Subepithelial Connective Tissue Graft Versus Amniotic Chorion Membrane for Peri-implant Mucosal Thickness Enhancement
Phase: NA
Conditions: Dental Implant
Sponsor: Tanta University

 Subepithelial Connective Tissue Graft versus 
Amniotic Chorion Membrane for Peri-implant 
Mucosal Thickness Enhancement. 
 
 
Protocol of thesis submitted for partial fulfillment of the requirements 
for a master's degree. 
In 
 Oral Medicine, Periodontology,  
Oral Diagnosis and Radiology 
 
Presented by 
Ahmed Aboelnagah Abdelsayed 
(B.D.S-2016) 
Faculty of dentistry, Alexandria university   
 
 
 
 
Tanta University 
Faculty of dentistry 
Oral Medicine, Periodontology,  
Oral Diagnosis and Radiology 
 Department 
2023 

Supervisors 
 
 
Dr. Hoda Mohamed Elguindy  
Professor of Oral Medicine, Periodontology, 
Oral Diagnosis & Radiology 
Faculty of Dentistry 
Tanta University 
(Main supervisor) 
 
 
Dr. Sahar Mahmoud Fawzy Ghoraba 
Professor and Head of Oral Medicine, Periodontology, 
Oral Diagnosis & Radiology 
Faculty of Dentistry 
Tanta University 
 
 
Dr. Mohamed Hamdy Helal 
Lecturer of Oral Medicine, Periodontology,  
Oral Diagnosis & Radiology 
 Faculty of Dentistry 
Tanta University 

 
1 
Introduction 
Soft tissue grafting procedures are increasingly performed for a 
number of indications in conjunction with dental implant therapy(1). Major 
clinical indications include gain of keratinized tissue (KT) and increase peri-
implant mucosal thickness (PMT) which improve functional, aesthetic, and 
biological outcomes after therapy(2). 
Various techniques and materials have been used to thicken the peri-
implant mucosa. These include connective tissue grafts (CTGs)(3), platelet-
rich fibrin (PRF)(4), acellular dermal matrix grafts(5) and xenogeneic collagen 
matrix(6). 
Soft tissue augmentation using subepithelial connective tissue graft 
(SCTG) harvested from the hard palate or tuberosity region has become the 
gold standard technique to thicken peri-implant tissue and to improve 
aesthetic outcomes(3). However, SCTG has been criticized to be associated 
with the risk of complications during and after surgery such as bleeding, 
infection or necrosis(7, 8). In addition, there is limitation in quality and 
quantity of tissue that is available for grafting. Moreover, the harvesting 
procedure at the donor site may be associated with increase morbidity due to 
graft harvesting(8). 
As a consequence, an acellular dermal matrix (ADM) allograft has 
been used as a SCTGs replacement. The ADM allograft is obtained from an 
allograft donor skin and produced by a carefully controlled process that 
removes the epidermis and dermis cells without altering the extracellular 
matrix structure which provides the basis for cellular in-growth and 
subsequent tissue remodelling(9). However, these grafts are very thin due to 
the manufacturing process and showed an increased shrinkage rate(10). 

 
2 
Besides, histological analysis of tissue augmented with ADM did not 
resemble native oral soft tissue, but had a more scar-like appearance(11). 
Recently, Amniotic Chorion membrane (ACM) has been proposed to 
augment keratinized tissues around teeth and dental implants. ACM is the 
inner most lining of the fetal membrane that is in contact with the developing 
fetus(12). The ACM has numerous advantages owing to its structure and 
composition. The extracellular matrix comprises collagen Types I, III, IV, V, 
and VI and cell-adhesion bioactive factors, such as fibronectin and 
laminin(13). 
Collagen is well tolerated and bioabsorbable, has haemostatic 
properties, and encourages migration of adjacent autogenous connective 
tissue. Fibronectin is involved in many cellular processes, including tissue 
repair, blood clotting, cell migration, and adhesion(14). 
Laminin and Laminin-5 has a high affinity for binding epithelial cells 
in contrast to traditionally available membranes(15) This biological factor 
allows the ACM to be left exposed to the oral environment. It is believed that 
the antibacterial property of ACM is related to the presence of lysozyme 
present in amniotic fluid(16) . 
According to Talmi and coworkers(17), lysozyme is a powerful 
bactericidal enzyme in high concentration in ACM, and it acts against many 
gram-negative microorganisms. Some studies have suggested that ACM is 
relatively well tolerated when used as a self-graft(18, 19). 
It has been also proven that the ACM has a potential for regeneration 
in periodontology as the matrix of the chorion contains abundant growth 
factors, such as keratinocyte growth factor, basic fibroblast growth factor, 

