我们承认美国国立卫生研究院拨款R01 DE021683，R01 DE019434，U01 HL099776，橡树基金会和Hagey实验室小儿再生医学的支持。

注:伦理声明:在批准的斯坦福大学行政专家组对实验室动物护理（APLAC）按照协议进行的所有涉及脊椎动物的研究。 1.准备<ol><li>允许10毫升市售的密度梯度的细胞分离介质（包含多聚蔗糖和泛影酸钠，调整至10.77克/毫升的密度）来RT在50ml锥形管中。 </li><li>制备流式细胞术（FACS）缓冲液用1×磷酸盐缓冲盐水（PBS）和2％胎牛血清（FCS）中。取50毫升的等分试样，并且在密度梯度的细胞分离介质步骤保持这种等分在RT下使用。 </li><li>有冰准备保持在分离过程中分离的组织和细胞的桶。 </li></ol> 2.准备文化传媒<ol><li>制备基础培养基:MEM（最低必需培养基），不含酚红（500毫升），谷氨酸（1×），FCS（10倍），10,000单位/ ml的青霉N（1X）。 </li><li>过滤基础培养基用0.22微米的过滤器。 </li><li>准备骨髓巨噬细胞诱导培养基: <ol><li>取50毫升步骤2.1准备的基础培养基中。 </li><li>添加前列腺素E2（PGE2，先生352.465克/摩尔）在10 -7 M终浓度。 </li><li>添加巨噬细胞集落刺激因子（M-CSF）在10纳克/毫升。 </li><li>不过滤骨髓巨噬细胞刺激媒体的最终解决方案。 </li></ol></li><li>准备破骨细胞诱导培养基: <ol><li>取50毫升步骤2.1准备的基础培养基中。 </li><li>添加前列腺素E2（PGE2，先生352.465克/摩尔）在10 -7 M终浓度</li><li>添加巨噬细胞集落刺激因子（M-CSF）在10纳克/毫升。 </li><li>添加RANKL在10纳克/毫升。 </li><li>不过滤破骨细胞诱导培养基的最终解决方案。 </li></ol></li></ol> 3.骨髓隔离<ol><li>安乐死1 C57BL / 6小鼠用二氧化碳吸入法，随后通过颈脱位。 注:鼠害必须由经过培训的人员使用适当的技术，设备和代理进行安乐死。 </li><li>位置并固定在鼠标上的夹层板，并喷以70％的酒精。 </li><li>剖析了股骨，胫骨，肱骨和脊柱。 <ol><li>首先用鼠标在仰卧位。做一个切口垂直地沿着下肢并通过除以软组织的附件沿股骨和胫骨的长度解剖开始。最后，脱臼从膝关节和髋关节的骨头从骨架完全释放的骨头。 </li><li>继续使沿上肢分离肱骨长度的切口。通过解剖软组织囊附着脱位的肱骨肩和肘关节。 </li><li>最后，把鼠标，让鼠标趴着。使皮肤切口沿着脊柱的长度，在中线。观察椎旁软组织对脊柱和切割沿脊柱的骨边缘的每一侧，通过椎旁软组织肿块解剖。使用剪刀，使整个脊柱的水平切口在颈部的基部，并在脊柱，刚好接近尾部插入，释放从软组织附着在脊柱的末端。 </li></ol></li><li>一旦骨头收获，让他们在FACS缓冲冰上。 </li><li>用薄纸从骨子里清除肌肉和软组织。 </li><li>将洗净的骨头成杵和研钵用3ml FACS缓冲液。 </li><li>在研钵轻轻粉碎的骨头。 </li><li>吸脱血染色流体和转移到50ml锥形管中通过使溶液通过70微米的细胞过滤网。 </li><li>加入5 ml FACS缓冲的碎骨头，并进一步挤压。再次，用移液管取出流体，并通过一个70微米的过滤器转移到50ml锥形管中。 </li><li>通常，粉碎的骨头TWI策，以新鲜FACS缓冲液三次，用研钵和研杵，每次加入另外5毫升新鲜的FACS缓冲液中，直到该流体不染色的红色。 </li><li>离心机在200×g离心骨髓溶液中5分钟，在4℃下，得到的细胞沉淀。 </li></ol>骨髓细胞4.梯度分离<ol><li>吸去上清，重悬在10ml的FACS缓冲液（保持在室温下）。 </li><li>以50毫升锥形管含有10ml市售的密度梯度细胞分离介质（RT）的。 </li><li>层的骨髓液涂布到密度梯度的细胞分离介质。慢慢地使用电动移液器做到这一点。