Titanium disc에 도포한 부착단백질이 사람 치은 조섬유세포의 부착 및 증식에 미치는 영향

Abstract

[한글]

치근형 골내 임프란트는 악골내에 매식하였을 때 생체조직과 접합하는 과정에서 골조직 계면·결합조직 계면·상피 계면을 형성하게 되며, 이 중 결합조직과 상피를 포함하는 연조직 계면은 임프란트의 유지와 예후에 중요한 역할을 하는 부분이다. 특히 결합조직계면은 결합조직 섬유에 의한 접합장치 (attachment apparatus)를 형성할 뿐만 아니라 상피의 하방성장을 억제함으로써 임프란트 주위조직을 건강하게 유지하는데 기여한다. 이러한 결합조직 부착부위에서의 조섬유세포 부착을 증진시키기 위한 방법으로는 표면조도(surface roughness) 조절과 부착단백질 (attachment protein)의 도포를 들 수 있으며, 이 중 부착단백질의 도포효과는 아직 정확히 밝혀지지 않고 있으며, Titanium상에서의 도포효과가 연구된 바 있는 단백질도 fibronectin과 type Ⅰ collagen 뿐이며 그 결과들이 일치하지 않고 있다. 더욱이 이들 연구의 실험환경과 단백질 도포방법 등에 차이가 있어 부착단

백질의 정확한 효용에 관한 평가가 어려운 실정이다. 따라서 일정하게 조절된 환경 하에서 이루어진 부착단백질의 부착효과에 대한 비교연구가 요구되고 있다. 본 연구에서는 부착단백질이 조섬유세포의 부착 및 중식에 미치는 영향을 평가하기 위하여 생체 내에서 합

성되는 부착단백질인 type Ⅰ collagen, type Ⅱ collagen, fibronectin, laminin, vitronectin을 사용하였다. 각각의 부착·단백질을 titanium disc에 도포하고, 부착단백질을 도포하지 않은 경우를 대조군으로 하여 그 위에 사람 치은 조섬유세포를 배양하였다. 배양 30, 60, 180분 후에 미부착 세포 수를 Coulter counter로 계수하여 부착단백질의 도포에 따른 조섬유세포의 부착도를 평가하였으며, 시편위에 부착된 세포들의 부착형태를 주사전자현미경으로 관찰하였다. 반점상 부착 (focal type adhesion)의 형성을 확인하기 위하여 배양 3, 24시간 후에 actin과 vinculin에 대한 세포화학 및 면역형광염색을 실시하였으며, (3)**H -thymidine을 첨가하여 부착단백질 도포에 따른 조섬유세포의 증식도를

평가하여 다음과 같은 곁과를 얻었다.

1. 조섬유세포의 부착도는 fibrunectin 도포군에서 시간 결과에 따라 가장 높았다 (p<0.05). Laminin과 vitronectin 및 type Ⅳ collagen 도포군은 그 다음으로 높은 부착도를 나타내었으며. type Ⅰ collagen도포군은 대조군과 차이를 보이지 않았다 (p>0.05).

2. Fib개nectin과 type Ⅳ collagen이 도포된 시편의 표면에서 가장 넓고 납작하게 부착된 조섬유세포들이 관찰되었다.

3. Fibronectin 도포군의 조섬유세포에서 actin 섬유가 가장 치밀하며 규칙적으로 배열되어 있었다.

4. Fibronetin을 도포한 경우에 세포질의 변연부에서 뚜렷한 반점상으로 vinculin이 염색되었다.

5. 조섬유세포의 증식도는 부착단백질의 도포에 따른 영향을 받지 않았다(p>0.05).

이상의 결과를 종합하면. titanium 임프란트 경부에 fibronectin을 도포함으로써 결합조직 부위의 부착을 촉진시키며 임프란트를 건강하게 유지시킬 수 있는 가능성을 시사하여주는 것으로 보여지며, 생체실험을 통하여 이를 임상에 응용할 수 있는 방법에 대한 연구와 상피계면과의 위치 및 기능적 관계를 고려하여 치은상피에서의 부착단백질 도포에 따른 효과에 대해서도 비교, 분석이 필요한 것으로 사료된다.

THE EFFECT OF ATTACHMENT PROTEINS COATED ON TITANIUM DISCS ON THE ADHESIVENESS AND

PROLIFERATION OF HUMAN GINGIVAL FIBROBLAST

Jeung Won Choi

Department of Dental Science, Graduate School, Yonsei University

(Directed by Assistant Professor Keun Woo Lee, D.D.S., M.S.D., Ph.D.)

Endosseous dental implants are interfaced with bone, connective tissue, and

epithelium when implanted into the jaw bone. The soft tissue interface including

connective tissue and epithelium is one of the mast critical factors in the

determination of implant maintenance and prognosis. The connective tissue interface

not only forms the attachment apparatus, but also contributes to the formation of

healthy peri-implant tissue by prevention of the epithelial downgrowth. There are

two ways to promote connective tissue fibroblasts adhesiveness to implanted

biomaterials, one is to control the surface roughness of the biomaterial and the

other is to coat the biomaterial with attachment proteins. Between them, coat the

biomaterials with attachment proteins is denounced. Fibronectin and type Ⅰ

collagen are the only attachment proteins studied to titanium biomaterials for

evaluation of their effectiveness, and the results were not united. And it is

difficult to evaluate the exact effect of the attachment proteins due to

experimental difficulties and methods of application. So the comparative analytical

studies of the effect of attachment proteins under controlled condition are needed.