 
3 
and transforming growth factor-β, that promote periodontal regeneration and 
provide a natural environment for accelerated healing(20). It was used in 
socket preservation, guided tissue regeneration and guided bone 
regeneration. Furthermore, the ability of this allograft to self-adhere 
eliminates the need for suturing(15, 21). 
To the best of our knowledge, there is still a scarcity of literature 
evaluating the feasibility of ACM. Furthermore, there have been no studies 
conducted to evaluate the clinical efficacy using SCTG compared to ACM 
simultaneously with dental implant placement. 
The hypothesis states that using ACM membranes will produce 
equivalent results in terms of PMT enhancement around dental implants in 
comparison to SCTG graft in humans. The present study will be performed 
to compare and evaluate clinically and radiographically the soft tissue 
healing around dental implants augmented with subepithelial connective 
tissue graft versus Amnion Chorion Membrane. 
 
 

 
4 
Aim of the work 
The objective of this study will be to assess the efficacy of the peri-
implant mucosal thickness enhancement following either subepithelial 
connective 
tissue 
grafts 
or 
Amnion 
Chorion 
Membrane 
placed 
simultaneously with dental implant placement. 
 
 

 
5 
Materials and Methods 
Study design: 
A 
Randomized 
clinical 
study, 
single 
blinded, 
with 
equal 
randomization, active controlled, with Allocation ratio 1:1. 
Study setting: 
The research will be carried out in the Periodontology clinic, Faculty 
of Dentistry, Tanta University. 
Ethical consideration: 
The Purpose of the present study will be explained to the patients and 
informed consent will be obtained according to guidelines on human 
research adopted by the Research Ethics Committee (REC) at Faculty of 
Dentistry, Tanta University. 
Sample size calculation: 
The minimal sample size is calculated based on a previous study aimed 
to evaluate the early volumetric changes after buccal soft tissue contour 
augmentation around implants with a porcine collagen matrix (CM) versus 
the subepithelial connective tissue graft (SCTG) from the palate.(22) Schmitt 
et al. (2021)(22) reported that the mean soft tissue thickness increase (mm) in 
the buccal contour after 6 months was 0.30 ± 0.16 mm (CM) and 0.80 ± 0.61 
mm (SCTG). The sample size was calculated to detect difference in increase 
in the soft tissue thickness. Based on Schmitt et al. (2021)(22) results, and 
adopting a power of 80% (=0.20) to detect a standardized effect size in 
increase in the soft tissue thickness (primary outcome) of 1.121, and level of 
significance 5% (α error accepted =0.05), the minimum required sample size 
was found to be 11 patients per group (number of groups=2) (Total sample 

 
6 
size=22 patients)(23, 24). After adjustment for a dropout rate of 10%, the 
sample size was increased to 13 patients per group (number of groups=2) 
(Total sample size=26 patients).(25) 
Software: 
The sample size was calculated using G Power version 3.1.9.2 (26). The 
equation is: 
𝑁= (𝑟+ 1)(𝑍∝/2 + 𝑍1−𝛽 )2𝜎2
𝑟𝑑2
 
Where Za is the normal deviate at a level of significance and Z1-b is the 
normal deviate at 1-b% power with b% of type II error 
Sites selection: 
Twenty-six sites that are indicated for peri-implant mucosal 
augmentation at the time of implant placement will be selected from the 
Periodontology Clinic, Faculty of Dentistry, Tanta University. Participants 
will be randomized to the control (subepithelial connective tissue graft) or 
test (amniotic chorion membrane) group. To include the patients in this study 
their written consent will be obtained, and all the procedure will be explained 
before the treatment. 
Inclusion criteria: 
− Patients with good systemic health with no contraindication for 
periodontal surgery. 
− No history of any medications in the previous 6 months that may interfere 
with periodontal tissue health or healing. 
− Ability to maintain good oral hygiene as evidenced in recall visits. 
− Aged 30 to 55 years. 

