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Journal of Oral Implantology
Histomorphometric Analysis of Bone Regeneration Using a dual-layer of membranes
(dPTFE placed over collagen) in fresh extraction sites:In Canine Model.
--Manuscript Draft-Manuscript Number:
AAID-JOI-D-13-00027R1
Full Title:
Histomorphometric Analysis of Bone Regeneration Using a dual-layer of membranes
(dPTFE placed over collagen) in fresh extraction sites:In Canine Model.
Short Title:
Histologic assessment of a novel socket grafting technique
Article Type:
Clinical Research
Keywords:
collagen membrane; high-density polytetrafluoroethylene membrane; histological
analysis; ridge preservation.
Corresponding Author:
Khalid Al-Hezaimi, MSc
King Saud University
Riyadh, SAUDI ARABIA
Corresponding Author Secondary
Information:
Corresponding Author's Institution:
King Saud University
Corresponding Author's Secondary
Institution:
First Author:
Khalid Al-Hezaimi, MSc
First Author Secondary Information:
Order of Authors:
Khalid Al-Hezaimi, MSc
Giovanna Iezzi
Ivan Rudek
Abdullah Al-Daafas
Khalid Al-Hamdan
Abdulaziz Al-Rasheed
Fawad Javed
Adriano Piatelli
Hom-Lay Wang
Order of Authors Secondary Information:
Abstract:
Background: In untreated extraction sockets, buccal bone remodeling compromises
the alveolar ridge width. The aim was to assess histologically the efficacy of using a
dual-layer of membranes (high density poly-tertrafluoroethylene [dPTFE] placed over
collagen) for ridge preservation (RP) in fresh extraction sites.
Material and methods: Eight beagle dogs were used. Following endodontic treatment
of mandibular bilateral second (P2)-, third (P3)- and fourth (P4) premolars, mandibular
bilateral first premolars and distal roots of P2, P3 and P4 were extracted atraumatically.
Animals were randomly divided into four treatment groups. Group-1: Control-no
treatment; Group-2: allograft placed in alveolum and socket covered with dPTFE
membrane; Group-3: allograft placed in alveolum, buccal plate overbuilt with allograft
and socket covered with dPTFE membrane; Group-4: allograft placed in alveolum and
covered with dual layer of membranes (dPTFE placed over collagen). No intent of
primary closure was performed for all groups. After 16-weeks, animals were sacrificed
and mandibular blocks were assessed histologically for buccolingual (BL) width of
alveolar ridge, percentage of bone formation and marrow spaces and the remaining
bone particles.
Results: BL width of the alveolar ridge was significantly higher among sockets in
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Group-4 than those in Group-1 (P<0.05). Amount of newly-formed bone existing in
each socket was higher among extraction sockets in Group-4 than those in Group-1,
Group-2 and Group-3 (P<0.001). There was a significant difference in the percentage
of bone marrow spaces between all groups (P<0.001). There was no significant
difference in the number of non-resorbed bone particles between the groups.
Conclusion: Using a dual layer of membrane was more effective in RP than
conventional socket augmentation protocols.
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*Article File
1
Title: Histomorphometric Analysis of Bone Regeneration Using a dual-layer of membranes
2
(dPTFE placed over collagen) in fresh extraction sites: In Canine Model.
3
Authors: Khalid Al-Hezaimi, BDS, MSc, FRCD (C),1,2 Giovanna Iezzi, DDS, PhD,3 Ivan
4
Rudek, DDS,4 Abdullah Al-Daafas, BDS, MSc,5 Khalid Al-Hamdan, BDS, MSc,1,2 Abdulaziz
5
Al-Rasheed, BDS, MSc,1,2 Fawad Javed, BDS, PhD,1 Adriano Piattelli, DDS, PhD,6 Hom-Lay
6
Wang, DDS, PhD7
7
Affiliations:
8
1. Engineer Abdullah Bugshan Research Chair for Growth Factors and Bone Regeneration, 3D
9
Imaging and Biomechanical Lab., College of Applied Medical Sciences; King Saud University,
10
Riyadh, Saudi Arabia;
11
2. Department of Periodontics and Community Dentistry, King Saud University, Riyadh, Saudi
12
Arabia;
13
3. Department of Medical, Oral and Biotechnological Sciences, University of Chieti-Pescara,
14
Chieti-Pescara, Italy;
15
4. Department of Periodontics and Oral Medicine, School of Dentistry, University of Michigan,
16
Ann Arbor, MI, USA.
