogenin Protein to theScaffold Surface by ELISATo provide a second method to quantify the adsorp-tion of amelogenin proteins to the surface of scaffoldscoated with either EMD liquid or EMD gel, ELISAprotein quantification was performed (Fig. 6). It wasshown that the coating of bone grafts with EMD liquidwas again superior for all bone grafts, but because ofthe variability in coating, a non-significant increasewas observed (Fig. 6A). Interestingly, when the
bone grafts were compared fortheir release of amelogenin pro-file with time, significant differ-ences were observed betweenEMD liquid and EMD gel (Figs.6B through 6D). For NBM parti-cles coated with either EMD liq-uid or EMD gel, it was observedthat 82.34 – 8.24 and 58.23 –13.25 ng, respectively, werecoated on the surface materials(Fig. 6B). After PBS rinsing, theresults demonstrated that, al-though NBM particles coatedwith EMD liquid decreasedslightly to 78.52 – 9.15 ng (from82.34 ng), surfaces coated withEMD gel decreased to 22.53 –14.54 ng (from 58.23 ng). Thus,one simple PBS rinse signifi-cantly reduced surface coatingof enamel matrix proteins frombone grafts coated with EMD gelbut had little effect on graftscoated with EMD liquid. There-after, only slight amounts ofamelogenin proteins were re-leased to the surrounding solu-tion during the time from 8 hoursto 10 days. This observation wasalso observed for DFDBA par-
ticles coated with either EMD liquid or EMD gel(Fig. 6C) and CaP particles (Fig. 6D). The resultsreveal that a single rinse with PBS is able to de-crease surface coating of amelogenin proteinscoated to bone grafting materials with EMD gel by>50% in all three bone grafting materials used in thisstudy (Fig. 6).
DISCUSSION
The aim of the current study is to investigate therelation between protein adsorption of enamel matrixproteins to grafting materials coated with either EMDin a liquid formulation or the commercially availableEMD gel. The use of EMD gel with a bone graft ina clinical setting has generated an array of mixedresults from clinical trials.22,23 Although a number ofstudies confirm an additional benefit, others havespeculated that only certain types of graftingmaterialsare advantageously mixed with EMD gel, whereasother grafts fail to provide additional benefits.22,23 Forthese reasons, the purpose of the present study is toinvestigate the patterns of enamel matrix proteinadsorption to the surface of grafting materials anddetermine what variability may exist between carriersystems for EMD.
Figure 2.SEM and TEM images of a NBM after coating with either EMD liquid or EMD gel. A) SEM image(·1,120) of NBM particles coated with EMD liquid demonstrates very similar surface characteristics asnative uncoatedparticles. The results from the TEM images, whichwere stained for EMDvia gold-labelingnanoparticles, demonstrate that all surface staining was either near the surface or penetrated withthe surface of the grafting material (C, arrows). B) In contrast, NBM particles coated with EMD geldemonstrated the presence of a much thicker coating by its PGA carrier.D) The surface coating with ananti-EMD antibody also revealed the presence of most of its coating far from the surface of graftingparticles (arrows). (·1,200.) Scale bars A and B) = 20 mm
Enamel Matrix Derivative Adsorption to Bone Grafts Volume 86 • Number 4
was observed. The most notable difference was thedistance from which the labeling was observed fromthe scaffold surface. On NBM + EMD liquid andDFDBA + EMD liquid, most adsorption of proteinswas done either within the scaffold or directly on thescaffold surface. Results from the TEM analysis re-vealed values of 8.45 and 44.85 nm from the scaffoldsurface, respectively (Fig. 5) (see supplementaryTable 1 in online Journal of Periodontology). Whenthese scaffolds were coated with EMD gel, this distanceincreased to 906.45 and 1,133.65 nm, respectively.This same trend was also observed on CaP scaffolds,although to a lesser extent (Fig. 5) (supplementaryTable 1 in online Journal of Periodontology).
Quantification of Amelogenin Protein to theScaffold Surface by ELISATo provide a second method to quantify the adsorp-tion of amelogenin proteins to the surface of scaffoldscoated with either EMD liquid or EMD gel, ELISAprotein quantification was performed (Fig. 6). It wasshown that the coating of bone grafts with EMD liquidwas again superior for all bone grafts, but because ofthe variability in coating, a non-significant increasewas observed (Fig. 6A). Interestingly, when the
bone grafts were compared fortheir release of amelogenin pro-file with time, significant differ-ences were observed betweenEMD liquid and EMD gel (Figs.6B through 6D). For NBM parti-cles coated with either EMD liq-uid or EMD gel, it was observedthat 82.34 – 8.24 and 58.23 –13.25 ng, respectively, werecoated on the surface materials(Fig. 6B). After PBS rinsing, theresults demonstrated that, al-though NBM particles coatedwith EMD liquid decreasedslightly to 78.52 – 9.15 ng (from82.34 ng), surfaces coated withEMD gel decreased to 22.53 –14.54 ng (from 58.23 ng). Thus,one simple PBS rinse signifi-cantly reduced surface coatingof enamel matrix proteins frombone grafts coated with EMD gelbut had little effect on graftscoated with EMD liquid. There-after, only slight amounts ofamelogenin proteins were re-leased to the surrounding solu-tion during the time from 8 hoursto 10 days. This observation wasalso observed for DFDBA par-
ticles coated with either EMD liquid or EMD gel(Fig. 6C) and CaP particles (Fig. 6D). The resultsreveal that a single rinse with PBS is able to de-crease surface coating of amelogenin proteinscoated to bone grafting materials with EMD gel by>50% in all three bone grafting materials used in thisstudy (Fig. 6).
DISCUSSION
The aim of the current study is to investigate therelation between protein adsorption of enamel matrixproteins to grafting materials coated with either EMDin a liquid formulation or the commercially availableEMD gel. The use of EMD gel with a bone graft ina clinical setting has generated an array of mixedresults from clinical trials.22,23 Although a number ofstudies confirm an additional benefit, others havespeculated that only certain types of graftingmaterialsare advantageously mixed with EMD gel, whereasother grafts fail to provide additional benefits.22,23 Forthese reasons, the purpose of the present study is toinvestigate the patterns of enamel matrix proteinadsorption to the surface of grafting materials anddetermine what variability may exist between carriersystems for EMD.
Figure 2.SEM and TEM images of a NBM after coating with either EMD liquid or EMD gel. A) SEM image(·1,120) of NBM particles coated with EMD liquid demonstrates very similar surface characteristics asnative uncoatedparticles. The results from the TEM images, whichwere stained for EMDvia gold-labelingnanoparticles, demonstrate that all surface staining was either near the surface or penetrated withthe surface of the grafting material (C, arrows). B) In contrast, NBM particles coated with EMD geldemonstrated the presence of a much thicker coating by its PGA carrier.D) The surface coating with ananti-EMD antibody also revealed the presence of most of its coating far from the surface of graftingparticles (arrows). (·1,200.) Scale bars A and B) = 20 mm
Enamel Matrix Derivative Adsorption to Bone Grafts Volume 86 • Number 4
In the present study, the choice was made toinvestigate three grafting materials commonly usedin dentistry. NBM was used as the xenograft of choicebecause it is studied extensively and the presentauthors have previous experience handling it. TheDFDBA used in this study was selected because ofits widespread use and osteoinductive advantagescompared to other DFDBA grafts.48-50 An alloplastbone graft fabricated from hydroxyapatite and b-TCPwas chosen as the synthetic material. To the best ofthe authors’ knowledge, in the present study, it is thefirst time observed by both SEM and TEM that largevariability existed with respect to the ability of enamelmatrix proteins to adsorb to these various bone grafts(Figs. 2 through 5). Interestingly, although the NBMparticles were able to adsorb the highest quantity ofprotein, the more interesting finding was the inabilityfor EMD gel to efficiently adsorb enamel matrix pro-teins to the surface of grafting materials at the presentsettings (Fig. 5). It was noted that the average distancein which amelogenin proteins were found from thesurface of bone grafting particles was >20 timesgreater in samples coated with EMD gel when com-pared to EMD liquid (see supplementary Table 1 inonline Journal of Periodontology).
The present study reveals thatthe adsorption of enamel matrixproteins may be attributed tothe material composition. BothDFDBA and NBM are derivedfrom natural bone, and their na-tive composition naturally con-tains specific sites for proteinadsorption. Because NBM parti-cles are completely devoid ofcells and proteins (deproteinizedmatrix), their ability to providespecific sites for new proteinadsorption make it an ideal graftchoice as a carrier for bioactivemolecules. A similar observationfor DFDBA was also observedwith a surprisingly high ability forenamel matrix proteins coatedwithin the interior surface ofgrafting material (Fig. 3A). Con-trary to this, no ability of EMDgel to penetrate the surface ofCaP molecules was observed.Because the surface containsno discernible pores able toallow any form of protein ad-sorption within the grafting ma-terial, it signifies that proteinadsorption is limited strictly tothe material surface, thus pro-
viding much less ability for the graft to carry ahigher load of bioactive molecules.
