이 연구는 국립 자연 과학 재단의 중국 (81572111), 과학 및 기술 프로젝트 재단의 장쑤 성 (BL2012002), 과학 연구 난징 프로젝트 (201402007), 자연 과학에 의해 지원 되었다 장쑤 성 (BK20161385)의 기초 그리고 중국 의사 협회 (2015COS0810)의 특별 한 기초.

여기에 설명 된 모든 메서드는 기관 동물 관리 및 사용 위원회 (IACUC)의 난징 대학에 의해 승인 되었습니다.
1. CoCrMo 입자 준비
<ol><li>조작된 높은 진공 3 전극 직류4를 사용 하 여 CoCrMo 입자를 얻을. 10-3 Pa 진공, 0.04 MPa 아르곤 및 수소 3:2 (v/v), 악기에서 장소 CoCrMo 합금 그리고 현재 650 A 음극 선.</li>
	<li>CoCrMo 입자의 직경을 측정 합니다.<ol>
<li>무수 에탄올의 1.5 mL에 CoCrMo 입자의 1 밀리 그램을 추가 합니다.</li>
		<li>무수 에탄올에 초음파 진동 28 kHz, 600 W 5 분에 의해 CoCrMo 입자를 resuspend.</li>
		<li>전송 전자 현미경 (TEM)의 객관적인 테이블에 결과 현 탁 액의 한 방울 (약 20 µ L)를 적용 합니다. 200 kV 가속 전압 및 0.24 nm 해상도에 가장 사진 시리즈를 캡처하십시오.</li>
		<li>TEM 현미경 사진에서 평균 직경 및 입자 크기 분포를 계산 하기 위해 제공 된 소프트웨어를 사용 합니다.</li>
	</ol>
</li>
	<li>오염을 부유<ol>
<li>고압 121 ° C와 15 psi에서 15 분 동안 입자의 50 g.</li>
		<li>Limulus Amebocyte Lysate (LAL) 분석 결과 양적으로 독 검색 (< 0.25 %EU / mL 내 부재를 나타내기 위해 여겨졌다)5.</li>
	</ol>
</li>
	<li>재고 솔루션650 mg/mL의 농도에서 인산 염 버퍼 염 (PBS)에 입자를 resuspend.</li>
</ol>2입니다. Calvarial Osteolysis 모델의 건설
<ol><li>6 주 된 C57BL/J6 쥐 (그룹 당 6 쥐) withpentobarbital (50 mg/kg) anesthetize 핀치 테스트를 사용 하 여 마 취의 수준을 평가 하기 위해. 일반적인 염 분으로 눈의 건조를 방지 합니다.</li>
	<li>경향이 위치에서 마우스를 놓습니다. 두개골에 모피는 면도기로 제거 하 고 75%의 에탄올을 포함 하는 의료 면봉을 사용 하 여 피부를 소독.</li>
	<li>포인트 지역화, 각각 두 눈과 귀 사이의 중간점을 포함 하 여 2 포인트를 식별 합니다. 다음, 포인트, 위의 두 사이의 라인을 확인 하 고가 위 (그림 1A)와 위의 라인을 따라 피부를 incise.</li>
