该项研究协议已获得长功夫纪念医院 (IRB99-1828B、100-3019C、99-3814B、102-1619C、101-4728B、103-4223C) 临床研究机构审查委员会批准。这项研究得到了长功夫纪念医院 (CMRPG392071、CMRPG3A1141、CMRPG3A1142 和 NMRPG3C0151) 的支持。这篇手稿是由华莱士的学术编辑编辑的。

该项研究协议获昌热心纪念医院机构评审委员会批准。所有与会者均提供书面知情同意。
1. 组织准备
<ol><li>在牙科手术中从病人身上收获骨膜组织 (图 1)。在局部麻醉下皮瓣反射后, 用骨膜分离器31从肺泡骨中取一块骨膜组织。</li>
	<li>收割后, 将5毫米 x 2 毫米的骨膜组织切片贮存在 Dulbecco 磷酸盐缓冲盐水 (DPBS) 中, 300 毫升青霉素和300毫克/毫升链霉素。在24小时内将组织转移到实验室。</li>
	<li>用手术刀将肺泡骨膜组织碎片切碎, 并保持0培养基中的样品 (由300毫升青霉素和300毫克/毫升的链霉素, α和5% 胎牛血清 [血清]) 在37摄氏度培养的孵化器湿润空气 (5% 二氧化碳)。在孵化器中, 准备一个水盆来保持湿度。</li>
	<li>3天后, 每周两次改变培养基。</li>
	<li>当细胞达到 subconfluence (80%), 释放黏附细胞与200µL 0.25% 胰蛋白酶在孵化器 (37 °c) 为3分钟, 然后使用4.5 毫升培养基终止反应。</li>
	<li>在新鲜的通道1培养基 (α, 10% 血清, 100 单位/毫升青霉素, 100 微克/毫升链霉素) 再次板块细胞。板是 5 x 103单元格 (按 hemocytometer 计数)。</li>
	<li>在单元格实现80% 汇流31之后, 执行每个后续通道。 注: 在开始时, 培养基将为橙色红色。大约三天后, 中等颜色应该变成黄色的阴影。细胞的加倍时间大约 30–40 h, 需要7天为3–5世代。</li>
	<li>培养分离的 PDCs 在α记忆补充与10% 个血清, 100 U/毫升青霉素和100毫克/毫升链霉素在孵化器 (37 °c, 5% CO2)。</li>
	<li>每天观察细胞生长, 每周两次更换生长培养基。使用第三到第五代细胞进行所有进一步的实验。</li>
</ol>2. 流式细胞仪
<ol><li>通过胰蛋白酶消化, 收获 1 x 106细胞:<ol>
<li>添加200µL 0.25% 胰蛋白酶。3分钟后, 使用4.5 毫升含有血清的培养基来终止胰蛋白酶的反应, 通过离心 (1500 rpm, 5 分钟, 22 °c) 来收集。</li>
		<li>用 1x DPBS 清洗细胞颗粒三次。并用重悬200µL 1x通透缓冲区中的单元格。用 hemocytometer 计数单元格。</li>
	</ol>
</li>
	<li>通过流式细胞术 (荧光活化细胞排序)32检查 PDCs 表达的表达表面标记。使用单克隆抗体对 CD19 (异硫氰酸荧光素 (FITC)), CD34 (FITC)、CD44 (藻红蛋白 [pe])、CD45 (FITC)、CD73 (pe)、CD90 (复方阿司匹林 [APC])、CD146 (pe)、STRO-1 (亚历克斯) 和 HLA-DR (FITC) 32。<ol>
<li>添加抗体的样本和文化在黑暗中的4°c 30 分钟。</li>
		<li>用 1x DPBS 清洗细胞三次。</li>
		<li>用2% 甲醛固定细胞, 进行流式细胞分析32。</li>
	</ol>
</li>
</ol>3. 细胞附着和活性与成骨、脂肪和软骨分化
通过在六-井板上用特异分化培养基培养三通道的骨膜细胞, 诱导细胞分化成成骨、脂肪和软骨血统。
