The International Journal of Periodontics & Restorative Dentistry © 2014 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY. NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.
375
The Feasibility of Using Coronally Advanced Flap with an Extracellular Matrix Membrane for Treating Gingival Recession Defects: A Preclinical Study Khalid Al Hezaimi, DDS, MSc1 Mansour Al Askar, DDS, MSc2 David M. Kim, DDS, DMSc3 Jamie Hyewon Chung, DDS4 Mindy S. Gil, DMD4/Myron Nevins, DDS5 The guided tissue regeneration (GTR) procedure has been demonstrated to successfully correct gingival recession (GR) defects. The aim of this study was to assess the feasibility of using the combination of a coronally advanced flap (CAF) with an extracellular membrane (ECM) to correct GR. GR defects were induced in the maxillary posterior region in five baboons. A 3-month healing period followed before the ECM was placed over the defect and covered with a CAF. Probing depth (PD), recession depth (RD), recession width (RW), and keratinized tissue width (KTW) were measured at baseline and 3 months postoperatively. Block biopsies of the treated areas were submitted for histologic review after a healing period of 3 months. There were no significant differences between the test (ECM + CAF) and control (CAF) groups in terms of changes in PD, RD, RW, and KTW after 3 months of treatment. However, significant differences have been noted for preoperation and postsurgery RD and RW values for both the control and test groups. Histomorphometric results showed minimal alveolar bone and connective tissue replacing the ECM membrane. CAF (either with or without the use of an ECM) is effective for the treatment of GR defects. (Int J Periodontics Restorative Dent 2014;34:375–380. doi: 10.11607/prd.2069)
Chairman and Associate Professor, Eng. A.B. Growth Factors and Bone Regeneration Research Chair, Department of Periodontics and Community Dentistry, College of Dentistry, King Saud University, Riyadh, Saudi Arabia. 2Assistant Professor, Department of Periodontics and Community Dentistry, College of Dentistry, King Saud University, Riyadh, Saudi Arabia. 3Associate Professor, Division of Periodontology, Department of Oral Medicine, Infection and Immunity, Division of Periodontics, Harvard School of Dental Medicine, Boston, USA. 4Postdoctoral Research Fellow, Department of Oral Medicine, Infection and Immunity, Division of Periodontics, Harvard School of Dental Medicine, Boston, USA. 5Associate Clinical Professor, Division of Periodontology, Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, Massachusetts, USA. 1
Correspondence to: Dr David M. Kim, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, MA 02115; fax: 617-432-1897; email: [email protected]. ©2014 by Quintessence Publishing Co Inc.
Successful gingival recession (GR) treatment is characterized by the elimination of exposed roots, reduction in probing depth, and increased clinical attachment. Numerous treatment strategies provided varying results depending on the degree of recession and treatment procedures selected.1,2 A mean of 68% to 91% of root coverage may be expected depending on the recession type and treatment procedure adopted, such as rotational flaps, coronally advanced flaps (CAF), connective tissue grafts, or free gingival grafts.3–5 Root coverage accomplished with the guided tissue regeneration procedure (GTR) using resorbable biocompatible barrier membranes can be successfully used to reestablish soft tissue dimensions over gingival recession defects.6–11 Their advantages include elimination of morbidity and discomfort associated with a palatal surgical harvest to procure graft material.12 Histologic evaluation illustrates limited new bone and cementum formation with Sharpey fiber attachment after GTR procedures.13–15 Clinical studies, however, have demonstrated
Volume 34, Number 3, 2014 © 2014 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY. NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.
376 promising outcomes in GTR with increased clinical attachment levels and width of keratinized gingiva (KG).8–11,15 Extracellular matrix membrane (ECM) (Dynamatrix, Cook Biotech) is obtained from the submucosa of the small intestine of pigs, using a process that retains the natural composition of matrix molecules such as collagens (types I, III, IV, and VI), glycosaminoglycans, glycoproteins, proteoglycans, and growth factors16,17 that play important roles in tissue repair and remodeling. The ECM membrane shelters periodontal progenitor ligament cells over defect sites, allowing their proliferation and new tissue formation around the defect. ECM membranes are mechanically durable and bioactive and are known to play essential roles in host tissue repair and remodeling.16,17 Natural ECM membranes blend well with the surrounding oral tissue and yield a natural postoperative clinical appearance when compared with the autogenous gingival grafted sites.18 The factors that influence the efficacy of GTR procedures include the interproximal height of adjacent bone, the dimensions of the adjacent interdental papilla, the size of the GR defect, flap thickness and tension, membrane exposure, and surgical skills of the operator.18–25 Soft tissue dehiscences resulting in membrane exposure may jeopardize the tissue regeneration process.26,27 The aim of this preclinical study was to assess the clinical and histologic feasibility of using CAF with an extracellular matrix membrane for treating gingival recession defects.
