Name: a novel in vivo gene transfer technique and in vitro cell based assays for the study of bone loss in musculoskeletal disorders
Uploaded: 2014-06-08T23:30:00.000+00:00
Duration: 11 min 47 s
Description: Watch this Scientific Journal Video about A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders at JoVE.com

Research was partly supported by NIH research grants R01 AR062173 and SHC 250862 to IEA. ES is the recipient of NIH T32 CTSC predoctoral fellowship.

sRANKL MC DNA의 1. 유체 역학 배달 <ol><li> 마우스의 꼬리 정맥을 통해 유체 역학 배달 <ol><li> 꼬리 정맥 주입하기 전에 마우스의 무게를. 마우스의 체중의 10 % ~의 총 부피에 sRANKL 또는 링거액에 녹색 형광 단백질 (GFP) MC (미리 따뜻한 37 ° C에서)를 희석. </li><li> 혈관을 팽창하고 눈에 보이는 측면 혈관 (정맥 주사)를 확인하기 위해 주사 전에 10 분 동안 새장에 마우스를 따뜻하게. 탈수와 고열을 피하기 위해 신중하게 마우스를 모니터링합니다. </li><li> 즉시 정맥 주사가 넓혀진 눈에 보이는대로, 제한 수단에 마우스를 전송하고 이동을 억제하는 통에 충분히 플러그를 삽입합니다. 같은 호흡으로 정상 활동에 마우스를 모니터링합니다. 필요한 경우 제한 기의 플러그를 조정합니다. </li><li> 와이프 주류로 꼬리의 끝으로부터 주입 영역 3 / 4을 소독. 마우스 꼬리 정맥 주입을 위해 27 ~ ​​30 G 바늘을 사용합니다. 한 손으로 단단히 꼬리를 잡고있는 바늘을 삽입꼬리 정맥에. 주사기에 압력을 적용하고 5-7 초 내에 주입을 완료합니다. </li><li> 꼬리 정맥에서 바늘을 제거하고 출혈을 멈추게하는 주사 부위에 면봉으로 압력을 적용합니다. 호흡 속도가 정상 속도로 감소되면 다음 동식물 사육장에 다시 마우스를 전송, 15 ~ 30 분 동안 마우스를 모니터링합니다. </li></ol></li><li> 마우스 혈청 TRACP5b 포스트 유전자 전송의 전신 마우스 sRANKL 표현과 정량화의 확인 <ol><li> 혈관을 팽창하고 정맥 주사를 볼 수 있도록하기 위해 5 ~ 10 분간의 장에 마우스를 따뜻하게. 탈수와 고열을 피하기 위해 신중하게 마우스를 모니터링합니다. </li><li> 꼬리의 끝에서 앞으로 사이트 한 곳에서 마우스의 꼬리에 잎을 가진 작은 절개를합니다. </li><li> 혈청 분리 튜브에 혈액의 약 100 μl를 수집합니다. </li><li> 실온에서 30 분 동안 혈청 분리 튜브를 배양한다. </li><li> 원심 분리기 5 분 10,000 XG에 샘플 및 혈청을 수집합니다. STOR추가 분석을 위해 -80 ° C에서 E 세럼. </li><li> 상용 가능한 키트를 사용하여 혈청 샘플에 sRANKL ELISA와 마우스 혈청 TRACP5b 분석을 수행합니다. </li></ol></li></ol> 마우스 BMMs 2. 체외 파골 세포 생성 <ol><li> 분리 및 BMMs의 배양 <ol><li> 경골과 대퇴골 뼈의 분리 : CO 2 기증자 마우스를 안락사, 70 % 에탄올로 다리를 소독. 그대로 대퇴골과 경골 뼈를 제거하기 위해, 종골 뼈에 치골에서 해부. </li><li> 뼈를 손상시키지 않고 조심스럽게 피부와 근육을 제거합니다. 인산염 완충 생리 식염수 (PBS)를 포함하는 배양 접시에서 처리 대퇴골과 경골 뼈를 놓습니다. </li><li> 골수 제거 : 대퇴골과 경골 뼈의 한쪽 끝을 잘라 및 α-MEM은 1 % 페니실린 / 스트렙토 마이신을 포함하는로드 25 G 바늘을 1 ㎖ 주사기를 사용하여 50 ㎖ 튜브에 골수를 플러시 (파골 세포 배양액 / OCM). </li><lI&gt; 프로세스 골수 : 혼합 및 70 μm의 나일론 셀 스트레이너를 통해 전달하여 새로운 50 ㎖ 튜브에 골수 현탁액을 전송합니다. 