<ol><li>为成骨分化, 在六-井培养板上, 在5000个细胞的密度下培养α-记忆细胞。<ol>
<li>在达到80% 汇合, 培养细胞在媒介包含α记忆, 5% 血清, β甘油 (10 毫米), 10−7 M 地塞米松 (0.1 毫米) 和5毫升抗坏血酸 (100 uM)。培养由α和5% 血清组成的媒介中的阴性控制。</li>
		<li>每周更改两次媒体。</li>
		<li>4周后, 评估细胞的潜能, 通过使用冯科萨检测细胞来鉴别成骨性的血统, 这就区分了在文化33中钙化沉积的存在。<ol>
<li>加10% 福尔马林固定。在30分钟内, 加入5% 硝酸银, 在室温下用紫外线 (紫外线) 照射1小时。</li>
			<li>添加 5% Na2, 因此4四次, 每个反应允许3分钟。最后, 用蒸馏水冲洗细胞两次。 注: 冯科萨染色是一种沉淀反应, 银离子和磷酸盐在酸性物质存在的情况下反应;这种染色技术不特定于钙。在这项研究中, 当被调查的细胞用硝酸银溶液处理时, 钙--由强的紫外光减少-被银替换, 变得可看见作为金属银。</li>
		</ol>
</li>
	</ol>
</li>
	<li>对于脂肪分化, 培养细胞密度为5000细胞每井在六井培养板含有α。<ol>
<li>在实现80% 汇流时, 培养细胞的培养基中含有α, 5%, 0.5 毫米3异丁基1甲基黄嘌呤 (100 毫克/毫升), 1% 青霉素,10 −6 M 地塞米松 (1 毫米), 胰岛素 (5 毫克/毫升) 和消炎痛 (60 毫米)。</li>
		<li>培养由α和5% 血清组成的媒介中的阴性控制。</li>
		<li>6–9周后, 使用油红色 O 染色细胞和突出脂滴, 从而确定存在的脂肪分化33:<ol>
<li>加10% 福尔马林固定。在30分钟内, 用0.5% 油红色 O 在室温下染色10分钟, 然后每一次用60% 分异丙醇清洗细胞三次;最后, 用蒸馏水冲洗细胞。 注: 油红色 O 是一种 lysochrome (脂溶性) 重氮染料, 用于染色中性脂, 主要是三酰甘油, 脂蛋白和胆固醇酯。染料溶解于细胞中的脂滴, 使脂滴变红。</li>
		</ol>
</li>
	</ol>
</li>
	<li>对于软骨细胞分化, 在六井培养板上培养出每一个含5000细胞的井。<ol>
<li>添加含有α-记忆的分化培养基, 5% 血清, 10−7 M 地塞米松 (0.1 毫米), 丙酮酸钠 (100 µg/毫升), 胰岛素转铁蛋白-硒 a (1 x 其), 转化生长因子β (10 ng/毫升) 和5毫升抗坏血酸 (100 µM)。</li>
		<li>培养由α和5% 血清组成的媒介中的阴性控制。</li>
		<li>第4-5 周后, 使用阿利新蓝染色突出酸性多糖, 如多糖或某些类型的粘多糖, 在细胞的软骨, 以确定存在软骨素分化 33.<ol>
<li>加10% 福尔马林固定。在30分钟内, 加入3% 乙酸, 并允许2分钟的反应。随后, 用蒸馏水冲洗三次, 加1% 阿利新蓝8GX 孵化30分钟, 然后用蒸馏水冲洗。最后, 加入3% 醋酸, 清洗3分钟, 然后用蒸馏水冲洗完成。 注意: 染色后, 特定染色的细胞部分会变成蓝绿色, 被称为 "alcianophilic"。</li>
		</ol>
</li>
	</ol>
</li>
</ol>4. 25-羟维生素 D 3 (125-(OH) 2 D 3) 对成骨作用的影响 
<ol><li>将 P3 P5 PDCs 分成六组, 并在6井板上培养不同的培养基:</li>
	<li>阴性组 (α和5% 血清);</li>
	<li>维生素 c 组 (α, 5% 血清, 10 毫米β甘油, 10−7 M 地塞米松 + 100 uM 维生素 c);</li>