17
5. Dental Clinics, King Fahad Security Force College, Riyadh, Saudi Arabia.
18
6. Department of Medical, Oral and Biotechnological Sciences, University of Chieti-Pescara,
19
Chieti-Pescara, Italy
1
20
7. Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry,
21
Ann Arbor, MI, USA.
22
Running title: Histologic assessment of a novel socket grafting technique
23
Conflict of interest and financial disclosure: The authors declare that they have no conflicts of
24
interest and there was no external source of funding for the present study.
25
Corresponding author: Dr. Khalid Al-Hezaimi. Engineer Abdullah Bugshan Research Chair
26
for Growth Factors and Bone Regeneration, 3D Imaging and Biomechanical Lab., College of
27
Applied Medical Sciences; and Department of Periodontics and Community Dentistry, King
28
Saud University, P.O.Box 60169, Riyadh 11545, Saudi Arabia. E-mail: [email protected]
29
Number of words (abstract): 249
Number of words (main text): 2694
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Number of figures: 5
Number of tables: 1
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Number of references: 29
32
Key words: Collagen membrane; high-density polytetrafluoroethylene membrane; histological
33
analysis; ridge preservation.
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2
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ABSTRACT
37
Background: In untreated extraction sockets, buccal bone remodeling compromises the alveolar
38
ridge width. The aim was to assess histologically the efficacy of using a dual-layer of membranes
39
(high density poly-tertrafluoroethylene [dPTFE] placed over collagen) for ridge preservation
40
(RP) in fresh extraction sites.
41
Material and methods: Eight beagle dogs were used. Following endodontic treatment of
42
mandibular bilateral second (P2)-, third (P3)- and fourth (P4) premolars, mandibular bilateral
43
first premolars and distal roots of P2, P3 and P4 were extracted atraumatically. Animals were
44
randomly divided into four treatment groups. Group-1: Control–no treatment; Group-2: allograft
45
placed in alveolum and socket covered with dPTFE membrane; Group-3: allograft placed in
46
alveolum, buccal plate overbuilt with allograft and socket covered with dPTFE membrane;
47
Group-4: allograft placed in alveolum and covered with dual layer of membranes (dPTFE placed
48
over collagen). No intent of primary closure was performed for all groups. After 16-weeks,
49
animals were sacrificed and mandibular blocks were assessed histologically for buccolingual
50
(BL) width of alveolar ridge, percentage of bone formation and marrow spaces and the
51
remaining bone particles.
52
Results: BL width of the alveolar ridge was significantly higher among sockets in Group-4 than
53
those in Group-1 (P<0.05). Amount of newly-formed bone existing in each socket was higher
54
among extraction sockets in Group-4 than those in Group-1, Group-2 and Group-3 (P<0.001).
55
There was a significant difference in the percentage of bone marrow spaces between all groups
56
(P<0.001). There was no significant difference in the number of non-resorbed bone particles
57
between the groups.
3
58
Conclusion: Using a dual layer of membrane was more effective in RP than conventional socket
59
augmentation protocols.
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4
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INTRODUCTION
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Studies1-6 have reported that buccal process of alveolar bone is largely composed of
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bundle bone (an immature bone supplied solely by ligaments and tendons) that makes it more
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susceptible to undergo resorption following tooth extraction than the lingual/palatal process. It is
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well-documented that extraction of multiple contiguous teeth is associated with a more extensive
81
buccal bone remodeling as compared to when a single tooth is extracted.4-6 In untreated
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extraction sockets, buccal bone remodeling may compromise the alveolar ridge width and
83
complicate future oral rehabilitative procedures such as implant therapy.1-5
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Various alveolar ridge preservation (RP) techniques have been proposed to minimize
85
post-extraction alveolar bone remodeling4, 7-9. Studies have proposed that overbuilding the buccal
86
plate with bone grafting materials minimizes post-extraction alveolar bone remodeling.10, 11 The
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authors hypothesized that addition of excessive bone grafting material in the areas where bone
88
resorption is significant (such as buccal and coronal surfaces of the socket); helps compensate
89
the natural bone resorption phenomenon.10, 11 The results showed that buccal overbuilding with
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excessive graft material is an ineffective technique for RP.10, 11.