Although the SEM and TEM analyses were ableto provide much qualitative analysis, the use of anELISA kit greatly enhanced the ability to accuratelyquantify the amount of amelogenin bound to thematerial surface. Although each of the bone graftswas able to adsorb either EMD liquid or EMD gel tothe surface of the bone grafting material, the no-ticeable difference was evident after rinsing with PBSat neutral pH. Because this solution is used in vitro torepresent physiologic pH, the finding that a simplerinse was able to wash >50% of amelogenin bound tothe surface of grafting materials coated with EMD gelwas quite surprising (Fig. 6). Although it is verydifficult to speculate what effect this might have ina clinical setting, the finding that such a large per-centage of protein is flushed so easily raises theclinical concern that after the placement of EMD gel +bone graft in a bone defect, a similar effect might beimagined whereby bodily fluids are able to dissociatethe adsorbed enamel matrix proteins from the scaf-fold surface. Noteworthy also is the fact that bone ap-position should be directly adjacent to the bone graftingmaterial surface. In the present study, it is found that
Figure 3.SEM and TEM images of DFDBA after coating with either EMD liquid or EMD gel. A) SEM imageof DFDBA particles coated with EMD liquid demonstrates its smooth surface properties with littleaccumulation of a thick surface coatingwith amelogenin proteins. The results fromTEM further confirmedthat most of the staining was present on the surface or slightly penetrated within the surface of thegrafting material (C, arrows). B) In contrast, DFDBA particles coated with EMD gel demonstrated thepresence of a much thicker coating caused by its PGA carrier. The surface coating with an anti-EMDantibody also revealed the presence of most of its coating far from the surface of grafting particles(D, arrows). (A) and B) ·1,600; scale bars = 20 mm.)
J Periodontol • April 2015 Miron, Bosshardt, Buser, et al.
In the present study, the choice was made toinvestigate three grafting materials commonly usedin dentistry. NBM was used as the xenograft of choicebecause it is studied extensively and the presentauthors have previous experience handling it. TheDFDBA used in this study was selected because ofits widespread use and osteoinductive advantagescompared to other DFDBA grafts.48-50 An alloplastbone graft fabricated from hydroxyapatite and b-TCPwas chosen as the synthetic material. To the best ofthe authors’ knowledge, in the present study, it is thefirst time observed by both SEM and TEM that largevariability existed with respect to the ability of enamelmatrix proteins to adsorb to these various bone grafts(Figs. 2 through 5). Interestingly, although the NBMparticles were able to adsorb the highest quantity ofprotein, the more interesting finding was the inabilityfor EMD gel to efficiently adsorb enamel matrix pro-teins to the surface of grafting materials at the presentsettings (Fig. 5). It was noted that the average distancein which amelogenin proteins were found from thesurface of bone grafting particles was >20 timesgreater in samples coated with EMD gel when com-pared to EMD liquid (see supplementary Table 1 inonline Journal of Periodontology).
The present study reveals thatthe adsorption of enamel matrixproteins may be attributed tothe material composition. BothDFDBA and NBM are derivedfrom natural bone, and their na-tive composition naturally con-tains specific sites for proteinadsorption. Because NBM parti-cles are completely devoid ofcells and proteins (deproteinizedmatrix), their ability to providespecific sites for new proteinadsorption make it an ideal graftchoice as a carrier for bioactivemolecules. A similar observationfor DFDBA was also observedwith a surprisingly high ability forenamel matrix proteins coatedwithin the interior surface ofgrafting material (Fig. 3A). Con-trary to this, no ability of EMDgel to penetrate the surface ofCaP molecules was observed.Because the surface containsno discernible pores able toallow any form of protein ad-sorption within the grafting ma-terial, it signifies that proteinadsorption is limited strictly tothe material surface, thus pro-
viding much less ability for the graft to carry ahigher load of bioactive molecules.
Although the SEM and TEM analyses were ableto provide much qualitative analysis, the use of anELISA kit greatly enhanced the ability to accuratelyquantify the amount of amelogenin bound to thematerial surface. Although each of the bone graftswas able to adsorb either EMD liquid or EMD gel tothe surface of the bone grafting material, the no-ticeable difference was evident after rinsing with PBSat neutral pH. Because this solution is used in vitro torepresent physiologic pH, the finding that a simplerinse was able to wash >50% of amelogenin bound tothe surface of grafting materials coated with EMD gelwas quite surprising (Fig. 6). Although it is verydifficult to speculate what effect this might have ina clinical setting, the finding that such a large per-centage of protein is flushed so easily raises theclinical concern that after the placement of EMD gel +bone graft in a bone defect, a similar effect might beimagined whereby bodily fluids are able to dissociatethe adsorbed enamel matrix proteins from the scaf-fold surface. Noteworthy also is the fact that bone ap-position should be directly adjacent to the bone graftingmaterial surface. In the present study, it is found that
Figure 3.SEM and TEM images of DFDBA after coating with either EMD liquid or EMD gel. A) SEM imageof DFDBA particles coated with EMD liquid demonstrates its smooth surface properties with littleaccumulation of a thick surface coatingwith amelogenin proteins. The results fromTEM further confirmedthat most of the staining was present on the surface or slightly penetrated within the surface of thegrafting material (C, arrows). B) In contrast, DFDBA particles coated with EMD gel demonstrated thepresence of a much thicker coating caused by its PGA carrier. The surface coating with an anti-EMDantibody also revealed the presence of most of its coating far from the surface of grafting particles(D, arrows). (A) and B) ·1,600; scale bars = 20 mm.)
J Periodontol • April 2015 Miron, Bosshardt, Buser, et al.
the use of EMD gel provided a much thicker surfacecoating, one that extends several micrometers, asviewed by SEM and TEM images. Although in someclinical studies the use of EMD gel has demonstratedpositive outcomes when combined with bone graftingmaterials, the present findings provide conclusiveevidence that a large variability exists betweenamelogenin adsorption to the various bone grafting
materials used in reconstructiveperiodontal surgery. Moreover,the present data provide therationale for the development ofan optimized delivery systemusing a liquid solution of EMDfor future combination with bonegrafting materials.
CONCLUSIONS
The results from the presentstudy demonstrate that surfacecoatings with enamel matrixproteins vary depending onthe coating of bone graftingmaterials with either EMD liquidor EMD gel. Surface coatingwith EMD gel increased drasti-cally the thickness of coating ofenamel matrix proteins to bonegrafting surfaces, which wereeasily dissolved after a simplePBS rinse. The use of EMD ina liquid formulation was able toprovide better surface coatingdirectly adjacent to scaffoldsurface, whereas protein adsorp-tion was also observed within theinterior of the bone grafting par-ticles when coated on NBM andDFDBA. When bone grafts werecoated with EMD in a liquid for-mulation, they demonstratedbetter long-term delivery ofenamel matrix proteins duringa 10-day period compared toEMD in a gel formulation. Thus,the liquid formulation of EMDallows for the following: 1) in-creased and more completesurface loading of porous graftmaterials; and 2) tighter andmore stable surface coating withenamel matrix proteins.
ACKNOWLEDGMENTS
The authors gratefully acknowl-edge the Robert K. Schenk Lab-
oratory of Oral Histology, Dental School at theUniversity of Bern, most notably Thuy Tran Nguyen,Monika Aeberhard, and David Reist for their consid-erable time and valuable insights into the histologicperformance of experiments. This work was fundedby the Department of Periodontology at the Univer-sity of Bern and Institute Straumann, for whichDrs. Tugulu, Gemperli, and Dard are employees.
Figure 4.SEM and TEM images of CaP bone grafting after coating with either EMD liquid or EMD gel. A) SEMimage of CaP particles coated with EMD liquid demonstrates accumulation of EMD on the surface ofgrafting particles. The results from TEM further confirmed that most of the staining was present at a slightdistance from the surface, with no presence of penetration within the bone grafting scaffold (C, arrows).B)Similarly, CaP particles coated with EMD gel demonstrated the presence of a thicker coating caused by itsPGA carrier. The surface coating with an anti-EMD antibody also revealed the presence of its coating ata distance from the surface of grafting particles (D, arrows). (A) and B) ·1,600; scale bars = 20 mm.)
Figure 5.A) Quantitative analysis of the number of gold-labeled particles that appear from TEM images revealeda slight increase in number on grafting particles coated with EMD liquid and EMD gel. B) The particleswere then quantified for their average distance from the scaffold surface, and results demonstrate a muchcloser binding affinity to scaffolds when grafts were coated with EMD liquid instead of EMD gel. * denotessignificant differences of P <0.05.
Enamel Matrix Derivative Adsorption to Bone Grafts Volume 86 • Number 4
the use of EMD gel provided a much thicker surfacecoating, one that extends several micrometers, asviewed by SEM and TEM images. Although in someclinical studies the use of EMD gel has demonstratedpositive outcomes when combined with bone graftingmaterials, the present findings provide conclusiveevidence that a large variability exists betweenamelogenin adsorption to the various bone grafting
materials used in reconstructiveperiodontal surgery. Moreover,the present data provide therationale for the development ofan optimized delivery systemusing a liquid solution of EMDfor future combination with bonegrafting materials.
CONCLUSIONS
The results from the presentstudy demonstrate that surfacecoatings with enamel matrixproteins vary depending onthe coating of bone graftingmaterials with either EMD liquidor EMD gel. Surface coatingwith EMD gel increased drasti-cally the thickness of coating ofenamel matrix proteins to bonegrafting surfaces, which wereeasily dissolved after a simplePBS rinse. The use of EMD ina liquid formulation was able toprovide better surface coatingdirectly adjacent to scaffoldsurface, whereas protein adsorp-tion was also observed within theinterior of the bone grafting par-ticles when coated on NBM andDFDBA. When bone grafts werecoated with EMD in a liquid for-mulation, they demonstratedbetter long-term delivery ofenamel matrix proteins duringa 10-day period compared toEMD in a gel formulation. Thus,the liquid formulation of EMDallows for the following: 1) in-creased and more completesurface loading of porous graftmaterials; and 2) tighter andmore stable surface coating withenamel matrix proteins.
ACKNOWLEDGMENTS
The authors gratefully acknowl-edge the Robert K. Schenk Lab-
oratory of Oral Histology, Dental School at theUniversity of Bern, most notably Thuy Tran Nguyen,Monika Aeberhard, and David Reist for their consid-erable time and valuable insights into the histologicperformance of experiments. This work was fundedby the Department of Periodontology at the Univer-sity of Bern and Institute Straumann, for whichDrs. Tugulu, Gemperli, and Dard are employees.