Method and materials
Surgical correction procedure
Study approval and outline
Coronal advancement of the gingival tissues was performed using a combination of full-thickness and partial-thickness flaps 3 months after the defects were created. Releasing incisions were made at the mesial of the first premolar and the distal of the second premolar (Fig 1a). Degranulation was performed, and the root surfaces were scaled using both ultrasonic device and hand instruments. An ECM membrane was placed over the defects, and the defects were then covered by CAF to the cemento enamel junction (CEJ) (Fig 1c) for three animals. Primary closure was achieved using interrupted resorbable sutures (Fig 1d). The same surgical procedure was performed for the control group (two animals) without the ECM membrane.
The experimental protocol was approved by the Research Ethics Review Committee of the Engineer Abdullah Bugshan Research Chair for Growth Factors and Bone Regeneration, 3D Imaging and Biomechanical Laboratory, College of Applied Medical Sciences and College of Dentistry Research Center (CDRC), King Saud University, Riyadh, Saudi Arabia. Five nonhuman primates (Papio hamadryas) with a minimum age of 12 months and weight of 8 to 10 kg were used. The animals were kept in individual cages and fed a soft diet throughout the study period. All surgical procedures were performed under general and local anesthesia in a sterile environment. The primates underwent full-mouth scaling and root planing using an ultrasonic scaler and hand curettes.
Recession defect induction
Full-thickness buccal flaps were reflected bilaterally for the maxillary premolar areas. Buccal root recession defects (5 mm × 3 mm) were surgically induced on the roots of the maxillary first and second premolars, and a notch was also placed at the base of the gingival recession defect.28 Tension-free primary flap closure was achieved using resorbable sutures. All animals underwent supragingival scaling once a week until euthanasia.
Clinical parameters
The following clinical parameters were assessed preoperatively and 3 months after treatment (just before euthanasia): (a) probing depth (PD),29 (b) recession depth (RD)— distance from the CEJ to the gingival margin,30 (c) recession width (RW)—distance from one end of the recession to another at the level of CEJ,30 and (d) width of keratinized tissue (KTW)—distance from the gingival margin to the mucogingival fold.30
The International Journal of Periodontics & Restorative Dentistry © 2014 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY. NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.
377
a
b
c
d
e
Fig 1 (a) Clinical presentation of the treatment protocol. (b) Placement of a notch at the base of the defect (arrows). (c) Placement of an ECM membrane over the critical size defect. (d) Primary closure using resorbable sutures and (e) after healing.
Euthanasia and specimen preparation
Three months after the surgical correction procedure, all animals were sacrificed under general anesthesia using an overdose of xylazine and ketamine hydrochloride. Jaw segments containing treatment sites were removed en bloc using an electric saw. The blocks were fixed and stored in 4% paraformaldehyde, dehydrated in step gradients of alcohol, and infiltrated and embedded in methyl methacrylate. Toluidine blue/pyronine G was used to stain the specimens. Histomorphometric analysis was performed using a microscope connected to a high-resolution video camera and a software program. Histomorphometric analysis was performed by a single investigator.
Statistical analysis
Clinical measurements
Statistical analysis was performed using the SPSS software (version 18.00, IBM). The paired t test was used to compare measurements performed at baseline and before euthanasia. The Student t test was used to compare the control and test groups.