角对着附近的锥形管顶部的内表面上的移液管尖和倾斜的锥形管中，以大约30°。上吸管用慢速设置，轻轻驱逐骨髓溶液，以便不打扰密度梯度的细胞分离介质。 注:最终会有是上述两种溶液（蜂窝溶液和密度梯度的细胞分离介质溶液）之间的明确定义。 </li><li>使用RT离心机，离心密度梯度细胞分离培养基和细胞溶液中的平衡离心机在200×g离心15分钟。此外，除去在离心机上的加速和减速，以确保有足够的相分离使用密度梯度的细胞分离介质梯度。 </li><li>离心后，吸断其包含感兴趣的骨髓细胞的混浊的中间层。 </li><li>转移这些细胞到新的离心管中。加入20毫升的附加FACS缓冲液（保持在冰上）洗涤细胞。 </li><li>离心机在200×g离心5分钟，在4℃下，得到的细胞沉淀。 </li><li>重悬在巨噬细胞刺激介质，以允许使用血球计数器进行细胞计数1毫升的最终的细胞沉淀。 </li></ol> 5.细胞计数<ol><li>取10微升的细胞溶液和混用10微升台盼蓝。负载10微升所得的混合物到血细胞计数器，将获得每毫升溶液中的细胞计数。 </li></ol> 6.细胞培养<ol><li>放置2毫升巨噬细胞刺激介质到24孔板的每个孔中。 </li><li>加入200,000细胞，以每孔中。 注意:根据我们的经验，细胞铺板密度是足够的破骨细胞的体外培养中最关键的要求之一。 </li><li>轻轻搅动板和放置在一个常规的培养箱中在37℃。 </li><li>不要更改媒体3天。 </li><li>后在巨噬细胞刺激媒体3天，改变媒体破骨细胞培养基。 </li><li>此后，为5-7天，在该点，大，多核破骨细胞应该在培养板上的可见每日更换介质。 </li></ol> 7.染色用抗酒石酸酸性磷酸酶（TRAP） <ol><li>经过5-7天的体外 CULTURE，从培养物中吸出培养基良好。 </li><li>用1×PBS三次，轻轻地洗井。 </li><li>通过加入4％多聚甲醛溶液固定细胞，并在4℃下离开1小时。 </li><li>在此期间，准备为TRAP染色（其可商购）通过预热到37℃。 </li><li> 1小时的多聚甲醛后，吸过和多聚甲醛轻轻洗净用1×PBS 3倍。 </li><li>加入1毫升的预温热的TRAP染色到板的每个孔被染色。 </li><li>将板在培养箱中在37℃下1小时，从光屏蔽。 </li><li> 1小时后，吸脱TRAP染色。 </li><li>用预热的去离子水洗涤各孔3次。 </li><li>染液井与吉尔的苏木（注意）1-2分钟。 </li><li>用亮视野显微镜图像的破骨细胞。 </li><li>洗孔用离子交换水，直至污迹的适当颜色强度来实现，典型地当晶核出现蓝Ë。 </li></ol> 8，破骨细胞骨吸收分析<ol><li>使用市售的24孔聚苯乙烯培养皿是预先涂有骨基质或无机晶体磷酸钙涂层。 </li><li>放置2毫升巨噬细胞刺激介质到每个孔中。 </li><li>添加在步骤2至各孔的端部获得200000新鲜分离骨髓细胞。 </li><li>轻轻之前搅动板以将其放置在培养箱中在37℃下3天。 </li><li>第3天，改变媒体破骨细胞分化的媒体。 </li><li> 5-7天执行与破骨细胞分化的媒体每日介质的变化。 </li></ol> 9.定量破骨细胞骨吸收活动的<ol><li> 5-7天后的体外培养破骨细胞的分化培养基上的矿化基质的，分析的矿化的表面，如下面所述，以评估破骨细胞的形成resorpti的能力上的凹坑，破骨细胞活性的指标。 </li><li>从再吸收测定板抽吸介质，并添加100微升10％的漂白剂溶液到每个孔中。 </li><li>孵育细胞15分钟，在室温下的10％漂白溶液中。 </li><li>用蒸馏水洗涤各孔3次。 </li><li>吸掉任何剩余的水，并从板甩掉多余的水，并允许在空气中干燥3-5小时。 </li><li>染液在板与市售冯Kossa染色试剂盒，以允许将未再吸收衬底更容易可视化。 </li><li>用亮视野显微镜采取的再吸收表面的代表性部分图像。 </li><li>利用图像分析软件，计算出再吸收表面的百分比，以允许破骨细胞骨吸收活性的定量。 </li></ol>