Figure 4.SEM and TEM images of CaP bone grafting after coating with either EMD liquid or EMD gel. A) SEMimage of CaP particles coated with EMD liquid demonstrates accumulation of EMD on the surface ofgrafting particles. The results from TEM further confirmed that most of the staining was present at a slightdistance from the surface, with no presence of penetration within the bone grafting scaffold (C, arrows).B)Similarly, CaP particles coated with EMD gel demonstrated the presence of a thicker coating caused by itsPGA carrier. The surface coating with an anti-EMD antibody also revealed the presence of its coating ata distance from the surface of grafting particles (D, arrows). (A) and B) ·1,600; scale bars = 20 mm.)
Figure 5.A) Quantitative analysis of the number of gold-labeled particles that appear from TEM images revealeda slight increase in number on grafting particles coated with EMD liquid and EMD gel. B) The particleswere then quantified for their average distance from the scaffold surface, and results demonstrate a muchcloser binding affinity to scaffolds when grafts were coated with EMD liquid instead of EMD gel. * denotessignificant differences of P <0.05.
Enamel Matrix Derivative Adsorption to Bone Grafts Volume 86 • Number 4
584
RESULTS
the use of EMD gel provided a much thicker surfacecoating, one that extends several micrometers, asviewed by SEM and TEM images. Although in someclinical studies the use of EMD gel has demonstratedpositive outcomes when combined with bone graftingmaterials, the present findings provide conclusiveevidence that a large variability exists betweenamelogenin adsorption to the various bone grafting
materials used in reconstructiveperiodontal surgery. Moreover,the present data provide therationale for the development ofan optimized delivery systemusing a liquid solution of EMDfor future combination with bonegrafting materials.
CONCLUSIONS
The results from the presentstudy demonstrate that surfacecoatings with enamel matrixproteins vary depending onthe coating of bone graftingmaterials with either EMD liquidor EMD gel. Surface coatingwith EMD gel increased drasti-cally the thickness of coating ofenamel matrix proteins to bonegrafting surfaces, which wereeasily dissolved after a simplePBS rinse. The use of EMD ina liquid formulation was able toprovide better surface coatingdirectly adjacent to scaffoldsurface, whereas protein adsorp-tion was also observed within theinterior of the bone grafting par-ticles when coated on NBM andDFDBA. When bone grafts werecoated with EMD in a liquid for-mulation, they demonstratedbetter long-term delivery ofenamel matrix proteins duringa 10-day period compared toEMD in a gel formulation. Thus,the liquid formulation of EMDallows for the following: 1) in-creased and more completesurface loading of porous graftmaterials; and 2) tighter andmore stable surface coating withenamel matrix proteins.
ACKNOWLEDGMENTS
The authors gratefully acknowl-edge the Robert K. Schenk Lab-
oratory of Oral Histology, Dental School at theUniversity of Bern, most notably Thuy Tran Nguyen,Monika Aeberhard, and David Reist for their consid-erable time and valuable insights into the histologicperformance of experiments. This work was fundedby the Department of Periodontology at the Univer-sity of Bern and Institute Straumann, for whichDrs. Tugulu, Gemperli, and Dard are employees.
Figure 4.SEM and TEM images of CaP bone grafting after coating with either EMD liquid or EMD gel. A) SEMimage of CaP particles coated with EMD liquid demonstrates accumulation of EMD on the surface ofgrafting particles. The results from TEM further confirmed that most of the staining was present at a slightdistance from the surface, with no presence of penetration within the bone grafting scaffold (C, arrows).B)Similarly, CaP particles coated with EMD gel demonstrated the presence of a thicker coating caused by itsPGA carrier. The surface coating with an anti-EMD antibody also revealed the presence of its coating ata distance from the surface of grafting particles (D, arrows). (A) and B) ·1,600; scale bars = 20 mm.)
Figure 5.A) Quantitative analysis of the number of gold-labeled particles that appear from TEM images revealeda slight increase in number on grafting particles coated with EMD liquid and EMD gel. B) The particleswere then quantified for their average distance from the scaffold surface, and results demonstrate a muchcloser binding affinity to scaffolds when grafts were coated with EMD liquid instead of EMD gel. * denotessignificant differences of P <0.05.
Enamel Matrix Derivative Adsorption to Bone Grafts Volume 86 • Number 4
the use of EMD gel provided a much thicker surfacecoating, one that extends several micrometers, asviewed by SEM and TEM images. Although in someclinical studies the use of EMD gel has demonstratedpositive outcomes when combined with bone graftingmaterials, the present findings provide conclusiveevidence that a large variability exists betweenamelogenin adsorption to the various bone grafting
materials used in reconstructiveperiodontal surgery. Moreover,the present data provide therationale for the development ofan optimized delivery systemusing a liquid solution of EMDfor future combination with bonegrafting materials.
CONCLUSIONS
The results from the presentstudy demonstrate that surfacecoatings with enamel matrixproteins vary depending onthe coating of bone graftingmaterials with either EMD liquidor EMD gel. Surface coatingwith EMD gel increased drasti-cally the thickness of coating ofenamel matrix proteins to bonegrafting surfaces, which wereeasily dissolved after a simplePBS rinse. The use of EMD ina liquid formulation was able toprovide better surface coatingdirectly adjacent to scaffoldsurface, whereas protein adsorp-tion was also observed within theinterior of the bone grafting par-ticles when coated on NBM andDFDBA. When bone grafts werecoated with EMD in a liquid for-mulation, they demonstratedbetter long-term delivery ofenamel matrix proteins duringa 10-day period compared toEMD in a gel formulation. Thus,the liquid formulation of EMDallows for the following: 1) in-creased and more completesurface loading of porous graftmaterials; and 2) tighter andmore stable surface coating withenamel matrix proteins.
ACKNOWLEDGMENTS
The authors gratefully acknowl-edge the Robert K. Schenk Lab-
oratory of Oral Histology, Dental School at theUniversity of Bern, most notably Thuy Tran Nguyen,Monika Aeberhard, and David Reist for their consid-erable time and valuable insights into the histologicperformance of experiments. This work was fundedby the Department of Periodontology at the Univer-sity of Bern and Institute Straumann, for whichDrs. Tugulu, Gemperli, and Dard are employees.
Figure 4.SEM and TEM images of CaP bone grafting after coating with either EMD liquid or EMD gel. A) SEMimage of CaP particles coated with EMD liquid demonstrates accumulation of EMD on the surface ofgrafting particles. The results from TEM further confirmed that most of the staining was present at a slightdistance from the surface, with no presence of penetration within the bone grafting scaffold (C, arrows).B)Similarly, CaP particles coated with EMD gel demonstrated the presence of a thicker coating caused by itsPGA carrier. The surface coating with an anti-EMD antibody also revealed the presence of its coating ata distance from the surface of grafting particles (D, arrows). (A) and B) ·1,600; scale bars = 20 mm.)
Figure 5.A) Quantitative analysis of the number of gold-labeled particles that appear from TEM images revealeda slight increase in number on grafting particles coated with EMD liquid and EMD gel. B) The particleswere then quantified for their average distance from the scaffold surface, and results demonstrate a muchcloser binding affinity to scaffolds when grafts were coated with EMD liquid instead of EMD gel. * denotessignificant differences of P <0.05.
Enamel Matrix Derivative Adsorption to Bone Grafts Volume 86 • Number 4
• この傾向はCaPでも見られたが、程度は小さかった(Fig. 5)。the use of EMD gel provided a much thicker surfacecoating, one that extends several micrometers, asviewed by SEM and TEM images. Although in someclinical studies the use of EMD gel has demonstratedpositive outcomes when combined with bone graftingmaterials, the present findings provide conclusiveevidence that a large variability exists betweenamelogenin adsorption to the various bone grafting
materials used in reconstructiveperiodontal surgery. Moreover,the present data provide therationale for the development ofan optimized delivery systemusing a liquid solution of EMDfor future combination with bonegrafting materials.
CONCLUSIONS
The results from the presentstudy demonstrate that surfacecoatings with enamel matrixproteins vary depending onthe coating of bone graftingmaterials with either EMD liquidor EMD gel. Surface coatingwith EMD gel increased drasti-cally the thickness of coating ofenamel matrix proteins to bonegrafting surfaces, which wereeasily dissolved after a simplePBS rinse. The use of EMD ina liquid formulation was able toprovide better surface coatingdirectly adjacent to scaffoldsurface, whereas protein adsorp-tion was also observed within theinterior of the bone grafting par-ticles when coated on NBM andDFDBA. When bone grafts werecoated with EMD in a liquid for-mulation, they demonstratedbetter long-term delivery ofenamel matrix proteins duringa 10-day period compared toEMD in a gel formulation. Thus,the liquid formulation of EMDallows for the following: 1) in-creased and more completesurface loading of porous graftmaterials; and 2) tighter andmore stable surface coating withenamel matrix proteins.
ACKNOWLEDGMENTS
The authors gratefully acknowl-edge the Robert K. Schenk Lab-
oratory of Oral Histology, Dental School at theUniversity of Bern, most notably Thuy Tran Nguyen,Monika Aeberhard, and David Reist for their consid-erable time and valuable insights into the histologicperformance of experiments. This work was fundedby the Department of Periodontology at the Univer-sity of Bern and Institute Straumann, for whichDrs. Tugulu, Gemperli, and Dard are employees.
Figure 4.SEM and TEM images of CaP bone grafting after coating with either EMD liquid or EMD gel. A) SEMimage of CaP particles coated with EMD liquid demonstrates accumulation of EMD on the surface ofgrafting particles. The results from TEM further confirmed that most of the staining was present at a slightdistance from the surface, with no presence of penetration within the bone grafting scaffold (C, arrows).B)Similarly, CaP particles coated with EMD gel demonstrated the presence of a thicker coating caused by itsPGA carrier. The surface coating with an anti-EMD antibody also revealed the presence of its coating ata distance from the surface of grafting particles (D, arrows). (A) and B) ·1,600; scale bars = 20 mm.)