At baseline, the mean preoperative PD, RD, RW, and KTW measurements for the control group were 1.31 ± 0.53 mm, 1.19 ± 0.46 mm, 1.50 ± 0.46 mm, and 2.31 ± 0.88 mm, respectively. After 3 months of treatment, the mean PD, RD, RW, and KTW measurements for the control group were 1.03 ± 0.21 mm, 0.31 ± 0.40 mm, 0.34 ± 0.44 mm, and 2.19 ± 0.75 mm, respectively. Significant differences were noted for RD (P = .001) and RW (P = .001) values when pre- and postoperative values were compared. For the test group, the mean preoperative PD, RD, RW, and KTW measurements were 1.38 ± 0.64 mm, 1.12 ± 0.57 mm, 1.08 ± 0.60 mm, and 2.12 ± 1.00 mm, respectively. The postoperative PD, RD, RW, and
Results Clinical observations
The clinical results were variable for both the control and test groups. For the test group, 7 of 12 roots (58%) achieved complete root coverage. For the control group, 4 of 8 roots (50%) achieved complete root coverage (Fig 1e).
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Table 1
Pre- and postoperative clinical measurements for control and test groups. Preoperative (mm)
Postoperative (mm)
PD
RD
RW
KTW
PD
RD
RW
KTW
Control (n = 8)
1.31
1.19
1.50
2.31
1.03
0.31
0.34
2.19
Test (n = 12)
1.38
1.12
1.08
2.12
1.25
0.42
0.38
2.50
PD = probing depth; RD = recession depth; RW = recession width; KTW = keratinized tissue width.
a
b
c
d
e
f
KTW measurements were 1.25 ± 0.34 mm, 0.42 ± 0.78 mm, 0.38 ± 0.61 mm, and 2.50 ± 0.77 mm, respectively. Significant differences were noted for RD (P = .007) and RW (P = .002) values when pre- and postoperative values were compared. There were no significant differences between the test and control groups in terms of their PD, RD, RW, and KTW measurements (P > .05) (Table 1).
Histology
An overview of the gingiva ending at the level of the notch is shown in Fig 2a (test group). Figure 2b shows the keratinized oral epithelium at a higher magnification. Collagen fiber bundles running in a vertical direction and remnants of the membrane are shown in Fig 2c. The arrowhead on Fig 2d shows the alveolar bone crest. Collagen
Fig 2 Histomorphometric results of the treatment following euthanasia (after 16 weeks). (a) Overview of the gingiva ending at the level of the notch. (b) Keratinized oral epithelium (arrow) at a higher magnification. (c) Collagen fiber bundles running in a vertical direction (arrow) and remnants of the membrane. (d) Alveolar bone crest (arrow). (e) Collagen fibers of the newly formed periodontal ligament at low resolution. (f) Collagen fibers of the newly formed periodontal ligament (arrow) at high resolution.
fibers of the newly formed periodontal ligament at low and high magnification are shown in Figs 2e and 2f, respectively.
Discussion This investigation simulates a chronic clinical scenario since the recession defects were planned to be treated 3 months following
The International Journal of Periodontics & Restorative Dentistry © 2014 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY. NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.
379 their induction. The results demonstrated successful outcomes in terms of root coverage using the ECM membrane plus GTR protocol or CAF alone. They support a previous study18 that compared the treatment efficacy of using a free gingival graft vs an ECM membrane for increasing the zone of keratinized gingiva and demonstrated that the matrix-generated tissue was comparable to the tissue derived from the autograft.18 It has been reported that early resorption of collagen membranes (crosslinked as well as noncrosslinked) may hamper tissue regeneration31; however, there is no consensus over the time duration for which matrix membranes should remain over the defects to yield maximum tissue regeneration. Clinical evidence32,33 has suggested that membrane exposure may negatively influence the overall efficacy of the GTR procedure. It may therefore be hypothesized that thicker ECM membranes placed on GR defects for longer time durations may play a role in the correction of GR. However, further in vivo studies are needed to assess the effect of adjunctive use of ECM on gingival thickness in root-coverage procedures. The present results showed a significant reduction in RD and RW measured at baseline compared with those measured after 3 months of treatment for both the control and test groups. Every effort was made to establish a regular plaque control regimen, but animals required general anesthesia for each event, which was impractical. Thus, the animals’ oral
hygiene status may have contributed to the incomplete root coverage for both groups. It has been reported that size of the induced defect may influence the outcome of root coverage protocols. Pini Prato and coauthors reported that the root coverage is greater when the recession is greater than 4.98 mm, which is approximately the size of the defect that was made in this model.34 However, further studies regarding the influence of GR size on root coverage are required.
