histomorphology of guided bone regeneration on …...intrabony, fenestration or dehiscence bony...

Histomorphology of Guided Bone

Regeneration on Dental Implant Dehiscence

Defects in Beagle dogs

Yoon-Sik Kim

The Graduate School

Yonsei University

Department of Dental Science

Histomorphology of Guided Bone

Regeneration on Dental Implant Dehiscence

Defects in Beagle dogs

A Dissertation ThesisSubmitted to the Department of Dental Scienceand the Graduate School of Yonsei University

in partial fulfillment of therequirements for the degree of

Doctor of Philosophy of Dental Science

Yoon-Sik Kim

June 2005

This certifies that the dissertation thesis

of Yoon-Sik Kim is approved

──────────────────────── Thesis Supervisor：Chong-Kwan Kim

────────────────────────

Kyoo-Sung Cho

────────────────────────

Seong-Ho Choi

────────────────────────

Keun-Woo Lee

────────────────────────

Hee-Jin Kim

The Graduate School

Yonsei University

June 2005

감감감사사사의의의 글글글

본 논문이 완성되기까지 시작부터 세심한 배려로 마칠 때까지 격려해주시고 지도하여 주신 김종관 교수님께 깊은 감사를 드리며 아울러 많은조언을 해주신 조규성 교수님,최성호 교수님,그리고 바쁘신 와중에도 예심,본심,종심까지 심사를 해주신 이근우 교수님,김희진 교수님께도 감사드립니다.또한 동물실험과 본 연구에 많은 도움을 주신 김태균,진미성,김동진

선생과 우수한 슬라이드 사진을 촬영해주며 갖은 심부름을 도맡아 해준 홍지연 선생과 실험내내 도움을 주신 치주과 교실원 여러분들께도 감사의 마음을 전합니다.부족한 영어를 보충해주신 최동훈 선생님께도 감사를 드립니다.그리고 지금까지 항상 사랑과 격려로 모든 학위과정을 마칠 때까지 보

살펴 주시고,이끌어주시며 같이 기뻐해 주신 양가 부모님께 감사드립니다.항상 건강하십시오.그리고 논문을 쓰는 동안 함께 해준 사랑하는 아들 민관이와 딸 내경,

그리고,아내 미정에게 이 논문을 바칩니다.

2005년 7월김 윤 식

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Table of Contents

Abstract (English) ·························································································· iii

I. Introduction ·································································································· 1

II. Materials and Methods ·············································································· 5

A. Animals & Materials ·········································································· 5

B. Experimental design ············································································ 6

C. Surgical protocol ·················································································· 7

D. Histologic Preparation & Findings ···················································· 9

III. Results ····································································································· 10

A. Clinical Findings ················································································ 10

B. Histologic findings ·············································································10

IV. Discussion ······························································································· 13

V. Conculsion ································································································ 22

VI. References ······························································································· 24

Legends ··········································································································· 35

Figures ············································································································ 39

Abstract (Korean) ·························································································· 45

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Table of Table

Table 1 : Table 1. Experimental Design .................................................... 5

List of Figures

Figure 1 : A schematic diagram depicting the experimental design ..............5

Figure 2 : Photograph of Surgically created dehiscence defect adjacent dental implant ............................................................. 31

Figure 3. A：Photograph of the chitosan membrane only (Group 1, Left) ······································································ 31 B：β-TCP+chitosan membrane (Group 2, Right) ···················· 31

Figure 4,5. Photomicrograph of Control (No treatment) ························ 40

Figure 6. Photomicrograph of Control (No treatment) ························ 41

Figure 7. Photomicrograph of the chitosan membrane only (Group 1) ·················································································· 41

Figure 8,9. Photomicrograph of the chitosan membrane only (Group 1) ·················································································· 42

Figure 10,11. Photomicrograph of β-TCP＋ chitosan membrane (Group 2) ··············································································· 43

Figure 12. Photomicrograph of autogenous bone＋＋＋β-TCP ＋ chitosan membrane (Group 3) ··············································· 44

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Abstract

Histomorphology of Guided Bone Regeneration on Dental Implant

Dehiscence Defects in Beagle dogs

Dehiscence bone defects, frequently observed on dental implants placed in

periodontitis-affected alveolar bone or extraction sockets were treated with β

-tricalcium phosphate and chitosan membrane for guided bone regeneration, and the

new bone formation on the treated sites were studied. Beagle dogs 18 to 24 month-old

weighing approximately 15kg were used for the experiment. First to fourth mandibular

premolars were extracted, and the post extraction alveolar bone surface was planed.

After 8 weeks of healing, 3 by 4 mm dehiscence defects were created using straight

fissure burs. Total of 16 oxidized titanium surface implants were placed on the bone

defects of the subjects, two on each side. Control sites were treated with implants only.

Experimental Group 1 sites were treated with implants and chitosan membrane.

Experimental Group 2 sites were treated with implants, β-tricalcium phosphate and

chitosan membrane. Experimental Group 3 sites were treated with implants, β

-tricalcium phosphate, autogenous bone and chitosan membrane. The animals were

sacrificed 12 weeks after implant placement, and the specimens from the treated sites

were histologically studied with following results.

1. Limited amount of new bone formation was observed in control group with

unexposed membrane.

