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 DNAの1。流体力学的送達<ol><li>マウス尾静脈を介して流体力学的送達<ol><li>尾静脈注射の前に、マウスを秤量する。リンゲル液中の希釈sRANKL又は緑色蛍光タンパク質（GFP）MC（37℃で予備加温）マウスの体重の約10％の総体積。 </li><li>血管を拡張し、横方向の静脈LVS（）が表示されるようにするために、注射の前に10分間ケージにマウスを温める。脱水と高体温を避けるために、慎重にマウスを監視します。 </li><li>とすぐのLVが拡張したと表示されているように、制止にマウスを移動し、動きを拘束するバレルに十分プラグを挿入します。このような呼吸などの正常な活動のためのマウスを監視します。必要に応じて、拘束具のプラグを調整します。 </li><li>拭いてアルコールで尾の先端から注入領域3月4日を消毒。マウス尾静脈注射用の27〜30 Gの針を使用してください。片手でしっかりと尾を持ち、針を挿入尾静脈へ。注射器に圧力を適用し、5〜7秒以内に注入を完了します。 </li><li>尾静脈から針を外し、出血を止めるために注射部位にコットンボールに圧力をかける。呼吸数は、通常の速度に低下した後、バックビバリウムにマウスを移動して、15〜30分間マウスを監視します。 </li></ol></li><li>マウス血清TRACP5bはポスト遺伝子導入の全身マウスsRANKLの発現および定量の確認<ol><li>血管を拡張と論理ボリュームが見えるようにするために5〜10分間ケージにマウスを温める。脱水と高体温を避けるために、慎重にマウスを監視します。 </li><li>尾の先端からサイト1項のマウスの尾の刃で小さな切開を行います。 </li><li>血清分離チューブ内の血液の約100μLを収集します。 </li><li> 30分間室温で血清分離管をインキュベートする。 </li><li> 5分間万×gで遠心分離サンプルと血清を収集します。 STORさらなる分析のために-80℃でのE血清。 </li><li>市販のキットを用いて血清サンプルにsRANKL ELISAおよびマウス血清TRACP5bはアッセイを行う。 </li></ol></li></ol> 2マウス BMMSからin vitro破骨細胞世代で <ol><li> BMMSの単離​​と培養<ol><li>脛骨および大腿骨の骨の単離：CO 2をドナーマウスを安楽死させると、70％エタノールで足を消毒。無傷で大腿骨と脛骨の骨を除去するために、踵骨の骨に恥骨から解剖。 </li><li>骨を損傷することなく、慎重に皮膚や筋肉を取り外します。リン酸緩衝生理食塩水（PBS）を含有するペトリ皿に処理し、大腿骨と脛骨を置く。 </li><li>骨髄の除去：1％ペニシリン/ストレプトマイシンを含有するα-MEMを装填し、大腿骨と脛骨の骨の一方の側に先端をカットし、25 G針を1ミリリットル注射器を用いて50ミリリットルチューブに骨髄を洗い流す（破骨細胞培養培地/ OCM）。 </li><lI&gt;プロセス骨髄：70ミクロンのナイロンセルストレーナーを通過させることにより、新しい50ミリリットルチューブに骨髄懸濁物を混合し、転送します。 300×gで遠心細胞及び1％ペニシリン/ストレプトマイシン及び10％FBS（破骨細胞培養培地/ OCM（+））を含有するα-MEM中に再懸濁する。 </li></ol></li><li>マクロファージの文化<ol><li>血球計または自動セルカウンターを用いて細胞を数える。 </li><li>プレート6ウェルプレート、ウェルあたり1×10 6個の細胞または96ウェルプレートに入れ、ガラス製カバースリップ（5mm）を又は象牙質切片に3×10 5個の細胞を、37℃でインキュベートする。 </li><li>吸引物のすべてのメディアは、非接着細胞を含有し、48時間、25 ng / mlのマウスM-CSFを含有する新鮮なOCM（+）37℃で細胞をインキュベートする。 </li><li>吸引物は、すべてのメディアと新鮮なOCM（+）メディアは、破骨細胞形成を開始する25 ng / mlのマウスのM-CSF、および30 ng / mLのマウスsRANKLを含む、37℃で細胞を培養する。必要に応じて、3日おきにメディアを補充する。 </li><li>細胞を育てる約6〜8日間、骨を再吸収することのできる巨大な多核細胞を形成する。とすぐに多核細胞が出現するように、市販のキットを用いて染色し、TRAP（酒石酸抵抗性酸性ホスファターゼ）を行い、完全に成熟したときに、機能的アッセイを行う。ファロイジン染色および画像先に説明したように19、走査電子顕微鏡（SEM）によってスライスを象牙質に付着細胞を使用してカバースリップ上でF-アクチンを可視化リング。 </li></ol></li></ol>ヒトPBMCから3。 インビトロ破骨細胞の生成<ol><li>ヒトPBMCを単離<ol><li> 50ミリリットルチューブに予め温めヒスト-1077の10ミリリットルを追加します。 </li><li>空の白血球フィルターは50mlのチューブ（PBSで血液を1:1の比）中に滅菌PBSで血液バンクから得た。血がヒストと混合しないことを確認しながら、非常にゆっくりとヒストソリューションより希釈した血液をレイヤー。 </li><li> 18℃で20分間1000×gで遠心分離サンプルフォロー遠心ING、慎重に白血球および新しい50mlチューブへの転送を含む白色バフィーコート層を単離する。 </li><li>細胞を回収し、5分間650×gで再度遠心PBSで細胞を希釈する。 </li><li>メディアや細胞カウンターを用いて細胞を数える - （）OCMで上清と再懸濁細胞を除去する。 </li></ol></li><li>メッキおよび培養したPBMC <ol><li> OCMにおける細胞（+）培地およびプレート8×10 6細胞/ ml当たり6ウェルプレートまたは1×10 6細胞/ ml当たりに96ウェルプレートにウェル再懸濁する。免疫蛍光イメージングのため、ならびに骨吸収アッセイに適した骨切片上のカバーガラス（5mm）をプレート上の細胞を、必要に応じて以前に20に記載のように。 </li><li> 2〜3日毎に又は必要に応じてヒトM-CSFで細胞を補充変化培養培地。その後、OCMにおける25 ng / mlのヒトM-CSF（+）と一緒に、人間のsRANKLの30 ng / mlのを追加して5日目の分化のステップに進んでosteoclastogenesを開始するメディアです。 </li><li>巨大多核細胞を形成するために約14〜21日間、細胞を増殖させる。できるだけ早く多核細胞が出現ように、これらの細胞は次いで、TRAPのために標識することができ、完全に成熟したときに、機能的アッセイを行うことができる。 F-アクチンリングはファロイジン染色を用いてカバーガラス上で可視化することができる。細胞形態は、SEMにより象牙質切片に付着した細胞において試験することができる。 </li></ol></li></ol>

