Name: obtaining primary osteocytes through murine calvarial fractionation of gfp-expressing osteocytes
Uploaded: 2020-06-02T15:00:00.000+00:00
Duration: 7 min 22 s
Description: Watch this Scientific Journal Video about Obtaining Primary Osteocytes Through Murine Calvarial Fractionation of GFP-Expressing Osteocytes at JoVE.com

这项工作得到了日本科学促进会的JSPS KAKENHI资助（香港第19K10397号和I.M.第18K09862号）的部分支持。

所有动物程序和动物护理均按照东北大学的规章制度进行。
1.解剖新生dmp1-黄玉小鼠颅骨
<ol><li>对于该协议，使用 6\u20127 天龄的 C57BL/6-Tg（Dmp1-Topaz）1Ikal/J 小鼠幼崽。用5%异氟醚吸入对小鼠实施安乐死，然后将幼崽转移到70%乙醇中。</li>
	<li>将安乐死的幼犬转移到未经处理的培养皿中。</li>
	<li>用剪刀和镊子抓住头骨底部的皮肤并做一个切口。</li>
	<li>以第一个切口为起点，在耳朵前面颅骨的两侧切开，去除皮肤，露出颅骨。</li>
	<li>握住鼻梁的头部，沿着λ样缝合线切开颅骨。沿着顶骨的侧边缘切割，并将其延伸至包括额部。</li>
	<li>将颅骨与下面的脑组织分开。 注意:为确保清洁的骨性颅骨和最少的软组织附着，请勿深入骨骼;作为参考，人们应该能够看到骨头下面剪刀的影子。</li>
	<li>将钙质转移到磷酸盐缓冲盐水中（PBS，整个方案中的pH 7.4）。确保凹面朝上。</li>
</ol>2.新生小鼠颅骨的分馏
<ol><li>将多达 5 个颅骨转移到含有 5 mL 2 mg/mL 胶原酶溶液的 50 mL 锥形管中。使用前准备的新鲜胶原酶溶液。<ol><li>要制备新鲜的胶原酶溶液，将胶原酶粉末加入分离缓冲液（70 mM NaCl、10 mM NaHCO 3、60 mM 山梨糖醇、3 mMK2 HPO4、1 mM CaCl 2、1 mg/mL 牛血清白蛋白 （BSA）、5 mg/mL 葡萄糖和 25 mM HEPES）中，浓度为2 mg/mL。将胶原酶粉末溶解在磁力搅拌器上。</li>
		<li>将胶原酶溶液通过0.22μm无菌过滤单元运行到50 mL管中，并在整个方案中将其保持在冰上。</li>
	</ol></li>
	<li>将Calvaria在37°C下以300rpm在摇床上孵育20分钟以获得馏分1。</li>
	<li>丢弃馏分 1 的消化物，用 5 mL 磷酸盐缓冲盐水 （PBS） 洗涤钙质并丢弃洗涤液。</li>
	<li>将钙瓦里亚在含有1mg / mL BSA的5mM乙二胺四乙酸（EDTA）中的PBS中孵育，PH 7.4在300rpm的振荡器上孵育15分钟。</li>
	<li>将消化物收集在 50 mL 锥形管中。用 5 mL PBS 洗涤颅骨，并将洗涤溶液添加到消化液中。 注意:在收集消化物和洗涤的每个点，将骨头移入其溶液中以将细胞与骨分离并防止双峰和细胞聚集。</li>
	<li>为了获得馏分2，将消化物在4°C下以300× g 离心5分钟。弃去上清液，将沉淀重悬于含有 10% 胎牛血清 （FBS）、100 IU/mL 青霉素 G 和 100 μg/mL 链霉素的 8 mL α最低必需培养基 （MEM） 中。在10厘米培养皿上播种。将馏分2在37°C下在5%CO2下孵育。</li>
	<li>将Calvaria在37°C的5mL胶原酶溶液中以300rpm在摇床上孵育20分钟。</li>
	<li>要收集分数 3，请重复步骤 2.5-6。将级分3在37°C下在5%CO2下孵育。 注意:此时，骨骼会变小，并减少到碎片。为确保不会意外吸入消化物中的骨头，从而导致在此过程中细胞丢失，请使用小型吸头移液器收集消化物（1000\u2012200 μL 移液器吸头）。</li>
	<li>将Calvaria在37°C的5mL胶原酶溶液中以300rpm在摇床上孵育20分钟。</li>
	<li>要收集分数 4，请重复步骤 2.5-6。将级分4在37°C下在5%CO2下孵育。</li>
	<li>将Calvaria在5mL含有1mg / mL BSA的5mM EDTA中的PBS（pH 7.4）在37°C下以300rpm的振荡器孵育15分钟。</li>
	<li>要收集分数 5，请重复步骤 2.5-6。将级分5在37°C下在5%CO2下孵育。骨细胞可以在同一天准备分选，也可以在分选前培养长达24小时。</li>
</ol>3. 用于荧光活化细胞分选的骨细胞制备
<ol><li>吸出每个细胞级分的培养基，并用 10 mL PBS 轻轻洗涤两次。</li>
	<li>在PBS中加入5mL的0.5%胰蛋白酶-EDTA，并将细胞在37°C孵育5分钟。 加入 5 mL 10% FBS α-MEM 并移液器以轻轻分离细胞。通过将 40 μm 细胞过滤器筛入 50 mL 锥形离心管中，将每个级分中的细胞合并。</li>
	<li>用 10 mL PBS 洗涤细胞，并通过 40 μm 细胞过滤器筛入 50 mL 锥形离心管中收集。将细胞在4°C下以300× g 离心5分钟。</li>
	<li>吸出上清液并向沉淀中加入10%FBS α-MEM。将细胞浓度调节至约 1 x 107 个细胞/mL。</li>
	<li>在35μm尼龙网盖管中过滤细胞。这些牢房现已准备好进行FACS。</li>
	<li>对于此协议，请使用100μm尺寸的喷嘴。收集液应由10%的FBS α-MEM组成。保持正在搅拌分选的样品，如果可能的话，在整个分选过程中保持在4°C。</li>
	<li>在排序之前，优化门控，通过调整侧向散射 （SSC） 区域和前向散射 （FSC） 区域来去除伪影和细胞。通过调整 SSC 宽度与 SSC 高度和 FSC 面积与 FSC 宽度来消除双峰。GFP阴性骨细胞应用作对照，以调整分选仪器的参数。</li>
	<li>细胞收集完成后，将细胞在4°C下以300× g 离心10分钟。用PBS清洗细胞。将细胞在4°C下以300× g 离心5分钟。</li>
	<li>重新悬浮细胞并使用10%FBS α-MEM根据需要调整数量。</li>
</ol>