	<li>和 10−7 M 125-(OH)2D3组 (α记忆, 5% 血清, 10 毫米β甘油, 10−7 m 地塞米松 + 10−7 m 125-(oh)2D3)。</li>
	<li>在每个井中添加2毫升培养基到一个孵化器 (37 °c), 并每星期更换一次培养基两次。</li>
</ol>5. 反向转录/定量实时聚合酶链反应
<ol><li>在 7 d 的成骨细胞分化后, 使用商业试剂将总 RNA 分离到冰上 (参见材料表):<ol>
<li>每井加1毫升隔离剂。添加200µL 的 bromochloropropane 和离开10分钟产生分层 RNA, 然后离心机在 1万 rpm 15 分钟在4°c。</li>
		<li>离心后, 添加500µL 2 丙醇到500µL 的上清含 RNA。沉淀在冰上的 RNA, 并允许15分钟的反应。</li>
		<li>在做法以后, 离心机在1.2万转每分钟15分钟在4°c, 在之后 RNA 位于管子的底部。随后, 使用1毫升75% 醇洗涤和离心机, 在4摄氏度的8分钟内, 取出上清液。</li>
		<li>去掉上清, 用真空浓缩器从液体底部产生 RNA。用水恢复。</li>
	</ol>
</li>
	<li>用禽 myeloblastosis 病毒逆转录酶反向转录 RNA (1 微克)。设置聚合酶链反应 (PCR) 跑在25°c 为10分钟跟随50°c 为60分钟和然后85°c 为5分钟, 在最后维护在4°c 之前。</li>
	<li>合成一股互补 DNA (cDNA)。用 5 ul 1:10 稀释 cDNA 进行定量 PCR (qPCR)。用底漆为磷酸、BSP、OCN、CBFA1 和 collagen-1 进行定量 rt-pcr (qRT pcr)。 注意: 为了避免信号的 DNA 污染, 在不同的外显子上设计了每个引物的正向和反向序列。</li>
	<li>使用商业 PCR 主组合 (参见材料表) 按照制造商的说明执行 qPCR。设置热循环仪在50°c 为2分钟跟随95°c 10 分钟然后40个周期每个在95°c 十五年代跟随60°c 六十年代。 注: SYBR 协议还需要一个熔化曲线阶段, 这是运行在95°c 十五年代, 60 °c 六十年代, 然后95°c 十五年代。</li>
	<li>规范化的周期阈值的磷酸, BSP, CBFA1, collagen-1 和 OCN 的管家基因 GAPDH。34
</li>
	<li>底漆对在材料表中列出。</li>
</ol>6. 碱性磷酸酶活性
<ol><li>使用前面描述的技术35来评估细胞的碱性酶活性, 该方法将p-硝基苯基磷酸盐转换为p-硝基苯酚;p-硝基苯基磷酸酯是一种磷酸酶基质, 它在去磷酸化时呈黄色, 以 405 nm 波长确定。根据布拉德福德检测确定的总蛋白, 对形成的总的p-硝基苯酚进行规范化。</li>
	<li>比较磷酸酶活性的控制 (α, 5% 血清), 维生素 C (α-记忆, 5% 血清, 10 毫米β甘油, 10−7米地塞米松 + 100 uM 维生素 C), 125-(OH)2D3 (α记忆, 5% 血清, 10 毫米β甘油和 10−7 M地塞米松 + 10−8 M 125-(OH)2D3), 和维生素 c + 125-(OH)2D3 (α记忆, 5% 血清, 10 毫米β甘油, 10−7米地塞米松 + 100 uM 维生素 C + 125-(oh)2D3)组在1、2、3和4周的文化。</li>
	<li>在冰上执行蛋白质提取过程:<ol>
<li>从6井板上刮出细胞, 加入50µL 的溶解缓冲液。随后, 将细胞放在冰上30分钟, 以摧毁细胞并释放蛋白质。</li>
		<li>30分钟后, 离心机在 13000 rpm 4 摄氏度, 15 分钟。离心后, 提取上清液进行贮存和使用。</li>
	</ol>
</li>
</ol>