원심 분리기 300 XG와의 세포를 1 % 페니실린 / 스트렙토 마이신과 10 % FBS (파골 세포 배양액 / OCM (+))를 포함하는 α-MEM에 다시 일시 중지합니다. </li></ol></li><li> 대 식세포의 문화 <ol><li> 혈구 또는 자동 세포 계수기를 사용하여 세포를 계산합니다. </li><li> 96 - 웰 플레이트에 넣고 37 ℃에서 부화 유리 커버 슬립 (5 ㎜) 또는 상아질 조각에 6 잘 판 3 × 10 5 세포를 잘 당 플레이트 1 X 10 6 세포 </li><li> 기음 비 부착 세포를 포함하는 48 시간 동안 25 NG / ML 마우스 M-CSF를 포함하는 신선한 OCM (+)와 37 ° C에서 세포를 품어 모든 미디어. </li><li> 25 NG / ML 마우스 M-CSF 및 파골 세포를 시작하는 30 NG / ML 마우스 sRANKL를 포함 기음 모든 미디어와 신선한 OCM으로 37 ° C에서 세포를 배양 (+) 미디어. 필요에 따라 매 3 일마다 미디어를 보충. </li><li> 세포 성장약 6~8일 뼈를 resorbing 할 수있는 거대한 멀티 핵 세포를 형성하는 데 필요한 권한입니다. 즉시 다핵 세포가 나타날 때, 상용 가능한 키트를 사용하여 염색 TRAP (주석 산염 저항하는 산성 인산 가수 분해 효소)를 수행하고, 완전히 성숙 할 때, 기능 분석을 수행합니다. phalloidin도 스테인 및 이미지 (19) 이전에 설명한 바와 같이 주사 전자 현미경 (SEM)에 의해 슬라이스 상아질 부착 세포를 이용하여 커버 슬립에 F-액틴 링 시각화. </li></ol></li></ol> 인간 PBMC를 3. 체외 파골 세포 생성 <ol><li> 인간 PBMC를의 분리 <ol><li> 50 ㎖ 튜브에 미리 예열 Histopaque-1077의 10 ML을 추가합니다. </li><li> 빈 백혈구 필터는 50 ML 튜브 (PBS와 혈액의 1:1 비율)로 멸균 PBS와 혈액 은행에서 얻을. 혈액이 histopaque와 혼합하지 않도록하고, 아주 천천히 histopaque 용액에 희석 된 혈액을 레이어. </li><li> 18 ° C에서 20 분 1000 XG에 원심 분리기 샘플 따라원심 분리를하여 보내고, 정중 백혈구 함유 백색 버피 코트 층을 분리하고 50 ㎖의 새로운 튜브로 옮긴다. </li><li> 세포를 수집하는 5 분 650 XG에 다시 PBS와 원심 분리기로 세포를 희석. </li><li> 미디어 및 셀 카운터를 사용하여 세포를 계산 (-) OCM에 뜨는에 resuspend 세포를 제거합니다. </li></ol></li><li> PBMC를 도금 및 배양 <ol><li> OCM에있는 세포를 재현 탁 (+) 미디어 판 (8) × 10 / ㎖ 당 잘 6 잘 플레이트 또는 1 X 10 6 세포 / ㎖ 당 잘 96 - 웰 플레이트에있는 6 개의 세포. 로 면역 형광 이미징뿐만 아니라 골 흡수 분석에 적합한 뼈 조각에 대한 필요에 따라 유리 커버 슬립 (5 ㎜)에 플레이트 세포는 이전에 (20)을 설명했다. </li><li> 변경 배양액 인간 M-CSF 2-3 일마다 또는 필요에 따라 세포를 보충. 그런 osteoclastogenes을 시작 OCM (+) 미디어 25 NG / ML 인간의 M-CSF와 함께 인간의 sRANKL 30 겨 / ㎖를 추가하여 5 일에 분화 단계로 진행이다. </li><li> 거대한 멀티 핵 세포를 형성하는 데 약 14-21일에 세포를 성장. 마자 다핵 세포가 나타나는 바와 같이, 이들 세포는 TRAP 표지 할 수 있고, 완전히 성숙 할 때, 기능적 분석법이 수행 될 수있다. F-액틴 링은 phalloidin도 얼룩을 사용하여 커버 슬립에 시각화 할 수 있습니다. 세포 형태는 SEM에 의해 조각을 상아질에 부착 된 세포에서 검사 할 수 있습니다. </li></ol></li></ol>