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2. Slightly greater amount of bone formation was observed on sites treated with β

-TCP+membrane or autogenous bone+β-TCP+membrane compared to control group.

3. Sites with early wound exposure showed signs of acute inflammation and limited

amount of new bone formation was observed regardless of membranes or bone

substitutes used.

4. Remnants of Chitosan membrane and β-TCP encapsulated with connective tissue

were observed during experimental periods.

These results suggest that further studies are needed on membrane rigidity and

infection control for space maintenance underneath the membrane and bone

substitutes in the treatment of dehiscence defects.

Key word : implant dehiscence defect, GBR, autogenous bone, β-tricalcium

phosphate, chitosan membrane

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Histomorphology of Guided Bone Regeneration on Dental Implant

Dehiscence Defects in Beagle dogs

Department of Dental science, The graduate School, Yonsei University

(Directed By Prof. Chong Kwan Kim, D.D.S., M.S.D., Ph.D)

Yoon-Sik Kim, D.D.S.

I. Introduction

Dental implants have been widely used for many years in the treatment of

esthetic and functional problems from alveolar bone loss following tooth

extraction. The success of implants essentially depends on qualities and

quantities of the surrounding bone during implant placement (Jovanovic et al.

1992; Johnson et al. 1997; Vicente et al. 2000). During implant placement,

thread exposure is often observed, especially in post-extraction sites or where

alveolar ridges are thin. The development of bone graft materials and

membrane techniques may give surgeons some options to solve these problems

(Hall et al. 1999; Piattelli et al. 1998; Dahlin et al 1989; Becker et al 1990;

Simion et al 1994, 1997; Becker et al. 1992, 1995; Mellonig & Nevins 1995;

Nyman et al. 1990; Palmer et al 1994).

Guided bone regeneration (GBR) has emerged as a treatment in the

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management of osseous defects associated with dental implants (Buser et al.

1994). Such defects may include supra-alveolar or lateral ridge defects, and

intrabony, fenestration or dehiscence bony defects. The placement of a barrier

membrane for GBR creates an isolated space for a blood clot to serve as a

matrix for alveolar bone regeneration. Dahlin et al.(1989) were able to show that

the degree of bone formation or fenestration defects in the maxilla is significantly

improved when the membrane technique is employed. In nonspace-making

defects, such as buccal dehiscence defects, adjunctive measures have been

suggested to support or tent the barrier membrane to provide a space for bone

regeneration to occur including placement of autograft bone, bone derivatives and

substitutes, or fixation screws or pins with or without adjunctive bone materials

(Becker et al. 1994; Buser et al. 1990; Jovanovic et al. 1992; Nevins & Mellonig

1992, 1994; Werbitt & Goldberg 1992; Shanaman 1992; Mellonig & Triplett

1993; Martin et al 1998).

E-PTFE membrane is effective to tissue integrity, but not resorbable, that is

needed secondary surgery. Recently many studies show the resorbable membrane

such as collagen, atelocollagen, polylactic acld (Guidor Matrix Barrier), polylactic

910(Vicryl Mesh), glycolide & lactide copoly-mer(Resolut Regenerative Material),

chitosan membrane, etc(Pitaru et al 1988; Laurell et al 1994; Caton et al 1994;

Caffesse et al 1994; Yeo et al 2005).

The use of chitosan (poly-N-acetyl glycosaminoglycan) in bone regeneration has

attracted a biodegradable natural biopolymer that has been shown to improve wound

healing and enhance bone formation (Lee et al., 2000; Madihally and Howard, 1999).

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Recent reports have noted chitosan's efficacy in the promotion of oral wound

healing in human studies(Klokkevold et al. 1999; Muzzarelli et al. 1989;

Sapelli et al. 1986).

Collapse of barrier membranes frequently encountered under pressure of the

overlying soft tissues often requires additional support via fillers placed

underneath the membrane. Mechanical strength is apparently insufficient, and a

space-making effect is completely lost in those cases where soft tissue

dehiscences leads to rupture of the membrane and subsequent collapse into the

defect. So the bone particles used for augmentation provide little additional

support to the membrane. Such studies have reported different degrees of success

of guided bone regeneration, depending upon the type of barrier selected,

presence or absence of an underlying graft material (Covani et al. 2003), types of

graft material, feasibility of technique, and clinician's preference (Becker et al

1994a, 1994b; Becker & Becker 1990; Carpio et al. 2000; Hall et al. 1999;

Hockers et al. 1999; Hutmacher et al. 1996; Jovanovic et al. 1992; Zablotskyet

al. 1991; Zitzmann et al. 1997).

The material used for augmentation should be osteoconductive and must

provide enough stability to resist soft tissue pressure above the membrane. For

these purposes, autogenous bone is still considered to be the material of choice

because of its unique biologic properties. The major drawback of autogenous

bone, however, is the additional surgical procedure. Alloplast, such as the

bioactive glass, may be an effective alternative to DFDB (Hall et al. 1999;

Johnson et al. 1997; Low et al. 1997; Vicente et al. 2000). Commonly used

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ceramic bioactive alloplastic bone grafting materials include hydroxyapatite and

β-tricalcium phosphate, which have been reported to form a chemical bond to

bone tissue (Baldock et al 1985; Furusawa et al 1997; Nery et al. 1975).