Figure 5.A) Quantitative analysis of the number of gold-labeled particles that appear from TEM images revealeda slight increase in number on grafting particles coated with EMD liquid and EMD gel. B) The particleswere then quantified for their average distance from the scaffold surface, and results demonstrate a muchcloser binding affinity to scaffolds when grafts were coated with EMD liquid instead of EMD gel. * denotessignificant differences of P <0.05.
Enamel Matrix Derivative Adsorption to Bone Grafts Volume 86 • Number 4
584
RESULTSQuantification of Amelogenin Protein to the Scaffold Surface by ELISA
Dr. Sculean has received lecture fees from InstituteStraumann. Institute Straumannmanufactured the bonegraft, EMD, and EMD gel used in this study. Drs. Miron,Bosshardt, Buser, Zhang, Caluseru, and Chandad re-port no conflicts of interest related to this study.
REFERENCES1. Buser D, Dula K, Belser U, Hirt HP, Berthold H. Localized
ridge augmentation using guided bone regeneration. 1.Surgical procedure in the maxilla. Int J PeriodonticsRestorative Dent 1993;13:29-45.
2. Jung RE, Fenner N, Hammerle CH, Zitzmann NU. Long-term outcome of implants placed with guided boneregeneration (GBR)using resorbable andnon-resorbablemembranes after 12-14 years. Clin Oral Implants Res2013;24:1065-1073.
3. Lorenzoni M, Pertl C, Keil C, Wegscheider WA. Treat-ment of peri-implant defects with guided bone regener-ation:A comparative clinical studywith variousmembranesand bone grafts. Int J Oral Maxillofac Implants 1998;13:639-646.
4. Wiltfang J, Schultze-Mosgau S, Merten HA, Kessler P,LudwigA,EngelkeW.Endoscopic andultrasonographicevaluation of the maxillary sinus after combined sinusfloor augmentation and implant insertion.Oral SurgOralMed Oral Pathol Oral Radiol Endod 2000;89:288-291.
5. Calori GM,Mazza E, ColomboM, Ripamonti C. The use ofbone-graft substitutes in large bone defects: Any specificneeds? Injury 2011;42(Suppl. 2):S56-S63.
6. Dragoo MR, Sullivan HC. Aclinical and histological evalu-ation of autogenous iliac bonegrafts in humans. I. Woundhealing 2 to 8 months. J Peri-odontol 1973;44:599-613.
7. Gross JS. Bone grafting mate-rials for dental applications: Apractical guide. Compend Con-tin Educ Dent 1997;18:1013-1018,1020-1022,1024,passim;quiz.
8. Hiatt WH, Schallhorn RG,Aaronian AJ. The induction ofnew bone and cementum for-mation. IV. Microscopic exam-ination of the periodontiumfollowing human bone andmarrow allograft, autograftand nongraft periodontal re-generative procedures. J Peri-odontol 1978;49:495-512.
9. Misch CE, Dietsh F. Bone-grafting materials in implantdentistry. Implant Dent1993;2:158-167.
10. Froum SJ, Wallace SS, ChoSC, Elian N, Tarnow DP. His-tomorphometric comparison ofa biphasic bone ceramic toanorganic bovine bone for si-nus augmentation: 6- to 8-month postsurgical assessmentof vital bone formation. A pilotstudy. Int J Periodontics Restor-ative Dent 2008;28:273-281.
11. Schwartz Z, Weesner T, van Dijk S, et al. Ability ofdeproteinized cancellous bovine bone to induce newboneformation. J Periodontol 2000;71:1258-1269.
12. Rios HF, Lin Z, Oh B, Park CH, Giannobile WV. Cell- andgene-based therapeutic strategies for periodontal regen-erative medicine. J Periodontol 2011;82:1223-1237.
13. Jepsen S, Heinz B, Jepsen K, et al. A randomizedclinical trial comparing enamel matrix derivative andmembrane treatment of buccal Class II furcation in-volvement in mandibular molars. Part I: Study designand results for primary outcomes. J Periodontol 2004;75:1150-1160.
14. Meyle J, Gonzales JR, Bodeker RH, et al. A randomizedclinical trial comparing enamel matrix derivative andmembrane treatment of buccal class II furcation in-volvement in mandibular molars. Part II: Secondaryoutcomes. J Periodontol 2004;75:1188-1195.
15. Modica F, Del Pizzo M, Roccuzzo M, Romagnoli R.Coronally advanced flap for the treatment of buccalgingival recessions with and without enamel matrixderivative. A split-mouth study. J Periodontol 2000;71:1693-1698.
16. Nemcovsky CE, Artzi Z, Tal H, Kozlovsky A, Moses O.A multicenter comparative study of two root coverageprocedures: Coronally advanced flap with addition ofenamel matrix proteins and subpedicle connectivetissue graft. J Periodontol 2004;75:600-607.
17. Sculean A, Donos N, Windisch P, et al. Healing ofhuman intrabony defects following treatment withenamel matrix proteins or guided tissue regeneration.J Periodontal Res 1999;34:310-322.
Figure 6.A)Quantitative analysis by ELISA to determine the quantity of adsorbed amelogenin proteins demonstratesa slight, non-significantpreference for grafting particles coatedwith EMD liquid.BthroughD) Interestingly,the surfaces coated with EMD gel demonstrated a much faster release profile of amelogeninproteins with time, demonstrating a more preferential binding of amelogenin to bone grafting particleswhen particles were coated with EMD liquid compared to EMD gel.
J Periodontol • April 2015 Miron, Bosshardt, Buser, et al.
585
RESULTSQuantification of Amelogenin Protein to the Scaffold Surface by ELISA
• 興味深いことに、骨移植材を経時的なアメロジェニンタンパクの放出について比較したところ、EMD liquidとEMD gelの間に有意差が見られた(Figs. 6B through 6D)。
Dr. Sculean has received lecture fees from InstituteStraumann. Institute Straumannmanufactured the bonegraft, EMD, and EMD gel used in this study. Drs. Miron,Bosshardt, Buser, Zhang, Caluseru, and Chandad re-port no conflicts of interest related to this study.
REFERENCES1. Buser D, Dula K, Belser U, Hirt HP, Berthold H. Localized
ridge augmentation using guided bone regeneration. 1.Surgical procedure in the maxilla. Int J PeriodonticsRestorative Dent 1993;13:29-45.
2. Jung RE, Fenner N, Hammerle CH, Zitzmann NU. Long-term outcome of implants placed with guided boneregeneration (GBR)using resorbable andnon-resorbablemembranes after 12-14 years. Clin Oral Implants Res2013;24:1065-1073.
3. Lorenzoni M, Pertl C, Keil C, Wegscheider WA. Treat-ment of peri-implant defects with guided bone regener-ation:A comparative clinical studywith variousmembranesand bone grafts. Int J Oral Maxillofac Implants 1998;13:639-646.
4. Wiltfang J, Schultze-Mosgau S, Merten HA, Kessler P,LudwigA,EngelkeW.Endoscopic andultrasonographicevaluation of the maxillary sinus after combined sinusfloor augmentation and implant insertion.Oral SurgOralMed Oral Pathol Oral Radiol Endod 2000;89:288-291.
5. Calori GM,Mazza E, ColomboM, Ripamonti C. The use ofbone-graft substitutes in large bone defects: Any specificneeds? Injury 2011;42(Suppl. 2):S56-S63.
6. Dragoo MR, Sullivan HC. Aclinical and histological evalu-ation of autogenous iliac bonegrafts in humans. I. Woundhealing 2 to 8 months. J Peri-odontol 1973;44:599-613.
7. Gross JS. Bone grafting mate-rials for dental applications: Apractical guide. Compend Con-tin Educ Dent 1997;18:1013-1018,1020-1022,1024,passim;quiz.
8. Hiatt WH, Schallhorn RG,Aaronian AJ. The induction ofnew bone and cementum for-mation. IV. Microscopic exam-ination of the periodontiumfollowing human bone andmarrow allograft, autograftand nongraft periodontal re-generative procedures. J Peri-odontol 1978;49:495-512.
9. Misch CE, Dietsh F. Bone-grafting materials in implantdentistry. Implant Dent1993;2:158-167.
10. Froum SJ, Wallace SS, ChoSC, Elian N, Tarnow DP. His-tomorphometric comparison ofa biphasic bone ceramic toanorganic bovine bone for si-nus augmentation: 6- to 8-month postsurgical assessmentof vital bone formation. A pilotstudy. Int J Periodontics Restor-ative Dent 2008;28:273-281.
11. Schwartz Z, Weesner T, van Dijk S, et al. Ability ofdeproteinized cancellous bovine bone to induce newboneformation. J Periodontol 2000;71:1258-1269.
12. Rios HF, Lin Z, Oh B, Park CH, Giannobile WV. Cell- andgene-based therapeutic strategies for periodontal regen-erative medicine. J Periodontol 2011;82:1223-1237.
13. Jepsen S, Heinz B, Jepsen K, et al. A randomizedclinical trial comparing enamel matrix derivative andmembrane treatment of buccal Class II furcation in-volvement in mandibular molars. Part I: Study designand results for primary outcomes. J Periodontol 2004;75:1150-1160.
14. Meyle J, Gonzales JR, Bodeker RH, et al. A randomizedclinical trial comparing enamel matrix derivative andmembrane treatment of buccal class II furcation in-volvement in mandibular molars. Part II: Secondaryoutcomes. J Periodontol 2004;75:1188-1195.