	<li>메스 (그림 1B)6calvaria에서 두개골과 제거 합니다.</li>
	<li>간단한 중단된 봉합으로 양쪽 끝에 봉합 피부.</li>
	<li>노팅 없이 절 개의 중간을 통해 봉합 라인을 확인 합니다. 봉합 선의 두 끝을 잡으십시오.</li>
	<li>Calvarias (그림 1C)2의 한가운데에 CoCrMo 입자 현 탁 액 (PBS에 50 mg/mL)의 50 µ L를 포함 합니다.</li>
	<li>간단한 중단된 봉합 (그림 1D) 내 마지막 스티치 매듭</li>
	<li>또 다른 2 주 동안 마우스를 유지.</li>
</ol>3. 마이크로-CT 스캔 하 여 Calvarial Osteolysis 모델의 평가
<ol><li>이산화탄소와 쥐를 희생. 수평 평면에 쥐 목을 벨. 뇌 조직 내부, 그리고 피부를 제거 하 고 밖에 서 모피. 추가 실험을 위한 calvarias을 수확.</li>
	<li>부드럽게 핀셋으로는 calvaria에 모든 연 조직 정리. 마이크로-CT 검사 전에 PBS 24 h calvarias 24 h. 담가 4 ° C에서 4 %paraformaldehyde 지운된 calvarias을 수정.</li>
	<li>마우스 calvarias 18 µ m의 아이소메트릭 해상도 및 45의 x 선 에너지 설정에서 고해상도 마이크로-CT 분석 kV 및 550 mA.</li>
	<li>소프트웨어와 마이크로-CT 데이터의 3 차원 재구성을 수행 합니다.</li>
	<li>정성 및 정량 분석입니다.<ol>
<li>먼저, 관심의 영역으로 중간 봉합 사 주위의 사각형 영역을 선택 합니다.</li>
		<li>둘째, 뼈 미네랄 밀도 (BMD)를 측정, 볼륨/총 뼈 볼륨 (BV/TV), 배수 수 (Tb.N), 배수 두께 (Tb.Th), 배수 분리/간격 (Tb.Sp), 그리고 마이크로-코네티컷에 대 한 제공 된 소프트웨어와 함께 총 다공성의 비율</li>
		<li>셋째, 일방통행 ANOVA에 의해 다양 한 측정을 위한 3 개 그룹을 비교할 수 있습니다. 포스트 hoc 분산 분석 적용 Bonferroni 방법2.</li>
	</ol>
</li>
</ol>4. Calvarial Osteolysis 모델 H & E 염색 법의 평가
<ol><li>15% 에틸렌 diamine tetraacetic 산 (EDTA) calvaria 샘플을 알려줍니다-4 ° c.에서 PBS 3 주 동안 매일 decalcification 솔루션을 변경 합니다.</li>
	<li>2 cm x 1 cm x 1 cm 큐브, 파라핀에 석된 샘플을 포함 하 고 입자 증 착의 영역에서 2 µ m 섹션으로 그들을 슬라이스.</li>
	<li>되며 섹션 얼룩 하 고 오신 앞7에서 설명한.</li>
	<li>가벼운 현미경 검사 법에 의해 전체 pathomorphism의 현미경을 캡처하십시오.</li>
</ol>