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The authors have nothing to disclose.

筋骨格条件は、罹患率と障害の原因をリードしているし、150以上の疾患や症候群で構成されます。今日は約90万人のアメリカ人に影響を与える。関節の炎症や骨の破壊性関節炎や骨粗しょう症などの筋骨格症状の主な機能です。骨粗しょう症は、多くの場合、骨の骨折につながる骨の整合性を弱める条件である。関節炎は腫れ、圧痛およびスティッフ制限通常の移動となり、障害につながる?...

筋骨格系疾患は、米国および国や地域の保健システムの1のために存在深刻な結果に何百万人もの人々に影響を与えます。これらの疾患は、骨の損失および広範な治療および回復の長い期間を必要とする関節機能によって特徴付けられる。一般に、数および/ ​​または骨粗鬆症および関節炎では、骨を吸収する破骨細胞特化し、細胞の活性の相対的増加が観察される2。生理学的条件下で破骨細胞の数および活性は、骨芽細胞により産生される核因子κ-Bリガンド（RANKL）の受容体活性化によって調節される。オステオプロテゲリン（OPG）、RANKLのためのデコイ受容体はまた、骨芽3によって生成される全身sRANKLの過剰発現、又はOPGの欠失を含むインビボ動物モデルは、骨粗しょう症の研究に非常に有用である。;しかしながら、これらの方法は、トランスジェニックマウス、4,5の発生を必要とする。ここでは、新たな代替筋骨格関連疾患の研究のためのsRANKLを過剰発現させる方法が記載されている。具体的には、ミニサークル（MC）DNA技術および流体力学的送達方法は、インビボで sRANKLの遺伝子導入を達成し、全身6マウスsRANKLを過剰発現するために使用した。 この方法はまた、低カルシウム食8により卵巣切除7および食事介入後の破骨細胞のホルモンの変調のような骨粗鬆症の他のin vivoモデルに対して相補的である。これらのモデルは、しかし、彼らは外科手術を必要とし、大幅なコスト9で、数ヶ月かかることがあり筋骨格関連障害のさまざまな側面を研究することは非常に便利です。卵巣摘出（OVX）げっ歯類モデルは卵巣の除去は、それによって人間の閉経後骨粗しょう症10を模倣するエストロゲン欠乏につながる実験動物モデルである。人間の閉経後骨粗しょう症、条件エストロゲン欠乏症encyは骨折のリスクの増加につながり、骨粗しょう症は、米国だけで約8万人の女性に影響を与えます。 OVXモデルは、閉経後の骨粗しょう症のために有用であるが、それは一般的に骨粗しょう症の研究に限定された利点を提供しています。エストロゲンは、したがって、その非存在下で破骨細胞活性の増加が観察される10〜12、破骨細胞を誘導し、骨芽細胞のアポトーシスを阻害することによって、骨損失を抑制する。骨吸収に有利RANKL-OPG比の不均衡13も観察される。しかしながら、 インビボでのエストロゲン欠乏はまた、を伴う成長因子β（TGFのβ）のレベルを減少させ、インターロイキン-7（IL-7）、TNF、IL-1およびIL-6 14,15が増加した。これらのサイトカインは、RANKL経路の骨リモデリングの調節機能が独立した知っていたとして、それは単に、RANKL-RANK軸に任意の破骨細胞活性化を帰属させることは不可能である。このホワイトペーパーで説明されたモデルは、VIVに留学する研究者を可能にしますO前炎症性サイトカインのない破骨細胞形成および骨量の減少におけるRANKL-RANK軸は、OVX齧歯類モデルと比較。 また、in vitroでの破骨細胞形成技術は、筋骨格疾患の潜在的な治療的処置のために、破骨細胞の活性化を研究するために不可欠なツールです。以前の研究は、マウスのマクロファージコロニー刺激因子（M-CSF）でマウス骨髄由来マクロファージ（BMMS）を培養し、マウスsRANKLが破骨細胞分化3,16,17につながることが示されている。ここで、マウスの骨髄から、ならびにインビトロ18ヒト末梢血単核細胞（PBMC）からの多核破骨細胞様細胞を生成するためのプロトコルが記載されている。成熟した終末分化して完全に機能する破骨細胞を定義するために必要な、細胞ベースのアッセイも簡単に説明している。これらのインビトロ技術は、生体内のアプローチで小説を補完し、一緒にPとして機能owerful調査ツールは、破骨細胞の分化および活性化を研究する。これらのシステムを使用して、科学者は、 インビボおよびインビトロで破骨細胞を生成し、それらの増殖および活性化に必要な刺激および信号を定義するだけでなく、薬理学的および生物学的阻害剤の有効性を試験することができる。

<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.

ここで、破骨細胞形成に対するサイトカインの効果を研究するための方法として、in vitroで破骨細胞への前駆細胞を分化するためのインビボおよび細胞培養プロトコルにおける破骨細胞の分化のための新規な遺伝子導入技術が記載されている。 図1では、マウスにおけるGFPおよびマウスsRANKL MCの成功した遺伝子導入の代表的な結果を示す。 図2にお...

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.

小説<em&gt; in vivoで</em&gt;遺伝子導入技術と<em&gt; in vitroで</em筋骨格障害における骨損失の検討のための&gt;細胞ベースのアッセイ

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

a-novel-vivo-gene-transfer-technique-vitro-cell-based-assays-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

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

11.6K ビュー.  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. 

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

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.

薬物治療と<em&gt;インビボ</em&gt;メダカ骨粗鬆症モデルにおける骨芽細胞破骨細胞の相互作用のイメージング

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

発生生物学

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.

骨転移モデル

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

医学

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.

シーケンシャル<em&gt;インビボ</em&gt;骨折修復中に、骨形成幹細胞/前駆細胞のイメージング

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

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

合わせた<em&gt;インビボ</em&gt;光とμCTイメージングマウスにおける整形外科インプラント感染で感染症、炎症、および骨解剖学を監視する

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

の応用<em&gt;インビボ</em組織工学骨格筋の修復を評価するためのラット前脛骨の&gt;機能テスト

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

生物工学

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.