 
7 
− Stable periodontal condition and missing one single tooth (maxillary 
anterior teeth and premolars) with adjacent teeth present and thin mucosal 
phenotype (< 2mm bucco- lingual thickness). 
Exclusion Criteria: 
− Medically compromised patients and systemic conditions precluding 
periodontal surgery. 
− Smokers. 
− Severe hematologic disorders (e.g., hemophilia or leukemia), uncontrolled 
infectious or metabolic diseases that could compromise normal healing, 
liver, or kidney dysfunction/failure. 
− Patients subjected to irradiation in the head and neck area. 
− Patients 
treated 
or 
under 
treatment 
with 
intravenous 
amino 
bisphosphonates. 
− Patient affected by active periodontitis or has poor oral hygiene and 
motivation. 
− Uncontrolled diabetes mellitus. 
− Pregnant women or planning to become pregnant, and nursing mothers. 
The clinical study design: 
1. Phase I therapy 
− All patients will receive a comprehensive periodontal examination, oral 
hygiene instructions and they will be subjected to full mouth scaling, root 
planning (SRP) and polishing. 
− Sites with occlusal trauma will be subjected to occlusal adjustment. 
− One month following phase I therapy, patients who qualify the inclusion 
criteria for surgical procedures in terms of oral hygiene measures and 
compliance will be selected. 

 
8 
2. Surgical Phase 
− The sites will be classified randomly by the sealed envelope technique 
into two treatment groups, 13 sites in each group. 
Group 1 (Control Group) 
Autogenous SCTGs + dental implants*. 
Group 2 (Test Group) 
ACM**+ dental implant 
Prior to the initiation of study procedures, Peri-implant Mucosal 
Thickness (PMT) at three different heights (1, 3, and 5 mm apical from the 
mucosal margin) will be measured using a custom stent and an endodontic 
spreader.  Following enrolment, each participant will be randomly assigned 
using the sealed envelope technique by a masked investigator, to one of two 
treatment groups: Control (implant placement with autogenous SCTG 
harvested from the palatal mucosa); or test (implant placement with ACM). 
Participant allocation will be withheld from the surgeon until shortly before 
the surgical visit. 
At the baseline surgical visit, local anaesthetic will be administered; 
the buccal PMT will be measured and recorded. A periodontal probe (UNC-
15) and an endodontic spreader will be gently inserted through the custom 
stent to the surface of the mucosa and the bone, respectively, at 
approximately 1, 3, and 5 mm apical to the estimated free mucosal margin, 
using the gingival zenith of the adjacent teeth as a reference. The difference 
 
* Biodem implant - Germany 
** BioXclude, USA 
 

 
9 
between the measurement from the stent to the mucosa (using the probe) and 
from the stent to the bone (using the endodontic spreader) as the PMT. 
Mid-buccal keratinized mucosal width (KMW) at the edentulous site 
using a periodontal probe (UNC-15) will be recorded, as well. 
Surgical procedure 
First surgery 
All patients will be premedicated with antibiotic prophylaxis 
(Augmentin 2,000 mg or Clindamycin 600mg), depending on the history of 
drug allergy, 1 hour prior to implant placement surgery. Clinical photographs 
will be taken prior to, during, and after surgery. 
All surgical procedures will be performed under strict aseptic 
conditions and all patients will be injected with local anaesthesia (Articaine 
4% 1:100 000 epinephrine). 
Paracrestal incisions extended through the papillae on either side of the 
implant site will be made. Vertical releasing incisions will be made, when it 
is necessary, on each side of the implant to facilitate the planned 
advancement of the flap tissue to cover the grafts. A combination of full 
thickness and partial thickness flap design will be employed to create a 
recipient bed for the graft and the implant will be placed in an ideal position 
according to the cone beam CT (CBCT) scan. 
Following baseline measurements, the implant will be placed 
according to the manufacturer's protocol, along with the provided cover 
screw. For the control group, a connective tissue graft will be harvested from 
the palatal mucosa. For the test groups, the ACM will be prepared according 
to the manufacturer's instructions and trimmed to appropriate size. 

 
10 
The grafts will be positioned over the coronal and buccal aspect of the 
alveolar ridge and secured to the recipient sites with non-absorbable sutures 
anchored in the buccal periosteum and the palatal flap, and then passively 
covered with the primary flap. The flaps will be sutured with multiple simple 
interrupted and double vertical loop sutures to achieve primary closure in all 
cases using non-absorbable sutures. 
3. Postoperative Care 
− Patients will be continued to take the same antibiotics for 7 days. They 
will be instructed to rinse twice daily with a chlorhexidine mouth-rinse 
(0.12%) during the first 2 weeks and to avoid brushing and interdental 
cleaning next to the treated area. They also will be told to avoid chewing 
on, or inflict any trauma, to the treated area. 
− Patients will return to the clinic 7 to 14 days post-surgery for suture 
removal, Final crown cementation after 6 months. 
4. Outcomes variables 
− The primary outcome of interest will be to measure changes (in mm) in 
horizontal PMT on the buccal aspect of the edentulous alveolar ridge from 
baseline (BL) (implant placement and grafting) to (3 and 6 month) of 
post-surgical healing.  
− Prior to surgery (baseline), at 3 and 6 month, impressions of the grafted 
sites will be taken including at least the two neighbouring teeth and using 
an Addition silicone impression material. Dental stone casts will be 
fabricated and optically scanned with a desktop 3D scanner. Digital 
models of each time-point per patient will be captured as 
stereolithography (STL) files. Subsequently, these STL files will be 
imported into a digital imaging software program for analysis of the 
volumetric changes in the grafted areas. The images of the baseline and 