<ol>
	<li>Dong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antisense+oligonucleotide+targeting+TNF-alpha+can+suppress+Co-Cr-Mo+particle-induced+osteolysis.">Antisense oligonucleotide targeting TNF-alpha can suppress Co-Cr-Mo particle-induced osteolysis.</a> J Orthop Res. 26 (8), 1114-1120 (2008).</li><li>Deng, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SIRT1+protects+osteoblasts+against+particle-induced+inflammatory+responses+and+apoptosis+in+aseptic+prosthesis+loosening.">SIRT1 protects osteoblasts against particle-induced inflammatory responses and apoptosis in aseptic prosthesis loosening.</a> Acta Biomater. 49, 541-554 (2017).</li><li>Langlois, J., Hamadouche, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+animal+models+of+wear-particle+osteolysis.">New animal models of wear-particle osteolysis.</a> Int Orthop. 35 (2), 245-251 (2011).</li><li>Wang, P., Zhao, F. X., Zhang, Z. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+ultrafine+zinc+powders+by+DC+arc+plasma+evaporation+method.">Preparation of ultrafine zinc powders by DC arc plasma evaporation method.</a> Chinese Journal of Nonferrous Metals. 21 (9), 2236-2241 (2011).</li><li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fibroblast+expression+of+RANKL+in+CoCrMo-particle-induced+osteolysis+is+mediated+by+ER+stress+and+XBP1s.">The fibroblast expression of RANKL in CoCrMo-particle-induced osteolysis is mediated by ER stress and XBP1s.</a> Acta Biomater. 24, 352-360 (2015).</li><li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+mediated+CoCrMo+particle-induced+peri-implant+osteolysis+by+promoting+osteoblast+apoptosis.">Autophagy mediated CoCrMo particle-induced peri-implant osteolysis by promoting osteoblast apoptosis.</a> Autophagy. 11 (12), 2358-2369 (2015).</li><li>Wang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle-induced+osteolysis+mediated+by+endoplasmic+reticulum+stress+in+prosthesis+loosening.">Particle-induced osteolysis mediated by endoplasmic reticulum stress in prosthesis loosening.</a> Biomaterials. 34 (11), 2611-2623 (2013).</li><li>Yang, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus-mediated+osteoprotegerin+gene+transfer+protects+against+particulate+polyethylene-induced+osteolysis+in+a+murine+model.">Adeno-associated virus-mediated osteoprotegerin gene transfer protects against particulate polyethylene-induced osteolysis in a murine model.</a> Arthritis Rheum. 46 (9), 2514-2523 (2002).</li><li>Liu, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+mediated+TiAl(6)V(4)+particle-induced+peri-implant+osteolysis+by+promoting+expression+of+TNF-alpha.">Autophagy mediated TiAl(6)V(4) particle-induced peri-implant osteolysis by promoting expression of TNF-alpha.</a> Biochem Biophys Res Commun. 473 (1), 133-139 (2016).</li><li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ER+Stress+Mediates+TiAl6V4+Particle-Induced+Peri-Implant+Osteolysis+by+Promoting+RANKL+Expression+in+Fibroblasts.">ER Stress Mediates TiAl6V4 Particle-Induced Peri-Implant Osteolysis by Promoting RANKL Expression in Fibroblasts.</a> PLoS One. 10 (9), e0137774 (2015).</li><li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TiAl6V4+particles+promote+osteoclast+formation+via+autophagy-mediated+downregulation+of+interferon-beta+in+osteocytes.">TiAl6V4 particles promote osteoclast formation via autophagy-mediated downregulation of interferon-beta in osteocytes.</a> Acta Biomater. 48, 489-498 (2017).</li><li>Chen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lycorine+suppresses+RANKL-induced+osteoclastogenesis+in+vitro+and+prevents+ovariectomy-induced+osteoporosis+and+titanium+particle-induced+osteolysis+in+vivo.">Lycorine suppresses RANKL-induced osteoclastogenesis in vitro and prevents ovariectomy-induced osteoporosis and titanium particle-induced osteolysis in vivo.</a> Sci Rep. 5, 12853 (2015).</li><li>Neuerburg, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+calcitonin+receptor+signalling+in+polyethylene+particle-induced+osteolysis.">The role of calcitonin receptor signalling in polyethylene particle-induced osteolysis.</a> Acta Biomater. 14, 125-132 (2015).</li><li>Catelas, I., Jacobs, J. 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저자는 공개 없다.

