Name: site-directed immobilization of bone morphogenetic protein 2 to solid surfaces by click chemistry
Uploaded: 2018-03-29T13:00:00.000+00:00
Duration: 11 min 20 s
Description: Watch this Scientific Journal Video about Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry at JoVE.com

作者感谢 m. Rubini 博士 (德国 Konstanz) 提供质粒编码 pyrrolysyl tRNA 和提供 pRSFduet pyrtRNAsynth 编码相应的氨酰 tRNA 合成酶。

1. BMP2 变型 BMP2-K3Plk 的生产
<ol><li>利用 PCR 方法对 BMP2-K3Plk 进行现场定向诱变克隆研究12<ol>
<li>用正向底漆 (5 ' GACCAGGACATATGGCTCAAGCCTAGCACAAACAGC 3 ') 和反向底漆 (5 ' CCAGGAGGATCCTTAGCGACACCCACAACCCT 3 '), 从 p25N-hmBMP2 矢量放大人类成熟 BMP2 (hmBMP2) (参见材料表), 引入琥珀色停止密码子 (标记) 在 BMP2´s 成熟部分的第一赖氨酸的位置。使用 H2O 的33.5 µL 进行 PCR 反应, 10 µL PCR 反应混合, 1.5 µL 10 µM dNTP 库存溶液, 1.5 µL 的前底漆 (10 pmol/µL), 1.5 µL 的反向底漆 (10 pmol/µL), 1 µL p25N-hmBMP2 (10 ng/µL)和1µL DNA 聚合酶 (参见材料表) 在热循环仪 (变性在95°c 为5分钟, 30 周期变性在95°c 为1分钟, 退火在60°c 为1分钟, 伸长在72°c 为1分钟和最后的引伸在72°c 为10分钟)。</li>
		<li>使用制造商推荐的商用工具包纯化 PCR 产品 (请参阅材料表)。</li>
		<li>用16µL H2O、3µL 消化缓冲液、10µL DNA (20 NdeI/µL) 和1µL 的限制酶, 以限制酶为45分钟消化 PCR 产物。热停用65°c 的酶5分钟. 用16µL h2O、3µL 消化缓冲液、10µL DNA (20 BamHI/µL) 和1µL 的限制酶, 用限制酶在37摄氏度上消化1小时的 PCR 产物。热停用酶80°c 5 分钟。</li>
		<li>使用16µL h2O、3µL 消化缓冲、10µL 的 DNA (20 ng/µL) 和1µL 的限制酶, 将 pET11a-pyrtRNA 向量与 NdeI 在37摄氏度的2小时和45分钟内消化。热停用65°c 为5分钟. 用 BamHI 37 °c 的 pET11a-pyrtRNA 向量消化 1 h, 使用16µL h2O, 3 µL 消化缓冲, 10 µL DNA (20 ng/µL), 1 µL 限制酶。热停用80摄氏度, 5 分钟。</li>
		<li>用琼脂糖凝胶电泳 (0.8% 琼脂糖, 60 分钟 100 V) 分离未消化的载体和消化残留物的消化载体。结扎在室温 (RT) BMP2-K3TAG 中消化 pET11a-pyrtRNA 骨干, 用100吴 pET11a-pyrtRNA 骨干和 34.5 ng BMP2-K3TAG 插入 (1:3 摩尔比) 进行消化后的插入。反应混合物包含 dna 骨干和插入, 2 µL 连接酶缓冲器, 1 µL 的 dna 连接酶,H2 O 到总容积20µL。</li>
		<li>在 BL21 (DE3) 细菌中进行 pET11a-pyrtRNA-BMP2-K3Plk 和 pSRF-pyrtRNAsynth 的共同转化:<ol>
<li>将每种质粒的 50 ng 添加到细菌中, 将混合物放在冰上30分钟, 热休克为42摄氏度, 五十年代, 在冰上放置5分钟, 在混合物中加入500µL 的溶源性汤 (磅) 培养基, 并在300分钟内摇动60分钟。</li>
			<li>将100µL 的细菌混合物涂在预热的卡那霉素 (50 µg/毫升)/氨苄西林 (100 µg/毫升) 板上, 并在37摄氏度一夜之间孵化。</li>
		</ol>
</li>
	</ol></li>
	<li>BMP2-K3Plk 的表达和纯化13,14<ol>