 
11 
follow-up datasets will be superimposed and matched using the best-fit 
algorithm at the adjacent tooth surfaces. After definition of specific 
regions of interest, the software will calculate the volumetric, changes 
will be measured in mm, which corresponded to the mean distance 
between the three surfaces representing the evaluated time-points (BL, 3 
and 6 month). 
− The secondary outcome of interest will include change (in mm) in mid-
buccal (KMW) in an apico-coronal direction. Assessment of wound 
healing at different post-surgical time points using a modified wound 
healing index that was introduced by Huang et al.,(18) which evaluates 
periodontal soft tissue wound healing with scores from 1 to 3. On this 
scale, wounds are scored at baseline of surgery and at 4,8,12,24 weeks 
post-operative. 
− The modified wound healing index (MWHI) included three categories:  
1) Uneventful wound healing with no or minimal mucosal edema or 
erythema, and no suppuration or graft exposure. 
2) Normal wound healing with slight to moderate mucosal edema, 
erythema and/or graft exposure, but no suppuration. 
3) Poor wound healing with significant mucosal edema, erythema, graft 
exposure, and suppuration. 
− Patient-reported outcome measures (PROMs): self-reported postoperative 
discomfort and overall satisfaction upon study completion using a 100-
point 
visual 
analog 
scale 
(VAS) 
recorded 
at 
1,3,7,15 
days 
post operatively. 
Statistical analysis: 
Data will be collected and analysed using Statistical Package for Social 
Science (SPSS) program for statistical analysis. Data validation for entry 

 
12 
mistakes will be carried out. The normality of distribution will be checked to 
determine the appropriate statistical approach to be adopted. Level of Alpha 
error will be set to 5% with a significance level of 95%. Statistical 
significance will be tested at p value <0.05. 
 
 

 
13 
References 
1. Chackartchi T, Romanos GE, Sculean A. Soft tissue‐related complications 
and management around dental implants. Periodontology 2000. 
2019;81(1):124-38. 
2. Thoma DS, Buranawat B, Hämmerle CH, Held U, Jung RE. Efficacy of 
soft tissue augmentation around dental implants and in partially 
edentulous areas: a systematic review. Journal of Clinical Periodontology. 
2014;41:S77-S91. 
3. Zucchelli G, Tavelli L, McGuire MK, Rasperini G, Feinberg SE, Wang 
HL, et al. Autogenous soft tissue grafting for periodontal and peri‐implant 
plastic surgical reconstruction. Journal of Periodontology. 2020;91(1):9-
16. 
4. Strauss FJ, Stähli A, Gruber R. The use of platelet‐rich fibrin to enhance 
the outcomes of implant therapy: A systematic review. Clinical oral 
implants research. 2018;29:6-19. 
5. Park J-B. Increasing the width of keratinized mucosa around endosseous 
implant using acellular dermal matrix allograft. Implant dentistry. 
2006;15(3):275-81. 
6. Moraschini V, Guimarães HB, Cavalcante IC, Calasans-Maia MD. 
Clinical efficacy of xenogeneic collagen matrix in augmenting keratinized 
mucosa round dental implants: a systematic review and meta-analysis. 
Clinical oral investigations. 2020;24:2163-74. 
7. Soileau KM, Brannon RB. A histologic evaluation of various stages of 
palatal healing following subepithelial connective tissue grafting 