Conclusion The present preclinical and histomorphometric experiment evaluated the efficacy of ECM membranes for the treatment of induced GR defects. In this context, it can be concluded that ECM membranes are feasible for GTR protocols.
Acknowledgements This study was supported by a grant from Cook Biotech and the College of Dentistry Research Center, King Saud University.
References 1. Oates TW, Robinson M, Gunsolley JC. Surgical therapies for the treatment of gingival recession. A systematic review. Ann Periodontol 2003;8:303–320. 2. Cheng YF, Chen JW, Lin SJ, Lu HK. Is coronally positioned flap procedure adjunct with enamel matrix derivative or root conditioning a relevant predictor for achieving root coverage? A systemic review. J Periodontal Res 2007;42:474–485.
3. Camelo M, Nevins M, Nevins ML, Schubach P, Kim D. Treatment of gingival recession defects with xenogenic collagen matrix: A histologic report. Int J Periodontics Restorative Dent 2012;32: 167–173. 4. Cairo F, Cortellini P, Tonetti M, et al. Coronally advanced flap with and without connective tissue graft for the treatment of single maxillary gingival recession with loss of inter-dental attachment. A randomized controlled clinical trial. J Clin Periodontol 2012;39:760–768. 5. Tinti C, Vincenzi G, Cortellini P, Pini Prato G, Clauser C. Guided tissue regeneration in the treatment of human facial recession. A 12-case report. J Periodontol 1992;63:554–560. 6. Kuru B, Yildirim S. Treatment of localized gingival recessions using gingival unit grafts: A randomized controlled clinical trial. J Periodontol 2013;84:41–50. 7. Malament KA, Nevins ML, Singh T. Interdisciplinary solutions for esthetic periodontal prosthodontic rehabilitation. Compend Contin Educ Dent 2011;32:33–40. 8. Rosetti EP, Marcantonio E Jr, Zuza EP, Marcantonio RA. Root coverage stability of the subepithelial connective tissue graft and guided tissue regeneration: A 30-month follow-up clinical trial. J Dent 2013;41:114–120. 9. Pini Prato G, Clauser C, Cortellini P, Tinti C, Vincenzi G, Pagliaro U. Guided tissue regeneration versus mucogingival surgery in the treatment of human buccal recessions. A 4-year follow-up study. J Periodontol 1996;67:1216–1223. 10. Harris RJ. A comparative study of root coverage obtained with guided tissue regeneration utilizing a bioabsorbable membrane versus the connective tissue with partial-thickness double pedicle graft. J Periodontol 1997;68:779–790. 11. Harris RJ. A comparison of 2 root coverage techniques: Guided tissue regeneration with a bioabsorbable matrix style membrane versus a connective tissue graft combined with a coronally positioned pedicle graft without vertical incisions. Results of a series of consecutive cases. J Periodontol 1998;69: 1426–1434. 12. Gottlow J, Nyman S, Lindhe J, Karring T, Wennström J. New attachment formation in the human periodontium by guided tissue regeneration. Case reports. J Clin Periodontol 1986;13:604–616.
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13. Cortellini P, DeSanctis M, Pini Prato G, Baldi C, Clauser C. Guided tissue regeneration procedure using a fibrinfibronectin system in surgically induced recession in dogs. Int J Periodontics Restorative Dent 1991;11:150–163. 14. Cortellini P, Clauser C, Prato GP. Histologic assessment of new attachment following the treatment of a human buccal recession by means of a guided tissue regeneration procedure. J Periodontol 1993;64:387–391. 15. Gottlow J, Karring T, Nyman S. Guided tissue regeneration following treatment of recession-type defects in the monkey. J Periodontol 1990;61:680–685. 16. Badylak SF. Small intestinal submucosa (SIS): A biomaterial conducive to smart tissue remodeling. In: Bell E (ed). Tissue Engineering: Current Perspectives. Cambridge: Burkhauser, 1993: 179–189. 17. Hodde J, Janis A, Ernst D, Zopf D, Sherman D, Johnson C. Effects of sterilization on an extracellular matrix scaffold: Part I. Composition and matrix architecture. J Mater Sci Mater Med 2007;18:537–543. 18. Nevins M, Nevins ML, Camelo M, Camelo JM, Schupbach P, Kim DM. The clinical efficacy of DynaMatrix extracellular membrane in augmenting keratinized tissue. Int J Periodontics Restorative Dent 2010;30:151–161. 19. Kim DM, Nevins M, Camelo M, et al. The feasibility of demineralized bone matrix and cancellous bone chips in conjunction with an extracellular matrix membrane for alveolar ridge preservation: A case series. Int J Periodontics Restorative Dent 2011;31:39–47.