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Traditionally, collagen membranes are used in guided bone regeneration (GBR) protocols
92
due to their hemostatic, chemostatic and cell adhesive characteristics;12,
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resorption rate following exposure to the oral environment has raised concerns over their usage
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in GBR. However, it has been reported that collagen cross-linking increases its biodurability and
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enables collagen-based membranes to support long-term osteogenic activity while preventing
96
other tissues from invading the defect.14,
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successfully
used
in
GBR
15
13
however, their fast
Cross-linked collagen membranes have been
procedures.16,
17
Advantages
of
using
high-density
5
98
polytetrafluoroethylene (dPTFE) membranes in GBR are that they do not require primary
99
closure, are easy to remove without the need for additional surgical interventions and minimize
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bacterial leakage;18-20 however, a limitation of dPTFE is that the membrane exhibits poor tissue
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adhesive properties, which may sequentially jeopardize GBR.21
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The present histologic study in dogs was based on the hypothesis that extraction sockets
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treated with bone graft and covered with a dual-layer of membranes (dPTFE placed over a cross-
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linked collagen membrane) show significantly higher percentage of trabecular bone and total
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mineralized tissue as compared to those treated with conventional socket augmentation using
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GBR concept. The aim of this histologic experiment was to assess the efficacy of using a dual-
107
layer membrane (dPTFE placed over collagen) for RP in fresh extraction sockets.
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MATERIALS AND METHODS
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Ethical guidelines
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The study protocol was approved by the research ethics review committee of the Eng. A.
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B. Research Chair for Growth Factors and Bone Regeneration, 3D Imaging and Biomechanical
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Lab., College of Dentistry, King Saud University, Riyadh, Saudi Arabia.
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Study animals
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Eight healthy female beagle dogs were used. The mean age and weight of the animals
117
were 24±0.83 months and 13.8±0.49 kilograms (Kg) respectively. The animals were vaccinated
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against hepatitis and rabies.
6
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All non-surgical and surgical procedures were performed under general anesthesia (Pfizer
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Limited, Sandwich, Kent, CT13 9NJ, UK) using ketamine (10 milligrams [mg] / Kg body-
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weight) and local anesthesia (AstraZeneca LP for DENTSPLY Pharmaceutical, York PA) with
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Xylocaine (with epinephrine 5 mg/millilitre).
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Preoperative management
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One week before tooth extraction, supragingival scaling was performed on all dogs using
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an ultrasonic scaler (NSK, Westborough, MA). At the day of surgery, intra-muscular (IM)
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injections of amoxicillin (25 mg/Kg body weight) (Betamox LA. Norbrook Laboratory Limited,
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Newry, County Down, Northern Ireland) were administered. The animals were draped and the
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surgical site was swabbed with an antiseptic solution (The Purdue Fredrick Company, Stamford,
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CT, USA).
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Root canal treatment and tooth extractions
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All surgical and non-surgical procedures were performed under general anesthesia using
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intramuscular injections of keramine (Pfizer Limited, Sandwich, Kent, CT13 9NJ, UK) with
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adjunct buccal infiltration of local anesthesia (AstraZeneca LP for DENTSPLY Pharmaceutical,
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York PA, USA). Non-surgical root canal treatment (RCT) was performed on bilateral
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mandibular second (P2), third (P3) and fourth premolars (P4). A No. 2 size round tungsten bur
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(Brassler, Savannah, GA, USA) mounted on a high-speed hand piece (Dentsply, York, PA,
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USA) was used to prepare the access cavity. Initial instrumentation of the root-canals was
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performed with K-type hand files (JS Dental, Ridgefield, CT, USA) following which rotary files
7
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(Profile, Dentsply, Addlestone, UK) were used for final root-canal preparation. The root-canals
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were irrigated with 5.25% sodium hypochlorite and obturated with vertically condensed gutta
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percha and sealer (Pulp Canal Sealer EWT, SybronEndo, Orange, CA, USA). Accuracy of root-
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canal length was verified by an apex locator (J. Morita USA, Inc.: Root ZX II- Apex Locator,
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Irvine, CA, USA) and periapical radiographs. Teeth with periapical lesions such as periapical
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granuloma were excluded.