 
14 
procedures: a comparison of eight cases. Journal of Periodontology. 
2006;77(7):1267-73. 
8. Del Pizzo M, Modica F, Bethaz N, Priotto P, Romagnoli R. The 
connective tissue graft: A comparative clinical evaluation of wound 
healing at the palatal donor site: A preliminary study. Journal of Clinical 
Periodontology. 2002;29(9):848-54. 
9. Wong AK, Schonmeyer BH, Singh P, Carlson DL, Li S, Mehrara BJ. 
Histologic analysis of angiogenesis and lymphangiogenesis in acellular 
human dermis. Plastic and reconstructive surgery. 2008;121(4):1144-52. 
10. 
Batista Jr EL, Batista FC, Novaes Jr AB. Management of soft tissue 
ridge deformities with acellular dermal matrix. Clinical approach and 
outcome after 6 months of treatment. Journal of Periodontology. 
2001;72(2):265-73. 
11. 
Wei PC, Laurell L, Geivelis M, Lingen MW, Maddalozzo D. 
Acellular dermal matrix allografts to achieve increased attached gingiva. 
Part 1. A clinical study. Journal of Periodontology. 2000;71(8):1297-305. 
12. 
Gulameabasse S, Gindraux F, Catros S, Fricain JC, Fenelon M. 
Chorion and amnion/chorion membranes in oral and periodontal surgery: 
A systematic review. Journal of Biomedical Materials Research Part B: 
Applied Biomaterials. 2021;109(8):1216-29. 
13. 
Bourne G. The foetal membranes: a review of the anatomy of normal 
amnion and chorion and some aspects of their function. Postgraduate 
medical journal. 1962;38(438):193. 

 
15 
14. 
Steinberg AD, LeBreton G, Willey R, Mukherjee S, Lipowski J. 
Extravascular clot formation and platelet activation on variously treated 
root surfaces. Journal of Periodontology. 1986;57(8):516-22. 
15. 
Gamal RM, Lotfy M, Taalab M. Evaluation of the efficacy of 
amniotic chorion membrane in management of grade II furcation defects 
(clinical study). Alexandria Dental Journal. 2018;43(3):102-8. 
16. 
Colocho G, Graham WP, Greene AE, Matheson DW, Lynch D. 
Human amniotic membrane as a physiologic wound dressing. Archives of 
Surgery. 1974;109(3):370-3. 
17. 
Inge E, Talmi YP, Sigler L, Finkelstein Y, Zohar Y. Antibacterial 
properties of human amniotic membranes. Placenta. 1991;12(3):285-8. 
18. 
Bennett J, Matthews R, Faulk WP. Treatment of chronic ulceration of 
the legs with human amnion. The Lancet. 1980;315(8179):1153-6. 
19. 
Gomes MF, Da Silva Dos Anjos MJ, de Oliveira Nogueira T, 
Guimarães SAC. Histologic evaluation of the osteoinductive property of 
autogenous demineralized dentin matrix on surgical bone defects in rabbit 
skulls using human amniotic membrane for guided bone regeneration. 
International Journal of Oral & Maxillofacial Implants. 2001;16(4). 
20. 
Holtzclaw DJ, Toscano NJ. Amnion–chorion allograft barrier used 
for guided tissue regeneration treatment of periodontal intrabony defects: 
A retrospective observational report. Clinical Advances in Periodontics. 
2013;3(3):131-7. 
21. 
Gomaa M, El Guindy H, Shoukheba M, Metwalli A. Healing of 
experimental gingival recession defects treated with amnion allograft: 

 
16 
Histologic and histometric analysis in dogs. Journal of Oral Biosciences. 
2022;64(1):93-9. 
22. 
Schmitt CM, Brückbauer P, Schlegel KA, Buchbender M, Adler W, 
Matta RE. Volumetric soft tissue alterations in the early healing phase 
after peri‐implant soft tissue contour augmentation with a porcine 
collagen matrix versus the autologous connective tissue graft: A 
controlled 
clinical 
trial. 
Journal 
of 
Clinical 
Periodontology. 
2021;48(1):146-63. 
23. 
Charan J, Biswas T. How to calculate sample size for different study 
designs in medical research? Indian Journal of Psychological Medicine. 
2013;35(2):121-6. 
24. 
Walters SJ, Jacques RM, dos Anjos Henriques-Cadby IB, Candlish J, 
Totton N, Xian MTS. Sample size estimation for randomised controlled 
trials with repeated assessment of patient-reported outcomes: what 
correlation between baseline and follow-up outcomes should we assume? 
Trials. 2019;20(1):1-16. 
25. 
Pannucci CJ, Wilkins EG. Identifying and avoiding bias in research. 
Plastic Reconstructive Surgery. 2010;126(2):619-25. 
26. 
Faul F, Erdfelder E, Lang AG, Buchner A. G*Power 3: a flexible 
statistical power analysis program for the social, behavioral, and 
biomedical sciences. Behavior Research Methods. 2007;39(2):175-91. 
 

 
17