20. Miller PD Jr. A classification of marginal tissue recession. Int J Periodontics Restorative Dent 1985;5:8–13. 21. Harris RJ. The connective tissue with partial thickness double pedicle graft: The results of 100 consecutively treated defects. J Periodontol 1994;65:448–461. 22. Pini Prato G, Pagliaro U, Baldi C, et al. Coronally advanced flap procedure for root coverage. Flap with tension versus flap without tension: A randomized controlled clinical study. J Periodontol 2000; 71:188–201. 23. Saletta D, Pini Prato G, Pagliaro U, Baldi C, Mauri M, Nieri M. Coronally advanced flap procedure: Is the interdental papilla a prognostic factor for root coverage? J Periodontol 2001;72:760–766. 24. Sullivan HC, Atkins JH. Free autogenous gingival grafts. 3. Utilization of grafts in the treatment of gingival recession. Periodontics 1968;6:152–160. 25. Clauser C, Nieri M, Franceschi D, Pagliaro U, Pini- Prato G. Evidence-based mucogingival therapy. Part 2: Ordinary and individual patient data meta-analyses of surgical treatment of recession using complete root coverage as the outcome variable. J Periodontol 2003;74:741–756. 26. Machtei EE, Dunford R, Grossi SG, Genco GJ. Gingival recession and exposure of barrier membrane: Effect on guided tissue regeneration of Class II furcation defects. Int J Periodontics Restorative Dent 1995;15:590–599. 27. Tal H, Pitaru S, Moses O, Kozlovsky A. Collagen gel and membrane in guided tissue regeneration in periodontal fenestration defects in dogs. J Clin Periodontol 1996;23:1–6.
28. Mlinek A, Smukler H, Buchner A. The use of free gingival grafts for the coverage of denuded roots. J Periodontol 1973; 44:248–254. 29. Armitage CC. Periodontal diagnoses and classification of periodontal diseases. Periodontol 2000 2004;34:9–21. 30. Nanditha S, Priya MS, Sabitha S, Arun KV, Avaneendra T. Clinical evaluation of the efficacy of a GTR membrane (HEALIGUIDE) and demineralised bone matrix (OSSEOGRAFT) as a space maintainer in the treatment of Miller’s Class I gingival recession. J Indian Soc Periodontol 2011;15:156–160. 31. Owens KW, Yukna RA. Collagen membrane resorption in dogs: A comparative study. Implant Dent 2001;10:49–58. 32. Tatakis DN, Trombelli L. Gingival recession treatment: Guided tissue regeneration with bioabsorbable membrane versus connective tissue graft. J Periodontol 2000;71:299–307. 33. Trombelli L, Scabbia A, Tatakis DN, Checchi L, Calura G. Resorbable barrier and envelope flap surgery in the treatment of human gingival recession defects. Case reports. J Clin Periodontol 1998;25:24–29. 34. Pini-Prato G, Baldi C, Pagliaro U, et al. Coronally advanced flap procedure for root coverage. Treatment of root surface: Root planning versus polishing. J Periodontol 1999;70:1064–1076.
The International Journal of Periodontics & Restorative Dentistry © 2014 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY. NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.