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After two months of RCT, none of the teeth showed signs of periapical pathosis. bilateral
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mandibular first premolars (P1) were extracted atraumatically. Bilateral mandibular P2, P3 and
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P4 were hemisected using piezosurgery (Mectron, Piezosurgery® Columbus, Ohio, USA) and
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the distal roots were atraumatically extracted using forceps. The distal alveolus was currettaged
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to stimulate bleeding and eliminate remnants of the periodontal ligaments.
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Animal grouping
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Randomization was performed by picking a paper marked either "Group 1", "Group 2",
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"Group 3" or "Group 4" from a brown bag. Group 1 (Control-group), comprised of an untreated
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extraction socket. In Group 2, particulate graft (Zimmer‫ ׀‬Dental, Puros® cancellous particulate
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allograft, Carlsbad, CA, USA) was placed in the socket and covered with dPTFE membrane
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(Cytoplast® Barrier Membranes, TXT-200, Lubbock, TX, USA). In Group 3, particulate graft
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(Zimmer‫ ׀‬Dental, Puros® cancellous particulate graft, Carlsbad, CA, USA) was placed in the
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socket, buccal plate was overbuilt with excess particulate graft and the socket was covered with
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dPTFE membrane (Cytoplast® Barrier Membranes, TXT-200, Lubbock, TX, USA). In Group 4,
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particulate graft (Zimmer‫ ׀‬Dental, Puros® cancellous particulate graft, Carlsbad, CA, USA) was
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placed in the alveolum and covered with a “dual-layer” of membranes (that is, a dPTFE
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membrane [Cytoplast® Barrier Membranes, TXT-200, Lubbock, TX] placed over a collagen
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membrane [Cytoplast® RTM Collagen, Osteogenics Biomedical, Inc. Germany]). In all groups,
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primary closure was not intended (Ethicon Inc. VICRYL [Polyglactin 910] suture, Somerville,
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NJ) (Figure 1).
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Postoperative management and euthanasia
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All subjects received IM injections of amoxicillin (Betamox LA. Norbrook Laboratory
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Limited, Newry, County Down, Northern Ireland) (5mg/Kg body weight once a day for 3 days).
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Plaque control procedures, which included topical application of a 0.2% chlorhexidine
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digluconate solution (GUM, Chicago, IL, USA) were performed twice a week for four months
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after surgery. IM antibiotics (Betamox LA. Norbrook Laboratory Limited, Newry, County
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Down, Northern Ireland) were continued for three days after surgery as 25-50 mg/Kg IM every 8
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hours.
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After 4-months, all subjects were sacrificed using an overdose of 3% sodium
pentobarbitol (Vortech Pharmaceuticals Limited, Dearborn, MI, USA).
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Jaw sectioning, measurement of vertical bone height and buccolingual width
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The jaw segments containing the extraction sockets were removed en block using an
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electric saw (Leica SP 1600, Bannockburn, IL, USA); and fixed in 10% neutral formalin
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solution.
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Histologic analysis
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The samples and surrounding tissues were washed in saline solution and were
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immediately fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in 0.15 mol/L cacodylate
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buffer at 4°C and pH 7.4, to be processed for histology. The specimens were processed to obtain
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thin ground sections and were dehydrated in ascending series of alcohol rinses and were
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embedded in a glycol (methyl methacrylate) resin. After polymerization, the specimens were
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sectioned with a high-precision diamond disk at about 150 mm and were ground to about 30 mm.
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The slides were stained with acid fuchsin and toluidine blue as described earlier.22 The following
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descriptive parameters were measured by one examiner (GI): (a) Buccolingual (BL) width of the
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alveolar ridge was defined as the distance from the most outer point of the buccal bone plate to
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the most outer point of the lingual bone plate. The measurement was taken at 2mm perpendicular
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from the CEJ and in the middle of the socket; (b) Percentage of bone/socket; (C) Percentage of
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bone marrow spaces and (d) Percentage of non-resorbed bone particles. The measurements were
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performed with a microscope linked to a video camera (Buehler, New jersey, USA) /computer
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(Buehler, New jersey, USA) and software (Buehler, New jersey, USA).
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Statistical analysis
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Statistical analysis was performed using a statistical software (SPSS, Version 18.00,
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Chicago, IL, USA). The descriptive analyses of each histomorphometric parameter in each dog
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was obtained after analyzing three sections per site, 50 µm apart for each position. Significance
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differences among the groups were determined using one-way analysis of variance. For multiple
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comparisons Bonferroni post hoc test was used. P-values less than 0.05 were considered
209
statistically significant.