마모 입자 유도 osteolysis 쥐에 대 한 두 가지 주요 방법이 있다: 공기 주머니 모델과 calvarial osteolysis 모델. 공기 주머니 모델에서 피하 생성 된 공기 주머니는 처음 설립, 마모 입자 소개 및 이식 뼈 조직8에 이어서. 주머니 벽 무 균 완화에 모방합니다. 그러나, 뼈 이식이 이다 nonvascular 생물 학적 활동 없이, 입자와 뼈 조직 간의 직접적인 상호 작용을 평가 하기 어렵습니다. Calvarial o...

가장 일반적인 원인은 이다 무 균을 풀면 총 엉덩이 관절 (THA) 교체 및 총 무릎 관절 (TKA) 교체 실패의 개정 수술1필요. 그러나, 기본 메커니즘 불분명2남아 있습니다. 긴 후속 입자 유도 osteolysis, 그 발생은 드문; 검색 하는 데 필요한 따라서, 그것은 임상의 경우에서 자사의 pathogenesis 탐험 도전 이다. 따라서, 복잡 한 세포와 조직 메커니즘에 초점을 맞추고 더 연구에 실험 비보에 모두 착용 입자 유도 osteolysis 모델 및 분석 실험 체 외에 세포 항상성3뼈에 관련 된 필요 합니다. 유효한 동물 모델 더 세포질 분석 실험에 대 한 증거를 제공 하는 뼈 손실에 마모 입자의 효과 드러내는에서 중요 하다.
CoCrMo 입자에 노출에 의해 건설 murine calvarial osteolysis 모델 입자와 무 균 완화에 다양 한 세포 간의 상호 작용을 평가 하기 위한 효과적이 고 유효한 방법입니다. 이 모델에서 CoCrMo 입자 발생할 calvarial osteolysis 유도 하는 대 식 세포에서 염증 성 cytokines, osteoclasts 활성화, osteoblast 확산 억제 및 osteoblast apoptosis를 홍보.
그것은이 모델을 설정 하려면 2 주 걸립니다. Osteolysis 수 수 시각 hematoxylin와 오신 (H & E) 얼룩 계량 및 마이크로 계산 된 단층 촬영 (마이크로 CT)2. 또한,이 모델은 상대적으로 저렴 한 비용, 그리고 유전자 변형 및 녹아웃 마우스 모델 다양 한 복용량3화합물의 많은 화면을 사용할 수 있습니다.
설정 하 고이 모델을 평가 절차는 간단 합니다. 첫째, CoCrMo 입자 높은 진공 3 전극 직류 여 되었고 50 mg/mL의 농도에서 인산 염 버퍼 염 (PBS)에서 resuspended. 다음, 후 분리 하 여 두개골과 날카로운 해 부 결과 서 스 펜 션의 50 µ L murine calvaria의 중간에 적용 되었습니다. 쥐 2 주 후, 희생 했다 및 calvaria 샘플 수확 했다; 질적 및 양적 분석 H & E andmicro-코네티컷 얼룩에 의해 수행 되었다
CoCrMo 입자에 노출에 의해 건설 murine calvarial osteolysis 모델 CoCrMo 입자와 대 식 세포, 섬유 아 세포, osteoblasts, osteoclasts에 무 균 완화 등 다양 한 세포 간의 상호 작용을 평가 하기 위한 이상적인 도구입니다.

