233 The Bone Grinding and Scaffold Grafting Techniques for Guide Bone Regeneration Induce the Stress on the Rat Brain Yeon Kyung Lee 1 , Ji Eun Kim 1 , So Hee Nam 1 , Sun il Choi 1 , Eon Pil Lee 1 , Hae Wook Choi 1 , Hong Sung Kim 1 , Jae Ho Lee 1 , Young Jin Jung 1 , Byeong Cheol Kang 2 , Jung Sik Cho 3 and Dae Youn Hwang 1 * Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University/ PNU-Laboratory Animal Resources Center, Miryang, Korea Department of Experimental Animal Research, Clinical Research Institute, Seoul National University Hospital, Seoul, Korea Multidisciplinary Technology Institute, Hoseo University, Asan, Korea Guided bone regeneration (GBR) is a technique that a barrier membrane is placed over the bone defect to prevent the cell growth from the connective tissue and epithelium. In this study, in order to determine whether GBR technique could induce stress in rats, the standardized bone defect in rat calvaria was covered with apatitte membrane. Bone and brain tissues were collected from rats at 3 days, 2, 4, and 16 weeks post-operation, and then alteration of the new bone formation at the defects and stress-related factors were detected with histological examination and Western blot, respectively. From 4 to 16 weeks after the operation, the apatitte membrane was attached to the region of regenerated bone and encapsulated with a thick fibrous layer. Furthermore, the concentration of cortisol, a good indicator of stress, significantly increased 3 days post-operation. However, the increase at 3 days was returned to the basal level in 2 weeks. In Western blot analysis, the highest phosphorylation level of extracellular signal- regulated kinase (ERK) was observed 3 day post-operation, while those of the c-jun N-terminal kinase (JNK) and p38 were detected 4 weeks post-operation. Taken together, the results suggest that GBR technique may induce the serious stress on the brain tissue via the induction of ERK phosphorylation during 2 weeks, and that the stress responses restored in 4 week via JNK and p38 signaling pathway. Key words: Guided bone regeneration, cortisol, mitogen-activated protein kinase (MAPK), stress, apatitte membrane Received 10 June 2010; Revised version received 21 July 2010; Accepted 28 August 2010 Guided bone regeneration (GBR) 은 조직공학분야에 새 롭게 도입된 개념으로서, 이식된 scaffold 의 구조물과 표 면을 따라 발생하는 조직의 재생(regeneration) 과 성장 (growth) 을 목적으로 시행되는 기술이다(Kellomaki et al ., 2000). 이러한 기술에서, 특정한 장치의 표면에서 골성장 은 bioceramics 과 같은 재질을 사용함으로써 혹은 이식물 질의 다공성에 의해 유도가 가능하다. 또한, 골의 파손부 위 혹은 골절부위에 scaffold 를 덮어주는 기술은 섬유조직 (fibrous tissue) 과 같이 원하지 않는 조직이 손상부위에 침 투하는 것을 막아주거나 가이드해 주는 방법으로 사용되 기도 한다. 이러한 기술에 적용되는 scaffold는 막 (membranes), 그물망(meshes), 평판(plates), 나사(screws), 마개(plugs), 막대(rods) 등의 형태로 사용이 가능하다 (Kellomaki et al ., 2000). Scaffold 의 재질로는 다양한 합 성 혹은 천연물유래 폴리머(polymers), 세라믹(ceramics), 유리(glasses) 그리고 복합재료(composites) 등이 사용되고 있으며(Hench and Wilson, 1999), 최근에는 생물학적으 로 안전하고, 생분해성(biodegradability) 이 있으며, 생활성 (bioactivity) 을 지니는 재질을 찾기 위한 연구가 활발히 진 행되고 있다(Kellomaki et al ., 2000). 최근에 GBR 은 치주골 결손(periodontal bone defects) 을 치료하기 위하여 사용되고 있다. 이러한 과정에 사용되는 차단막(barrier membrane) 은 치주골 손상부위를 차단함으 로써 치주결합조직(gingival connective tissues), 치주상피 (gingival epithelium) 또는 치근막(periodontal ligament) 이 손상부위로 침투하여 성장하지 못하도록 억제하는 기능 을 수행하게 된다(Aslan et al ., 2004). 또한, GBR 기술은 골손상(intra-bony defects) 부위에 골의 재생에도 사용되 *Corresponding author: Dae Youn Hwang, Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University, 50 Cheonghak-ri, Samnangjin- eup, Miryang, Gyeongnam 627-706, Korea Tel: +82-55-350-5388 Fax: +82-55-350-5389 E-mail: [email protected]Lab. Anim. Res. 2010: 26(3), 233-239