作者没有什么可透露的。

最近发展起来的治疗方式, 即组织工程所涉及的 MSCs, 有许多优点。MSCs 是存在于几种组织类型中的多能细胞, 可分化成多种功能性胚层组织细胞37和其他细胞, 如成骨体。
骨膜充当祖细胞的利基, 作为骨骼的丰富的血管供应, 它包裹着38。在我们的研究中, 34 份调查样本中, 20 成功地取得了 MSC 的菌落, 成功的隔离率为58.8%。本研究根据其增殖和?...

虽然近年来已开发了许多相关技术, 但骨重建仍然受到多种限制因素的限制, 估计必要重建的程度往往是不可能的。除了获得良好的长期成功率外, 还需要硬组织增强来实现审美和功能目标。这种手术常用的方法包括自体和同种异体骨移植、xenografting 和异体骨移植。在各种类型的骨移植中, 自体骨移植被认为是最有效的。然而, 供体部位的发病率, 受损的血管, 和有限的组织可用性,1一直是主要的缺点, 自生骨移植。另外, 同种异体骨移植和异基因移植也与疾病传播有关。目前, 人工骨移植广泛用于解决这些问题。然而, 由于缺乏成骨潜能, 临床结局也有很大差异。纤维素等材料与体积波动、感染和强度缺乏有关。
用组织工程学进行骨隆增引起了相当大的兴趣。在这种技术中, 骨髓间充质干细胞 (MSCs) 最初用于促进成骨细胞分化, 然后移植到骨质丢失部位以实现骨修复。本程序目前已应用于细胞治疗。与其他方法相比, 提取有限数量的组织来实现骨重建更简单、更少侵入。
MSCs 作为一种用于牙科再生的细胞疗法的工具的潜在作用, 是各研究小组日益关注的问题之一。研究证实, MSCs 可以区别于以下类型的组织: 骨髓, 脂肪, 滑膜, 周细胞, 小梁骨, 人脐带和牙科组织 2, 3.MSCs 的常见来源包括骨髓、脂肪组织和牙科组织。与脂肪组织和骨髓间的干细胞相比, 牙干细胞的优点是容易获得, 收获后发病率较低。与胚胎干细胞相比, 来自牙科组织的 MSCs 出现 nonimmunogenic, 与复杂的伦理问题没有关联3。
在 2006年, 国际细胞治疗学会建议使用以下标准来识别 MSCs: 首先, mscs 必须能够附着在塑料上。其次, MSCs 必须是阳性的表面抗原 CD105, CD73, CD90 和阴性的标记, 单核, 巨噬细胞, 和 B 淋巴细胞除了造血抗原 CD45 和 CD344。作为最终标准, MSCs 必须能够在体外分化的标准条件下区分成以下三种类型的细胞: 成骨细胞、脂肪细胞和软骨组织4。迄今为止, 已有六种类型的人牙干细胞被分离和表征。第一种是从人牙髓组织中分离出来的, 称为产后牙髓干细胞5。随后, 三种其他类型的牙科干细胞被隔离并被描述为: 脱落的落叶牙齿的干细胞6、牙周韧带7和顶端的乳突8。最近, 牙科卵泡衍生的9, 牙龈组织衍生的10, 口腔芽干细胞 (DBSCs)11, 和根尖囊肿 mscs (hPCy-mscs)12也已确定。
Friedenstein 是第一个定义 MSCs13的。MSCs 具有很高的增殖潜能, 可以在移植前进行区分, 这表明它们是再生过程的理想候选者10。