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RESULTS
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1-Alveolar ridge width
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The BL widths of the alveolar ridge in Group-1, 2, 3 and 4 were 5.27±1.22 mm,
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5.81±1.11 mm, 6.41±0.99 mm and 6.64±1.64 mm respectively. The BL width of the alveolar
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ridge was significantly higher among sockets in Group-4 (6.64±1.64 mm) as compared to those
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in Group-1 (5.27±1.22 mm) (P<0.05).
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2-Percentage of bone formation/socket
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The amount of bone existed in each socket was significantly higher among extraction
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sockets in Group-4 (92.5±10.4%) as compared to those in Group-1 (34±19.35%) (P<0.001),
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Group-2 (43±29.41%) (P<0.001) and Group-3 (56.5±25.01%) (P<0.001). These results are
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shown in Table 1.
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3- Bone marrow space percentage
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The percentage of bone marrow spaces in Groups 1, 2, 3 and 4 were 54.4217.97%,
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50.64 26.51%, 35.64 22.7% and 7.510.40% respectively. The difference was significance
11
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between all the groups (P<0.001).
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4- Percentage of non-resorbed bone particles
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The remaining non-resorbed bone particles were counted and calculated per socket. Since
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the first group had no bone graft material, it was not included. The remaining particles for groups
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2, 3 and 4 were 13.0  4.2%, 14.8  4.49% and 10.5  3.34% respectively. There was no
234
significance difference between the groups (P<0.633).
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Histological analysis
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In Group-1 (at 10X), mandibular cortical bone surrounded by oral mucosa was observed
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in the area of the first premolar tooth with periodontal ligament surrounding the apical portion of
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the canine. Mature compact bone with large amounts of osteons can be observed (Figure 2a and
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2b).
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In Group-2, osteons cells and cortical bone with mature compact and trabecular bone can
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be seen (Figure 3a). At 40X magnification, particulate graft, compact and mature bone was
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evident on the buccal surface was observed with newly formed bone with small marrow spaces
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(Figure 3b). At 40X magnification, newly formed bone with small marrow spaces and cement
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lines can be observed between pre-existing bone and newly formed bone
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same magnification (40X) no residual particles of biomaterial can be detected inside the bone
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defect (Figure 3d).
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(Figure 3c). At the
In Group-3, cortical, compact and mature bone were observed with fibrointegrated
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residual particulate graft particles next to oral mucosa (Figure 4a-4c). At 100X magnification,
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fibrointegrated particulate graft particles with dense connective tissue in tight contact with
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particulate graft were observed (Figure 4d-4f). At 40X magnification, newly formed bone with
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small marrow spaces can be observed. Cement lines can be observed between pre-existing bone
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and newly formed bone (Figure 4a-4f).
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In Group-4, at 10X magnification (Figure 5a), large amount of trabecular bone were
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observed in the socket site. At 40X magnification, significant mature remodeled bone where the
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areas of remodeling can be observed (Figure 5b). At higher magnification where evidence of
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remaining particles and evidence of modeling and remodeling was taking place (Figure 5c and
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5d). At another view, it can be seen the bonding between the newly formed bone and native
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bone (Figure 5e and 5f).
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DISCUSSION
262
The present histologic results showed that RP (in fresh extraction sites) using a dual-layer
263
of membranes (dPTFE placed over collagen membrane) enhances BL width of the alveolar ridge
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as compared to when regenerative protocols were performed using either a single layer of
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membrane or without a barrier membrane. In addition, the present experimental histologic results
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also demonstrated that the amount of new bone formed in extraction sockets in Group-4 was
267
significantly higher than that formed in Groups 1, 2 and 3 and that the number of non-resorbed
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bone particles were significantly less among extraction sockets in Group-4 as compared to those
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in Groups 2, 3 and 4. Furthermore, scanning electron microscopy (SEM) results by Yun et al.