Recently, Studies about the use of β-tricalcium phosphate with dental implant

placement were reported (Merten et al. 2001; Nkenke et al. 2002; Palti &

Hoch 2002; Trisi et al. 2003; von Arx et al 2001; Zerbo et al. 2001). The

pure β-TCP was resorbed simultaneously with new bone formation, without

interference with the bone matrix formation.

The purpose of this study is to histologically evaluate and compare the healing

of the dehiscence defects using chitosan membrane, β-TCP, autogenous bone

around submerged oxidizing surface dental implant in beagle dogs. Histologic

analysis provides the clinician with better understanding for selecting the most

suitable graft materials.

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II. Materials and Methods

A. Animals & Materials

1. Animals

Four male, lab-bred beagle dogs, approximately 18 to 24 months old and

weighing about 15 kg, were used. The animals had intact dentition and healthy

periodontium. Porous β-tricalcium phosphate particles1), newly developed chitosan

membrane2) were used in dehiscence defects around oxidizing surface dental

implant (8.5mm in length, 3.3mm in diameter)3). Animal selection and

management, surgical protocol, and preparation followed routines approved by the

Animal Care and Use Committee, Yonsei Medical Center, Seoul, Korea.

2. Chitosan non-woven membrane

a. Manufacturing Chitosan Fibers

In order to prepare the chitosan dope, biomedical grade chitosan (degree of

deacetylation 100 %, MW 540,000, polydispersity 1.20) was dissolved in 2 % acetic

acid with a concentration of 3 % (w/w). The dope was then filtered and de-aerated for

further continuous spinning through a nozzle assembly with 0.1 mm ø×1500 holes

using a metering gear pump.

The coagulation-bath was composed of a 10% NaOH solution. The bundle of

filaments were neutralized and coagulated in the bath. These were then stretched

1) CerasorbⓇ, Curasan AG, Kleinostheim, Germany.2) Department of Clothing & Textiles, Ewha Womans University, Seoul, Korea.3) Bio-PlantⓇ, for the experimental usage, manufactured by Cowell-Medi Co. Seoul,

Korea.

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approximately 20 % and rinsed in 100°C water with the subsequent treatment of a

spinning oil emulsion and drying resulting in the chitosan fiber product.

b. Manufacturing Needle-punched Nonwoven Fabrics made of chitosan fibers

The chitosan fibers were steam treated and subjected to a stuffer box treatment in

order for the fibers to have the appropriate crimp level. These were then cut to 50 µm

length staple fibers. The chitosan fibers were open with the opening machine, which

were then carded by roll carding machine to manufacture a chitosan fiber web. Five

or six layers of the manufactured webs were arranged, and subjected to an additional

needle punching process to obtain the nonwoven fabrics. The thickness of the

manufactured nonwoven fabrics was adjusted to 2 mm using a calender with the

proper setting of the machine pressure.

B. Experimental design

Animals were divided in four groups. Animals in the sugical control group

were given the flap operation only. (Table 1, Fig 1)

Table 1. Experimental Design

Group Treatment

Control

Group 1

Group 2

Group 3

Flap only

Chitosan membrane

β-TCP＋Chitosan membrane

Autograft＋β-TCP＋Chitosan membrane

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Fig 1. A schematic diagram depicting the experimental design

C. Surgical protocol

Prestudy preparation included scaling and plaque control to obtain gingival

health.

Teeth were extracted under general anesthesia and sterile conditions in an

operating room using Atropine4) 0.05㎎ /㎏ SQ, RompunⓇ5) 2㎎ /㎏ , Ketamine6) 10

㎎ /㎏ IV as a premedication. The dogs were placed on a heating pad, intubated,

and inhalated with 2% enflurane and monitored with an electrocardiogram. After

disinfection of the surgical sites, 2% lidocane HCl with epinephrine 1:100,000

was used by infiltration at the surgical sites. The animals received lactated

Ringer's solution7) intravenously during surgeries. Elevation of buccal and lingual

4) Kwangmyung Parm. Co.,Ltd, Seoul. Korea5) Bayer Korea Ltd, Seoul, Korea6) Korea United Pharm, Seoul. Korea7) Lactated Ringer's Inj., Jung-Wae Pharmaceutical Company, Seoul. Korea

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mucoperiosteal gingival flaps were made following crevicular incisions from the

distal aspect of the mandibular canine to the mesial aspect of the first molar. The

roots of the mandibular premolar teeth were separated with dental burs and

chisels, and were carefully extracted. Flaps were sutured with vertical mattress

5-0 resorbable sutures. The dogs received antibiotics Cefazoline8) 10㎎ /㎏ IV the

day of surgery.

The implants were placed under the same surgical conditions as the tooth

extraction after a healing period of 8 weeks. A crestal incision was made

preservation of keratinized tissue on each side of the incision. Mucoperiosteal flaps

were carefully reflected on the buccal and lingual aspect. The edentulous ridge

was carefully flattened with an surgical bur and irrigation with sterile saline. Four

implant sites (2 per jaw quadrant) were prepared. Two implants were placed on

each side of the mandible. The implant osteotomy was performed at 800 rpm

under irrigation of chilled saline. Before placement, a standardized dehiscence

defect (3☓4mm) was created at the buccal of the each implant site with a straight

fissure bur(Fig2.). Placement was made without tapping. The implants were

positioned such that the most coronal portion of the implant body was level with

the osseous crest and centered in a buccal - lingual position. Each animal received

2 dental implants per jaw quadrant for a total of 16 implants for the 4 animals.

