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.

中sRANKL MC DNA 1。水动力传递<ol><li>通过小鼠尾静脉流体力学交货<ol><li>尾静脉注射前称量鼠标。中的小鼠体重的10％〜的总体积稀释sRANKL或绿色荧光蛋白（GFP）的MC在林格氏溶液（预暖，在37℃）。 </li><li>热身鼠标在一个笼子10分钟前注射，以扩张血管，使侧脉（LVS）可见。监视鼠标小心，以避免脱水和高热。 </li><li>只要LV的被扩张，可见，鼠标转移到阻挡并将插头远远不够倒入桶内，以抑制运动。监视鼠标正常活动如呼吸。如果需要调整限制器的插头。 </li><li>从尾尖用酒精擦拭消毒注射区的3/4。使用27-30号针头的小鼠尾静脉注射。紧紧握住尾巴用一只手插入针到尾静脉。施加压力的注射器，并在5-7秒完成注射。 </li><li>从尾静脉中取出针头并用棉球将压力施加于注射部位止血。监视鼠标15-30分钟，再用鼠标转回动物饲养，一旦呼吸率降低到正常速度。 </li></ol></li><li>的小鼠血清TRACP5b的帖子基因转移系统性鼠标sRANKL的表达和定量确认<ol><li>热身鼠标在一个笼子里5-10分钟，以扩张血管，使的LV可见。监视鼠标小心，以避免脱水和高热。 </li><li>作一小切口用刀片将鼠标的尾部在一个站点1列从尾部的尖端。 </li><li>收集约100微升的血液中的血清分离管中。 </li><li>孵育血清分离管在室温下放置30分钟。 </li><li>以10,000×g离心5分钟，离心样品并收集血清。 STOR在-80°C，E无血清作进一步的分析。 </li><li>使用市售的试剂盒血清样本进行sRANKL ELISA和小鼠血清TRACP5b的检测。 </li></ol></li></ol> 2，体外破骨细胞生成的鼠标桥梁养护管理<ol><li>隔离和桥梁养护管理的培养<ol><li>胫骨和股骨骨隔离:安乐死的供体小鼠与CO 2，消毒腿用70％乙醇。从耻骨解剖到跟骨，以除去股骨和胫骨完好无损。 </li><li>小心地取出皮肤和肌肉不破坏骨。放置处理股骨和胫骨中含有磷酸盐缓冲盐水（PBS）的培养皿中。 </li><li>去除骨髓:切断对股骨和胫骨的骨骼的一侧的前端和冲洗出骨髓入50毫升的管中，使用1ml注射器用25号针头，装有含1％青霉素/链霉素的α-MEM （破骨细胞培养基/ OCM）。 </li><l我&gt;过程骨髓:混合并通过一个70微米尼龙细胞过滤器传递给它的骨髓悬浮液转移至新的50ml管中。离心将细胞以300×g离心并重新悬浮在α-MEM中含有1％青霉素/链霉素和10％胎牛血清（破骨细胞培养基/ OCM（+））。 </li></ol></li><li>巨噬细胞的培养<ol><li>使用血球或自动细胞计数器计数细胞。 </li><li>板1×10 6每孔细胞的6孔板或3×10 5细胞在玻璃盖玻片（5毫米）或牙本质片放置在96孔板中孵育在37℃下</li><li>吸出物含有非贴壁细胞和培养细胞在37℃下用含有25纳克/毫升鼠M-CSF 48小时新鲜OCM（+）的所有介质。 </li><li>含有25毫微克/毫升鼠标M-CSF和30毫微克/毫升小鼠sRANKL启动破骨细胞吸出所有媒体和培养细胞在37℃的新鲜OCM（+）的媒体。根据需要补充媒体每3天。 </li><li>细胞生长约6-8天以形成能够再吸收骨巨多核细胞。只要多核细胞出现，执行TRAP（抗酒石酸酸性磷酸酶），使用市售的试剂盒染色，而当完全成熟，进行功能分析。直观上用鬼笔环肽染色和图像附着用扫描电子显微镜（SEM）如先前19描述牙本质片的细胞的盖玻片的F-肌动蛋白环。 </li></ol></li></ol> 3， 在体外破骨细胞生成从人PBMC <ol><li>人外周血单个核细胞的分离<ol><li>加入10 ml预热的Histopaque-1077的入50毫升的试管中。 </li><li>从用无菌PBS血库获得到50ml试管（1:1比例的血用PBS）空白细胞过滤器。层中的稀释血液在HISTOPAQUE溶液非常缓慢，并确保血液不与HISTOPAQUE混合。 </li><li>在1000×g离心20分钟，离心样品在18°C。跟随ING离心，小心分离含有白细胞的白色血沉棕黄层和层转移到新的50ml管中。 </li><li>在650×g离心5分钟，再稀释细胞，用PBS离心以收集细胞。 </li><li>在OCM去除上清，重悬细胞（ - ）介质，并使用细胞计数器计数细胞。 </li></ol></li><li>电镀与外周血单个核细胞培养<ol><li>重悬的细胞在OCM（+）介质和板8×10 6个细胞/ ml，每孔中×10 6个细胞/ ml，每孔在96孔板的6孔板或1。根据需要为免疫荧光成像，以及对骨片适于骨吸收测定法作为在玻璃盖玻片（5毫米）的板的细胞先前所描述20。 </li><li>改变培养基，补充细胞与人M-CSF，每2-3天或按要求。然后通过添加人sRANKL的30纳克/毫升连同25毫微克/毫升的人M-CSF在OCM（+）媒体发起osteoclastogenes进行到第5天的分化步骤是。 </li><li>生长细胞为约14至21天，以形成巨型多核细胞。只要多核细胞出现，这些细胞可以被标记为TRAP，并且当完全成熟，功能分析可以被执行。 F-肌动蛋白环可以可视化使用鬼笔环肽染色盖玻片。细胞的形态，可以检查连接在用SEM对牙本质切片的细胞​​。 </li></ol></li></ol>