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M., 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=Tartrate-resistant+acid+phosphatase+5b:+a+novel+serum+marker+of+bone+resorption.">Tartrate-resistant acid phosphatase 5b: a novel serum marker of bone resorption.</a> Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 15, 1337-1345 (2000).</li><li>Adamopoulos, I. E., 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=IL-23+is+critical+for+induction+of+arthritis,+osteoclast+formation,+and+maintenance+of+bone+mass.">IL-23 is critical for induction of arthritis, osteoclast formation, and maintenance of bone mass.</a> J Immunol. 187, 951-959 (2011).</li><li>Suda, T., Takahashi, N., Martin, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+osteoclast+differentiation.">Modulation of osteoclast differentiation.</a> Endocrine reviews. 13, 66-80 (1992).</li><li>Takahashi, 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=Osteoblastic+cells+are+involved+in+osteoclast+formation.">Osteoblastic cells are involved in osteoclast formation.</a> Endocrinology. 123, 2600-2602 (1988).</li><li>Bradley, E. W., Oursler, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclast+culture+and+resorption+assays.">Osteoclast culture and resorption assays.</a> Methods Mol Biol. 455, 19-35 (2008).</li><li>Arai, F., 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=Commitment+and+differentiation+of+osteoclast+precursor+cells+by+the+sequential+expression+of+c-Fms+and+receptor+activator+of+nuclear+factor+kappaB+(RANK)+receptors.">Commitment and differentiation of osteoclast precursor cells by the sequential expression of c-Fms and receptor activator of nuclear factor kappaB (RANK) receptors.</a> The Journal of experimental medicine. 190, 1741-1754 (1999).</li><li>Fuller, K., 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=Macrophage+colony-stimulating+factor+stimulates+survival+and+chemotactic+behavior+in+isolated+osteoclasts.">Macrophage colony-stimulating factor stimulates survival and chemotactic behavior in isolated osteoclasts.</a> The Journal of experimental medicin. 178, 1733-1744 (1993).</li><li>Edwards, J. R., Mundy, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+osteoclast+biology:+old+findings+and+new+insights+from+mouse+models.">Advances in osteoclast biology: old findings and new insights from mouse models.</a> Nature reviews. Rheumatology. 7, 235-243 (2011).</li><li>Weinstein, R. 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=Promotion+of+osteoclast+survival+and+antagonism+of+bisphosphonate-induced+osteoclast+apoptosis+by+glucocorticoids.">Promotion of osteoclast survival and antagonism of bisphosphonate-induced osteoclast apoptosis by glucocorticoids.</a> The Journal of clinical investigation. 109, 1041-1048 (2002).</li></ol>

The authors have nothing to disclose.