15. Modica F, Del Pizzo M, Roccuzzo M, Romagnoli R.Coronally advanced flap for the treatment of buccalgingival recessions with and without enamel matrixderivative. A split-mouth study. J Periodontol 2000;71:1693-1698.
16. Nemcovsky CE, Artzi Z, Tal H, Kozlovsky A, Moses O.A multicenter comparative study of two root coverageprocedures: Coronally advanced flap with addition ofenamel matrix proteins and subpedicle connectivetissue graft. J Periodontol 2004;75:600-607.
17. Sculean A, Donos N, Windisch P, et al. Healing ofhuman intrabony defects following treatment withenamel matrix proteins or guided tissue regeneration.J Periodontal Res 1999;34:310-322.
Figure 6.A)Quantitative analysis by ELISA to determine the quantity of adsorbed amelogenin proteins demonstratesa slight, non-significantpreference for grafting particles coatedwith EMD liquid.BthroughD) Interestingly,the surfaces coated with EMD gel demonstrated a much faster release profile of amelogeninproteins with time, demonstrating a more preferential binding of amelogenin to bone grafting particleswhen particles were coated with EMD liquid compared to EMD gel.
J Periodontol • April 2015 Miron, Bosshardt, Buser, et al.
585
RESULTSQuantification of Amelogenin Protein to the Scaffold Surface by ELISA
Dr. Sculean has received lecture fees from InstituteStraumann. Institute Straumannmanufactured the bonegraft, EMD, and EMD gel used in this study. Drs. Miron,Bosshardt, Buser, Zhang, Caluseru, and Chandad re-port no conflicts of interest related to this study.
REFERENCES1. Buser D, Dula K, Belser U, Hirt HP, Berthold H. Localized
ridge augmentation using guided bone regeneration. 1.Surgical procedure in the maxilla. Int J PeriodonticsRestorative Dent 1993;13:29-45.
2. Jung RE, Fenner N, Hammerle CH, Zitzmann NU. Long-term outcome of implants placed with guided boneregeneration (GBR)using resorbable andnon-resorbablemembranes after 12-14 years. Clin Oral Implants Res2013;24:1065-1073.
3. Lorenzoni M, Pertl C, Keil C, Wegscheider WA. Treat-ment of peri-implant defects with guided bone regener-ation:A comparative clinical studywith variousmembranesand bone grafts. Int J Oral Maxillofac Implants 1998;13:639-646.
4. Wiltfang J, Schultze-Mosgau S, Merten HA, Kessler P,LudwigA,EngelkeW.Endoscopic andultrasonographicevaluation of the maxillary sinus after combined sinusfloor augmentation and implant insertion.Oral SurgOralMed Oral Pathol Oral Radiol Endod 2000;89:288-291.
5. Calori GM,Mazza E, ColomboM, Ripamonti C. The use ofbone-graft substitutes in large bone defects: Any specificneeds? Injury 2011;42(Suppl. 2):S56-S63.
6. Dragoo MR, Sullivan HC. Aclinical and histological evalu-ation of autogenous iliac bonegrafts in humans. I. Woundhealing 2 to 8 months. J Peri-odontol 1973;44:599-613.
7. Gross JS. Bone grafting mate-rials for dental applications: Apractical guide. Compend Con-tin Educ Dent 1997;18:1013-1018,1020-1022,1024,passim;quiz.
8. Hiatt WH, Schallhorn RG,Aaronian AJ. The induction ofnew bone and cementum for-mation. IV. Microscopic exam-ination of the periodontiumfollowing human bone andmarrow allograft, autograftand nongraft periodontal re-generative procedures. J Peri-odontol 1978;49:495-512.
9. Misch CE, Dietsh F. Bone-grafting materials in implantdentistry. Implant Dent1993;2:158-167.
10. Froum SJ, Wallace SS, ChoSC, Elian N, Tarnow DP. His-tomorphometric comparison ofa biphasic bone ceramic toanorganic bovine bone for si-nus augmentation: 6- to 8-month postsurgical assessmentof vital bone formation. A pilotstudy. Int J Periodontics Restor-ative Dent 2008;28:273-281.
11. Schwartz Z, Weesner T, van Dijk S, et al. Ability ofdeproteinized cancellous bovine bone to induce newboneformation. J Periodontol 2000;71:1258-1269.
12. Rios HF, Lin Z, Oh B, Park CH, Giannobile WV. Cell- andgene-based therapeutic strategies for periodontal regen-erative medicine. J Periodontol 2011;82:1223-1237.
13. Jepsen S, Heinz B, Jepsen K, et al. A randomizedclinical trial comparing enamel matrix derivative andmembrane treatment of buccal Class II furcation in-volvement in mandibular molars. Part I: Study designand results for primary outcomes. J Periodontol 2004;75:1150-1160.
14. Meyle J, Gonzales JR, Bodeker RH, et al. A randomizedclinical trial comparing enamel matrix derivative andmembrane treatment of buccal class II furcation in-volvement in mandibular molars. Part II: Secondaryoutcomes. J Periodontol 2004;75:1188-1195.
15. Modica F, Del Pizzo M, Roccuzzo M, Romagnoli R.Coronally advanced flap for the treatment of buccalgingival recessions with and without enamel matrixderivative. A split-mouth study. J Periodontol 2000;71:1693-1698.
16. Nemcovsky CE, Artzi Z, Tal H, Kozlovsky A, Moses O.A multicenter comparative study of two root coverageprocedures: Coronally advanced flap with addition ofenamel matrix proteins and subpedicle connectivetissue graft. J Periodontol 2004;75:600-607.
17. Sculean A, Donos N, Windisch P, et al. Healing ofhuman intrabony defects following treatment withenamel matrix proteins or guided tissue regeneration.J Periodontal Res 1999;34:310-322.
Figure 6.A)Quantitative analysis by ELISA to determine the quantity of adsorbed amelogenin proteins demonstratesa slight, non-significantpreference for grafting particles coatedwith EMD liquid.BthroughD) Interestingly,the surfaces coated with EMD gel demonstrated a much faster release profile of amelogeninproteins with time, demonstrating a more preferential binding of amelogenin to bone grafting particleswhen particles were coated with EMD liquid compared to EMD gel.
J Periodontol • April 2015 Miron, Bosshardt, Buser, et al.
585
RESULTSQuantification of Amelogenin Protein to the Scaffold Surface by ELISA
Dr. Sculean has received lecture fees from InstituteStraumann. Institute Straumannmanufactured the bonegraft, EMD, and EMD gel used in this study. Drs. Miron,Bosshardt, Buser, Zhang, Caluseru, and Chandad re-port no conflicts of interest related to this study.
REFERENCES1. Buser D, Dula K, Belser U, Hirt HP, Berthold H. Localized
ridge augmentation using guided bone regeneration. 1.Surgical procedure in the maxilla. Int J PeriodonticsRestorative Dent 1993;13:29-45.
2. Jung RE, Fenner N, Hammerle CH, Zitzmann NU. Long-term outcome of implants placed with guided boneregeneration (GBR)using resorbable andnon-resorbablemembranes after 12-14 years. Clin Oral Implants Res2013;24:1065-1073.
3. Lorenzoni M, Pertl C, Keil C, Wegscheider WA. Treat-ment of peri-implant defects with guided bone regener-ation:A comparative clinical studywith variousmembranesand bone grafts. Int J Oral Maxillofac Implants 1998;13:639-646.
4. Wiltfang J, Schultze-Mosgau S, Merten HA, Kessler P,LudwigA,EngelkeW.Endoscopic andultrasonographicevaluation of the maxillary sinus after combined sinusfloor augmentation and implant insertion.Oral SurgOralMed Oral Pathol Oral Radiol Endod 2000;89:288-291.
5. Calori GM,Mazza E, ColomboM, Ripamonti C. The use ofbone-graft substitutes in large bone defects: Any specificneeds? Injury 2011;42(Suppl. 2):S56-S63.
6. Dragoo MR, Sullivan HC. Aclinical and histological evalu-ation of autogenous iliac bonegrafts in humans. I. Woundhealing 2 to 8 months. J Peri-odontol 1973;44:599-613.
7. Gross JS. Bone grafting mate-rials for dental applications: Apractical guide. Compend Con-tin Educ Dent 1997;18:1013-1018,1020-1022,1024,passim;quiz.
8. Hiatt WH, Schallhorn RG,Aaronian AJ. The induction ofnew bone and cementum for-mation. IV. Microscopic exam-ination of the periodontiumfollowing human bone andmarrow allograft, autograftand nongraft periodontal re-generative procedures. J Peri-odontol 1978;49:495-512.
9. Misch CE, Dietsh F. Bone-grafting materials in implantdentistry. Implant Dent1993;2:158-167.
10. Froum SJ, Wallace SS, ChoSC, Elian N, Tarnow DP. His-tomorphometric comparison ofa biphasic bone ceramic toanorganic bovine bone for si-nus augmentation: 6- to 8-month postsurgical assessmentof vital bone formation. A pilotstudy. Int J Periodontics Restor-ative Dent 2008;28:273-281.
11. Schwartz Z, Weesner T, van Dijk S, et al. Ability ofdeproteinized cancellous bovine bone to induce newboneformation. J Periodontol 2000;71:1258-1269.
12. Rios HF, Lin Z, Oh B, Park CH, Giannobile WV. Cell- andgene-based therapeutic strategies for periodontal regen-erative medicine. J Periodontol 2011;82:1223-1237.
13. Jepsen S, Heinz B, Jepsen K, et al. A randomizedclinical trial comparing enamel matrix derivative andmembrane treatment of buccal Class II furcation in-volvement in mandibular molars. Part I: Study designand results for primary outcomes. J Periodontol 2004;75:1150-1160.