375
The Feasibility of Using Coronally Advanced Flap with an Extracellular Matrix Membrane for Treating Gingival Recession Defects: A Preclinical Study Khalid Al Hezaimi, DDS, MSc1 Mansour Al Askar, DDS, MSc2 David M. Kim, DDS, DMSc3 Jamie Hyewon Chung, DDS4 Mindy S. Gil, DMD4/Myron Nevins, DDS5 The guided tissue regeneration (GTR) procedure has been demonstrated to successfully correct gingival recession (GR) defects. The aim of this study was to assess the feasibility of using the combination of a coronally advanced flap (CAF) with an extracellular membrane (ECM) to correct GR. GR defects were induced in the maxillary posterior region in five baboons. A 3-month healing period followed before the ECM was placed over the defect and covered with a CAF. Probing depth (PD), recession depth (RD), recession width (RW), and keratinized tissue width (KTW) were measured at baseline and 3 months postoperatively. Block biopsies of the treated areas were submitted for histologic review after a healing period of 3 months. There were no significant differences between the test (ECM + CAF) and control (CAF) groups in terms of changes in PD, RD, RW, and KTW after 3 months of treatment. However, significant differences have been noted for preoperation and postsurgery RD and RW values for both the control and test groups. Histomorphometric results showed minimal alveolar bone and connective tissue replacing the ECM membrane. CAF (either with or without the use of an ECM) is effective for the treatment of GR defects. (Int J Periodontics Restorative Dent 2014;34:375–380. doi: 10.11607/prd.2069)
Chairman and Associate Professor, Eng. A.B. Growth Factors and Bone Regeneration Research Chair, Department of Periodontics and Community Dentistry, College of Dentistry, King Saud University, Riyadh, Saudi Arabia. 2Assistant Professor, Department of Periodontics and Community Dentistry, College of Dentistry, King Saud University, Riyadh, Saudi Arabia. 3Associate Professor, Division of Periodontology, Department of Oral Medicine, Infection and Immunity, Division of Periodontics, Harvard School of Dental Medicine, Boston, USA. 4Postdoctoral Research Fellow, Department of Oral Medicine, Infection and Immunity, Division of Periodontics, Harvard School of Dental Medicine, Boston, USA. 5Associate Clinical Professor, Division of Periodontology, Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, Massachusetts, USA. 1
Correspondence to: Dr David M. Kim, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, MA 02115; fax: 617-432-1897; email: [email protected]. ©2014 by Quintessence Publishing Co Inc.
Successful gingival recession (GR) treatment is characterized by the elimination of exposed roots, reduction in probing depth, and increased clinical attachment. Numerous treatment strategies provided varying results depending on the degree of recession and treatment procedures selected.1,2 A mean of 68% to 91% of root coverage may be expected depending on the recession type and treatment procedure adopted, such as rotational flaps, coronally advanced flaps (CAF), connective tissue grafts, or free gingival grafts.3–5 Root coverage accomplished with the guided tissue regeneration procedure (GTR) using resorbable biocompatible barrier membranes can be successfully used to reestablish soft tissue dimensions over gingival recession defects.6–11 Their advantages include elimination of morbidity and discomfort associated with a palatal surgical harvest to procure graft material.12 Histologic evaluation illustrates limited new bone and cementum formation with Sharpey fiber attachment after GTR procedures.13–15 Clinical studies, however, have demonstrated
Volume 34, Number 3, 2014 © 2014 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY. NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.
376 promising outcomes in GTR with increased clinical attachment levels and width of keratinized gingiva (KG).8–11,15 Extracellular matrix membrane (ECM) (Dynamatrix, Cook Biotech) is obtained from the submucosa of the small intestine of pigs, using a process that retains the natural composition of matrix molecules such as collagens (types I, III, IV, and VI), glycosaminoglycans, glycoproteins, proteoglycans, and growth factors16,17 that play important roles in tissue repair and remodeling. The ECM membrane shelters periodontal progenitor ligament cells over defect sites, allowing their proliferation and new tissue formation around the defect. ECM membranes are mechanically durable and bioactive and are known to play essential roles in host tissue repair and remodeling.16,17 Natural ECM membranes blend well with the surrounding oral tissue and yield a natural postoperative clinical appearance when compared with the autogenous gingival grafted sites.18 The factors that influence the efficacy of GTR procedures include the interproximal height of adjacent bone, the dimensions of the adjacent interdental papilla, the size of the GR defect, flap thickness and tension, membrane exposure, and surgical skills of the operator.18–25 Soft tissue dehiscences resulting in membrane exposure may jeopardize the tissue regeneration process.26,27 The aim of this preclinical study was to assess the clinical and histologic feasibility of using CAF with an extracellular matrix membrane for treating gingival recession defects.