<li>在50毫升的 LB 培养基中以50µg/毫升卡那霉素和100µg/毫升氨苄西林在37摄氏度的情况下, 在一夜之间繁殖一只蜂群。</li>
		<li>稀释隔夜文化1:20 到800毫升的了不起的肉汤 (TB) 培养基补充50µg/毫升卡那霉素和100µg/毫升氨苄西林, 并增长37°c, 直到达到 OD600 0.7。添加炔丙基赖氨酸到最终浓度为10毫米。在异丙基β-D-1-thiogalactopyranoside (IPTG) 诱导前, 从培养基中收集100µL 样品。</li>
		<li>通过添加 IPTG 诱导基因表达, 最终浓度为1毫米。在 180 rpm 的轨道振动筛中, 以16小时为37摄氏度的培养。从文化中收集100µL 样本。</li>
		<li>离心机的整个文化在 9000 x g 30 分钟丢弃上清, 并并用重悬细菌颗粒在30毫升的 TBSE 缓冲 (10 毫米三, 150 毫米氯化钠, 1 毫米乙二胺乙酸 (EDTA)) 与 1:1000 (v/v) 2-巯基乙醇 (新鲜添加)。 注意事项: 在通风罩下处理2巯基乙醇。避免接触皮肤和眼睛。避免吸入蒸汽或薄雾。使用包含 2-巯基乙醇的缓冲器在通风罩下执行所有泥沙步骤。</li>
		<li>称空离心机烧杯并将悬浮颗粒转移到空烧杯中。离心机在 6360 x g 为20分钟, 丢弃上清液, 用颗粒称量烧杯。减去空烧杯的重量来计算颗粒的重量。 注意: 协议可以在这里暂停。在短期内或在-80 摄氏度的长期冷冻颗粒在-20 °c。恢复该协议后, 在 RT 中解冻颗粒。</li>
		<li>并用重悬中的颗粒 (10 毫米三 pH 8.0; 150 毫米氯化钠; 1 毫米 EDTA, 375 毫米蔗糖; 1:1000 (v/v) 2-巯基乙醇 (新鲜添加))。每10克球团使用200毫升的圣凯瑟缓冲器。</li>
		<li>油脂实验在冰上悬浮 (10 分钟以四十年代脉冲, 二十年代刹车和30% 振幅)。离心机在 6360 x g 为20分钟, 丢弃上清液并称量颗粒。重复超声波和离心步骤4次。</li>
		<li>并用重悬100毫升的 tb 缓冲器 (10 毫米三, 150 毫米氯化钠) 的颗粒。离心机在 6360 x g 为20分钟, 丢弃上清和重粒。</li>
		<li>并用重悬颗粒在核酸酶缓冲器 (100 毫米三, 1 毫米 EDTA, 3毫米氯化镁 2) 与新鲜添加 80 U/毫升核酸酶 (例如, Benzonase) 使用10毫升/克丸。在搅拌板 (30 rpm) 上在 RT 上孵化过夜。</li>
		<li>添加海卫四缓冲器 (60 毫米 EDTA, 1.5 米氯化钠, 6% (v/v) 4-(11, 33-Tetramethylbutyl) 苯基聚乙二醇) 悬浮。增加的海卫的缓冲容量对应于悬浮的0.5 容量部分。在 RT 中孵育10分钟 (不搅拌)。离心机在 6360 x g 为20分钟, 丢弃上清液并称量颗粒。</li>
		<li>并用重悬颗粒在 TE 缓冲器 (100 毫米三, 20 毫米 EDTA) 使用8毫升/克颗粒。离心机在 6360 x g 为20分钟, 丢弃上清液并称量颗粒。</li>
		<li>并用重悬颗粒加入4毫升/克25毫米 NaAc pH 5.0 和5毫升/克6米 GuCl 与1毫米 dithiothreitol (新添加)。在搅拌板 (30 rpm) 上, 在4°c 处孵化过夜。</li>
		<li>离心机在 75500 x g 20 分钟。上清液现在包含展开的单体 BMP2 蛋白。收集上清, 并把这个提取物浓缩到 20 OD/毫升使用 3 kDa 分子量切断 (截留分子量) 膜在浓缩细胞 (见材料表)。</li>
		<li>将浓缩单体加入复缓冲液 (2 米 LiCl, 50 毫米三, 25 毫米, 5 毫米 EDTA, 1 毫米谷胱甘肽 (GSSG), 2 毫米谷胱甘肽 (GSH)) 搅拌 (30 rpm)。在黑暗中在 RT 中孵化120小时。 注意: 在此潜伏期内, 复 occours。从这个步骤开始的解决方案包含折叠二聚体 BMP2-K3Plk。</li>
		<li>使用浓缩的 hcl 将溶液的 pH 值调整为 3.0. 透析解决方案对1毫米 hcl. 用 10 kDa 分子量切断 (截留分子量) 膜在浓缩细胞中浓缩透析溶液。</li>
		<li>通过添加缓冲 A (20 毫米 NaAc, 30% 异丙醇) 平衡解决方案。添加与解决方案的30% 卷对应的缓冲区容量。离心溶液在 4700 x g 15 分钟。</li>