14. Meyle J, Gonzales JR, Bodeker RH, et al. A randomizedclinical trial comparing enamel matrix derivative andmembrane treatment of buccal class II furcation in-volvement in mandibular molars. Part II: Secondaryoutcomes. J Periodontol 2004;75:1188-1195.
15. Modica F, Del Pizzo M, Roccuzzo M, Romagnoli R.Coronally advanced flap for the treatment of buccalgingival recessions with and without enamel matrixderivative. A split-mouth study. J Periodontol 2000;71:1693-1698.
16. Nemcovsky CE, Artzi Z, Tal H, Kozlovsky A, Moses O.A multicenter comparative study of two root coverageprocedures: Coronally advanced flap with addition ofenamel matrix proteins and subpedicle connectivetissue graft. J Periodontol 2004;75:600-607.
17. Sculean A, Donos N, Windisch P, et al. Healing ofhuman intrabony defects following treatment withenamel matrix proteins or guided tissue regeneration.J Periodontal Res 1999;34:310-322.
Figure 6.A)Quantitative analysis by ELISA to determine the quantity of adsorbed amelogenin proteins demonstratesa slight, non-significantpreference for grafting particles coatedwith EMD liquid.BthroughD) Interestingly,the surfaces coated with EMD gel demonstrated a much faster release profile of amelogeninproteins with time, demonstrating a more preferential binding of amelogenin to bone grafting particleswhen particles were coated with EMD liquid compared to EMD gel.
J Periodontol • April 2015 Miron, Bosshardt, Buser, et al.
585
RESULTSQuantification of Amelogenin Protein to the Scaffold Surface by ELISA
Dr. Sculean has received lecture fees from InstituteStraumann. Institute Straumannmanufactured the bonegraft, EMD, and EMD gel used in this study. Drs. Miron,Bosshardt, Buser, Zhang, Caluseru, and Chandad re-port no conflicts of interest related to this study.
REFERENCES1. Buser D, Dula K, Belser U, Hirt HP, Berthold H. Localized
ridge augmentation using guided bone regeneration. 1.Surgical procedure in the maxilla. Int J PeriodonticsRestorative Dent 1993;13:29-45.
2. Jung RE, Fenner N, Hammerle CH, Zitzmann NU. Long-term outcome of implants placed with guided boneregeneration (GBR)using resorbable andnon-resorbablemembranes after 12-14 years. Clin Oral Implants Res2013;24:1065-1073.
3. Lorenzoni M, Pertl C, Keil C, Wegscheider WA. Treat-ment of peri-implant defects with guided bone regener-ation:A comparative clinical studywith variousmembranesand bone grafts. Int J Oral Maxillofac Implants 1998;13:639-646.
4. Wiltfang J, Schultze-Mosgau S, Merten HA, Kessler P,LudwigA,EngelkeW.Endoscopic andultrasonographicevaluation of the maxillary sinus after combined sinusfloor augmentation and implant insertion.Oral SurgOralMed Oral Pathol Oral Radiol Endod 2000;89:288-291.
5. Calori GM,Mazza E, ColomboM, Ripamonti C. The use ofbone-graft substitutes in large bone defects: Any specificneeds? Injury 2011;42(Suppl. 2):S56-S63.
6. Dragoo MR, Sullivan HC. Aclinical and histological evalu-ation of autogenous iliac bonegrafts in humans. I. Woundhealing 2 to 8 months. J Peri-odontol 1973;44:599-613.
7. Gross JS. Bone grafting mate-rials for dental applications: Apractical guide. Compend Con-tin Educ Dent 1997;18:1013-1018,1020-1022,1024,passim;quiz.
8. Hiatt WH, Schallhorn RG,Aaronian AJ. The induction ofnew bone and cementum for-mation. IV. Microscopic exam-ination of the periodontiumfollowing human bone andmarrow allograft, autograftand nongraft periodontal re-generative procedures. J Peri-odontol 1978;49:495-512.
9. Misch CE, Dietsh F. Bone-grafting materials in implantdentistry. Implant Dent1993;2:158-167.
10. Froum SJ, Wallace SS, ChoSC, Elian N, Tarnow DP. His-tomorphometric comparison ofa biphasic bone ceramic toanorganic bovine bone for si-nus augmentation: 6- to 8-month postsurgical assessmentof vital bone formation. A pilotstudy. Int J Periodontics Restor-ative Dent 2008;28:273-281.
11. Schwartz Z, Weesner T, van Dijk S, et al. Ability ofdeproteinized cancellous bovine bone to induce newboneformation. J Periodontol 2000;71:1258-1269.
12. Rios HF, Lin Z, Oh B, Park CH, Giannobile WV. Cell- andgene-based therapeutic strategies for periodontal regen-erative medicine. J Periodontol 2011;82:1223-1237.
13. Jepsen S, Heinz B, Jepsen K, et al. A randomizedclinical trial comparing enamel matrix derivative andmembrane treatment of buccal Class II furcation in-volvement in mandibular molars. Part I: Study designand results for primary outcomes. J Periodontol 2004;75:1150-1160.
14. Meyle J, Gonzales JR, Bodeker RH, et al. A randomizedclinical trial comparing enamel matrix derivative andmembrane treatment of buccal class II furcation in-volvement in mandibular molars. Part II: Secondaryoutcomes. J Periodontol 2004;75:1188-1195.
15. Modica F, Del Pizzo M, Roccuzzo M, Romagnoli R.Coronally advanced flap for the treatment of buccalgingival recessions with and without enamel matrixderivative. A split-mouth study. J Periodontol 2000;71:1693-1698.
16. Nemcovsky CE, Artzi Z, Tal H, Kozlovsky A, Moses O.A multicenter comparative study of two root coverageprocedures: Coronally advanced flap with addition ofenamel matrix proteins and subpedicle connectivetissue graft. J Periodontol 2004;75:600-607.
17. Sculean A, Donos N, Windisch P, et al. Healing ofhuman intrabony defects following treatment withenamel matrix proteins or guided tissue regeneration.J Periodontal Res 1999;34:310-322.
Figure 6.A)Quantitative analysis by ELISA to determine the quantity of adsorbed amelogenin proteins demonstratesa slight, non-significantpreference for grafting particles coatedwith EMD liquid.BthroughD) Interestingly,the surfaces coated with EMD gel demonstrated a much faster release profile of amelogeninproteins with time, demonstrating a more preferential binding of amelogenin to bone grafting particleswhen particles were coated with EMD liquid compared to EMD gel.
J Periodontol • April 2015 Miron, Bosshardt, Buser, et al.
the use of EMD gel provided a much thicker surfacecoating, one that extends several micrometers, asviewed by SEM and TEM images. Although in someclinical studies the use of EMD gel has demonstratedpositive outcomes when combined with bone graftingmaterials, the present findings provide conclusiveevidence that a large variability exists betweenamelogenin adsorption to the various bone grafting
materials used in reconstructiveperiodontal surgery. Moreover,the present data provide therationale for the development ofan optimized delivery systemusing a liquid solution of EMDfor future combination with bonegrafting materials.
CONCLUSIONS
The results from the presentstudy demonstrate that surfacecoatings with enamel matrixproteins vary depending onthe coating of bone graftingmaterials with either EMD liquidor EMD gel. Surface coatingwith EMD gel increased drasti-cally the thickness of coating ofenamel matrix proteins to bonegrafting surfaces, which wereeasily dissolved after a simplePBS rinse. The use of EMD ina liquid formulation was able toprovide better surface coatingdirectly adjacent to scaffoldsurface, whereas protein adsorp-tion was also observed within theinterior of the bone grafting par-ticles when coated on NBM andDFDBA. When bone grafts werecoated with EMD in a liquid for-mulation, they demonstratedbetter long-term delivery ofenamel matrix proteins duringa 10-day period compared toEMD in a gel formulation. Thus,the liquid formulation of EMDallows for the following: 1) in-creased and more completesurface loading of porous graftmaterials; and 2) tighter andmore stable surface coating withenamel matrix proteins.
ACKNOWLEDGMENTS
The authors gratefully acknowl-edge the Robert K. Schenk Lab-
oratory of Oral Histology, Dental School at theUniversity of Bern, most notably Thuy Tran Nguyen,Monika Aeberhard, and David Reist for their consid-erable time and valuable insights into the histologicperformance of experiments. This work was fundedby the Department of Periodontology at the Univer-sity of Bern and Institute Straumann, for whichDrs. Tugulu, Gemperli, and Dard are employees.
Figure 4.SEM and TEM images of CaP bone grafting after coating with either EMD liquid or EMD gel. A) SEMimage of CaP particles coated with EMD liquid demonstrates accumulation of EMD on the surface ofgrafting particles. The results from TEM further confirmed that most of the staining was present at a slightdistance from the surface, with no presence of penetration within the bone grafting scaffold (C, arrows).B)Similarly, CaP particles coated with EMD gel demonstrated the presence of a thicker coating caused by itsPGA carrier. The surface coating with an anti-EMD antibody also revealed the presence of its coating ata distance from the surface of grafting particles (D, arrows). (A) and B) ·1,600; scale bars = 20 mm.)
Figure 5.A) Quantitative analysis of the number of gold-labeled particles that appear from TEM images revealeda slight increase in number on grafting particles coated with EMD liquid and EMD gel. B) The particleswere then quantified for their average distance from the scaffold surface, and results demonstrate a muchcloser binding affinity to scaffolds when grafts were coated with EMD liquid instead of EMD gel. * denotessignificant differences of P <0.05.
Enamel Matrix Derivative Adsorption to Bone Grafts Volume 86 • Number 4
the use of EMD gel provided a much thicker surfacecoating, one that extends several micrometers, asviewed by SEM and TEM images. Although in someclinical studies the use of EMD gel has demonstratedpositive outcomes when combined with bone graftingmaterials, the present findings provide conclusiveevidence that a large variability exists betweenamelogenin adsorption to the various bone grafting
materials used in reconstructiveperiodontal surgery. Moreover,the present data provide therationale for the development ofan optimized delivery systemusing a liquid solution of EMDfor future combination with bonegrafting materials.
