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

Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength and flexibility and allows it to fulfill its functions. Bone is continuously exposed to a variety of stimuli, which in pathological conditions can deregulate bone remodeling. To study bone biology and diseases and evaluate potential therapeutic agents, it has been necessary to develop in vitro and in vivo models.
This manuscript describes the dissection process and culture conditions of calvarias isolated from neonatal mice to study bone formation and the bone tumor microenvironment. In contrast to in vitro and in vivo models, this ex vivo model allows preservation of the three-dimensional environment of the tissue as well as the cellular diversity of the bone while culturing under defined conditions to simulate the desired microenvironment. Therefore, it is possible to investigate bone remodeling and its mechanisms, as well as the interactions with other cell types, such as the interactions between cancer cells and bone.
The assays reported here use calvarias from 5-7 day old BALB/C mice. The hemi-calvarias obtained are cultured in the presence of insulin, breast cancer cells (MDA-MB-231), or conditioned medium from breast cancer cell cultures. After analysis, it was established that insulin induced new bone formation, while cancer cells and their conditioned medium induced bone resorption. The calvarial model has been successfully used in basic and applied research to study bone development and cancer-induced bone diseases. Overall, it is an excellent option for an easy, informative, and low-cost assay.

The ex vivo culture of bone explants can be a valuable tool for the study of bone physiology and the potential evaluation of drugs in bone remodeling and bone diseases. The presented protocol describes the preparation and culture of calvarias isolated from newborn mice skulls, as well as its applications.

Calvaria 공동 배양 및 조직 학을 사용하여 뼈 리모델링 및 종양 - 뼈 미세 환경 재현

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength ...

연구

암 연구학

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

BMPR-IB +의 신속한 격리 두개골 결함 치유 모델에서 사용을위한 기질 세포를 지방이는 유래

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

발생학

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

Calvarial 중요 한 뼈 결함 수리 다 현미경 Vivo에서 유전자 변형 3D-PLGA/nHAp 비 계에 의해 신생 시각화

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

생체공학

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

마우스의 간단한 임계 크기의 대퇴 결함 모델

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

의학

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

마우스의 분절 대퇴 임계 크기 결함 모델의 설립은 플레이트 고정술에 의해 안정화

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

Endochondral 골절 표준화 된 Biomechanical 조건 하에서 쥐에서 치유를 분석 하는 최소 침 습 모델

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

Protocol for Developing a Femur Osteotomy Model in Wistar Albino Rats

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. Osteoanabolic drugs have the potential to augment the repair of fractures but have constraints like high costs or undesirable side effects. The bone healing potential of a drug can initially be determined by in vitro studies, but in vivo studies are needed for the final proof of concept. Our objective was to develop a femur osteotomy rodent model that could help researchers understand the development of callus formation following fracture of the shaft of the femur and that could help establish whether a potential drug has bone healing properties. Adult male Wistar albino rats were used after Institutional Animal Ethics Committee clearance. The rodents were anesthetized, and under aseptic conditions, complete transverse fractures at the middle one-third of the shafts of the femurs were created using open osteotomy. The fractures were reduced and internally fixed using intramedullary K-wires, and secondary fracture healing was allowed to take place. After surgery, intraperitoneal analgesics and antibiotics were given for 5 days. Sequential weekly x-rays assessed callus formation. The rats were sacrificed based on radiologically pre-determined time points, and the development of the fracture callus was analyzed radiologically and using immunohistochemistry.

Here, we present a protocol to iatrogenically fracture the shaft of the femur of Wistar albino rats and follow up on the development of the callus. This femur osteotomy model can help researchers evaluate the process of fracture healing and to study how a drug could influence fracture healing.

Wistar 알비노 쥐에서 대퇴골 절골술 모델을 개발하기 위한 프로토콜

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. ...

쥐 대퇴골 상자의 확립 및 평가: 공동 결함 모델

Creating a Box-Cavity Defect Model in the Cortical Bone of Rat Femora

Severe bone defects or complex fractures can result in serious complications such as nonunion or insufficient bone healing. Tissue engineering, which involves the application of cells, scaffolds, and cytokines, is considered a promising solution for bone regeneration. Consequently, various animal models that simulate bone defects play a crucial role in exploring the therapeutic potential of tissue engineering for bone healing. In this study, we established a box-shaped cortical bone defect model in the mid-femur of rats, which could serve as an ideal model for assessing the function of biomaterials in promoting bone healing. This box-shaped cortical bone defect was drilled using an oral low-speed handpiece and shaped by a lathe needle. Post-operative micro-CT analysis was immediately conducted to confirm the successful establishment of the box-cavity cortical bone defect. The femurs on the operated side of the rats were then harvested at multiple time points post-surgery (0 days, 2 weeks, 4 weeks, and 6 weeks). The healing process of each sample&#39;s defect area was evaluated using micro-CT, hematoxylin and eosin (H&#38;E) staining, and Masson trichrome staining. These results demonstrated a healing pattern consistent with intramembranous ossification, with healing essentially complete by 6 weeks. The categorization of this animal model&#39;s healing process provides an effective in vivo method for investigating novel biomaterials and drugs that target intramembranous ossification during bone tissue defect healing.

저자 스포트라이트: 생체 재료 및 뼈 재생의 향상된 평가를 위한 Box-Cavity Cortical Approach

Here, we present a protocol for creating a box-cavity defect in rat femoral diaphysis tissue. This model can assess biomaterial performance under biomechanical stress and explore mechanisms of bone regeneration related to intramembranous osteogenesis.

Rat Fetora의 피질 뼈에서 Box-Cavity Defect 모델 생성

Severe bone defects or complex fractures can result in serious complications such as nonunion or insufficient bone healing. Tissue engineering, which ...

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

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