		<li>平衡在快速蛋白质液相色谱 (FPLC) 系统上进行离子交换的柱状体15与150毫升缓冲器 a. 将蛋白质溶液加载到平衡柱上。使用100毫升的缓冲器 b (20 毫米 NaAc, 30% 异丙醇, 2 米氯化钠) 的线性梯度洗脱分数 b. 收集2毫升的分数。 注: 根据总柱容积将蛋白质按入列前浓缩。最后的蛋白质体积应 < 总柱容积的5%。</li>
		<li>通过 sds 聚丙烯酰胺凝胶 (12%) 电泳 (sds 页) 16和考马斯亮蓝染色17 (参见材料表) 分析了每一个分数的20µL。 注: 考马斯亮蓝色染色 SDS 页凝胶显示含有脂肪酸 (26 kDa) 和含有单体的分数 (13 kDa) 的分数。</li>
		<li>池中含有二聚体的分数。透析含馏分的二聚体池, 隔夜在4°c 对1毫米 HCl (5 公升容量)。使用 10 kDa 浓缩离心过滤装置集中最终产品 (请参阅材料表)。用 sds 聚丙烯酰胺 (12%) 凝胶电泳 (sds 页) 和考马斯蓝染色法对最终产物进行分析。 注: 考马斯亮蓝染色的 SDS 页凝胶上的带子应该只显示一个波段在 26 kDa。这是最后的 BMP2-K3Plk 产品。</li>
	</ol></li>
</ol>2. 优化铜 (I)-催化炔烃-叠氮化物 Cycloaddition (CuAAC) 条件
<ol><li>抗坏血酸钠 (NaAsc) 和铜 (II) 硫酸盐 (丘索4 )对野生型 BMP2 (BMP2-WT)的影响<ol>
<li>孵化20µM BMP2-WT 与 NaAsc 的不同比率丘索4。在50µL 卷中使用 0.5 mm NaAsc 和 0.5 mm 丘索4 (起始浓度)。继续以下 NaAsc 到丘索4: (1:1), (1.7: 1), (10:1), (20: 1), 保持丘索4浓度常数在0.5 毫米和调整 NaAsc 的浓度相应。在 RT 上一夜之间在旋转搅拌机 (20 rpm) 上执行 H2O 的反应。</li>
		<li>通过添加含有 5% 2-巯基乙醇和孵育样品的载入缓冲剂, 在95摄氏度进行6分钟, 制备减少的样品. 用 sds 聚丙烯酰胺凝胶 (12%) 电泳 (sds 页) 和考马斯蓝染色法分析减少和不减少的样品。Coomasie 染色的 SDS 页凝胶显示带在 13 kDa (减少条件) 到 26 kDa 和更高的分子量 (不减少的条件) 的范围。</li>
	</ol>
</li>
	<li>
三 (3-hydroxypropyltriazolylmethyl) 胺 (THPTA) 对 CuAAC 反应的影响
	<ol>
<li>孵化20µM BMP2-K3Plk 与 THPTA 的不同比率丘索4。使用5毫米 NaAsc 和200µM 3-叠氮基 7-hydroxycoumarin (保持 NaAsc 和 3-叠氮基-7-hydroxycoumarin 常数)。使用50µM THPTA 和0.5 毫米的丘索4作为起始浓度。使用 THPTA 与丘索的不同比率4: (7:1);(10:1);(12:1);(15:1);(20:1). 在 H2O (50 µL 总容积) 中对旋转混合器 (20 rpm) 上的 RT 执行 24 h 的反应。</li>
		<li>在联轴器上, 3-叠氮基-7-hydroxycoumarin 成为荧光染料。在减少和不减少的条件下准备样品, 并通过 SDS 页面进行分析。用特定波长为 3-叠氮基-7-hydroxycoumarin (λ abs = 404 毫微米;λ em = 477 nm) 在激发通道下可视化凝胶。</li>
	</ol>
</li>
</ol>3. BMP2-K3Plk 对叠氮化物功能性琼脂糖珠的共价耦合技术
<ol><li>孵育20µM 的 BMP2-K3Plk 或 BMP2-WT (用作负控制) 与20µL 的叠氮化物活化琼脂糖珠在总容积500µL 的反应缓冲 (0.1 米 HEPES pH 7.0, 3.9 米尿素, 50 µM 丘索4, 250 µM THPTA, 5 毫米抗坏血酸钠) 为 2 h 在 RT 在 rotating 搅拌机 (20 rpm)。通过添加5毫米 EDTA (最终浓度) 停止反应。</li>
	<li>在旋转搅拌机上孵育15分钟 (20 rpm)。离心机样品在 2万 x g 为1分钟在 RT. 收集上清液。</li>