Graft materials were then inserted into the dehiscence defect sites.

The flap were closed with 5-0 resorbable sutures. Periosteal releasing incisions

were performed to achieve tension free wound closure. Vertical mattress and

8) Yuhan Co. Seoul. Korea

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interrupted sutures were used. The post-operative care was the same as the tooth

extraction. Throughout the study, the animals were fed with a soft diet to prevent

mechanical trauma during healing.

The dogs were sacrificed at 12 weeks following placement of the graft

materials. Euthanasia was performed with an overdose of the drugs used in the

process of anesthesia. Block sections including segments with implants were

preserved and fixed in 10 % neutral buffered formalin for histologic preparation

and analysis.

D. Histologic Preparation & Findings

48 hours after sacrifice, the specimens were cut into block sections on a low

speed saw and placed in fresh 10% formalin for complete fixation before

dehydration. The specimens were then dehydrated through graded alcohols of

70%, 80%, 95%, 95%, 100%, 100% at 2 hour intervals for 1 week. Infiltration

was completed by using monomer and embedded in methyl-methacrylate resin.

Polymerization of the specimens lasted seven days. Methyl-methacrylate blocks

were trimmed with a band saw and mounted on a plastic mount with adhesive.

Block was reduced with the grinding unit to approximately 40㎛ thickness, and

stained with Hematoxylin and Eosin.

Sections were analyzed under microscope for new bone formation and bone to

implant contact and residual graft particle content.

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III. Results

A. Clinical Findings

During the postoperative healing periods, there were pre-exposure of the tissue

covering the remaining implants. Three implants were lost because of severe

inflammation.

B. Histologic findings

Evaluation of the histologic sections revealed similar findings in all treatment

group. Similar aspect showed in control and Group1, but Group2 and Group 3

were different. There was no evidence of acute inflammatory reaction around

unexposed dental implants. Premature exposed implants showed acute

inflammation and bone loss. Grafted particles retained adjacent to the implant

could be seen. The treatment groups in this study showed histologic evidence of

a little new bone formation from the base of the bone defects. Slightly bone loss

of distal side of implant (no bony defect) was observed.

1. The control ( flap only )

Upper parts of bone defects were completely filled with connective tissue

which consisted of some fiber, blood vessels, and inflammatory cells. Collapsed

defect space were observed in this specimen(Fig 4). There was little or no

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inflammatory cell infiltration. However, there was a little amount of newly

formed bone adjacent to the surface of the implants, originating from the existing

base of the bone defects. Small amount of new bone-to-implant contact was

revealed and direct bone-to-implant contact at the lingual side of implant(Fig 5).

Vertical oriented CT fibers. Separation from implant surface is an artifact(Fig 6).

2. Group 1 ( Chitosan membrane )

Minimal amount of newly formed bone originating from existing bone was

found only in the proximity of the base of the bone defects. New bone-to-implant

surface contact was not seen. Such findings were similar to those of the control.

In the more basal area of bone regeneration direct bone-to-implant contact

without intervening soft tissue was present. Chitosan membrane remnants with

connective tissue and fibrous tissue above new bone and a little amount of new

bone formation near the surface of dental implant(Fig 7). Separation from implant

surface is an artifact(Fig 8). Parallel oriented osteoblasts far from implant were

shown. Thick staining on osteoblast surround blood vessel, vertical oriented

osteoblast near the surface of dental implant(Fig 9). There were inflammatory cells

exist in peripheral tissues.

3. Group 2 ( ββββ -TCP＋＋＋Chitosan membrane )

Most of the β-TCP granule in the dehisence defects were surrounded with

connective tissue. New bone formation around graft materials was not present. A

little amount of new bone formation originating from the existing base of bone

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defects was observed at the surface of dental implant, new bone-to-implant

surface contact was not present. Detachment of the trabeculae from the implant

surface was likely an artifact created during biopsy retrieval and specimen

preparation. More apically, bone was denser, with large marrow spaces(Fig 10).

There was no evidence of osseointegration at the grafted area. Chitosan membrane

remnants and granules surrounded with connective tissue and inflammatory

cells above new bone were shown(Fig 11). There was direct bone-to-implant contact

under chitosan membrane.

4. Group 3 ( Autograft＋＋＋ββββ -TCP＋＋＋Chitosan membrane )

The biopsies from these sites consisted of grafted bone particles enmeshed in

connective tissue. Most of the β-TCP was observed at the middle part of the

defects. Some amount of newly formed bone adjacent surface of the implants

was observed, originating from the existing base of bone defects. However, new

bone- to-implant surface contact was not revealed. Fibrous connective tissue

distinguish the bone and the surface of implant. A large amount of inflammatory

cells near the surface of dental implant(Fig 12). New bone formation under

autogenous bone and chitosan membrane. Implant-to-CT interface, parallel fiber.

Separation from implant surface is an artifact.