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+and+targeting+osteoclastic+activity+in+prostate+cancer+bone+metastases.">Understanding and targeting osteoclastic activity in prostate cancer bone metastases.</a> Current molecular medicine. 13, 626-639 (2013).</li></ol>

没有一个作家有披露或有利益冲突申报。

一个很容易分离，培养大量的破骨细胞在体外的能力一直负责帮助推动骨生物学和破骨细胞介导的疾病的认识。这是RANKL的识别，导致此，当它最近被鉴定为破骨细胞的形成，分化和存活16-18的主要调节剂。 据我们的经验是， 在体外培养骨髓破骨细胞在很大程度上依赖于接种密度。我们观察到，该协议得到的破骨细胞的失败通常是由于一个不理想的接种?...

骨重建是动态的，并且涉及骨形成与骨吸收1的耦合。这个严格调节的过程是负责维持正常的体内平衡中的骨架，并响应于损伤和疾病。 破骨细胞是独特的，多核细胞，其能够再吸收二者的有机和无机骨基质。破骨细胞是来源于骨髓的2-5的单核细胞/巨噬细胞系。在破骨细胞的功能，或形成的异常可导致多种临床病症，包括骨质疏松症象常见的情况的。 在体外产生的破骨细胞的能力已经允许在我们的骨生物学6的理解显著进步。其结果是，新的治疗剂的新兴治疗破骨细胞有关的疾病，这是负责显著发病率和死亡率7 </sUP&gt;。稳态维持骨量和骨强度需要骨形成的成骨细胞和骨吸收破骨细胞8,9的一致行动人。骨骼平衡被改变中的一些疾病，包括绝经后骨质疏松症，其中增加的破骨细胞活性导致骨质量和密度10的致病损失。随着人类疾病的转基因小鼠模型的可用性，有更多的机会来破译人骨疾病11-13的破骨细胞的作用。 对破骨细胞的培养技术大量协议，出现在文献中，有许多变型描述9,12,14。性和同事描述了类似的方法，以如下所述，在小鼠骨髓细胞中破骨测定中对它们的描述的协议。然而，释放以下长骨收获骨髓细胞，性等冲洗骨髓腔的α-MEM完全培养基14。Catalfamo检查高血糖对破骨细胞功能的影响，并描述了其中的所有细胞动员骨髓潮红的方法进行培养24小时，在该点处的非贴壁细胞被丢弃12，一种技术也可用于由Boyle 等人的9，这些先前公布的协议必要冲洗骨髓，一个繁琐的实践中，其中还引入了针刺伤害和有价值的骨髓损失的风险，因为一个人必须切骨的两端部的做法。的协议中，我们描述了，实现了使用研钵和研杵的分离的破骨细胞，其类似于由Weischenfeldt 等人所述的巨噬细胞分离的方法。15 我们的经验，但是，是使用在破骨细胞的生产方面先前公布的技术的结果变量的结果即破骨细胞的分离和体外培养，常常导致在无法培养的破骨细胞。因此，我们设计了一个协议，该协议允许对小鼠骨髓的一贯隔离，以产生大量的体外多核破骨细胞，具有细胞最初镀形成的巨噬细胞和破骨细胞随后的70-80％的产率约，在存在破骨细胞诱导培养基。

<table><tbody><tr><td>MEM, no glutamine, no phenol red</td><td>Gibco</td><td>51200-038</td><td></td></tr><tr><td>M-CSF, recombinant mouse</td><td>Gibco</td><td>PMC2044</td><td></td></tr><tr><td>Recombinant Mouse TRANCE/RANK L/TNFSF11 (&lt;em&gt;E. coli&lt;/em&gt; expressed)</td><td>R&amp;amp;D Systems</td><td>462-TEC-010</td><td></td></tr><tr><td>Prostaglandin E2</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Histopaque-1077</td><td>Sigma-Aldrich</td><td>10771</td><td></td></tr><tr><td>Acid Phosphatase, Lekocyte (TRAP) kit</td><td>Sigma-Aldrich</td><td>387A</td><td></td></tr><tr><td>Osteoassay bone resorption plates, 24 well plates</td><td>Corning Life Sciences</td><td>3987</td><td></td></tr></tbody></table>