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作者没有什么可透露的。

第一个分离的骨细胞来自使用（OB7.3）或PHEX的禽变种分离的鸡颅骨7 ;然而，这种方法受到可行抗体可用性的限制，因为必须制造具有特异性的骨细胞特异性抗体。研究人员使用顺序酶促过程的不同修饰从小鼠和大鼠长骨中获得骨细胞;据报道，这些培养物的纯度设定为约70%9。cre小鼠模型的发展允许工程骨细胞，其在其表面表达GFP。该小鼠模型与FACS一起用于获得?...

骨细胞是来自成骨细胞祖细胞的终末分化细胞，嵌入其分泌基质1。它们是骨细胞群中最丰富和最长寿的细胞。它们位于腔隙内，具有特征性的星状形态，树突通过称为小管的通道延伸，形成与周围环境和骨表面的广泛沟通和代谢交换网络2。骨细胞在骨重塑中的作用是成骨细胞和破骨细胞，它们是赋予适应机械负荷的主要机械感觉细胞3，参与磷酸盐稳态4和骨基质矿化5，并与腔隙小管系统一起充当内分泌器官，向远处组织发出信号6。
骨细胞位于矿化基质内，这限制了可及性并使其分离具有挑战性，从而阻碍了体外研究。最早的分离方法之一描述了使用骨细胞特异性单克隆抗体（OB7.3）7从鸡颅骨中分离出骨细胞，该抗体后来被称为PHEX（PHosphate调节基因与X染色体上的内肽酶同源）的禽变体8。其他研究人员使用大鼠9或小鼠10长骨的顺序消化来获得富含骨细胞的部分，其中骨细胞纯度据报道约为70%9。该程序的局限性包括被骨细胞以外的其他细胞类型污染的培养物纯度欠佳，并且由于骨细胞已失去分裂能力，骨细胞可能被培养物中的其他细胞过度生长。这些挑战限制了长期文化的可用性。
为了克服这些限制，开发了不同的骨细胞系。MLO-Y4细胞系11和MLO-A5细胞系12是研究最广泛的细胞系，可用于研究早期骨细胞;然而，它们对于研究成熟骨细胞信号传导不太有用，因为它们表达低水平的硬化素和FGF2313，两者都是成熟的骨细胞标志物。其他细胞系包括IDG-SW3 14和Ocy45415，表达高水平的硬化素和FGF23，可用于研究晚期骨细胞阶段。细胞系被证明是有用的研究工具;然而，它们并非没有限制，因为它们不能完全代表原代细胞的生物学。不同的细胞系代表骨细胞成熟谱的不同发育阶段，细胞系不能代表原代骨细胞的异质性16,17。
为了获得原代骨细胞的纯培养物，研究人员利用了CRE小鼠模型，其中8kb dmp1启动子用于驱动骨细胞中的绿色荧光蛋白（GFP）表达18,19。Paic等人的双转基因小鼠（pOB-Col 2.3-GFP-青色和DMP1-GFP-托帕石）19和Nakashima等人的dmp1-黄玉转基因小鼠已被用于获得骨细胞群。其中他们采用表达GFP的骨细胞的顺序消化和FACS来获得原代骨细胞的培养物19,20。激活tdTomato蛋白的10 kb dmp1报告小鼠Ai9中的cre方向被证明存在于骨髓内的骨细胞，成骨细胞，肌肉和细胞中。8 kb dmp1启动子具有相同的表达模式，但只有一部分成骨细胞和骨髓细胞表达该蛋白，这表明8 kb dmp1启动子更具特异性21。尽管如此，应谨慎解释使用8 kb dmp1启动子获得的结果，并且应常规使用骨细胞与成骨细胞特异性标记进行基因表达谱，以确保获得的群体具有足够高的纯度。
破骨细胞标志物OSCAR和Dcstamp在造血未耗尽与缺氧骨细胞群体中发现，这一发现使作者得出结论，从8 kb dmp1-黄玉新生儿颅骨分馏和GFP分选中获得的消化物被造血细胞污染。造血细胞的污染可以通过收紧GFP分选门来减轻，因为GFP阳性造血细胞的GFP强度比GFP阳性间充质细胞（骨细胞）低22。