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IV. Discussion

The purpose of this study was to histologically evaluate and compare the

healing of the dehiscence defects treated with chitosan membrane, autogenous

bone, and β-tricalcium phosphate (β-TCP) around submerged oxidized titanium

surface dental implant in beagle dogs. Exposure of a part of the implant suface

is a common feature of implant placement, especially in postextraction sites or

where there are thin alveolar ridges. It is often necessary to apply the principles

of guided bone regeneration to improve bone regeneration with or without

grafting materials because of bone defect such as dehiscence fenestration,

circumferential bone defect (Jovanovic et al. 1992; Kohal et al. 1998). An ideal

method to treat such defects is to regenerate alveolar bone through GBR

procedures. There are many graft materials and technique that are used recently

in attempt to repair osseous defects around implants (Artzi et al. 1997; Becker

et al. 1990; Dahlin et al. 1989; Hall et al. 1999; Hṻrzeler et al. 1997; Lazzara

1989; Vicente et al. 2000; Zablotsky et al. 1991). The first experimental study

evaluating GBR in conjunction with root form dental implants was published by

Dahlin and coworkers (1989). The membranes most often used for guided bone

regeneration are bioinert membranes made of expanded polytetrafluoroethylene

(e-PTFE). Non-resorbable membranes, however, require surgical removal after

4-6 weeks, posing risks such as possible damage to newly-formed bone tissue

through mechanical interruption or failure to achieve flap coverage over the new

tissue. Resorbable membranes eliminate such problems in addition to the

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financial burden of secondary surgical procedure for patients. A few studies

have demonstrated the incapacity of resorbable membranes to promote bone

growth in a vertical direction, since these materials lack sufficient rigidity and

stability to protect the underlying regenerative area. Cortellini et al. (1996)

compared resorbable membranes (Resolut; W.L.GORE) and non-resorbable

membranes (e-PTFE) and reported no difference in attachement gain. Becker et

al. (1996) studied attachment changes for one year using resorbable membrane

(Resolut; W.L.GORE) with positive results. Resorbable membranes, once

exposed, are difficult to remove, and spread of infection of newly formed tissue

under the membrane cannot be prevented. For these reasons, Becker et al.

(1996) suggested that non- resorbable membranes should be used in cases where

early phase wound closure was uncertain. They also recommended more strict

attention to factors that might affect healing such as plaque control and smoking

habit should be used in resorbable membrane cases.

Consequently, researchers have recently grown interested in the search for a

new non-toxic, biodegradable material that would be free from any side effect.

Among them, the most representative are natural biopolymers, especially,

dextrin, mucopolysaccharide, and chitin. Among those considered, chitin and its

extract, chitosan (poly-N-acetyl glucosaminoglycan), are attracting particular

attention. In addition to biological merits, chitosan is a superior implantation

material because of its versatile physical property: its availability in a variety

of forms-such as solutions, powders, flakes, gels, sponges, fibers and

film-facilitates its use. It is also easy to combine chitosan with other materials

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(Park et al. 2003). Lab and animal studies have proved chitosan's ability to

reduce the weight by absorbing and combining fat, control the cholesterol

level, reinforce the immune reaction against bacteria, virus, and cancerous

cells, and enhance the healing of injured connective tissue and the bleeding

control (Sapelli et al. 1986; Moon et al. 1996; Klokkevold et al. 1999). Ito et

al(1991). have developed self-hardening mixtures of chitosan and

hydroxyapatite as well as chitosan and β-tricalcium phosphate and proposed

their use as bone filling pastes of edentulous ridge. Newly developed chitosan

membrane is a biodegradable barrier membrane which is proven to be safe in

animal and human studies. Regeneration potential of the membrane is also

expected to be comparable to e-PTFE membrane (Yeo et al. 2003).

Resorbable barriers generally lack sufficient rigidity to maintain the space

and tend to collapse into the defect. The use of filling materials has recently

gained new interest coupled with the use of the barrier membranes, since this

allows for the space-making effect when the membrane is not stiff enough.

Jovanovic et al. (1995) suggested that in large defects where stable

maintenance of blood clots might be difficult, use of bone graft could be

helpful in protecting the blood clots. The bone graft can induce bone

formation and provide space-maintenance. Nevin et al. (1994) and Mellonig et

al. (1995) showed that allograft used under GBR membrane successfully

provided space for new bone formation.

Among the numerous filling materials, autologous bone seems to be the gold

standard, but its use is limited by the necessity of an additional surgical

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procedure, the difficulty of obtaining large amounts of donor tissue, and the

risk of complications. For these reasons, many attempts have been made to

find a synthetic graft material that matches the features of autologous bone.

Researchers have paid attention to the development of bone substitutes

including synthetic materials, which could be used to secure a space for bone

growth. Recently, Studies about the use of β-tri-calcium phosphate with dental

implant placement were reported (Nkenke et al. 2002; Palti & Hoch 2002;

Trisi et al. 2003; von Arx et al 2001; Zerbo et al. 2001).