CONCLUSIONS
The results from the presentstudy demonstrate that surfacecoatings with enamel matrixproteins vary depending onthe coating of bone graftingmaterials with either EMD liquidor EMD gel. Surface coatingwith EMD gel increased drasti-cally the thickness of coating ofenamel matrix proteins to bonegrafting surfaces, which wereeasily dissolved after a simplePBS rinse. The use of EMD ina liquid formulation was able toprovide better surface coatingdirectly adjacent to scaffoldsurface, whereas protein adsorp-tion was also observed within theinterior of the bone grafting par-ticles when coated on NBM andDFDBA. When bone grafts werecoated with EMD in a liquid for-mulation, they demonstratedbetter long-term delivery ofenamel matrix proteins duringa 10-day period compared toEMD in a gel formulation. Thus,the liquid formulation of EMDallows for the following: 1) in-creased and more completesurface loading of porous graftmaterials; and 2) tighter andmore stable surface coating withenamel matrix proteins.
ACKNOWLEDGMENTS
The authors gratefully acknowl-edge the Robert K. Schenk Lab-
oratory of Oral Histology, Dental School at theUniversity of Bern, most notably Thuy Tran Nguyen,Monika Aeberhard, and David Reist for their consid-erable time and valuable insights into the histologicperformance of experiments. This work was fundedby the Department of Periodontology at the Univer-sity of Bern and Institute Straumann, for whichDrs. Tugulu, Gemperli, and Dard are employees.
Figure 4.SEM and TEM images of CaP bone grafting after coating with either EMD liquid or EMD gel. A) SEMimage of CaP particles coated with EMD liquid demonstrates accumulation of EMD on the surface ofgrafting particles. The results from TEM further confirmed that most of the staining was present at a slightdistance from the surface, with no presence of penetration within the bone grafting scaffold (C, arrows).B)Similarly, CaP particles coated with EMD gel demonstrated the presence of a thicker coating caused by itsPGA carrier. The surface coating with an anti-EMD antibody also revealed the presence of its coating ata distance from the surface of grafting particles (D, arrows). (A) and B) ·1,600; scale bars = 20 mm.)
Figure 5.A) Quantitative analysis of the number of gold-labeled particles that appear from TEM images revealeda slight increase in number on grafting particles coated with EMD liquid and EMD gel. B) The particleswere then quantified for their average distance from the scaffold surface, and results demonstrate a muchcloser binding affinity to scaffolds when grafts were coated with EMD liquid instead of EMD gel. * denotessignificant differences of P <0.05.
Enamel Matrix Derivative Adsorption to Bone Grafts Volume 86 • Number 4
In the present study, the choice was made toinvestigate three grafting materials commonly usedin dentistry. NBM was used as the xenograft of choicebecause it is studied extensively and the presentauthors have previous experience handling it. TheDFDBA used in this study was selected because ofits widespread use and osteoinductive advantagescompared to other DFDBA grafts.48-50 An alloplastbone graft fabricated from hydroxyapatite and b-TCPwas chosen as the synthetic material. To the best ofthe authors’ knowledge, in the present study, it is thefirst time observed by both SEM and TEM that largevariability existed with respect to the ability of enamelmatrix proteins to adsorb to these various bone grafts(Figs. 2 through 5). Interestingly, although the NBMparticles were able to adsorb the highest quantity ofprotein, the more interesting finding was the inabilityfor EMD gel to efficiently adsorb enamel matrix pro-teins to the surface of grafting materials at the presentsettings (Fig. 5). It was noted that the average distancein which amelogenin proteins were found from thesurface of bone grafting particles was >20 timesgreater in samples coated with EMD gel when com-pared to EMD liquid (see supplementary Table 1 inonline Journal of Periodontology).
The present study reveals thatthe adsorption of enamel matrixproteins may be attributed tothe material composition. BothDFDBA and NBM are derivedfrom natural bone, and their na-tive composition naturally con-tains specific sites for proteinadsorption. Because NBM parti-cles are completely devoid ofcells and proteins (deproteinizedmatrix), their ability to providespecific sites for new proteinadsorption make it an ideal graftchoice as a carrier for bioactivemolecules. A similar observationfor DFDBA was also observedwith a surprisingly high ability forenamel matrix proteins coatedwithin the interior surface ofgrafting material (Fig. 3A). Con-trary to this, no ability of EMDgel to penetrate the surface ofCaP molecules was observed.Because the surface containsno discernible pores able toallow any form of protein ad-sorption within the grafting ma-terial, it signifies that proteinadsorption is limited strictly tothe material surface, thus pro-
viding much less ability for the graft to carry ahigher load of bioactive molecules.
Although the SEM and TEM analyses were ableto provide much qualitative analysis, the use of anELISA kit greatly enhanced the ability to accuratelyquantify the amount of amelogenin bound to thematerial surface. Although each of the bone graftswas able to adsorb either EMD liquid or EMD gel tothe surface of the bone grafting material, the no-ticeable difference was evident after rinsing with PBSat neutral pH. Because this solution is used in vitro torepresent physiologic pH, the finding that a simplerinse was able to wash >50% of amelogenin bound tothe surface of grafting materials coated with EMD gelwas quite surprising (Fig. 6). Although it is verydifficult to speculate what effect this might have ina clinical setting, the finding that such a large per-centage of protein is flushed so easily raises theclinical concern that after the placement of EMD gel +bone graft in a bone defect, a similar effect might beimagined whereby bodily fluids are able to dissociatethe adsorbed enamel matrix proteins from the scaf-fold surface. Noteworthy also is the fact that bone ap-position should be directly adjacent to the bone graftingmaterial surface. In the present study, it is found that
Figure 3.SEM and TEM images of DFDBA after coating with either EMD liquid or EMD gel. A) SEM imageof DFDBA particles coated with EMD liquid demonstrates its smooth surface properties with littleaccumulation of a thick surface coatingwith amelogenin proteins. The results fromTEM further confirmedthat most of the staining was present on the surface or slightly penetrated within the surface of thegrafting material (C, arrows). B) In contrast, DFDBA particles coated with EMD gel demonstrated thepresence of a much thicker coating caused by its PGA carrier. The surface coating with an anti-EMDantibody also revealed the presence of most of its coating far from the surface of grafting particles(D, arrows). (A) and B) ·1,600; scale bars = 20 mm.)
J Periodontol • April 2015 Miron, Bosshardt, Buser, et al.
the use of EMD gel provided a much thicker surfacecoating, one that extends several micrometers, asviewed by SEM and TEM images. Although in someclinical studies the use of EMD gel has demonstratedpositive outcomes when combined with bone graftingmaterials, the present findings provide conclusiveevidence that a large variability exists betweenamelogenin adsorption to the various bone grafting
materials used in reconstructiveperiodontal surgery. Moreover,the present data provide therationale for the development ofan optimized delivery systemusing a liquid solution of EMDfor future combination with bonegrafting materials.
CONCLUSIONS
The results from the presentstudy demonstrate that surfacecoatings with enamel matrixproteins vary depending onthe coating of bone graftingmaterials with either EMD liquidor EMD gel. Surface coatingwith EMD gel increased drasti-cally the thickness of coating ofenamel matrix proteins to bonegrafting surfaces, which wereeasily dissolved after a simplePBS rinse. The use of EMD ina liquid formulation was able toprovide better surface coatingdirectly adjacent to scaffoldsurface, whereas protein adsorp-tion was also observed within theinterior of the bone grafting par-ticles when coated on NBM andDFDBA. When bone grafts werecoated with EMD in a liquid for-mulation, they demonstratedbetter long-term delivery ofenamel matrix proteins duringa 10-day period compared toEMD in a gel formulation. Thus,the liquid formulation of EMDallows for the following: 1) in-creased and more completesurface loading of porous graftmaterials; and 2) tighter andmore stable surface coating withenamel matrix proteins.
ACKNOWLEDGMENTS
The authors gratefully acknowl-edge the Robert K. Schenk Lab-
oratory of Oral Histology, Dental School at theUniversity of Bern, most notably Thuy Tran Nguyen,Monika Aeberhard, and David Reist for their consid-erable time and valuable insights into the histologicperformance of experiments. This work was fundedby the Department of Periodontology at the Univer-sity of Bern and Institute Straumann, for whichDrs. Tugulu, Gemperli, and Dard are employees.
Figure 4.SEM and TEM images of CaP bone grafting after coating with either EMD liquid or EMD gel. A) SEMimage of CaP particles coated with EMD liquid demonstrates accumulation of EMD on the surface ofgrafting particles. The results from TEM further confirmed that most of the staining was present at a slightdistance from the surface, with no presence of penetration within the bone grafting scaffold (C, arrows).B)Similarly, CaP particles coated with EMD gel demonstrated the presence of a thicker coating caused by itsPGA carrier. The surface coating with an anti-EMD antibody also revealed the presence of its coating ata distance from the surface of grafting particles (D, arrows). (A) and B) ·1,600; scale bars = 20 mm.)
Figure 5.A) Quantitative analysis of the number of gold-labeled particles that appear from TEM images revealeda slight increase in number on grafting particles coated with EMD liquid and EMD gel. B) The particleswere then quantified for their average distance from the scaffold surface, and results demonstrate a muchcloser binding affinity to scaffolds when grafts were coated with EMD liquid instead of EMD gel. * denotessignificant differences of P <0.05.