<table border="0" cellpadding="0" cellspacing="0" width="684">
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">CoCrMo alloy from prosthesis</td>
			<td style="width:183px;">Waldemar Link GmbH &#38; Co</td>
			<td style="width:119px;">GEMINI MK II</td>
			<td style="width:167px;">Raw material to obtain CoCrMo nanoparticles</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Fabricated high-vacuum three-electrode direct current</td>
			<td style="width:183px;">College of Materials Science &#38; Engineering , Nanjing University of Technology</td>
			<td style="width:119px;"></td>
			<td>Self designed machine</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">6 week old male C57BL/6J mice</td>
			<td style="width:183px;">Model animal research center of Nanjing University</td>
			<td style="width:119px;">N000013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100% Ethanol</td>
			<td style="width:183px;">Nanjing Reagent</td>
			<td>C0691514023</td>
			<td>Solvent of CoCrMo nanoparticles for transmission electron microscope scanning</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1.5 ml Microcentrifuge tubes</td>
			<td style="width:183px;">Taizhou Weierkang Medical Supplies co., LTD</td>
			<td>W603</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Microanalytical balance</td>
			<td style="width:183px;">Shenzhen Qun long Instrument Equipment Co,. LTD</td>
			<td>EX125DZH</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultrasonic shaker</td>
			<td style="width:183px;">Shanghai Yuhao scientific instrument co., LTD</td>
			<td style="width:119px;">YH-200DH</td>
			<td>To suspend CoCrMo nanoparticles</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Transmission Electron Microscope</td>
			<td style="width:183px;">FEI</td>
			<td style="width:119px;">Tecnai G20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SimplePCI software</td>
			<td style="width:183px;">Compix Inc.</td>
			<td style="width:119px;">6.6 version</td>
			<td>To calculate the mean diameter and particle size distribution.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">High-handed sterilization pan</td>
			<td style="width:183px;">QIULONGYIQI</td>
			<td style="width:119px;">KYQL-100DS</td>
			<td>To decontaminate endotoxin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Limulus Amebocyte Lysate (LAL) Assay</td>
			<td style="width:183px;">Charles River</td>
			<td style="width:119px;">R13025</td>
			<td>To detect endotoxin&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 ml Microcentrifuge tubes</td>
			<td style="width:183px;">Taizhou Suyi Medical</td>
			<td style="width:119px;">B122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phosphate-buffered saline</td>
			<td style="width:183px;">Boster Biological Technology</td>
			<td style="width:119px;">AR0030</td>
			<td>Solvent of CoCrMo nanoparticles stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pentobarbital Sodium</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:119px;">P3761</td>
			<td>To anesthetize mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Normal saline</td>
			<td style="width:183px;">SACKLER</td>
			<td style="width:119px;">SR8572EP-15</td>
			<td>To prevent drying of mice eyes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">75% Ethanol</td>
			<td style="width:183px;">Nanjing Reagent</td>
			<td style="width:119px;">C0691560275</td>
			<td>Disinfection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Medical cotton ball</td>
			<td style="width:183px;">Shuitao</td>
			<td style="width:119px;">1278298933</td>
			<td>Disinfection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Shaver</td>
			<td style="width:183px;">Kemei</td>
			<td style="width:119px;">KM-3018</td>
			<td>To shave the fur</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scissor</td>
			<td style="width:183px;">RWD LIFE SCIENCE</td>
			<td style="width:119px;">S12005-10</td>
			<td>To incise skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Suture</td>
			<td style="width:183px;">RWD LIFE SCIENCE</td>
			<td style="width:119px;">F34001-01</td>
			<td>To suture skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Needle holder</td>
			<td style="width:183px;">RWD LIFE SCIENCE</td>
			<td style="width:119px;">F33001-01</td>
			<td>To suture skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Needle</td>
			<td style="width:183px;">RWD LIFE SCIENCE</td>
			<td style="width:119px;">R14003-12</td>
			<td>To suture skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vessel forceps</td>
			<td style="width:183px;">RWD LIFE SCIENCE</td>
			<td style="width:119px;">F22003-09</td>
			<td>To suture skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scalpel</td>
			<td style="width:183px;">RWD LIFE SCIENCE</td>
			<td style="width:119px;">S31010-01</td>
			<td>To harvest calvaria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tweezers</td>
			<td style="width:183px;">RWD LIFE SCIENCE</td>
			<td style="width:119px;">F12006-10</td>
			<td>To harvest calvaria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100 &#181;L pipettes</td>
			<td style="width:183px;">Eppendorf</td>
			<td style="width:119px;">3120000240</td>
			<td>To embed particles suspension in the calvatias</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100 &#181;L pipette tips</td>
			<td style="width:183px;">AXYGEN</td>
			<td style="width:119px;">T-200-Y</td>
			<td>To embed particles suspension in the calvatias</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">5 ml Microtubes</td>
			<td style="width:183px;">Taizhou Weierkang Medical Supplies co., LTD</td>
			<td>W621</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4% Paraformaldehyde</td>
			<td style="width:183px;">Servicebio</td>
			<td style="width:119px;">G1101</td>
			<td>Fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micro Computed Tomography&#160;</td>
			<td style="width:183px;">SkyScan</td>
			<td style="width:119px;">SkyScan1176</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethylene Diamine Tetraacetic Acid</td>
			<td style="width:183px;">Servicebio</td>
			<td style="width:119px;">G1105</td>
			<td>Decalcification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraffin</td>
			<td style="width:183px;">Servicebio</td>
			<td style="width:119px;">#0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraffin slicing machine</td>
			<td style="width:183px;">Leica</td>
			<td style="width:119px;">RM2125RTS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass slide</td>
			<td style="width:183px;">Servicebio</td>
			<td style="width:119px;">G6004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cover glass</td>
			<td style="width:183px;">Servicebio</td>
			<td style="width:119px;">200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HE staining kit</td>
			<td style="width:183px;">Servicebio</td>
			<td style="width:119px;">#1-5</td>
			<td>HE staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Light microscope</td>
			<td style="width:183px;">Nikon</td>
			<td style="width:119px;">E200</td>
			<td></td>
		</tr>
	</tbody>
</table>

construction and evaluation of a murine calvarial osteolysis model by exposure to cocrmo particles in aseptic loosening