	<li>用三倍于哈佛商学院500缓冲器 (50 毫米 HEPES, 500 毫米 NaCl), 三倍, 1000 µL 4 米氯化镁2和两次与1000µL 磷酸盐缓冲盐水 (PBS) 清洗含有珠子的球团, µL 1000。在每一个洗涤步骤, 离心机在 2万 x g 1 分钟的 RT 和收集上清液。在1000µL 的 PBS 中存储耦合珠, 4 摄氏度。 注意: 清除上清液时请勿扰乱珠粒。</li>
</ol>4. 使用德州红标记的 BMP 受体 I A 胞外区 ( BMPR欧共体) 验证固定化 BMP2-K3Plk 的存在和生物活性
<ol><li>孵化50µL 的 BMP2-K3Plk 功能性珠子与1µM 德州红标记 BMPR IAEC与150µL 的哈佛商学院500缓冲器 (50 毫米 HEPES, 500 毫米 NaCl) 在 RT 为 1 h 在旋转搅拌机 (20 rpm)。</li>
	<li>将珠子离心 2万 x 克, 在 RT 上1分钟. 丢弃上清。用1000µL 的哈佛商学院500缓冲区来清洗珠子。重复洗涤步骤额外的3次。每洗一步后, 2万 x 克离心机在 RT 1 分钟。</li>
	<li>并用重悬在500µL 的 PBS 和吸管50µL 在玻璃封面幻灯片的珠子。将显微镜过滤器设置在561或 594 nm 之间。检测德州 Red–BMPREC-BMP2-K3Plk 功能化珠子使用荧光显微镜和拍照。</li>
</ol>5. 测定碱性磷酸酶 (磷酸) 表达式, 以证明在偶联反应前后产生的 BMP2-K3Plk 的体外生物活性。
<ol><li>
碱性磷酸酶 (ALP) 测定法
	<ol>
<li>培养 promyoblastic C2C12 细胞 (ATCC CRL-172) 在 Dulbecco 的改良鹰的培养基 (DMEM) 与10% 胎小牛血清 (FCS), 100 U/毫升青霉素 G 和100µg/毫升链霉素在37°c 在湿润气氛 5% CO2。</li>
		<li>种子 C2C12 细胞在密度为 3 x 104细胞/以及到96井微板块。让细胞整夜附着。在 100 CO100 37 h 的湿润气氛中, 去除培养基和孵化 C2C12 细胞, 在100µL/井 DMEM (2% FCS, 5% U/毫升青霉素 G 和2µg/毫升链霉素) 存在 0.5-200 nM BMP2-WT 或 BMP2-K3Plk 在72摄氏度。</li>
		<li>用100µL 的 PBS 去除培养基和洗涤单元。溶解细胞在 RT 1 小时与100µL/井溶解缓冲 (1% NP-40, 0.1 米甘氨酸, pH 9.6, 1 毫米氯化镁2, 1 毫米 ZnCl2) 在震动板 (220 rpm)。</li>
		<li>添加100µL/井的磷酸酶缓冲 (0.1 米甘氨酸, pH 9.6, 1 毫米氯化镁2, 1 毫米 ZnCl2) 与1毫克/毫升 p 硝基苯磷酸 (新鲜添加) 到细胞裂解。</li>
		<li>测量吸收 405 nm 在 multiplate 读取器添加的碱性磷酸酶缓冲, 并每5分钟, 直到颜色的发展完成。使用逻辑模型生成剂量响应曲线和 EC50 值, y = a2 + (12)/(1 + (x/x0)p)。</li>
	</ol>
</li>
	<li>
碱性磷酸酶 (磷酸) 染色
	<ol>
<li>培养 promyoblastic C2C12 细胞 (ATCC CRL-172) 在 Dulbecco 的改良鹰培养基 (DMEM) 与10% 胎小牛血清 (FCS), 100 U/毫升青霉素 G 和100µg/毫升链霉素在37°c 的湿润气氛在 5% CO2。</li>
		<li>种子 C2C12 细胞在密度为 3 x 104细胞/以及在96井微板块。让细胞整夜附着。移除介质并添加20µL/井 BMP2-K3Plk 耦合珠。BMP2-K3Plk 耦合珠是在1000年µL 的 PBS 悬浮。</li>
		<li>在 DMEM 中制备了0.4% 低熔点琼脂糖溶液。熔化在微波 (600 W 为3分钟) 并且让它凉快在水浴在37°c 直到使用。</li>
		<li>在每个井中添加20µL 0.4% 低熔点琼脂糖 (覆盖珠和细胞)。离心机在 2000 x g 5 分钟在20摄氏度。添加80µL DMEM (2% FCS, 100 U/毫升青霉素 G 和100µg/毫升链霉素) 和孵化在37摄氏度的湿润气氛在 5% CO2 72 h。</li>
		<li>去除培养基, 小心不要分离凝固的0.4% 琼脂糖。添加100µL/井1步 NBT/BCIP (硝基蓝四氮唑氯化物, 5-溴-4-氯-3 '-indolyphosphate 对甲苯胺盐) 基质溶液。</li>