The purpose of this in vitro study was to evaluate the adhesiveness and

proliferation of human gingival fibroblasts to attachment protein-coated and

non-coated endosseous titanium biomaterials. Well-known attachment proteins were

used, they were type Ⅰ collagen, type Ⅳ collagen, fibmnectin, laminin, and

vitronectin. Each attachment proteins applied onto the commercially pure titanium

discs. In this study, the protein-coated and non-coated titanium discs were

classified as each groups. Human gingival fibreblasts cultured onto each groups.

After 30, 60, 180 mins incubation time, unattached cells counted with Coulter

counter for evaluation of the adhesiveness of human gingival fibroblasts. The

configurations of attached human gingival flbroblasts were done by SEM observation.

To confirm the focal contact areas, actin cytochemical and vinculin

immunofluorescent staning were done after 3, 24 hours of incubation. To evaluate

the effect of attachment proteins on the proliferation of human gingival

fibroblasts, (3)**H -thymidine were added after 72 hours incubation. The results

were as follows.

1. Adhesiveness of human gingival fibroblasts were best in fibronectin-coated

group among groups (p<0.05). Then laminin, vitrenectin, and type Ⅳ collagen groups

were better, and there were no statistically significant difference between type Ⅰ

collagen group and control (p>0.05).

2. Fibronectin and type Ⅳ collagen groups showed well spread human gingival

fibroblasts in SEM observation.

3. Fibronectin group showed most dense and regular arrangement of actin filaments

among groups.

4. Fibrenectin group showed most distinct vinculin patches along the cytoplasmic

precess among groups.

5. In the proliferation of human gingival fibroblasts, there were no

stasistically significant difference among groups (p>0.75).

On the bases of these findings, strongly suggest that fibronectin coating at the

neck portion of titanium biomaterial promotes adhesiveness of human gingival

fibroblasts and suggest the possibility of healthy implant maintenance. Clinical

application of the attachment proteins are further studied and comparative analysis

of attachment proteins applied on implant biomaterials against epithelial cells are

needed.

[영문]

Endosseous dental implants are interfaced with bone, connective tissue, and epithelium when implanted into the jaw bone. The soft tissue interface including connective tissue and epithelium is one of the mast critical factors in the determination of implant maintenance and prognosis. The connective tissue interface not only forms the attachment apparatus, but also contributes to the formation of healthy peri-implant tissue by prevention of the epithelial downgrowth. There are two ways to promote connective tissue fibroblasts adhesiveness to implanted biomaterials, one is to control the surface roughness of the biomaterial and the

other is to coat the biomaterial with attachment proteins. Between them, coat the biomaterials with attachment proteins is denounced. Fibronectin and type Ⅰ collagen are the only attachment proteins studied to titanium biomaterials for

evaluation of their effectiveness, and the results were not united. And it is difficult to evaluate the exact effect of the attachment proteins due to experimental difficulties and methods of application. So the comparative analytical studies of the effect of attachment proteins under controlled condition are needed.

The purpose of this in vitro study was to evaluate the adhesiveness and proliferation of human gingival fibroblasts to attachment protein-coated and non-coated endosseous titanium biomaterials. Well-known attachment proteins were used, they were type Ⅰ collagen, type Ⅳ collagen, fibmnectin, laminin, and

vitronectin. Each attachment proteins applied onto the commercially pure titanium discs. In this study, the protein-coated and non-coated titanium discs were classified as each groups. Human gingival fibreblasts cultured onto each groups.

After 30, 60, 180 mins incubation time, unattached cells counted with Coulter counter for evaluation of the adhesiveness of human gingival fibroblasts. The configurations of attached human gingival flbroblasts were done by SEM observation.

To confirm the focal contact areas, actin cytochemical and vinculin immunofluorescent staning were done after 3, 24 hours of incubation. To evaluate the effect of attachment proteins on the proliferation of human gingival fibroblasts, (3)**H -thymidine were added after 72 hours incubation. The results were as follows.

1. Adhesiveness of human gingival fibroblasts were best in fibronectin-coated group among groups (p<0.05). Then laminin, vitrenectin, and type Ⅳ collagen groups were better, and there were no statistically significant difference between type Ⅰ

collagen group and control (p>0.05).

2. Fibronectin and type Ⅳ collagen groups showed well spread human gingival fibroblasts in SEM observation.

3. Fibronectin group showed most dense and regular arrangement of actin filaments among groups.

4. Fibrenectin group showed most distinct vinculin patches along the cytoplasmic precess among groups.

5. In the proliferation of human gingival fibroblasts, there were no stasistically significant difference among groups (p>0.75).

On the bases of these findings, strongly suggest that fibronectin coating at the neck portion of titanium biomaterial promotes adhesiveness of human gingival fibroblasts and suggest the possibility of healthy implant maintenance. Clinical

application of the attachment proteins are further studied and comparative analysis of attachment proteins applied on implant biomaterials against epithelial cells are needed.