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The authors have nothing to disclose.

肌肉骨骼条件，导致发病率和致残的主要原因，并包含超过150个疾病和证候;影响大约90万美国人。关节的炎症和骨质破坏是骨骼肌肉条件的主要功能，包括关节炎和骨质疏松症。骨质疏松症是骨弱化完整性，常常导致骨的骨折的情况。关节炎是一种慢性的，衰弱的疾病，其特征是变得肿胀，招标和僵硬，限制正常运动，并可能导致残疾的关节发炎。虽然肌肉骨骼疾病的发病机制大大不同于彼此，?...

肌肉骨骼疾病影响到千百万人在美国和现在的严重后果为国家和地方卫生系统1。这些疾病的特点是骨损失和关节功能，需要广泛的治疗和恢复很长时间。通常，在破骨细胞的数量和/或活性的相对增加，细胞专门的重吸收骨骼，在骨质疏松和关节炎，观察2。在生理条件下的破骨细胞的数量和活性是通过核因子κ-B配体（RANKL），它是由成骨细胞产生的受体活化调节。护骨素（OPG），诱饵受体RANKL由成骨细胞3也产生涉及sRANKL全身过度表达，或删除OPG的体内动物模型是非常有价值的骨质疏松症的研究。然而，这些方法需要转基因小鼠4,5的产生。这里，一种新型的替代过表达sRANKL为肌肉骨骼相关的疾病的研究的方法进行说明。具体地，小环（MC）DNA技术和流体动力传递方法被用来实现sRANKL的体内基因转移和过表达小鼠sRANKL全身6。 这种方法也补充其他体内模型的骨质疏松，如由低钙饮食8以下卵巢切除7和饮食干预破骨细胞的激素调节。这些模型是研究的但是他们需要外科手术，可能需要长达数月，在一个显著的成本9肌肉骨骼相关疾病的不同方面非常有用。卵巢切除（OVX）啮齿动物模型的实验动物模型，其中卵巢切除导致雌激素缺乏，从而模仿人类绝经后骨质疏松症10。人绝经期后骨质疏松症，一个条件，其中的雌激素deficiENCY导致骨折的风险增加和骨质疏松症影响约800万妇女在美国就有。虽然绝经后骨质疏松症的去势模型是非常有用的它提供了有限的优势，在一般的研究骨质疏松症。雌激素可以抑制骨质流失，通过诱导破骨细胞，抑制成骨细胞凋亡，因此，在其没有增加破骨细胞活性观察10-12。一个RANKL-OPG比例失衡有利于骨吸收也观察到13。然而， 在体内的雌激素缺乏也伴随着降低的转化生长因子β（TGFβ），增加白细胞介素-7（IL-7）和肿瘤坏死因子，IL-1和IL-6的水平14,15。由于这些细胞因子已知的骨重塑调节功能独立于RANKL通路，这是不可能的属性的任何破骨细胞的活化，仅与RANKL-RANK轴。在本文中描述的模型使研究人员在VIV研究ØRANKL-RANK轴的破骨细胞和骨量丢失无促炎细胞因子相比，去卵巢啮齿类动物模型。 此外， 体外破骨细胞技术是必不可少的工具来研究破骨细胞活化肌肉骨骼疾病的潜在治疗方法。以前的研究还表明，与小鼠巨噬细胞集落刺激因子（M-CSF）和小鼠sRANKL培养小鼠骨髓衍生的巨噬细胞（桥梁养护）能导致破骨细胞分化3,16,17。在此，协议来生成从小鼠骨髓以及从人外周血单核细胞（PBMC） 在体外 18多核破骨细胞样细胞进行了描述。都定义一个成熟的终末分化和功能齐全的破骨细胞所需的基于细胞的测定也简单说明。这些体外技术补充体内的方法小说一起用于为powerful调查工具，研究破骨细胞分化和活化。使用这些系统中，科学家们能够产生在体内和体外的破骨细胞和确定所需的增殖和活化的刺激和信号，以及测试的药理和生物抑制剂的功效。

<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.

这里， 在体内和细胞培养协议破骨细胞分化的新基因转移技术，用于差分前体细胞在体外破骨细胞作为研究细胞因子对破骨细胞形成的影响的方法进行了描述。在图1中，绿色荧光蛋白和小鼠sRANKL MC小鼠成功的基因转移的代表性结果示。在图2中，示出的小鼠骨髓或前体细胞向破骨细胞通过明场显微术的人PBMC细胞分化时程文化的代表性图像， 图3?...

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.