Enamel Matrix Derivative Adsorption to Bone Grafts Volume 86 • Number 4
Dr. Sculean has received lecture fees from InstituteStraumann. Institute Straumannmanufactured the bonegraft, EMD, and EMD gel used in this study. Drs. Miron,Bosshardt, Buser, Zhang, Caluseru, and Chandad re-port no conflicts of interest related to this study.
REFERENCES1. Buser D, Dula K, Belser U, Hirt HP, Berthold H. Localized
ridge augmentation using guided bone regeneration. 1.Surgical procedure in the maxilla. Int J PeriodonticsRestorative Dent 1993;13:29-45.
2. Jung RE, Fenner N, Hammerle CH, Zitzmann NU. Long-term outcome of implants placed with guided boneregeneration (GBR)using resorbable andnon-resorbablemembranes after 12-14 years. Clin Oral Implants Res2013;24:1065-1073.
3. Lorenzoni M, Pertl C, Keil C, Wegscheider WA. Treat-ment of peri-implant defects with guided bone regener-ation:A comparative clinical studywith variousmembranesand bone grafts. Int J Oral Maxillofac Implants 1998;13:639-646.
4. Wiltfang J, Schultze-Mosgau S, Merten HA, Kessler P,LudwigA,EngelkeW.Endoscopic andultrasonographicevaluation of the maxillary sinus after combined sinusfloor augmentation and implant insertion.Oral SurgOralMed Oral Pathol Oral Radiol Endod 2000;89:288-291.
5. Calori GM,Mazza E, ColomboM, Ripamonti C. The use ofbone-graft substitutes in large bone defects: Any specificneeds? Injury 2011;42(Suppl. 2):S56-S63.
6. Dragoo MR, Sullivan HC. Aclinical and histological evalu-ation of autogenous iliac bonegrafts in humans. I. Woundhealing 2 to 8 months. J Peri-odontol 1973;44:599-613.
7. Gross JS. Bone grafting mate-rials for dental applications: Apractical guide. Compend Con-tin Educ Dent 1997;18:1013-1018,1020-1022,1024,passim;quiz.
8. Hiatt WH, Schallhorn RG,Aaronian AJ. The induction ofnew bone and cementum for-mation. IV. Microscopic exam-ination of the periodontiumfollowing human bone andmarrow allograft, autograftand nongraft periodontal re-generative procedures. J Peri-odontol 1978;49:495-512.
9. Misch CE, Dietsh F. Bone-grafting materials in implantdentistry. Implant Dent1993;2:158-167.
10. Froum SJ, Wallace SS, ChoSC, Elian N, Tarnow DP. His-tomorphometric comparison ofa biphasic bone ceramic toanorganic bovine bone for si-nus augmentation: 6- to 8-month postsurgical assessmentof vital bone formation. A pilotstudy. Int J Periodontics Restor-ative Dent 2008;28:273-281.
11. Schwartz Z, Weesner T, van Dijk S, et al. Ability ofdeproteinized cancellous bovine bone to induce newboneformation. J Periodontol 2000;71:1258-1269.
12. Rios HF, Lin Z, Oh B, Park CH, Giannobile WV. Cell- andgene-based therapeutic strategies for periodontal regen-erative medicine. J Periodontol 2011;82:1223-1237.
13. Jepsen S, Heinz B, Jepsen K, et al. A randomizedclinical trial comparing enamel matrix derivative andmembrane treatment of buccal Class II furcation in-volvement in mandibular molars. Part I: Study designand results for primary outcomes. J Periodontol 2004;75:1150-1160.
14. Meyle J, Gonzales JR, Bodeker RH, et al. A randomizedclinical trial comparing enamel matrix derivative andmembrane treatment of buccal class II furcation in-volvement in mandibular molars. Part II: Secondaryoutcomes. J Periodontol 2004;75:1188-1195.
15. Modica F, Del Pizzo M, Roccuzzo M, Romagnoli R.Coronally advanced flap for the treatment of buccalgingival recessions with and without enamel matrixderivative. A split-mouth study. J Periodontol 2000;71:1693-1698.
16. Nemcovsky CE, Artzi Z, Tal H, Kozlovsky A, Moses O.A multicenter comparative study of two root coverageprocedures: Coronally advanced flap with addition ofenamel matrix proteins and subpedicle connectivetissue graft. J Periodontol 2004;75:600-607.
17. Sculean A, Donos N, Windisch P, et al. Healing ofhuman intrabony defects following treatment withenamel matrix proteins or guided tissue regeneration.J Periodontal Res 1999;34:310-322.
Figure 6.A)Quantitative analysis by ELISA to determine the quantity of adsorbed amelogenin proteins demonstratesa slight, non-significantpreference for grafting particles coatedwith EMD liquid.BthroughD) Interestingly,the surfaces coated with EMD gel demonstrated a much faster release profile of amelogeninproteins with time, demonstrating a more preferential binding of amelogenin to bone grafting particleswhen particles were coated with EMD liquid compared to EMD gel.
J Periodontol • April 2015 Miron, Bosshardt, Buser, et al.
Dr. Sculean has received lecture fees from InstituteStraumann. Institute Straumannmanufactured the bonegraft, EMD, and EMD gel used in this study. Drs. Miron,Bosshardt, Buser, Zhang, Caluseru, and Chandad re-port no conflicts of interest related to this study.
REFERENCES1. Buser D, Dula K, Belser U, Hirt HP, Berthold H. Localized
ridge augmentation using guided bone regeneration. 1.Surgical procedure in the maxilla. Int J PeriodonticsRestorative Dent 1993;13:29-45.
2. Jung RE, Fenner N, Hammerle CH, Zitzmann NU. Long-term outcome of implants placed with guided boneregeneration (GBR)using resorbable andnon-resorbablemembranes after 12-14 years. Clin Oral Implants Res2013;24:1065-1073.
3. Lorenzoni M, Pertl C, Keil C, Wegscheider WA. Treat-ment of peri-implant defects with guided bone regener-ation:A comparative clinical studywith variousmembranesand bone grafts. Int J Oral Maxillofac Implants 1998;13:639-646.
4. Wiltfang J, Schultze-Mosgau S, Merten HA, Kessler P,LudwigA,EngelkeW.Endoscopic andultrasonographicevaluation of the maxillary sinus after combined sinusfloor augmentation and implant insertion.Oral SurgOralMed Oral Pathol Oral Radiol Endod 2000;89:288-291.
5. Calori GM,Mazza E, ColomboM, Ripamonti C. The use ofbone-graft substitutes in large bone defects: Any specificneeds? Injury 2011;42(Suppl. 2):S56-S63.
6. Dragoo MR, Sullivan HC. Aclinical and histological evalu-ation of autogenous iliac bonegrafts in humans. I. Woundhealing 2 to 8 months. J Peri-odontol 1973;44:599-613.
7. Gross JS. Bone grafting mate-rials for dental applications: Apractical guide. Compend Con-tin Educ Dent 1997;18:1013-1018,1020-1022,1024,passim;quiz.
8. Hiatt WH, Schallhorn RG,Aaronian AJ. The induction ofnew bone and cementum for-mation. IV. Microscopic exam-ination of the periodontiumfollowing human bone andmarrow allograft, autograftand nongraft periodontal re-generative procedures. J Peri-odontol 1978;49:495-512.
9. Misch CE, Dietsh F. Bone-grafting materials in implantdentistry. Implant Dent1993;2:158-167.
10. Froum SJ, Wallace SS, ChoSC, Elian N, Tarnow DP. His-tomorphometric comparison ofa biphasic bone ceramic toanorganic bovine bone for si-nus augmentation: 6- to 8-month postsurgical assessmentof vital bone formation. A pilotstudy. Int J Periodontics Restor-ative Dent 2008;28:273-281.
11. Schwartz Z, Weesner T, van Dijk S, et al. Ability ofdeproteinized cancellous bovine bone to induce newboneformation. J Periodontol 2000;71:1258-1269.
12. Rios HF, Lin Z, Oh B, Park CH, Giannobile WV. Cell- andgene-based therapeutic strategies for periodontal regen-erative medicine. J Periodontol 2011;82:1223-1237.
13. Jepsen S, Heinz B, Jepsen K, et al. A randomizedclinical trial comparing enamel matrix derivative andmembrane treatment of buccal Class II furcation in-volvement in mandibular molars. Part I: Study designand results for primary outcomes. J Periodontol 2004;75:1150-1160.
14. Meyle J, Gonzales JR, Bodeker RH, et al. A randomizedclinical trial comparing enamel matrix derivative andmembrane treatment of buccal class II furcation in-volvement in mandibular molars. Part II: Secondaryoutcomes. J Periodontol 2004;75:1188-1195.
15. Modica F, Del Pizzo M, Roccuzzo M, Romagnoli R.Coronally advanced flap for the treatment of buccalgingival recessions with and without enamel matrixderivative. A split-mouth study. J Periodontol 2000;71:1693-1698.
16. Nemcovsky CE, Artzi Z, Tal H, Kozlovsky A, Moses O.A multicenter comparative study of two root coverageprocedures: Coronally advanced flap with addition ofenamel matrix proteins and subpedicle connectivetissue graft. J Periodontol 2004;75:600-607.
17. Sculean A, Donos N, Windisch P, et al. Healing ofhuman intrabony defects following treatment withenamel matrix proteins or guided tissue regeneration.J Periodontal Res 1999;34:310-322.
Figure 6.A)Quantitative analysis by ELISA to determine the quantity of adsorbed amelogenin proteins demonstratesa slight, non-significantpreference for grafting particles coatedwith EMD liquid.BthroughD) Interestingly,the surfaces coated with EMD gel demonstrated a much faster release profile of amelogeninproteins with time, demonstrating a more preferential binding of amelogenin to bone grafting particleswhen particles were coated with EMD liquid compared to EMD gel.
J Periodontol • April 2015 Miron, Bosshardt, Buser, et al.