尽管大多数研究都用骨髓作为干细胞的来源, 但最近14也使用了骨膜衍生细胞 (PDCs)。骨膜比骨髓更容易接近。因此, 在这项技术中, 我们使用肺泡骨膜, 以消除在手术中需要额外的切口和减少手术发病率的病人。骨膜是形成长骨骼外衬的结缔组织, 包括两个不同的层: 由成纤维细胞、胶原蛋白和弹性纤维组成的外层纤维层15, 直接接触的内富细胞形成层骨表面。形成层包含混合细胞种群, 主要是成纤维细胞16, 成骨细胞17, 毛细血管18, 以及一个关键亚群, 确定为 MSCs19,20,21。大多数研究报告说, PDCs 是可比的, 如果不是优越, 骨髓源干细胞的骨骼愈合和再生22,23,24。骨膜容易接近, 具有良好的再生效果。然而, 很少有研究聚焦于骨膜25、26、27。
在骨修复方面, 目前的临床实践涉及在支持性支架内扩增的骨膜祖细胞移植。最近的研究重点是获取有缺陷区域的干细胞, 并使用祖细胞进行组织再生20。牙医还预测牙周骨再生在牙周治疗和牙种植体中的应用前景。对于供体部位, 骨膜可以很容易地收获一般牙科外科医生。这与骨髓基质细胞比较有利, 因为在常规的口腔手术中可以访问骨膜。因此, 本研究的目的是建立一个捕捞 PDCs 的协议, 并评估人骨膜干细胞的形态、附着性、活力和增殖。
维生素 D 代谢物影响体内骨-矿物质动态平衡。一项研究报告说, 24 r、25-(OH)2D3活性维生素 D 对人骨髓间充质干细胞 (hMSCs)28的成骨细胞分化至关重要。骨骼稳态和修复由维生素 D3代谢产物网络调节, 其中 125-(OH)2D3 (骨化三醇) 是最具生物学活性和相关的骨骼健康的调节。维生素 D3对于钙化29至关重要。在一项使用2维昆明白鼠的研究中, 小鼠的胚体显示维生素 C 和维生素 d 补充剂有效促进了 ESC 型成骨细胞30的分化。在它的其他生物活动中, 125-(OH)2D3刺激体外hMSCs 与成骨细胞的分化, 可以根据碱性磷酸酶 (磷酸) 酶活性或 OCN 基因的增加来监测。表达。
很少有研究发现, 与维生素 C 和 125-(OH)2D3的联合治疗在人类 PDCs 中的剂量反应关系, 特别注重骨组织工程。因此, 在本研究中, 我们检查 125-(OH)2D3和维生素 C 的单一或联合治疗的最佳浓度, 以诱导人 PDCs 的成骨分化。本议定书的目的是确定从牙槽骨膜中分离出来的细胞是否包含有 MSC 表型的细胞, 以及这些细胞是否可以在培养中被扩展 (体外) 并分化形成所需的组织。.此外, 我们评估 PDCs 的能力, 以分化为骨, 软骨细胞和脂肪。研究的第二部分评估维生素 C 和 10−10的影响, 10−9, 10−8, 和 10−7 M 125-(OH)2D3 PDCs 的成骨活动。本研究的主要目的是评估维生素 C 和 125 (OH)2D3在 PDCs 的成骨细胞分化过程中的作用, 以及支持成骨细胞的基因, 如碱性磷酸酶、collagen-1、OCN、BSP 和 CBFA1。此外, 本研究根据这些结果确定了人类 PDCs 的最佳骨诱导条件。