Method and materials
Surgical correction procedure
Study approval and outline
Coronal advancement of the gingival tissues was performed using a combination of full-thickness and partial-thickness flaps 3 months after the defects were created. Releasing incisions were made at the mesial of the first premolar and the distal of the second premolar (Fig 1a). Degranulation was performed, and the root surfaces were scaled using both ultrasonic device and hand instruments. An ECM membrane was placed over the defects, and the defects were then covered by CAF to the cemento enamel junction (CEJ) (Fig 1c) for three animals. Primary closure was achieved using interrupted resorbable sutures (Fig 1d). The same surgical procedure was performed for the control group (two animals) without the ECM membrane.
The experimental protocol was approved by the Research Ethics Review Committee of the Engineer Abdullah Bugshan Research Chair for Growth Factors and Bone Regeneration, 3D Imaging and Biomechanical Laboratory, College of Applied Medical Sciences and College of Dentistry Research Center (CDRC), King Saud University, Riyadh, Saudi Arabia. Five nonhuman primates (Papio hamadryas) with a minimum age of 12 months and weight of 8 to 10 kg were used. The animals were kept in individual cages and fed a soft diet throughout the study period. All surgical procedures were performed under general and local anesthesia in a sterile environment. The primates underwent full-mouth scaling and root planing using an ultrasonic scaler and hand curettes.
Recession defect induction
Full-thickness buccal flaps were reflected bilaterally for the maxillary premolar areas. Buccal root recession defects (5 mm × 3 mm) were surgically induced on the roots of the maxillary first and second premolars, and a notch was also placed at the base of the gingival recession defect.28 Tension-free primary flap closure was achieved using resorbable sutures. All animals underwent supragingival scaling once a week until euthanasia.
Clinical parameters
The following clinical parameters were assessed preoperatively and 3 months after treatment (just before euthanasia): (a) probing depth (PD),29 (b) recession depth (RD)— distance from the CEJ to the gingival margin,30 (c) recession width (RW)—distance from one end of the recession to another at the level of CEJ,30 and (d) width of keratinized tissue (KTW)—distance from the gingival margin to the mucogingival fold.30
The International Journal of Periodontics & Restorative Dentistry © 2014 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY. NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.
377
a
b
c
d
e
Fig 1 (a) Clinical presentation of the treatment protocol. (b) Placement of a notch at the base of the defect (arrows). (c) Placement of an ECM membrane over the critical size defect. (d) Primary closure using resorbable sutures and (e) after healing.
Euthanasia and specimen preparation
Three months after the surgical correction procedure, all animals were sacrificed under general anesthesia using an overdose of xylazine and ketamine hydrochloride. Jaw segments containing treatment sites were removed en bloc using an electric saw. The blocks were fixed and stored in 4% paraformaldehyde, dehydrated in step gradients of alcohol, and infiltrated and embedded in methyl methacrylate. Toluidine blue/pyronine G was used to stain the specimens. Histomorphometric analysis was performed using a microscope connected to a high-resolution video camera and a software program. Histomorphometric analysis was performed by a single investigator.
Statistical analysis
Clinical measurements
Statistical analysis was performed using the SPSS software (version 18.00, IBM). The paired t test was used to compare measurements performed at baseline and before euthanasia. The Student t test was used to compare the control and test groups.
At baseline, the mean preoperative PD, RD, RW, and KTW measurements for the control group were 1.31 ± 0.53 mm, 1.19 ± 0.46 mm, 1.50 ± 0.46 mm, and 2.31 ± 0.88 mm, respectively. After 3 months of treatment, the mean PD, RD, RW, and KTW measurements for the control group were 1.03 ± 0.21 mm, 0.31 ± 0.40 mm, 0.34 ± 0.44 mm, and 2.19 ± 0.75 mm, respectively. Significant differences were noted for RD (P = .001) and RW (P = .001) values when pre- and postoperative values were compared. For the test group, the mean preoperative PD, RD, RW, and KTW measurements were 1.38 ± 0.64 mm, 1.12 ± 0.57 mm, 1.08 ± 0.60 mm, and 2.12 ± 1.00 mm, respectively. The postoperative PD, RD, RW, and
Results Clinical observations
The clinical results were variable for both the control and test groups. For the test group, 7 of 12 roots (58%) achieved complete root coverage. For the control group, 4 of 8 roots (50%) achieved complete root coverage (Fig 1e).