		<li>在紫色染色后立即使用光镜分析碱性磷酸酶染色。使用明亮的现场显微镜和拍照。</li>
	</ol>
</li>
</ol>

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作者声明没有竞争的财政利益。

通过遗传密码子扩展生成标记的蛋白质变体允许在主要蛋白质序列的任何位置引入各种非天然氨基酸类似物。如果像 BMP2 这样的 BMPs, 常见的标签, 如 6-组氨酸 (他) 的标签只能被引入 n-末期, 因为 protein´s 的 C 末端是埋在三级蛋白质结构, 因此不能从外部进入。在其他位置, 引入标签的大小很可能会导致结构的改变, 从而抹杀 BMP´s 生物活性。此外, 引入突变的 BMP2 序列可以影响复性的功效, 也可以改变其他蛋白质参数, 如等电点的修饰蛋白。因此, 建立的蛋白质生产协议中的每一步都可能需要根据改变的蛋白质 variant´s 特性进行调整。BMP2-K3Plk 的生产确实需要修改已建立的方法来表达野生型 BMP2。对不同的培养时间或炔丙基赖氨酸 (保良局朱正贤) 浓度进行了测试 (未显示数据), 以达到最高的表达率。复用, 分离和纯化步骤幸运地不需要进一步 adaptions。我们已经报告, 如果在我们的实验室生产的其他 BMP2 变种, 一些蛋白质特性发生了显著变化, 这需要修改 BMP 生产方法的几个步骤18。所生产的 BMP2-K3Plk 显示生物活性与野生型蛋白相当。差异发生在高 BMP2 浓度, 可能是由于较低的溶解度的 BMP2-K3Plk 变异在培养基相比, 野生型 BMP2。
尽管铜催化叠氮化物-炔烃 cycloaddition (CuAAC)19的已知优势, CuAAC 反应应小心地适应特定的应用。我们展示了 BMP2 是如何被分散或产生聚集或多聚体由于强烈的还原反应条件。因此, 所有反应参数必须详细分析, 以找出条件, 使蛋白质不受影响。
我们的方法, 以共价键夫妇的 BMP2 变异叠氮化物功能性琼脂糖珠通过点击化学可以实现高效, 使功能性珠触发成骨分化在体外。将野生型 BMP2 蛋白用于与珠子的反应时, 只能观察到碱性磷酸酶表达的弱染色, 表明偶联在 BMP2-K3Plk 的炔丙基噬菌体残留物中确实发生了高度的特异性。
这种位置耦合特异性反映了一个伟大的改善与固定程序涉及经典的 NHS/化学。在这种情况下, 发生随机耦合, 主要涉及赖氨酸残留物中存在的主要胺类。由于可能的连接的多样性与支架, 导致蛋白质的不同的取向对细胞感受器官, 被结合的蛋白质有一个可变的成骨活动。
使用现场直接固定化 BMP2 可能克服与高剂量可溶性 BMP2 有关的缺点, 这是需要诱导骨形成。此外, 结果清楚地表明, 某些细胞反应, 如 C2C12 细胞的成骨分化, 是完全由固定化的 BMP2 启动, 尽管最近的研究声称, 信号转导需要吞的配体/配体-受体复合体20,21。我们的研究结果与其他研究相一致, 证明 BMP2 (但不是以站点为导向) 到非 endocytosable 表面, 仍然能够诱导成骨细胞分化为22。
考虑到在偶联反应中使用了 BMP2 的超生理浓度, 可进一步降低固定化 BMP2 的用量, 同时仍能保存珠的成骨性质。然而, 在这种优化步骤之前, BMP2-K3Plk-functionalized 脚手架需要在动物实验中进行测试, 以证明其成骨潜能在体内。

骨组织工程和骨再生的最终目的是克服常规治疗非愈合骨折的缺点和局限性。自动或异体移植主要用作当前的治疗策略, 尽管它们都有几个缺点。理想的植骨应由诱导成骨和 osteoconduction 诱导成骨, 导致移植 osteointegration 骨。目前, 只有自动移植被认为是 "金标准", 因为它提供了理想的骨移植的所有特点。不幸的是, 它还提出了一些重要的负面方面, 例如长时间的手术时间和第二个创伤部位, 通常需要更多的并发症 (例如, 慢性疼痛, 血肿形成, 感染, 美容缺陷,等等)。同种异体移植, 另一方面具有所有一般方面的次优特征1。在过去几年中, 替代植骨技术得到了改善, 目的是生产骨诱导、osteoconductive、生物相容性和 bioresorbable 的支架。由于许多生物材料不显示所有这些成骨特征, 不同的生长因子, 主要 BMP2 和 BMP7, 已被纳入, 以改善其成骨潜能的特殊支架2。