osteoclast derivation from mouse bone marrow

<html>破骨细胞是从骨髓中的单核细胞/巨噬细胞系衍生的高度特化的细胞。其重吸收两者的有机和无机骨基质的独特能力意味着它们在调节骨骼重塑中发挥关键作用。在一起，成骨细胞和破骨细胞是负责动态耦合的过程，涉及既骨吸收和骨形成的共同行动，以维持健康和疾病过程中的正常骨架。 如在体内主要骨吸收细胞，改变破骨细胞的分化或功能可能导致在体内产生深远的影响。具有改变破骨细胞功能有关的疾病的范围可以在严重程度从致命新生儿疾病由于不能形成对造血骨髓的空间，更通常观察到的疾病，如骨质疏松症，其中过度的破骨细胞骨吸收易患断裂形成。 ENT"&gt;一个孤立的破骨细胞的数量高在体外的能力也允许在骨重建周期的理解显著的进展，并铺平了道路，以应对这些疾病的新的治疗策略的发现。 在这里，我们描述了一个协议，分离培养小鼠骨髓，将产生大量的破骨细胞的破骨细胞。

<html>该方法的目的是要在一个星期容易分离大量破骨细胞的体外培养 ，典型。大量破骨细胞的成功分离用抗酒石酸酸性磷酸酶染色（ 图1A）中得到证实。大破骨细胞被可视化为紫色大细胞细胞核多个（通常≥3核）。使用该协议，它是常见的，分离的破骨细胞与破骨细胞每多达30细胞核（ 图1B）。 使用矿化的表面被认为是评估在体外破骨...

Watch this Scientific Journal Video about Osteoclast Derivation from Mouse Bone Marrow at JoVE.com

Osteoclast Derivation from Mouse Bone Marrow

破骨细胞是体内的主要骨吸收细胞。一个分离的破骨细胞中大量的能力已经导致破骨细胞生物学的理解显著进步。在这个协议中，我们描述了一种用于分离，培养和定量破骨细胞的体外活性。

小鼠骨髓破骨细胞的推导

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

osteoclast-derivation-from-mouse-bone-marrow

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24.0K 次观看.  Stanford University School of Medicine. 破骨细胞是体内的主要骨吸收细胞。一个分离的破骨细胞中大量的能力已经导致破骨细胞生物学的理解显著进步。在这个协议中，我们描述了一种用于分离，培养和定量破骨细胞的体外活性。 

视频: Osteoclast Derivation from Mouse Bone Marrow

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

骨肉瘤的三维骨细胞外基质模型

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

科研

癌症研究

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

在鼠标破骨细胞前体Notch信号的刺激

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

发育生物学

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

RANKL 小鼠骨髓破骨细胞培养法研究 mTORC1 在破骨细胞形成中的作用

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

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 ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

鼠后肢长骨解剖和骨髓隔离

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

免疫与感染

Biological Compatibility Profile on Biomaterials for Bone Regeneration

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such lesions, there are potential complications with their use. Thus, material scientists are developing synthetic, biocompatible biomaterials to overcome these problems. In this study, we present a multidisciplinary platform for evaluating biomaterials for bone repair. We combined expertise from bone biology and immunology to develop a platform including in vitro osteoclast (OC) and osteoblast (OB) assays and in vivo mouse models of bone repair, immunogenicity, and allergenicity. We demonstrate how to perform the experiments, summarize the results, and report on biomaterial biocompatibility. In particular, we tested OB viability, differentiation, and mineralization and OC viability and differentiation in the context of &#946;-tricalcium phosphate (&#946;-TCP) disks. We also tested a &#946;-TCP/Collagen (&#946;-TCP/C) foam which is a commercially available material used clinically for bone repair in a critical-sized calvarial bone defect mouse model to determine the effects on the early phase of bone healing. In parallel experiments, we evaluated immune and allergic responses in mice. Our approach generates a biological compatibility profile of a bone biomaterial with a range of parameters necessary for predicting the biocompatibility of biomaterials used for bone healing and repair in patients.

The number of novel biomaterials engineered for repairing large bone lesions is continuously expanding with the aim to enhance bone healing and overcome the complications associated with bone transplantation. Here, we present a multidisciplinary strategy for pre-clinical biocompatibility testing of biomaterials for bone repair.

骨再生生物材料的生物相容性曲线

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such ...