근골격계 조건은 병적 상태와 장애의 원인을 선도하고 150 개 이상의 질병 및 증후군 구성되어 있습니다; 현재 약 90 만 명의 미국인에 영향을 미치는. 관절 염증 및 골 파괴는 관절염과 골다공증 등의 근골격 조건의 주된 기능이다. 골다공증은 주로 뼈의 골절로 이어지는 뼈의 무결성을 약화 조건이다. 관절염은 부어, 부드럽고 뻣뻣한 제한 정상적인 운동이되어 장애로 이어질 수있는 관절의 염증?...

근골격계 질환은 국가 및 지역 보건 시스템 1에 대한 미국과 현재의 심각한 결과에있는 사람들의 수백만에 영향을 미칩니다. 이 질환은 뼈의 손실과 광범위한 치료 및 회복의 오랜 기간을 필요로 관절 기능을 특징으로한다. 일반적으로, 파골 세포의 수 및 / 또는 활성의 상대적 증가는 세포가 골다공증, 뼈 재 흡수하도록 특수화 및 관절염이 관찰된다. 생리적 조건에서 파골 세포의 수와 활성을 골아 세포에 의해 생산되는 핵 인자 κ B-리간드 (RANKL)의 수용체 활성에 의해 조절된다. 게린 (OPG), RANKL에 대한 미끼 수용체는 조골 세포 (3)에 의해 생성되는 조직 sRANKL의 과발현 또는 OPG의 삭제를 포함하는 생체 내 동물 모델 골다공증 연구에 매우 유용합니다.; 그러나,이 방법은 형질 전환 마우스 4,5의 생성을 필요로합니다. 여기서, 새로운 대안근골격계 관련 질환의 연구 sRANKL를 과발현하는 방법이 설명된다. 특히, minicircle (MC) DNA 기술 및 유체 전달 방법은 생체 내에서 sRANKL의 유전자 전달을 달성하고 체계적으로 6 마우스 sRANKL를 과발현하는 데 사용되었습니다. 이 방법은 또한 낮은 칼슘 다이어트 (8)에 의해 난소 7식이 개입을 다음 파골 세포의 호르몬 변조 골다공증의 다른 생체 모델을 보완. 이 모델들은 수술 절차가 필요하고 상당한 비용 9에서 몇 달까지 걸릴 수 있습니다 그러나 근골격계 관련 질환의 다양한 측면을 연구하는 것은 매우 유용하다. 난소 절제 (OVX) 설치류 모델은 난소를 제거함으로써 인간의 폐경 후 골다공증 (10)을 흉내 낸 에스트로겐 결핍에 이르게 실험 동물 모델입니다. 인간의 폐경 후 골다공증, 에스트로겐 defici 조건ency은 골절의 위험이 증가로 연결 골다공증은 미국에서만 약 8 백만명의 여자에 영향을 미칩니다. OVX 모델은 폐경 후 골다공증에 유용하지만 그것은 일반적으로 골다공증을 연구하는 제한된 이점을 제공합니다. 에스트로겐 따라서 그것의 부재에서 증가 된 파골 세포 활성은 10-12을 관찰하고, 파골 세포 및 조골 세포 유도 세포 사멸을 억제함으로써, 골 손실을 억제한다. 골 흡수 호의 RANKL-OPG 비율 불균형도 13를 관찰된다. 성장 인자 β (TGF β의), 증가 된 인터루킨 12 (IL-12) 및 TNF, IL-1 및 IL-6 (14, 15)를 변형의 수준을 감소함으로써 그러나, 생체 내에서 에스트로겐 결핍도 수반한다. 이러한 사이토 카인은 RANKL 경로의 뼈 리모델링 조절 성 기능을 독립적 알고있다, 그것은 전적으로 RANKL-RANK 축에있는 파골 세포의 활성화를 속성 수 없다. 이 문서에 설명 된 모델은 비브에서 공부하는 연구자 수오 전 염증성 사이토 카인없이 파골 세포와 뼈 손실에 RANKL-RANK 축이 OVX 쥐 모델에 비해. 또한, 체외 파골 세포 기술은 근골격계 질환의 잠재적 인 치료 트리트먼트의 파골 세포의 활성을 연구하는 중요한 도구입니다. 이전 연구는 또한 마우스 대 식세포 집락 자극 인자 (M-CSF)와 마우스 sRANKL와 마우스 골수 유래 대 식세포 (BMMs)를 배양하는 파골 세포의 분화 3,16,17로 이어질 수있는 것으로 나타났습니다. 여기서, 마우스 골수로부터뿐만 아니라 체외 (18)에서 인간의 말초 혈 단핵 세포 (PBMC를)까지 다핵 파골 세포 - 유사 세포를 생성하는 프로토콜이 설명된다. 성숙한 말기 차별화 된 완벽한 기능 파골 세포를 정의하는 데 필요한 세포 기반 분석도 간략하게 설명되어 있습니다. 체외 기술은 생체 방식의 소설을 보완하고 함께 P 역할owerful 조사 도구는 파골 세포의 분화 및 활성화를 공부한다. 이러한 시스템을 사용하여, 과학자들은 생체 내 및 시험관 내에서 파골 세포 생성 및 증식 및 활성화에 필요한 자극 및 신호를 정의 할뿐만 아니라 약리학 적 및 생물학적 억제제의 효능을 시험 할 수있다.