<table>
	<tbody>
		<tr>
			<td>0.25% trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>2-phospho-L-ascorbic acidtrisodium salt</td>
			<td>Sigma</td>
			<td>49752</td>
			<td></td>
		</tr>
		<tr>
			<td>35-mm culture dishes</td>
			<td>Corning</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr>
			<td>3-isobutyl-1-methylxanthine</td>
			<td>Sigma</td>
			<td>I5879</td>
			<td></td>
		</tr>
		<tr>
			<td>6&#160; well plate</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline phosphatase</td>
			<td>ABI</td>
			<td>Hs01029144_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase Activity Colorimetric Assay Kit</td>
			<td>BioVision</td>
			<td>K412-500</td>
			<td></td>
		</tr>
		<tr>
			<td>avian myeloblastosis virus reverse transcriptase</td>
			<td>Roche</td>
			<td>10109118001</td>
			<td></td>
		</tr>
		<tr>
			<td>CD146</td>
			<td>BD</td>
			<td>561013</td>
			<td></td>
		</tr>
		<tr>
			<td>CD19</td>
			<td>BD</td>
			<td>560994</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34</td>
			<td>BD</td>
			<td>560942</td>
			<td></td>
		</tr>
		<tr>
			<td>CD44</td>
			<td>BD</td>
			<td>561858</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45</td>
			<td>BD</td>
			<td>561088</td>
			<td></td>
		</tr>
		<tr>
			<td>CD73</td>
			<td>BD</td>
			<td>561014</td>
			<td></td>
		</tr>
		<tr>
			<td>CD90</td>
			<td>BD</td>
			<td>561974</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell banker1</td>
			<td>ZEAOAQ</td>
			<td>11888</td>
			<td></td>
		</tr>
		<tr>
			<td>core binding factor alpha-1</td>
			<td>ABI</td>
			<td>Hs00231692_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>dexamethasone</td>
			<td>Sigma</td>
			<td>D4902</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH</td>
			<td>ABI</td>
			<td>Hs99999905_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>HLA-DR</td>
			<td>BD</td>
			<td>562008</td>
			<td></td>
		</tr>
		<tr>
			<td>indomethacin</td>
			<td>Sigma</td>
			<td>I7378</td>
			<td></td>
		</tr>
		<tr>
			<td>insulin</td>
			<td>sigma</td>
			<td>91077C</td>
			<td></td>
		</tr>
		<tr>
			<td>insulin&#8211;transferrin&#8211;selenium-A</td>
			<td>Sigma</td>
			<td>I1884</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroAmp Fast 96 well reaction plate(0.1ml)</td>
			<td>Life</td>
			<td>4346907</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroAmp optical adhesive film</td>
			<td>Life</td>
			<td>4311971</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium 1X Alpha Modification</td>
			<td>HyClone</td>
			<td>SH30265.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Permeabilization buffer</td>
			<td>eBioscience</td>
			<td>00-8333-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Gibco</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>STRO-1</td>
			<td>BioLegend</td>
			<td>340103</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBER Green PCR Master Mix</td>
			<td>AppliedBiosystems</td>
			<td>4309155</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Master Mix</td>
			<td>Life</td>
			<td>4304437</td>
			<td></td>
		</tr>
		<tr>
			<td>transforming growth factor-&#946;</td>
			<td>Sigma</td>
			<td>T7039&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol reagent (for RNA isolation)</td>
			<td>Life</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate</td>
			<td>Sigma</td>
			<td>G9422</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">collagen-1</td>
			<td rowspan="2">Invitrogen</td>
			<td>forward primer 5&#39; CCTCAAGGGCTCCAACGAG-3</td>
			<td></td>
		</tr>
		<tr>
			<td>reverse primer 5&#39;-TCAATCACTGTCTTGCCCCA-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">OCN</td>
			<td rowspan="2">Invitrogen</td>
			<td>forward primer 5&#39;-GTGCAGCCTTTGTGTCCAAG-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>reverse primer 5&#39;-GTCAGCCAACTCGTCACAGT-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">BSP</td>
			<td rowspan="2">Invitrogen</td>
			<td>forward primer 5&#39; AAAGTGAGAACGGGGAACCT-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>reverse primer 5&#39;-GATGCAAAGCCAGAATGGAT-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>Commercial ALP primers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Commercial CBFA1 primers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of mesenchymal stem cells from human alveolar periosteum and effects of vitamin d on osteogenic activity of periosteum-derived cells