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(2011) showed that a dual layer of membrane (dPTFE placed over collagen membrane) is an
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effective way for RP around immediate implants. Various explanations may be posed in this
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regard. Firstly, since collagen- and dPTFE membranes exhibit advantageous properties (such as
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optimal behavior toward soft-tissue responses and optimal durability respectively); it is
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postulated that using these membranes (collagen and dPTFE) as a dual-layer further augments
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new bone formation then when each membrane type is used individually. Next, it may also be
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proposed that placement of a dPTFE augments new bone formation by stabilizing the graft at the
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defect site/s. Moreover, bacterial count has been reported to be significantly lower on the inner
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aspect of the dPTFE membrane as compared to the outer surface (Yun et al. 2011). This suggests
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that the dPTFE membrane provides a reasonably bacteria-free environment to the underlying
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collagen membrane that may in turn facilitate its chemostatic and cell adhesive properties. The
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present histologic study supports the SEM results reported by Yun et al. (2011). However,
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further studies are warranted to identify the microbial species that may be associated with
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extraction barrier membranes placed in fresh extraction sites.
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Various bone grafting materials with different densities are used in RP procedures. 23-25 It
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is hypothesized that bone mineral density of the bone graft material used during RP protocols
286
may influence the overall outcome of the procedure. In the present experiment, a standardized
287
bone particulate graft was used in all extraction sockets; which reflects that the outcome was
288
chiefly associated with the dual-layer technique and not the type of particulate graft used. An
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interesting finding in the present experiment was that the percentage of non-resorbed bone
290
particles was significantly less in extraction sockets belonging to Group-4 as compared to
291
Groups 2, 3 and 4. A possible explanation that may be proposed in this regard is that placement
292
of a dual-layer of membrane helps stack the bone minerals in the region of interest thereby
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preventing dispersion of the bone matrix. This may in turn have helped augment the undergoing
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bone regeneration as compared to other groups where early resorption as well as collapse of
295
collagen membrane may have been liable for reduced bone formation and demonstrating
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increased number of non-resorbed bone mineral particles.
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Tissue biotypes have been associated with the outcomes of periodontal surgical
298
interventions.26-28 Thin soft tissue biotypes have been associated with thinner underlying alveolar
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bone and angular bone defects as compared to thick soft tissue biotypes.26,27 In a recent clinical
300
study, Le et al.27 investigated the relationship between buccal/labial bone thickness and crestal
301
labial soft tissue thickness around dental implants. The results demonstrated a significant
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relationship between the amount of buccal bone and thickness of soft tissues. Since canine
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models exhibit a thin periodontal tissue biotype, it is tempting to speculate that the outcomes of
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bone regenerative protocols may also be compromised compared to those performed in clinical
305
scenarios. However, further studies are warranted in this regard.
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It is pertinent to mention that there are a few limitations of the present experiment. It is
307
noteworthy that in the present experiment, all walls of the extraction sockets were intact. It is
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therefore hypothesized that presence of an osseous defect or a pathological lesion within or
309
around the extraction socket may jeopardize new bone formation. Likewise, clinical studies29-32
310
have shown that systemic disease (such as poorly-controlled diabetes mellitus and acquired
311
immune deficiency syndrome) and tobacco habits (such as cigarette smoking and tobacco
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chewing) jeopardize the alveolar bone and also negatively influence the outcome of periodontal
313
therapy. It is tempting to speculate that the efficacy of novel RP techniques (such as those
314
described in the present study) may be compromised in immunocompromised individuals and in
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tobacco-smokers. It has also been reported that the severity of periodontal disease increases with
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advancing age.26 It may therefore be argued that the outcomes of ridge preservative techniques in
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elderly individuals may be compromised as compared to those in younger patients. However,
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further studies are required in this regard.
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CONCLUSION
322
Within the limits of the present histologic investigation, it is concluded that ridge
323
preservation using a dual layer of membrane following tooth extraction enhances the alveolar
324
ridge width as compared to when a single layer of membrane is used.
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Acknowledgement
This study is registered at the College of Dentistry Research Center (CDRC No: FR
0026), King Saud University, Riyadh, Saudi Arabia.
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FIGURE LEGENDS
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Figure 1. Clinical and radiographic pictures of the treatment protocol
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(a) Preoperative clinical photograph. (b) Distal roots were hemisectioned and extracted with no
340
dehiscence. (c) A measuring surgical stent was fabricated to ensure repeat measurements
341
overtime. (d) Shows the different sites with application of membrane alone, membrane and bone
342
graft, dual-layer membrane, and bone graft material. (e) Primary closure was achieved. (f)
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Radiograph showing endodontically treated distal roots filled with gutta percha.
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Figure 2. Histologic evaluation of extraction sockets in Group-1 (no treatment group) using
346
Toluidine blue acid fuchsin.