In this study, we used β-TCP. A small amount of β-TCP were visible and

surrounded with connective tissue in this histologic specimen. A lesser newly

formed bone was found only in the proximity of the base of bone defects,

originating from the pre-existing bone. There was no evidence of osseointegration

at the grafted area. Implant-bone interface below the defects showed osseointegration

with no signs of inflammation. Zerbo et al. (2001) showed considerable replacement

of the bone substitute by bone and bone marrow. In biopsy at 9.5 months of the

mandible, 34% of the biopsy consisted of mineralized bone tissue and 29% of

remaining β-TCP, while the biopsy at 8 months after sinus floor augmentation

consisted of 20% mineralized bone and 44% remaining β-TCP. This report

suggest that this graft material, possibly by virtue of its porosity and chemical

nature, may be a suitable bone substitute that can biodegradable and be replaced

by new mineralizing bone tissue. β-TCP proved to be resorbable in 6 months

without interfering with the new bone matrix formation, but it did not enhance

spontaneous bone regeneration in an infrabony defects (Trisi, 2003). New bone

- 17 -

formation most likely occurred from apical wound margin in dehiscence defect.

Therefore, we expected that evaluation of rapid bone healing could be avaliable

within a 12 weeks healing period of dehiscence defects in beagle dogs. In this

study, most remnants of β-TCP granules in the dehiscence defects were

surrounded with connective tissue. Small amount of newly formed bone tissue

was observed at the base of the bone defects, originating from pre-existing bone.

No signs of acute inflammation were observed on the unexposed implant surface. Sites

treated with chitosan membrane-only showed limited bone formation similar to control

sites. The membrane was expected to stablize the β-TCP and contribute to their

initial fixation. Some amount of bone formation was observed on sites treated with β

-TCP+membrane or autogenous bone＋β-TCP＋membrane. Varying amounts of

vertical bone growth were observed in the histological analysis. However, bone

regeneration never reached the shoulder of the implants in the buccal aspect. The

use of β-TCP did not appear to provide more bone to implant contact and new

bone formation. We feel that more evidence for new bone formation from β-TCP

grafting materials may be needed. There was no observation of autogenous bone,

that was already resorbed and maybe replaced with new bone formation.

Premature exposure of non-resorbable and resobable membranes leads to

extensive reorption of bone graft and lack of continuity between the graft and the

recipient bed (Donos et al. 2002). Exposure does not necessarily lead to a

complete failure of the treatment, but it hampers the predictability of positive

outcomes (Gotfredsen et al. 1993; Lang et al. 1994; Warrer et al. 1991).

Obviously, the use of grafting materials seemed to compensate for the lack of

- 18 -

stiffness of the chitosan membrane.

Sites with early wound exposure showed signs of acute inflammation and extensive

bone loss with little to no new bone formation. The clinical significance of having

exposed implant threads was evaluated in a retrospective study by Lekholm et

al. (1996). These authors reported that a few exposed threads would not have

adverse effects on the overall long term prognosis of an implant. In our study,

it was difficult to see new bone to implant surface contact in spite of

oxidized titanium surface implants. Dahlin et al. (1995) showed that peri-implant

dehiscence defects in human with mean depth of 4.7mm treated with barrier

membranes had 3.6mm of new bone formation. Becker et al. (1995) in an

animal study using 5mm dehiscence defects reported 4.2mm of bone formation

in membrane-only cases, 3.8mm in membrane-DFDB cases, and 5mm in

membrane-autograft cases, suggesting negative effect of DFDB in new bone

formation. Cho et al. (1998) showed that peri-implant dehiscence defects

treated with barrier membrane and DFDB had larger area of new bone

formation, while the rate of new bone formation was lower compared to

membrane-only cases suggesting possible delaying of healing from DFDB.

Simion et al. (1994, 1998) using miniscrews reported 3-4mm of new bone

formation using membrane-only. This result differs from the result of the

present study. Possible explanation is, first, the difference in healing period.

The present study used 12 weeks while Simion et al. used more than 9-11

months. Second, miniscrews of 1.3mm diameter may offer better healing

potential compared to relatively larger 3.3mm implant fixtures. Martin et al.

- 19 -

(1998) reported that membrane-bone graft therapy and TR-GTAM therapy for

space provision were effective in increasing the bone regeneration. They also

showed that exposure of membrane resulted in marked decrease in bone

formation, and resorbable membranes showed less amount of new bone

formation compared to non-resorbable membranes. Also, if exposed, an

installed membrane can cause inflammation around the wound. Finally, the

success of the operation is subject to the influence of other factors such as

the overall condition of the patient, the state of oral hygiene, smoking, the

material and location of the membrane, the extent of gingival recession, the

operator's efficiency, and the like. Moses et al. (2005) compared clinical results of

premature exposure of various membranes. Premature exposure of membranes used

in GBR results in decreased bone formation. Premature exposure of membranes and

subsequent and consequent exposure of implants result in impaired bone healing.

When exposed, exposed portion of resorbable membranes is completely resorbed in

3-4 weeks. Bioabsorbable membranes rapidly dis integrate when exposed to the oral

environment, resulting in a shortened barrier function (Machtei 2001). Plaque

contamination, therefore, may be less of a factor compared to exposed non-resorbable

membranes, which may be considered a clinical advantage. Tissue tention may result

from the additional space underneath the gingival tissue, and barrier membrane is

frequently exposed where the tention from suturing is the greatest. Incision method

and suturing method to lessen this problem are required. Initiation of membrane

resorption is ideally 8 weeks after placement considering the maturation of

newly formed bone. Chitosan membranes in the present study were frequently

- 20 -

exposed prematurely, and more studies are needed to ascertain whether

chitosan membrane itself is a factor in this premature wound exposure.