生物工程

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a single-cell suspension for further use in embryoid body induction. Following mesodermal induction, embryoid bodies undergo hematopoietic differentiation, producing a floating hematopoietic cell population. Subsequently, the harvested hematopoietic cells undergo a macrophage colony-stimulating factor maturation step and, finally, osteoclast differentiation. After osteoclast differentiation, osteoclasts are characterized by staining for TRAP in conjunction with a methyl green nuclear stain. Osteoclasts are observed as multinucleated, TRAP+ polykaryons. Their identification can be further supported by Cathepsin K staining. Bone and mineral resorption assays allow for functional characterization, confirming the identity of bona fide osteoclasts. This protocol demonstrates a robust and versatile method to differentiate human osteoclasts from iPSCs and allows for easy adoption in applications requiring large quantities of functional human osteoclasts. Applications in the areas of bone research, cancer research, tissue engineering, and endoprosthesis research could be envisioned.

This protocol presents the differentiation of human osteoclasts from induced pluripotent stem cells (iPSCs) and describes methods for the characterization of osteoclasts and osteoclast precursors.

破骨细胞与人诱导多能干细胞的分化和表征

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a ...

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused by increased differentiation and activation of OCs, so the inhibition of this pathobiology is a key therapeutic principle.Two key factors drive the differentiation of OCs from myeloid precursors: macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Human circulating CD14+ monocytes have long been known to differentiate into OCs in vitro. However, the exposure time and the concentration of RANKL influence the differentiation efficiency. Indeed, protocols for the generation of human OCs in vitro have been described, but they often result in a poor and lengthy differentiation process. Herein, a robust and standardized protocol for generating functionally active mature human OCs in a timely manner is provided. CD14+ monocytes are enriched from human peripheral blood mononuclear cells (PBMCs) and primed with M-CSF to upregulate RANK. Subsequent exposure to RANKL generates OCs in a dose- and time-dependent manner. OCs are identified and quantified by staining with tartrate acid-resistant phosphatase (TRAP) and light microscopy analysis. Immunofluorescence staining of nuclei and F-actin is used to identify functionally active OCs. In addition, OSCAR+CD14&#8722; mature OCs are further enriched via flow cytometry cell sorting, and OC functionality quantified by mineral (or dentine/bone) resorption assays and actin ring formation. Finally, a known OC inhibitor, rotenone, is used on mature OCs, demonstrating that adenosine triphosphate (ATP) production is essential for actin ring integrity and OC function. In conclusion, a robust assay for differentiating high numbers of OCs is established in this work, which in combination with actin ring staining and an ATP assay&#160;provides a useful in vitro model to evaluate OC function and to screen for novel therapeutic compounds that can modulate the differentiation process.

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts are key bone-resorbing cells in the body. This protocol describes a reliable method for the in vitro differentiation of osteoclasts from human peripheral blood monocytes. This method can be used as an important tool to further understand osteoclast biology in homeostasis and in diseases.

功能性破骨细胞与人外周血CD14+ 单核细胞的鉴别

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

大鼠骨髓破骨衣原体的分离、纯化及鉴别

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

生物学

<meta charset="utf8"></head><body>用于膜内骨修复研究的改进骨髓损伤模型

Improved Methodology for Studying Postnatal Osteogenesis via Intramembranous Ossification in a Murine Bone Marrow Injury Model

Long bone injuries heal through either endochondral or intramembranous bone formation pathways. Unlike the endochondral pathway that requires a cartilage template, the process of intramembranous ossification involves the direct conversion of skeletal stem and progenitor cells (SSPCs) into bone-forming osteoblasts. There are limited surgical methods to model this process in experimental mice. Here, we have improved upon a bone marrow injury model in mice to facilitate the study of bone repair via intramembranous ossification and to assess postnatal regulators of osteogenesis. This method is highly reproducible and user-friendly, and it allows temporal assessment of new bone formation in a short period (3-7 days post-injury) using micro-computed tomography (&#181;CT) and frozen section histology. Furthermore, the contributions of SSPCs and mature osteoblasts can be readily assessed using a combination of fluorescent reporter mice and this intramembranous bone marrow injury model. In clinical contexts, intramembranous bone formation is relevant for healing critical size defects, stabilized fractures, cortical defects, trauma from tumor resections, and joint replacements.

<meta charset="utf8"></head><body>在小鼠骨髓损伤模型中通过膜内骨化研究产后成骨的改进方法

The murine bone marrow injury model is a useful tool for studying postnatal osteogenesis through intramembranous ossification. This simplified surgical protocol describes the bone marrow ablation procedure for downstream assessment of new bone formation and cellular responses following injury.

在小鼠骨髓损伤模型中通过膜内骨化研究产后成骨的改进方法

Long bone injuries heal through either endochondral or intramembranous bone formation pathways. Unlike the endochondral pathway that requires a cartilage ...