Volume 34, Number 3, 2014 © 2014 BY QUINTESSENCE PUBLISHING CO, INC. PRINTING OF THIS DOCUMENT IS RESTRICTED TO PERSONAL USE ONLY. NO PART MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM WITHOUT WRITTEN PERMISSION FROM THE PUBLISHER.
378
Table 1
Pre- and postoperative clinical measurements for control and test groups. Preoperative (mm)
Postoperative (mm)
PD
RD
RW
KTW
PD
RD
RW
KTW
Control (n = 8)
1.31
1.19
1.50
2.31
1.03
0.31
0.34
2.19
Test (n = 12)
1.38
1.12
1.08
2.12
1.25
0.42
0.38
2.50
PD = probing depth; RD = recession depth; RW = recession width; KTW = keratinized tissue width.
a
b
c
d
e
f
KTW measurements were 1.25 ± 0.34 mm, 0.42 ± 0.78 mm, 0.38 ± 0.61 mm, and 2.50 ± 0.77 mm, respectively. Significant differences were noted for RD (P = .007) and RW (P = .002) values when pre- and postoperative values were compared. There were no significant differences between the test and control groups in terms of their PD, RD, RW, and KTW measurements (P > .05) (Table 1).
Histology
An overview of the gingiva ending at the level of the notch is shown in Fig 2a (test group). Figure 2b shows the keratinized oral epithelium at a higher magnification. Collagen fiber bundles running in a vertical direction and remnants of the membrane are shown in Fig 2c. The arrowhead on Fig 2d shows the alveolar bone crest. Collagen
Fig 2 Histomorphometric results of the treatment following euthanasia (after 16 weeks). (a) Overview of the gingiva ending at the level of the notch. (b) Keratinized oral epithelium (arrow) at a higher magnification. (c) Collagen fiber bundles running in a vertical direction (arrow) and remnants of the membrane. (d) Alveolar bone crest (arrow). (e) Collagen fibers of the newly formed periodontal ligament at low resolution. (f) Collagen fibers of the newly formed periodontal ligament (arrow) at high resolution.
fibers of the newly formed periodontal ligament at low and high magnification are shown in Figs 2e and 2f, respectively.
Discussion This investigation simulates a chronic clinical scenario since the recession defects were planned to be treated 3 months following
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379 their induction. The results demonstrated successful outcomes in terms of root coverage using the ECM membrane plus GTR protocol or CAF alone. They support a previous study18 that compared the treatment efficacy of using a free gingival graft vs an ECM membrane for increasing the zone of keratinized gingiva and demonstrated that the matrix-generated tissue was comparable to the tissue derived from the autograft.18 It has been reported that early resorption of collagen membranes (crosslinked as well as noncrosslinked) may hamper tissue regeneration31; however, there is no consensus over the time duration for which matrix membranes should remain over the defects to yield maximum tissue regeneration. Clinical evidence32,33 has suggested that membrane exposure may negatively influence the overall efficacy of the GTR procedure. It may therefore be hypothesized that thicker ECM membranes placed on GR defects for longer time durations may play a role in the correction of GR. However, further in vivo studies are needed to assess the effect of adjunctive use of ECM on gingival thickness in root-coverage procedures. The present results showed a significant reduction in RD and RW measured at baseline compared with those measured after 3 months of treatment for both the control and test groups. Every effort was made to establish a regular plaque control regimen, but animals required general anesthesia for each event, which was impractical. Thus, the animals’ oral
hygiene status may have contributed to the incomplete root coverage for both groups. It has been reported that size of the induced defect may influence the outcome of root coverage protocols. Pini Prato and coauthors reported that the root coverage is greater when the recession is greater than 4.98 mm, which is approximately the size of the defect that was made in this model.34 However, further studies regarding the influence of GR size on root coverage are required.
Conclusion The present preclinical and histomorphometric experiment evaluated the efficacy of ECM membranes for the treatment of induced GR defects. In this context, it can be concluded that ECM membranes are feasible for GTR protocols.
Acknowledgements This study was supported by a grant from Cook Biotech and the College of Dentistry Research Center, King Saud University.
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