347
(a) Mandibular cortical bone surrounded by oral mucosa in the area of the first premolar tooth.
348
The apex of the canine root surrounded by periodontal ligament can also be seen at 10X
349
magnification. (b) At 40X magnification, mature compact bone with osteons (Arrow). Fibrous
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connective tissue of oral mucosa (Arrow head).
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Figure 3. Histologic evaluation of extraction sockets in Group-2 (particulate graft placed in
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alveolum and socket covered with a high density polytetrafluoroethylene membrane) using
354
Toluidine blue acid fuchsin.
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(a) At 10X magnification, cortical bone with mature compact bone can be seen. Inside the
356
section trabecular bone can also be observed.
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mature bone belonging to the external portion of the jaw (Arrow).
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particulate graft surrounded by connective tissue both located outside the defect (Arrow head).
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(c) At 100X magnification, newly formed bone with small marrow spaces and cement lines can
(b) At 40X magnification, cortical, compact,
At 40X magnification,
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be observed between pre-existing bone and newly formed bone (Arrow). At 100X magnification,
361
connective tissue in close proximity to newly formed bone (Arrow head).
362
magnification, mature bone tissue can be observed (Arrow) with no residual particles of
363
particulate graft inside the bone defect. At 40X magnification, newly formed bone in tight
364
contact to pre-existing bone can be observed (Arrow heads).
(d) At 40X
365
366
Figure 4. Histologic evaluation using Toluidine blue acid fuchsin of extraction sockets in Group-
367
3 (particulate graft placed in alveolum, buccal plate overbuilt with particulate graft and socket
368
covered with a high density polytetrafluoroethylene membrane).
369
370
(a) At 10X magnification, cortical, compact and mature bone can be observed with
371
fibrointegrated residual particulate graft particles next to oral mucosa. In the central portion of
372
the sample trabecular bone can be observed in the central portion. (b) At 40X magnification, a
373
small marrow space inside the cortical bone can be observed (Arrow) with particulate graft
374
particles surrounded by fibrous tissue (Arrowhead). (c) At 40X magnification, preexisting bone
375
(Arrow) and newly-formed bone (Arrowhead) can be observed. (d) At 40X magnification,
376
cortical bone (Arrow) and fibrointegrated residual particulate graft particles, located outside the
377
defect can be observed (Arrow head). (e) At 100X magnification, fibrointegrated particulate graft
378
particles (Arrow head) with dense connective tissue in tight contact with particulate graft can be
379
observed. (f) At 100X magnification, dense connective tissue (Arrow) and particulate graft
380
particles (Arrow Head) can be seen.
381
18
382
Figure 5. Histologic evaluation using Toluidine blue acid fuchsin of extraction sockets in Group-
383
4 (particulate graft placed in alveolum and covered with a dual-layer of membrane [high density
384
polytetrafluoroethylene membrane placed over a collagen membrane]).
385
386
(a) At 10X magnification, Trabecular bone can be observed in the area where defect was
387
induced. (b) At 10X magnification, bone trabeculae in the medullary portion of the section can
388
be observed. (c) At 100X magnification, particulate graft particles surrounded by connective
389
tissue can be seen. (d) At 40X magnification, newly formed bone in tight contact to pre-existing
390
bone (Arrow) with fibrointegrated particulate graft can be observed (Arrow Head). (e) Arrow: At
391
40X magnification, mature bone with areas of remodeling can be observed (Arrow) with
392
fibrointegrated particulate graft particles inside the defect (Arrow Head). (f) Bonding between
393
the newly formed bone and native.
394
395
396
397
398
399
400
401
19
402
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Table
Click here to download Table: Table 1 (Dual layer Histo).doc
1
Table 1. Mean (± standard deviation) bone volume and buccolingual widths of extraction
2
sockets in Groups 1-, 2-, 3- and 4.
Group-1
Group-2
(mean±SD) (mean±SD)
Group-3
Group-43
(mean±SD)
(mean±SD)
4
8
Percentage of bone
formation/socket
34±19.35
Buccolingual width
(mm)
5.27±1.22
* P<0.001
43±29.41
*
56.5±25.01†
92.5±10.4†
6.41±0.99
6.64±1.646
*
† P<0.001
5.81±1.11
‡
‡ P<0.05
mm: millimeters
5
7
SD: Standard deviation
9
10
1
Figure
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Figure
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