体外研究骨细胞的方法为最近关于骨细胞生物学的丰富信息做出了贡献。然而，骨细胞分离仍然是一个劳动密集型和漫长的过程，细胞产量低。所描述的使用胶原酶和EDTA的骨消化方法通常高达823级分，需要几个小时，其中骨细胞活力被征税。研究人员报告使用级分（2\u20125）进行细胞分选20，并表明与骨细胞相关的基因与成骨细胞相关的基因的表达谱证实了分离纯骨细胞群的成功20。在本文中，我们描述了获得分数（2\u20125）的过程，并比较了从分数1到8开始的每个分数的骨细胞产量，以确定每个分数中骨细胞的返回。我们还描述了使用胶原酶和乙二胺四乙酸（EDTA）解剖新生dmp1-topaz小鼠颅骨和颅骨消化，以及制备FACS细胞。

<table><tbody><tr><td>BD FACSDiva software</td><td>BD Biosciences</td><td></td><td>Data aquisition and analysis</td></tr><tr><td>BD Falcon Tube</td><td>BD Biosciences</td><td>352235</td><td>12 x 75 mm Tube with Cell Strainer Cap, 35 &amp;mu;m nylon mesh.</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich, MO, USA</td><td></td><td></td></tr><tr><td>Collagenase</td><td>Wako, Osaka, Japan</td><td>034-22363</td><td>0.2% (w/v), crude collagenase mix sourced from C. histolyticum.</td></tr><tr><td>EDTA</td><td>Dojindo, Kumamoto, Japan</td><td></td><td>5mM EDTA prepared with 0.1% BSA</td></tr><tr><td>FACSAriaTM II</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Biowest, Nuaill&amp;eacute;, France</td><td></td><td></td></tr><tr><td>Isolation buffer</td><td></td><td></td><td>70mM NaCl, 10mM NaHCO, 60mM sorbitol, 3mM K2HPO4, 1mM CaCl2, 0.1% (w/v) BSA, 0.5% (w/v) glucose and 25 mM HEPES</td></tr><tr><td>Millex Sterile Filter Unit</td><td>Merck Millipore, Ireland</td><td>SLGV033RS</td><td>0.22&amp;mu;m</td></tr><tr><td>Nylon cell strainer</td><td>FALCON, NY, USA</td><td></td><td>40&amp;mu;m</td></tr><tr><td>Trypsin-EDTA</td><td>Life Technologies, NY, USA</td><td></td><td>0.5% x10. diluted to x1 in PBS</td></tr><tr><td>&amp;alpha;-MEM</td><td>Wako, Osaka, Japan</td><td></td><td>Containing 10% fetal bovine serum, 100 IU/ml penicillin G, and 100 &amp;mu;g/ml streptomycin</td></tr></tbody></table>

obtaining primary osteocytes through murine calvarial fractionation of gfp-expressing osteocytes