间充质干细胞 (MSCs) 存在于各种组织中, 可分化为多种细胞类型, 包括成骨细胞。在骨髓间充质干细胞的牙科来源中, 骨膜是一种易于接触的组织, 被确定为包含层间的 mscs。然而, 这个来源还没有得到广泛的研究。
维生素 D3和 125-(OH)2D3已被证明可以刺激体外诱导干细胞分化为成骨细胞。此外, 维生素 C 有助于胶原蛋白的形成和骨骼细胞的生长。然而, 还没有研究维生素 D3和维生素 C 对 MSCs 的影响。
本文提出了一种从人肺泡骨膜中分离 MSCs 的方法, 并对 125-(OH)2D3可能对这些细胞施加骨诱导影响的假说进行了研究。我们还研究了骨髓间充质干细胞在人肺泡骨膜中的存在, 并评估了干细胞黏附和增殖。要评估维生素 C (作为控制) 和各种浓度的 125-(OH)2D3 (10−10) 的能力, 10−9, 10−8, 10−7 M) 改变关键 mRNA 生物标志物采用实时聚合酶链反应 (rt-pcr) 测定了碱性磷酸酶、骨涎蛋白 (BSP)、核结合因子 alpha-1 (CBFA1)、collagen-1 和骨钙素 (OCN) 的分离 MSCs mRNA 表达。

对于所有的定量分析, 这些数据被显示为平均的标准偏差 (SD)。所有统计分析都是使用学生的t测试进行的。共获得34份样本, 平均参加者年龄为 48.1, 12.3 y 十一的样本是从男性患者和23从女性患者获得的。从磨牙区和六的前区获得二十八份样品;26从上颌骨和8从下颌骨获得。平均期限在牙科做法和文化之间是 0.5, 0.1 h。在34份调查样本中, 20 成功地取得了 MSC 的菌落, 成功?...

Watch this Scientific Journal Video about Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells at JoVE.com

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

人肺泡骨膜间充质干细胞的分离及维生素 D 对骨膜衍生细胞成骨活性的影响

本文提出了一种研究维生素 c (维生素 c) 和125二羟基维生素 D [125-(OH)2d3] 诱导的骨膜衍生细胞 (PDCs) mRNA 表达标志物的协议。此外, 我们评估 PDCs 的能力, 以分化为骨, 软骨细胞和脂肪。

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

isolation-mesenchymal-stem-cells-from-human-alveolar-periosteum

Research

JoVE Journal

Bioengineering

9.3K 次观看.  Chang Gung University. 本文提出了一种研究维生素 c (维生素 c) 和125二羟基维生素 D [125-(OH)2d3] 诱导的骨膜衍生细胞 (PDCs) mRNA 表达标志物的协议。此外, 我们评估 PDCs 的能力, 以分化为骨, 软骨细胞和脂肪。

视频: Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity1. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible2. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers3. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay4. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes3,4. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of &gt;50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.

Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.