作为一个基本的标准, 这种生长因子传递系统应提供一个控制剂量释放一段时间, 以促进必要的事件, 如细胞的招募和依恋, 细胞肉芽和血管生成。然而, BMPs 和其他成骨生长因子通常是固定的非共价键的3。诱捕和吸附技术需要使用超生理量的蛋白质, 由于最初的爆裂释放, 导致严重的缺点在体内,通常影响周围组织通过诱导骨过度增长,骨溶解, 肿胀和炎症4。因此, 可通过共价固定化方法, 在交付部位保留较长时间的生长因子。经过化学修饰的 BMP2 (琥珀酰化5、乙酰6 或生物素化 7)、工程化 heterodimers 8 或BMP2派生寡肽9已设计并用于克服与吸收.然而, 这些构造的生物活性是不可预知的, 因为这种安排有可能抑制固定配体对细胞受体的束缚。如前所示, 所有四受体链参与形成激活配体-受体配合物与固定化 BMP2 互动, 以充分激活所有下游信号瀑布10。
为了克服由于固定化因子的生物活性、稳定性和生物利用度等因素而产生的不均匀产品结果的问题, 我们设计了一种能以站点为导向的共价键结合支架的 BMP 变体。这个变种称为 BMP2-K3Plk, 包括由遗传密码子扩展11引入的人工氨基酸。该变种已成功地链接到脚手架使用共价键耦合战略, 同时保持其生物活性。

<table><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1-Step NBT/BCIP</td><td>Thermo Fisher</td><td>34042</td><td>Add solution to cells</td></tr><tr><td>3-Azido-7-hydroxycoumarin</td><td>BaseClick</td><td>BCFA-047-1</td><td>Chemical used for click reaction</td></tr><tr><td>Agarose low melting point</td><td>Biozym</td><td>840101</td><td>Agarose for ALP assay&amp;nbsp;</td></tr><tr><td>Azide agarose beads</td><td>Jena Bioscience</td><td>CLK-1038-2</td><td>Beads used for reaction</td></tr><tr><td>BamHI (Fast Digest enzyme)</td><td>Thermo Fisher Scientific</td><td>FD0054</td><td>Restriction enzyme</td></tr><tr><td>BMP receptor IA (BMPR-IA&lt;sub&gt;EC)&lt;/sub&gt;</td><td>--</td><td>--</td><td>Produced in our lab</td></tr><tr><td>Coomassie Brilliant Blue G-250 Dye</td><td>Thermo Fisher Scientific</td><td>20279</td><td>Chemical used for Coomassie Brilliant blue staining of SDS PAGE</td></tr><tr><td>Copper (II) sulfate anhydrous (CuSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Alfa Aesar</td><td>A13986</td><td>Chemical used for click reaction</td></tr><tr><td>DNA Polymerase and reaction buffer&amp;nbsp;</td><td>Kapabiosystems</td><td>KK2102</td><td>KAPA HiFi PCR Kit</td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM) GlutaMAX</td><td>Gibco</td><td>61965-026</td><td>Cell culture media</td></tr><tr><td>ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma Aldrich GmbH</td><td>E5134-1kg</td><td>Chemical used to stop