마모 입자 유도 osteolysis 관절 교체 실패, 무 균 완화의 주요 원인 이지만 기본 메커니즘 불분명 남아. 검색 및 산발적 발생에 필요한 긴 후속 인 그것 병 인 ofparticle 유도 osteolysis 임상의 경우에서 평가 하기 위해 도전적 이다. 따라서, 최적의 동물 모델은 추가 연구 필요. CoCrMo 입자에 노출에 의해 설립 calvarial osteolysis murine 모델은 입자와 무 균 완화에 다양 한 세포 간의 상호 작용을 평가 하기 위한 효과적이 고 유효한 도구. 이 모델에서 CoCrMo 입자 처음 높은 진공 3 전극 직류 여 되었고 50 mg/mL의 농도에서 인산 염 버퍼 염 분에 resuspended. 다음, 후 분리 하 여 두개골과 날카로운 해 부 결과 서 스 펜 션의 50 µ L murine calvaria의 중간에 적용 되었습니다. 2 주 후, 생쥐 희생 했다, 및 calvaria 표본 수확 했다; 질적 및 양적 평가 되며 고 오신 얼룩 및 마이크로 컴퓨터 단층 촬영에 의해 수행 되었습니다. 이 모델의 장점 절차 단순, 뼈 손실의 양적 평가, osteolysis 개발, 잠재적인 사용 유전자 변형 또는 녹아웃 모델, 그리고 상대적으로 저렴 한 비용의 속도 포함 한다. 그러나,이 모델은 기계적인 힘 및 입자 무 균 완화의 만성 효과 평가 하는 데 사용할 수 없습니다. Calvarial osteolysis murine 모델 CoCrMo 입자에 노출에 의해 생성 된 마모 입자 및 다양 한 셀, 예를 들면, 대 식 세포, 섬유 아 세포, osteoblasts 및 osteoclasts, 무 균 완화에 사이 상호 작용을 평가 하기 위한 이상적인 도구입니다.

사내 생산된 nanoscale CoCrMo 입자 했다 약 50 nm (3.56의 표준 오차) 직경, 가장 (그림 2)에 의해 정량으로. 마우스 calvarias의 CoCrMo 입자, 동물에 노출 된 후 (n = 그룹 당 6) 또 다른 2 주 동안 유지 되었다. 2 주 이내에 calvarial 절 개는 완전 하 게 치유 하 고 봉합 떨어질 수 있습니다. 모든 로컬 감염 또는 뼈 손실 평가 영향을 없앤 5 월. 마우스 희생 후 calvaria 샘?...

Watch this Scientific Journal Video about Construction and Evaluation of a Murine Calvarial Osteolysis Model by Exposure to CoCrMo Particles in Aseptic Loosening at JoVE.com

Construction and Evaluation of a Murine Calvarial Osteolysis Model by Exposure to CoCrMo Particles in Aseptic Loosening

건설 및 무 균 완화에 CoCrMo 입자에 노출에 의해 Calvarial Osteolysis Murine 모델의 평가

이 원고는 마모 입자 및 무 균 완화에 다양 한 세포 간의 상호 작용을 평가 하기 위한 이상적인 동물 모델을 구성 하는 CoCrMo 입자에 노출에 의해 calvarial osteolysis murine 모델을 설명 합니다.

Wear particle-induced osteolysis is a major cause of aseptic loosening in arthroplasty failure, but the underlying mechanism remains unclear. Due to long ...

construction-evaluation-murine-calvarial-osteolysis-model-exposure-to

Research

JoVE Journal

Medicine

7.8K 조회수.  Nanjing University. 이 원고는 마모 입자 및 무 균 완화에 다양 한 세포 간의 상호 작용을 평가 하기 위한 이상적인 동물 모델을 구성 하는 CoCrMo 입자에 노출에 의해 calvarial osteolysis murine 모델을 설명 합니다.