骨细胞曾经被认为是骨骼的被动驻留，具有感知机械负荷的后台功能，现在被带到聚光灯下，并已被证明具有多种主要功能，例如主动修改细胞外基质并与腔隙小管系统形成内分泌器官，该系统包围它向远处发送信息。由于可以从分离原代骨细胞到骨细胞样细胞系的体外测试骨细胞的方法，骨细胞现在正经历着巨大的兴趣和对结构和功能的知识激增。骨细胞生物学的许多方面以及与其他分子成分的相互作用尚未被发现。在该协议中，我们详细描述了从dmp1-topaz新生小鼠颅骨中有效分离原代骨细胞，其通过细胞分离并随后通过FACS获得原代骨细胞的培养物在骨细胞中表达绿色荧光蛋白。

该协议的目的是通过分离过程从dmp1-topaz新生小鼠颅骨中获得原代骨细胞培养物的过程，该过程使用胶原酶降解胶原基质和EDTA进行钙螯合，之后准备用于FACS的细胞以将骨细胞与其他细胞群分离。
从新生小鼠颅骨获得原代骨细胞的方法通常描述使用馏分（1\u20128）进行分类23。为了测试该方法的效率，我们比较了从组分1到8开始的从dmp1-topaz小鼠颅骨中获得的骨?...

Watch this Scientific Journal Video about Obtaining Primary Osteocytes Through Murine Calvarial Fractionation of GFP-Expressing Osteocytes at JoVE.com

Obtaining Primary Osteocytes Through Murine Calvarial Fractionation of GFP-Expressing Osteocytes

该协议描述了新生儿dmp1-黄玉小鼠颅骨的解剖和通过细胞消化和分级分离表达绿色荧光蛋白的骨细胞，以及用于荧光活化细胞分选（FACS）的骨细胞制备。

通过表达GFP的骨细胞的小鼠颅骨分割获得原代骨细胞

The osteocyte, once thought to be a passive resident of the bone given the backstage function of sensing mechanical loading, is now brought to the ...

obtaining-primary-osteocytes-through-murine-calvarial-fractionation

Research

JoVE Journal

Biology

2.1K 次观看.  Tohoku University Graduate School of Dentistry. 该协议描述了新生儿dmp1-黄玉小鼠颅骨的解剖和通过细胞消化和分级分离表达绿色荧光蛋白的骨细胞，以及用于荧光活化细胞分选（FACS）的骨细胞制备。

视频: Obtaining Primary Osteocytes Through Murine Calvarial Fractionation of GFP-Expressing Osteocytes

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

小鼠骨髓基质细胞与骨破骨祖的分离、培养及分化

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

科研

发育生物学

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.

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 ...

医学

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of therapies to enhance fracture healing.&#160; Periosteal cells rapidly migrate to an injury to supply new chondrocytes and osteoblasts for fracture healing. Traditionally, the efficacy of a cytokine to induce cell migration has only been conducted in vitro by performing a transwell or scratch assay. With advancements in intravital microscopy using multiphoton excitation, it was recently discovered that 1) P-SSCs express the migratory gene CCR5 and 2) treatment with the CCR5 ligand known as CCL5 improves fracture healing and the migration of P-SSCs in response to CCL5. These results have been captured in real-time. Described here is a protocol to visualize P-SSC migration from the calvarial suture skeletal stem cell (SSC) niche towards an injury after treatment with CCL5. The protocol details the construction of a mouse restraint and imaging mount, surgical preparation of the mouse calvaria, induction of a calvaria defect, and acquisition of time-lapse imaging.

This protocol describes the detection of CCL5-mediated periosteal skeletal stem cell migration in real-time using live animal intravital microscopy.

CCL5诱导小鼠骨膜骨骼干细胞迁移的实时成像

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of ...