In the present study, chitosan membrane remnants were observed even after

12 weeks, suggesting that barrier effect is maintained for sufficient period of

time.

Clinically, the average initially exposed implant surface is larger, and

incomplete wound closure is prevalent compared to the long term delayed

implants (Zitzmann et al. 1997). Of course, the defects were surgically created

dehiscence defects which could exhibit a different wound healing than that seen

in naturally occurring defects. We used the dehiscence defects design that was

surgically created around implant (3×4mm) to mimic small amount of exposed

implant thread. Such defect design of the present experiment may result in the

collapse of chitosan membrane, and more studies are needed to provide sufficient

rigidity to the chitosan membranes. Thin formation trabecular bone along implant

is easily destructive when inflammation is exists around implant. So We need

bone grafts materials in the use of membranes for space making.

In summary, Results from this limited animal study demonstrated that a small

amount of new bone formation in β-TCP was observed. There was no significant

difference in new bone formation between Group 1 (Chitosan membrane) and

control defects. Most of the residual graft particles adjacent to the implant, were

found embeded in connective tissue. There was a slight difference in the amount

of new bone formation between sites grafted with autogenous bone and β-TCP.

The new bone may be the result of dense connective tissue reinforced by the

- 21 -

bone graft rather than replacement of the graft with true bone (Carpio et al.

2000). Past studies indicated that all guided bone regeneration procedures (GBR

alone and GBR/graft combination) equally produced the greatest amount of

clinical 'hard tissue fill' by bone graft alone (Hṻṻṻṻrzeler et al 1995,1997). The

results in this study were obtained with dehiscence defect morphology.

Dehiscence defects treated without membrane and space-making GBR material

may result in bone resorption. More studies on rigidity reinforcement of chitosan

membrane may be needed. We feel that additional studies should be undertaken

to examine the long term use of grafting materials adjacent to the implant surface

which were used in this studies. It is concluded that a chitosan membrane

enhanced bone regeneration, in particular in conjunction with the use of grafting

materials.

- 22 -

V. Conculsion

The aim of this study was to histologically evaluate and compare the healing

of the dehiscence defects using chitosan membrane, autogenous bone, and β

-tricalcium phosphate (β-TCP) around submerged oxidized titanium surface dental

implant. Sixteen oxidized titanium surface dental implants were used. Defects

were then grafted with either combination of autogenous bone, β-TCP and chitosan

membrane or not filled (control). The experimental animals were sacrificed 12

weeks after implant placement.

Result from this limited study demonstrated followings;

1. Limited amount of new bone formation was observed in control group with

unexposed membrane.

2. Slightly greater amount of bone formation was observed on sites treated with β

-TCP+membrane or autogenous bone＋β -TCP＋membrane compared to control

group.

3. Sites with early wound exposure showed signs of acute inflammation and limited

amount of new bone formation was observed regardless of membranes or bone

substitutes used.

4. Remnants of Chitosan membrane and β -TCP encapsulated with connective tissue

- 23 -

were observed during experimental periods.

These results suggest that further studies are needed on membrane rigidity and

infection control for space maintenance underneath the membrane and bone

substitutes in the treatment of dehiscence defects.

- 24 -

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- 35 -

Legends

Figure 2 : Photograph of Surgically created dehiscence defect adjacent dental

implant.

Figure 3 A, B : Photograph of the chitosan membrane only (Group 1, Left)

and β-TCP+chitosan membrane (Group 2, Right)

Figure 4 A: Photomicrograph of Bucco-lingual ground section at 12weeks after

implantation (Control, No treatment), Bone defect were completely

filled with connective tissue. There was a little amount of newly

formed bone near implant at the apical area of the defect. (original

magnification×10)

B: Photomicrograph of fig A, showing a little amount of new bone

formation and direct bone-to-implant contact at the bottom in spite

of small amount.(original magnification×20)

C: Photomicrograph of fig A, showing cervical area of the lingual side

of implant. CT fibers perpendicular to implant surface. Direct

bone-to-implant contact at the threads.(original magnification×100)


implantation (Control, No treatment), showing middle area of the

lingual side of implant. Direct bone-to-implant contact at the

threads.(original magnification×100)

- 36 -

B: Photomicrograph of fig A, showing vertical oriented osteoblast(OB)

near the threads of the lingual side of implant. Direct bone-to

-implant contact. (original magnification×200)

C: Photomicrograph of fig A, showing osteocyte(OC) far from implant.

(original magnification×200)


implantation (Control, No treatment), Bone defect were completely

filled with connective tissue. Implant-to-soft tissue interface. vertical

oriented CT fibers. Separation from implant surface is an artifact.