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233
The Bone Grinding and Scaffold Grafting Techniques for Guide Bone Regeneration Induce the Stress on the Rat Brain
Yeon Kyung Lee1, Ji Eun Kim1, So Hee Nam1, Sun il Choi1, Eon Pil Lee1, Hae Wook Choi1, Hong Sung Kim1, Jae Ho Lee1, Young Jin Jung1, Byeong Cheol Kang2, Jung Sik Cho3 and Dae Youn Hwang1*
1Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University/PNU-Laboratory Animal Resources Center, Miryang, Korea
2Department of Experimental Animal Research, Clinical Research Institute, Seoul National University Hospital, Seoul, Korea3Multidisciplinary Technology Institute, Hoseo University, Asan, Korea
Guided bone regeneration (GBR) is a technique that a barrier membrane is placed over the bone defectto prevent the cell growth from the connective tissue and epithelium. In this study, in order to determinewhether GBR technique could induce stress in rats, the standardized bone defect in rat calvaria wascovered with apatitte membrane. Bone and brain tissues were collected from rats at 3 days, 2, 4, and 16weeks post-operation, and then alteration of the new bone formation at the defects and stress-relatedfactors were detected with histological examination and Western blot, respectively. From 4 to 16 weeksafter the operation, the apatitte membrane was attached to the region of regenerated bone andencapsulated with a thick fibrous layer. Furthermore, the concentration of cortisol, a good indicator ofstress, significantly increased 3 days post-operation. However, the increase at 3 days was returned to thebasal level in 2 weeks. In Western blot analysis, the highest phosphorylation level of extracellular signal-regulated kinase (ERK) was observed 3 day post-operation, while those of the c-jun N-terminal kinase(JNK) and p38 were detected 4 weeks post-operation. Taken together, the results suggest that GBRtechnique may induce the serious stress on the brain tissue via the induction of ERK phosphorylationduring 2 weeks, and that the stress responses restored in 4 week via JNK and p38 signaling pathway.
Key words: Guided bone regeneration, cortisol, mitogen-activated protein kinase (MAPK), stress, apatittemembrane
Received 10 June 2010; Revised version received 21 July 2010; Accepted 28 August 2010
Guided bone regeneration (GBR)은 조직공학분야에 새
롭게 도입된 개념으로서, 이식된 scaffold의 구조물과 표
면을 따라 발생하는 조직의 재생(regeneration)과 성장
(growth)을 목적으로 시행되는 기술이다(Kellomaki et al.,
2000). 이러한 기술에서, 특정한 장치의 표면에서 골성장
은 bioceramics과 같은 재질을 사용함으로써 혹은 이식물
질의 다공성에 의해 유도가 가능하다. 또한, 골의 파손부
위 혹은 골절부위에 scaffold를 덮어주는 기술은 섬유조직
(fibrous tissue)과 같이 원하지 않는 조직이 손상부위에 침
투하는 것을 막아주거나 가이드해 주는 방법으로 사용되
기도 한다. 이러한 기술에 적용되는 scaffold는 막
(membranes), 그물망(meshes), 평판(plates), 나사(screws),
마개(plugs), 막대(rods) 등의 형태로 사용이 가능하다
(Kellomaki et al., 2000). Scaffold의 재질로는 다양한 합
성 혹은 천연물유래 폴리머(polymers), 세라믹(ceramics),
유리(glasses) 그리고 복합재료(composites) 등이 사용되고
있으며(Hench and Wilson, 1999), 최근에는 생물학적으
로 안전하고, 생분해성(biodegradability)이 있으며, 생활성
(bioactivity)을 지니는 재질을 찾기 위한 연구가 활발히 진
행되고 있다(Kellomaki et al., 2000).