A Fluorescent Intravital Imaging Approach to Study Load-Induced Calcium Signaling Dynamics in Mouse Osteocytes

Bone tissue is exquisitely sensitive to differences in mechanical load magnitude. Osteocytes, dendritic cells that form a syncytium throughout the bone, are responsible for the mechanosensory function of bone tissue. Studies employing histology, mathematical modeling, cell culture, and ex vivo bone organ cultures have greatly advanced the understanding of osteocyte mechanobiology. However, the fundamental question of how osteocytes respond to and encode mechanical information at the molecular level in vivo is not well understood. Intracellular calcium concentration fluctuations in osteocytes offer a useful target for learning more about acute bone mechanotransduction mechanisms. Here, we report a method for studying osteocyte mechanobiology in vivo, combining a mouse strain with a fluorescently genetically encoded calcium indicator expressed in osteocytes with an in vivo loading and imaging system to directly detect osteocyte calcium levels during loading. This is achieved with a three-point bending device that can deliver well-defined mechanical loads to the third metatarsal of living mice while simultaneously monitoring fluorescently indicated calcium responses of osteocytes using two-photon microscopy. This technique allows for direct in vivo observation of osteocyte calcium signaling events in response to whole bone loading and is useful in the endeavor to reveal mechanisms in osteocyte mechanobiology.

<meta charset="utf8"></head><body>一种荧光活体成像方法研究小鼠骨细胞中负荷诱导的钙信号动力学

The current article describes performing an intravital imaging approach to observe mechanically induced calcium signaling of embedded osteocytes in vivo in real-time in response to tissue-level mechanical loading of the mouse third metatarsal.

一种荧光活体成像方法研究小鼠骨细胞中负荷诱导的钙信号动力学

Bone tissue is exquisitely sensitive to differences in mechanical load magnitude. Osteocytes, dendritic cells that form a syncytium throughout the bone, ...

生物工程

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line.
The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features.
After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules.
Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. 
The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models.&#160;

维甲酸的应用获取从初级小鼠成骨细胞骨细胞培养

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

生物学

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

<meta charset="utf8"></head><body>用于 CFD 分析的骨细胞 3D 建模以研究骨骼中的机械感觉

Analysis and Imaging of Osteocytes

Osteocytes are the bone cells that are thought to respond to mechanical strains and fluid flow shear stress (FFSS) by activating various biological pathways in a process known as mechanotransduction. Confocal image-derived models of osteocyte networks are a valuable tool for conducting Computational Fluid Dynamics&#160;(CFD) analysis to evaluate shear stresses on the osteocyte membrane, which cannot be determined by direct measurement. Computational modeling using these high-resolution images of the microstructural architecture of bone was used to numerically simulate the mechanical loading exerted on bone and understand the load-induced stimulation of osteocytes.
This study elaborates on the methods to develop 3D single osteocyte models using confocal microscope images of the Lacunar-Canalicular Network&#160;(LCN) to perform CFD analysis utilizing various computational modeling software. Prior to confocal microscopy, the mouse bones are sectioned and stained with Fluorescein isothiocyanate (FITC) dye to label the LCN. At 100x resolution, Z-stack images are collected using a confocal microscope and imported into MIMICS software (3D image-based processing software) to construct a surface model of the LCN and osteocyte-dendritic processes.
These surfaces are then subtracted using a Boolean operation in 3-Matic software (3D data optimization software) to model the lacunar fluidic space around the osteocyte cell body and canalicular space around the dendrites containing lacunocanalicular fluid. 3D volumetric fluid geometry is imported into ANSYS software (simulation software) for CFD analysis. ANSYS CFX (CFD software) is used to apply physiological loading on the bone as fluid pressure, and the wall shear stresses on the osteocytes and dendritic processes are determined. The morphology of the LCN affects the shear stress values sensed by the osteocyte cell membrane and cell processes. Therefore, the details of how confocal image-based models are developed can be valuable in understanding osteocyte mechanosensation and can lay the groundwork for future studies in this area.

<meta charset="utf8"></head><body>骨细胞的分析和成像

This study outlines the method to visualize and develop three-dimensional (3D) models of osteocytes within the lacunar-canalicular network (LCN) for computational fluid dynamics (CFD) analysis. The generated models using this method help to understand osteocyte mechanosensation in healthy or diseased bones.

骨细胞的分析和成像

Osteocytes are the bone cells that are thought to respond to mechanical strains and fluid flow shear stress (FFSS) by activating various biological ...

通过表达GFP的骨细胞的小鼠颅骨分割获得原代骨细胞

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小鼠骨髓基质细胞与骨破骨祖的分离、培养及分化

顺序

CCL5诱导小鼠骨膜骨骼干细胞迁移的实时成像

一种荧光活体成像方法研究小鼠骨细胞中负荷诱导的钙信号动力学

一种荧光活体成像方法研究小鼠骨细胞中负荷诱导的钙信号动力学

维甲酸的应用获取从初级小鼠成骨细胞骨细胞培养

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

骨细胞的分析和成像

骨细胞的分析和成像

骨细胞的分析和成像