一种新型的<em&gt;在体内</em&gt;基因转移技术和<em&gt;在体外</em&gt;细胞分析为肌肉骨骼疾病骨质流失的研究

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

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 次观看.  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. 

视频: 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.

鼠标使用新型脊髓损伤的后肢骨质流失的纵向评价，在体内，方法论

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

科研

医学

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.

戒毒治疗和<em&gt;在体内</em&gt;在青鳉鱼骨质疏松模型成骨破骨细胞相互作用的成像

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

发育生物学

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.

骨转移模型

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.

通过实体瘤移植植入骨模拟原发性骨肿瘤和骨转移

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

癌症研究

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.

顺序<em&gt;在体内</em&gt;成骨干/祖细胞在骨折修复的影像学

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.

多显微镜下的血管生成术在基因修饰的 3 d-PLGA/nHAp 支架上进行颅骨临界骨缺损修复

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

生物工程

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.

股骨窗口商会模型<em&gt;在体内</em在小鼠骨髓&gt;手机跟踪

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

免疫与感染

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.

半自动纵 Microcomputed Tomography-based 裸鼠骨质疏松症相关椎体骨折模型的定量结构分析

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>骨类器官衍生细胞在辐照小鼠中进行有效 HSCT

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>用于收获治疗性造血干细胞/祖细胞的体内骨类器官方法

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.

用于收获治疗性造血干细胞/祖细胞的体内骨类器官方法

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

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鼠标使用新型脊髓损伤的后肢骨质流失的纵向评价，在体内，方法论

戒毒治疗和

骨转移模型

通过实体瘤移植植入骨模拟原发性骨肿瘤和骨转移

顺序

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