비디오: Construction and Evaluation of a Murine Calvarial Osteolysis Model by Exposure to CoCrMo Particles in Aseptic Loosening

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

태아 알코올 노출의 Zebrafish 모델에서 Teratogenic 변경 사항 평가

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

연구

의학

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening6, angiogenesis7-9, growth factor secretion9-11, and muscle precursor cell activation12 are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see 13). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner14, offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. 
Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

신경근육학 전기적 자극에 의​​해 근육 트레이닝의 Murine 모델

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

지주막 하 출혈의 생쥐 모델

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

Background 
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered advancements to decrease the occurrence this injury.&#160;The current model demonstrates reproducible lower&#160;extremity paralysis following thoracic aortic occlusion.
Methods 
Adult male C57BL6 mice were anesthetized with isoflurane. Through a cervicosternal incision the aorta was exposed. The descending thoracic aorta and left subclavian arteries were identified without entrance into pleural space. Skeletonization of these arteries was followed by immediate closure (Sham) or occlusion for 4 min (moderate ischemia) or 8 min (prolonged ischemia). The sternotomy and skin were closed and the mouse was transferred to warming bed for recovery.&#160; Following recovery, functional analysis was obtained at 12 hr intervals until 48 hr.
Results 
Mice that underwent sham surgery showed no observable hind&#160;limb deficit. Mice subjected to moderate ischemia for 4 min had minimal functional deficit at 12 hr followed by progression to complete paralysis at 48 hr. Mice subjected to prolonged ischemia had an immediate paralysis with no observable hind-limb movement at any point in the postoperative period. There was no observed intraoperative or post&#160;operative mortality.
Conclusion 
Reproducible lower extremity paralysis whether immediate or delayed can be achieved in a murine model. Additionally, by using a median sternotomy and careful dissection, high survival rates, and reproducibility can be achieved.

The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.

척수 허혈 - 재관류의 생쥐 모델에서 흉부 대동맥 폐색에 의해 재생산 할 하반신 마비

Background
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

피하 종양 모델에서 종양 침투 백혈구 하위 집합의 평가

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

정량 실시간 PCR에 의해 마우스 유방 종양 모델에서 폐 전이의 평가

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

쥐 모델에서 Infrarenal 복부 대동맥 클램핑에 의해 재현 동맥 발가 벗김 부상

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Light Sheet Microscopy에 의한 심근 성 심근염의 Murine 모델에서의 백혈구 침윤의 정량적 시각화

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high incidence in aging populations. The current conventional therapies for COPD focus mainly on symptom-modifying drugs; thus, the development of new therapies is urgently needed. Qualified animal models of COPD could help to characterize the underlying mechanisms and can be used for new drug screening. Current COPD models, such as lipopolysaccharide (LPS) or the porcine pancreatic elastase (PPE)-induced emphysema model, generate COPD-like lesions in the lungs and airways but do not otherwise resemble the pathogenesis of human COPD. A cigarette smoke (CS)-induced model remains one of the most popular because it not only simulates COPD-like lesions in the respiratory system, but it is also based on one of the main hazardous materials that causes COPD in humans. However, the time-consuming and labor-intensive aspects of the CS-induced model dramatically limit its application in new drug screening. In this study, we successfully generated a new COPD model by exposing mice to high levels of ozone. This model demonstrated the following: 1) decreased forced expiratory volume 25, 50, and 75/forced vital capacity (FEV25/FVC, FEV50/FVC, and FEV75/FVC), indicating the deterioration of lung function; 2) enlarged lung alveoli, with lung parenchymal destruction; 3) reduced fatigue time and distance; and 4) increased inflammation. Taken together, these data demonstrate that the ozone exposure (OE) model is a reliable animal model that is similar to humans because ozone overexposure is one of the etiological factors of COPD. Additionally, it only took 6 - 8 weeks, based on our previous work, to create an OE model, whereas it requires 3 - 12 months to induce the cigarette smoke model, indicating that the OE model might be a good choice for COPD research.

This study describes the successful generation of a new chronic obstructive pulmonary disease (COPD) animal model by repeatedly exposing mice to high concentrations of ozone.

반복된 오존 노출에 의해 쥐에 만성 폐쇄성 폐 질환 모델의 생성

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

외과 마우스 모형에 있는 골관절염의 표준화한 조직 형측정 평가

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...