<table><tbody><tr><td>alpha-MEM</td><td>Life Technologies&amp;nbsp;</td><td>12561-056</td><td></td></tr><tr><td>Human M-CSF</td><td>Miltenyi Biotec</td><td>130-096-492</td><td></td></tr><tr><td>Mouse M-CSF</td><td>Miltenyi Biotec</td><td>130-094-643</td><td></td></tr><tr><td>Human RANK-Ligand - soluble</td><td>Miltenyi Biotec</td><td>130-094-631</td><td></td></tr><tr><td>Mouse RANK-Ligand - soluble</td><td>Miltenyi Biotec</td><td>130-094-076</td><td></td></tr><tr><td>Tailveiner Restrainer for mice</td><td>Braintree</td><td>TV-150 STD</td><td></td></tr><tr><td>Mouse TRANCE/RANK L/TNFSF11 Quantikine ELISA Kit&amp;nbsp;</td><td>R&amp;amp;D systems</td><td>MTR00</td><td></td></tr><tr><td>Acid Phosphatase, Leukocyte (TRAP) Kit</td><td>Sigma</td><td>387A</td><td></td></tr><tr><td>MouseTRAP assay&amp;nbsp;</td><td>immunodiagnostic systems</td><td>SB-TR103</td><td></td></tr></tbody></table>

a novel in vivo gene transfer technique and in vitro cell based assays for the study of bone loss in musculoskeletal disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

여기서, 파골 세포에 대한 사이토 카인의 효과를 연구하기위한 방법으로 체외로 파골 전구 세포 분화에 대한 생체 내 및 세포 배양 프로토콜에서 파골 세포의 분화를위한 신규 한 유전자 전달 기술이 설명된다. 그림 1에서, 마우스에있는 GFP 마우스 sRANKL MC의 성공적인 유전자 전달의 대표적인 결과가 표시됩니다. 그림 2에서, 마우스의 골수 또는 시야 ?...

Watch this Scientific Journal Video about A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders at JoVE.com

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

소설<em&gt; 생체</em&gt; 유전자 전달 기술과<em&gt; 시험관</em근골격계 질환의 뼈 손실의 연구를위한&gt; 셀 기반 분석 실험

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

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A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

11.6K 조회수.  University of California, Davis. Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described. 

비디오: A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

소설을 사용하여 척수 상해 후에 마우스 뒷다리 뼈 손실의 세로 평가, 생체내에 방법론

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

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Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

약물 치료와<em&gt; 생체</em&gt;은 메다카 물고기 골다공증 모델에서 조골 파골 세포 상호 작용의 영상

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

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Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

뼈 전이의 모델

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

원발성 뼈 종양 및 뼈에 이식된 고형 종양을 통한 뼈 전이 모델링

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...