以下のための大腿骨のウィンドウチャンバーモデル<em&gt;インビボ</emマウス骨髄内&gt;細胞トラッキング

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

免疫・感染学

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.

半自動縦マイクロ トモグラフィーを用いた定量的モデルの構造解析、裸ラット骨粗鬆症による椎体骨折

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

A Lab-On-A-Chip Platform for Stimulating Osteocyte Mechanotransduction and Analyzing Functional Outcomes of Bone Remodeling

Bone remodeling is a tightly regulated process that is required for skeletal growth and repair as well as adapting to changes in the mechanical environment. During this process, mechanosensitive osteocytes regulate the opposing responses between the catabolic osteoclasts and anabolic osteoblasts. To better understand the highly intricate signaling pathways that regulate this process, our lab has developed a foundationary lab-on-a-chip (LOC) platform for analyzing functional outcomes (formation and resorption) of bone remodeling within a small scale system. As bone remodeling is a lengthy process that occurs on the order of weeks to months, we developed long-term cell culturing protocols within the system. Osteoblasts and osteoclasts were grown on functional activity substrates within the LOC and maintained for up to seven weeks. Afterward, chips were disassembled to allow for the quantification of bone formation and resorption. Additionally, we have designed a 3D printed mechanical loading device that pairs with the LOC platform and can be used to induce osteocyte mechanotransduction by deforming the cellular matrix. We have optimized cell culturing protocols for osteocytes, osteoblasts, and osteoclasts within the LOC platform and have addressed concerns of sterility and cytotoxicity. Here, we present the protocols for fabricating and sterilizing the LOC, seeding cells on functional substrates, inducing mechanical load, and disassembling the LOC to quantify endpoint results. We believe that these techniques lay the groundwork for developing a true organ-on-a-chip for bone remodeling.

Here, we present protocols for analyzing bone remodeling within a lab-on-a-chip platform. A 3D printed mechanical loading device can be paired with the platform to induce osteocyte mechanostransduction by deforming the cellular matrix. The platform can also be used to quantify bone remodeling functional outcomes from osteoclasts and osteoblasts (resorption/formation).

骨造形メカノダクタ化を刺激し、骨改造の機能的成果を解析するためのラボオンチッププラットフォーム

Bone remodeling is a tightly regulated process that is required for skeletal growth and repair as well as adapting to changes in the mechanical ...

<meta charset="utf8"></head><body>放射線照射マウスにおける効果的な造血幹細胞移植のための骨オルガノイド由来細胞

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>治療用造血幹細胞/前駆細胞を回収するためのin vivoオステオオルガノイドアプローチ

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.

治療用造血幹細胞/前駆細胞を回収するためのin vivoオステオオルガノイドアプローチ

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

小説

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薬物治療と

骨転移モデル

シーケンシャル

合わせた

の応用

以下のための大腿骨のウィンドウチャンバーモデル

半自動縦マイクロトモグラフィーを用いた定量的モデルの構造解析、裸ラット骨粗鬆症による椎体骨折

骨造形メカノダクタ化を刺激し、骨改造の機能的成果を解析するためのラボオンチッププラットフォーム

治療用造血幹細胞/前駆細胞を回収するためのin vivoオステオオルガノイドアプローチ

治療用造血幹細胞/前駆細胞を回収するためのin vivoオステオオルガノイドアプローチ

小説

要約

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薬物治療と

骨転移モデル

シーケンシャル

合わせた

の応用

以下のための大腿骨のウィンドウチャンバーモデル

半自動縦マイクロ トモグラフィーを用いた定量的モデルの構造解析、裸ラット骨粗鬆症による椎体骨折

骨造形メカノダクタ化を刺激し、骨改造の機能的成果を解析するためのラボオンチッププラットフォーム

治療用造血幹細胞/前駆細胞を回収するためのin vivoオステオオルガノイドアプローチ

治療用造血幹細胞/前駆細胞を回収するためのin vivoオステオオルガノイドアプローチ

半自動縦マイクロトモグラフィーを用いた定量的モデルの構造解析、裸ラット骨粗鬆症による椎体骨折