최근에 GBR은 치주골 결손(periodontal bone defects)을
치료하기 위하여 사용되고 있다. 이러한 과정에 사용되는
차단막(barrier membrane)은 치주골 손상부위를 차단함으
로써 치주결합조직(gingival connective tissues), 치주상피
(gingival epithelium) 또는 치근막(periodontal ligament)이
손상부위로 침투하여 성장하지 못하도록 억제하는 기능
을 수행하게 된다(Aslan et al., 2004). 또한, GBR 기술은
골손상(intra-bony defects) 부위에 골의 재생에도 사용되
*Corresponding author: Dae Youn Hwang, Department ofBiomaterials Science, College of Natural Resources & LifeScience, Pusan National University, 50 Cheonghak-ri, Samnangjin-eup, Miryang, Gyeongnam 627-706, KoreaTel: +82-55-350-5388Fax: +82-55-350-5389E-mail: [email protected]
kinase (ERK), c-jun N-terminal kinase (JNK) 및 p38 단
백질의 인산화 정도를 Western blot을 이용하여 관찰하였
다. 먼저 실험동물로부터 뇌조직 100 mg에 cell lysis
solution (iNtRON Co., Seoul, Korea)을 첨가하여 분쇄한
후 13,000 rpm에서 5분 동안 원심분리하여 단백질을 분
리하고, bincinchoninic acid assay 방법을 이용하여 단백
질을 정량하여 Western blot에 사용하였다. 4-20% SDS-
PAGE gel에 50 µg의 단백질을 전기영동 한 후 hybond-
enhanced chemiluminescence (ECL) membrane (GE
healthcare, Pittsburgh, PA, USA)에 transfer하고, 3%
skim milk에서 2시간 동안 blocking하였다. Anti-ERK (sc-
94, Santa Cruz Biotechnology, Santa Cruz, CA, USA),
anti-p-ERK (sc-7383, Santa Cruz Biotechnology), anti-
Figure 1. Experimental scheme and procedure of guided bone regeneration of bone defect. A. Two cavities were created using adrill. A round defect of 3-5 mm in diameter and 0.1-0.2 mm in depth was formed in the calvaria and covered with apatite membrane.B. After the operation of apatite membrane, bone and brain tissue were collected from rats at 3 days and 2, 4 and 16 weeks toexamine histological alterations and Western blot analysis. C. Rat cranium showing two full-thickness bone defects with 3-5 mm indiameter.
Figure 2. Representative histological findings of control (A) and3 days (B) and 2 (C), 4 (D) and 16 weeks (E) post-operation.The arrow in Figure 2B indicates regenerative film.
Effect of bone grinding and scaffold grafting on the brain 237
Figure 4. Effects of guided bone regeneration on MAPKsignaling pathway. Fifty micrograms of the protein per samplewere immunoblotted with the antibodies for ERK, p-ERK, JNK,p-JNK, p38, p-p38 and β-actin, respectively. The samples wereassayed in triplicate. The values are mean±SD. *P<0.05 is thesignificance level compare to control group.
Figure 3. Changes of the plasma cortisol concentration in ratswith regenerated bone. The serum cortisol level was measuredby radioimmunoassay. Values are mean±SD. *P<0.05 is thesignificance level compare to control group.
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