암 연구학

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

순차<em&gt; 생체 내</em&gt; 골절 치료시 골 형성 줄기 / 전구 세포의 이미징

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

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

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

대한 대퇴골 창 회의소 모델<em&gt; 생체</em쥐의 골수에서&gt; 셀 추적

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

면역학 및 감염병학

Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ages. Animal OVCF models are essential to the preclinical development of translational tissue engineering strategies. While a number of models currently exist, this protocol describes an optimized method for inducing multiple highly reproducible vertebral defects in a single nude rat. A novel longitudinal semiautomated microcomputed tomography (&#181;CT)-based quantitative structural analysis of the vertebral defects is also detailed. Briefly, rats were imaged at multiple time points post-op. The day 1 scan was reoriented to a standard position, and a standard volume of interest was defined. Subsequent &#181;CT scans of each rat were automatically registered to the day 1 scan so the same volume of interest was then analyzed to assess for new bone formation. This versatile approach can be adapted to a variety of other models where longitudinal imaging-based analysis could benefit from precise 3D semiautomated alignment. Taken together, this protocol describes a readily quantifiable and easily reproducible system for osteoporosis and bone research. The suggested protocol takes 4 months to induce osteoporosis in nude ovariectomized rats and between 2.7 and 4 h to generate, image, and analyze two vertebral defects, depending on tissue size and equipment.

The goal of this protocol is to generate a nude rat osteoporosis-related vertebral compression fracture model that can be longitudinally evaluated in vivo using a semiautomated microcomputed tomography-based quantitative structural analysis.

반자동 경도 Microcomputed Tomography 기반 정량적 구조 분석 누드 쥐 척추 골절 골다공증 관련 모델의

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ...

<meta charset="utf8"></head><body>방사선 조사된 마우스에서 효과적인 HSCT를 위한 골오가노이드 유래 세포

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an adequate source of HSPCs, we developed an in vivo osteo-organoid by implanting scaffolds loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) into an internal muscle pouch near the femur in mice. After 12 weeks of implantation, we retrieved the in vivo osteo-organoids and conducted flow cytometry analysis on HPSCs, revealing a significant presence of HSPC subsets within the in vivo osteo-organoids.
We then established a sublethal model of hematopoietic/immune system injury in mice through radiation and performed hematopoietic stem cell transplantation (HSCT) by injecting the extracted osteo-organoid-derived cells into the peripheral blood of radiated mice. The effect of hematopoietic recovery was evaluated through hematological, peripheral blood chimerism, and solid organ chimerism analyses. The results confirmed that in vivo osteo-organoid-derived cells can rapidly and efficiently reconstruct damaged peripheral and solid immune organs in irradiated mice. This approach holds potential as an alternative source of HSPCs for HSCT, offering benefits to a larger number of patients.

<meta charset="utf8"></head><body>치료용 조혈 줄기세포/전구세포를 채취하기 위한 In vivo Osteo-organoid 접근법

Here, we established in vivo osteo-organoids triggered by bone morphogenetic protein-2-loaded gelatin scaffolds to harvest therapeutic hematopoietic stem/progenitor cells for the reconstruction of a damaged hematopoietic and immune system. Overall, this approach can provide a promising cell source for cell therapies.

치료용 조혈 줄기세포/전구세포를 채취하기 위한 In vivo Osteo-organoid 접근법

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an ...

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뼈 전이의 모델

원발성 뼈 종양 및 뼈에 이식된 고형 종양을 통한 뼈 전이 모델링

순차

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

대한 대퇴골 창 회의소 모델

반자동 경도 Microcomputed Tomography 기반 정량적 구조 분석 누드 쥐 척추 골절 골다공증 관련 모델의

치료용 조혈 줄기세포/전구세포를 채취하기 위한 In vivo Osteo-organoid 접근법

치료용 조혈 줄기세포/전구세포를 채취하기 위한 In vivo Osteo-organoid 접근법