从病人的血液样本可行的循环肿瘤细胞的快速分离

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

科研

生物工程

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

开发，扩展，<em&gt;在体内</em&gt;从人类胚胎干细胞（hESCs）监测人类NK细胞和诱导多能干细胞（iPS细胞）

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

优化人骨髓间充质干细胞附着在聚（ε己内酯）静电纺纱线

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

人骨髓间充质干细胞吸收含有法卡林多醇的新型脂质包覆纳米颗粒

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Construction and Use of an Electrical Stimulation Chamber for Enhancing Osteogenic Differentiation in Mesenchymal Stem/Stromal Cells In Vitro

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) has been demonstrated to increase MSC osteogenic differentiation in vitro and promote bone healing in clinical settings. Here we describe the construction of an EStim cell culture chamber and its use in treating rat bone-marrow-derived MSC to enhance osteogenic differentiation. We found that treating MSCs with EStim for 7 days results in a significant increase in the osteogenic differentiation, and importantly, this pro-osteogenic effect persists long after (7 days) EStim is discontinued. This approach of pretreating MSCs with EStim to enhance osteogenic differentiation could be used to optimize bone tissue engineering treatment outcomes and, thus, help them to achieve their full therapeutic potential. In addition to this application, this EStim cell culture chamber and protocol can also be used to investigate other EStim-sensitive cell behaviors, such as migration, proliferation, apoptosis, and scaffold attachment.

Here we present a protocol for the construction of a cell culture chamber designed to expose cells to various types of electrical stimulation, and its use in treating mesenchymal stem cells to enhance osteogenic differentiation.

电刺激室的构建与应用, 促进间充质茎间质细胞体外成骨分化

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

成纤维细胞衍生的人类工程结缔组织用于筛查应用

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded during arthroscopic procedures and knee replacement surgeries. Additionally, its removal is associated with minimal donor site morbidity. Recent studies have demonstrated that IFP-MSCs do not lose their proliferation capacity during in vitro expansion and have age-independent osteogenic differentiation potential. IFP-MSCs possess superior chondrogenic differentiation potential compared to bone marrow-derived MSCs (BMSCs) and adipose-derived stem cells (ADSCs). Although these cells are easily obtainable from aged and diseased patients, their effectiveness is limited. Hence, using IFP-MSCs from healthy donors is important to determine their efficacy in biomedical applications. As access to a healthy human donor is challenging, animal models could be a better alternative to enable fundamental understanding. Large animals such as dogs, horses, sheep, and goats play a crucial role in translational research. Amongst these, the goat could be a preferred model since the stifle joint of the goat has the closest anatomy to the human knee joint. Moreover, goat-IFP can fulfill the higher MSC numbers needed for tissue regeneration applications. Furthermore, low cost, availability, and compliance with the 3R principles for animal research make them an attractive model. This study demonstrates a simple protocol for isolating IFP-MSCs from the stifle joint of goats and in vitro culture conditions for their expansion and differentiation. The aseptically isolated IFP from the goat was washed, minced, and digested enzymatically. After filtration and centrifugation, the collected cells were cultured. These cells were adherent, had MSCs-like morphology, and demonstrated remarkable clonogenic ability. Further, they differentiated into adipogenic, chondrogenic, and osteogenic lineages, demonstrating their multipotency. In conclusion, the study demonstrates the isolation and expansion of MSCs, which show potential in tissue engineering and regenerative medicine applications.

Infrapatellar fat pad mesenchymal&#160;stem cells (IFP-MSCs) can be isolated easily from the infrapatellar fat pad of the knee joint. They proliferate well in vitro, form CFU-F colonies, and differentiate into adipogenic, chondrogenic, and osteogenic lineages. Herein, the methodology for the isolation, expansion, and differentiation of IFP-MSCs from goat stifle joint is provided.

山羊窒息关节髌下脂肪垫间充质干细胞的分离、扩增和分化

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded ...

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

In this manuscript, we demonstrate the preparation of a biohybrid hydrogel bioink containing graphene for use in peripheral tissue engineering. Using this 3D biohybrid material, the neural differentiation protocol of stem cells is performed. This can be an important step in bringing similar biomaterials to the clinic.

用于外周神经工程的石墨烯基3D生物杂化水凝胶生物墨水的制备与表征

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global ...