B: Photomicrograph of Bucco-lingual ground section at 12weeks after

implantation (Control, No treatment), showing a direct bone-

to-implant contact at the bottom in spite of small amount. (original

magnification×200)


implantation (Group 1, the chitosan membrane only), There was small

amount of newly formed bone near implant at the apical area of the

defect. (original magnification×20)

B: Photomicrograph of fig A, showing chitosan membrane remnants

(RC) with connective tissue and fibrous tissue above new bone and

a little amount of new bone formation near the surface of dental

implant. (original magnification×100)

- 37 -


implantation (Group 1, the chitosan membrane only), showing

chitosan membrane remnants(RC) with connective tissue and fibrous

tissue above new bone. There was small amount of newly formed

bone near implant at the apical area of the defect. (original

magnification×100)

B: Photomicrograph of fig A, showing vertical oriented osteoblast near

the surface of dental implant. Separation from implant surface is an

artifact. (original magnification×200)


implantation (Group 1, the chitosan membrane only), showing parallel

oriented osteoblast far from implant. Thick staining on osteoblast

surround blood vessel (original magnification×100)

B: Photomicrograph of Bucco-lingual ground section at 12weeks after

implantation (Group 1, the chitosan membrane only), showing CT to

bone interface in the periosteum. (original magnification×200)

Figure 10 A : Photomicrograph of Bucco-lingual ground section at 12weeks after

implantation (Group 2, β-TCP+chitosan membrane), showing

granules surrounded with connective tissue and chitosan membrane

remnants above new bone and a small amount of new bone

formation near the surface of dental implant. There was no evidence

of osseointegration at the grafted area. (original magnification×20)

- 38 -

B : Photomicrograph of fig A, showing chitosan membrane remnants

and granules surrounded with connective tissue above new bone.



implantation (Group 2, β-TCP+chitosan membrane), showing

chitosan membrane remnants and granules surrounded with

connective tissue above new bone. (original magnification×100)

B : Photomicrograph of fig A, there was direct bone-to-implant contact

under chitosan membrane. (original magnification×100)


implantation (Group 3, autogenous bone＋β-TCP＋ chitosan

membrane), New bone formation under autogenous bone and

chitosan membrane (original magnification×200).

B : Photomicrograph of another slide, showing new bone formation and

no obervation of chitosan membrane and β-TCP granules. Implant-

to-CT interface, parallel fiber. Separation from implant surface is an

artifact. (original magnification×100).

C : Photomicrograph of another slide, showing fibrous tissue between new

bone and the surface of dental implant. There were many

inflammatory cells between bone and implant. There was no

bone-to-implant contact (original magnification×20).

- 39 -

Figures

Figure 2 : Photograph of Surgically created dehiscence defect

adjacent dental implant.

A B

Figure 3-A, B : Photograph of the chitosan membrane only (Group 1, Left)

and β -TCP+chitosan membrane (Group 2, Right)

- 40 -

Histologic findings

A B C

Figure 4. Photomicrograph of Control

A B C


- 41 -

A B


A B

Figure 7. Photomicrograph of Group 1

- 42 -

A B


A B


- 43 -

A B


A B


- 44 -

A B C


- 45 -

국문요약

임임임플플플란란란트트트의의의 열열열개개개형형형 결결결손손손부부부에에에서서서 골골골유유유도도도재재재생생생에에에 관관관한한한 조조조직직직학학학적적적 연연연구구구(Histomorphology of Guided Bone Regeneration on Dental Implant

Dehiscence Defects in Beagle dogs)

연세대학교 대학원 치의학과(지도 김 종 관 교수)

김 윤 식

치주염으로 인해 파괴되어 얇아진 치조골이나 발치 직후 즉시 식립한임플란트의 경우 자주 발생하는 열개형 골결손 부위에 β-tricalcium

Phosphate와 키토산 차단막의 사용에 따른 신생골조직의 형성양상을 관찰하였다 . 실험동물로는 18개월에서 24개월 사이의 15kg 정도 되는 성견을사용하였다 . 16개의 Oxidized Titanium surface implant가 사용되었으며 , 성견하악의 양측에 각각 2개씩 사용되었다 . 제1,2,3,4 소구치를 발치하고 편평한골면을 형성한뒤 8주후 straight fissure bur를 이용하여 3x4mm의 균일한 열개형 골결손부를 형성하였다 . 임플란트를 식립한후 판막을 바로 봉합한 것을 대조군 , 골결손부에 키토산 차단막을 피개한 것을 실험1군 , β-tricalcium

Phosphate를 축조하고 키토산 차단막을 피개한 것을 실험2군 , 자가골과 β

-tricalcium Phosphate를 축조한후 키토산차단막을 피개한 것을 실험3군으로하여 매식술 시행 12주후에 실험동물을 희생시켜 조직학적 소견을 관찰하

- 46 -

였다 .

1. 대조군에서 차단막이 노출되지 않은 경우에도 신생골 형성은 미미하였다

2. β-TCP와 차단막을 사용한 경우와 자가골과 β-TCP를 함께 차단막과 사용한 경우 대조군과 비교하여 좀더 많은 양의 신생골이 관찰된다 .

3. 조기노출되는 경우 , 염증상태의 발현과 함께 차단막 , 골대체물질의 사용에 관계없이 대조군과 비슷하게 신생골 형성은 미약하였다 .

4. 실험기간동안 키토산 차단막과 β-TCP는 완전히 흡수되지 않고 잔사가 일부 관찰되었다 .

이상의 결과로 미루어 볼때 열개형 골결손부의 치료에 있어서 공간유지를위한 차단막이나 골대체물질 사용시 강도와 감염방지에 대한 많은 연구가더 이루어져야 할것으로 사료된다 .

핵핵핵심심심 되되되는는는 말말말：：： implant dehiscence defect, GBR, autogenous bone,

ββββ-tricalcium phosphate, chitosan membrane

histomorphology of guided bone regeneration on …...intrabony, fenestration or dehiscence bony...

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