click reaction</td></tr><tr><td>Isopropyl &amp;szlig;-D-1-thiogalactopyranoside (IPTG)</td><td>Carl Roth GmbH</td><td>2316.5</td><td>Bacteria induction (1mM final concentration)&amp;nbsp;</td></tr><tr><td>NdeI (Fast Digest enzyme)</td><td>Thermo Fisher Scientific</td><td>ER0581</td><td>Restriction enzyme</td></tr><tr><td>NHS-activated Texas Red</td><td>Life technologies</td><td>T6134</td><td>Coupled to receptor</td></tr><tr><td>P- Nitrophenyl Phosphate</td><td>Sigma Aldrich GmbH</td><td>N4645-1G</td><td>Alkaline Phosphatase</td></tr><tr><td>p25N-hmBMP2&amp;nbsp;</td><td>--</td><td>--</td><td>Plasmid kindly provided from Walter Sebald to J. Nickel</td></tr><tr><td>pET11a-pyrtRNA</td><td>--</td><td>--</td><td>Provided by the Chair for Pharmaceutics and Biopharmacy, University Wuerzburg</td></tr><tr><td>propargyl-L-lysine (Plk)</td><td>--</td><td>--</td><td>Provided by the Chair for Pharmaceutics and Biopharmacy, University Wuerzburg</td></tr><tr><td>pSRFduet-pyrtRNAsynth</td><td>--</td><td>--</td><td>Provided by the Chair for Pharmaceutics and Biopharmacy, University Wuerzburg</td></tr><tr><td>Qiagen Gel Extraction Kit</td><td>Qiagen</td><td>28704</td><td>Gel Purification</td></tr><tr><td>Qiagen PCR purification Kit</td><td>Qiagen</td><td>28104</td><td>PCR Purification&amp;nbsp;</td></tr><tr><td>Sodium L-ascorbate</td><td>Sigma Aldrich GmbH</td><td>A7631-100G</td><td>Chemical used for click reaction</td></tr><tr><td>T4 DNA Ligase</td><td>ThermoScientific</td><td>EL0011</td><td>Ligation&amp;nbsp;</td></tr><tr><td>tris(3-hydroxypropyltriazolylmethyl)amine (THPTA)</td><td>BaseClick</td><td>BCMI-006-100</td><td>Chemical used for click reaction</td></tr><tr><td>4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol</td><td>Sigma Aldrich GmbH</td><td>X100-1L</td><td>Triton X 100&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Amicon concentrating cell 400 ml&amp;nbsp;</td><td>Merck KGaA</td><td>UFSC40001</td><td>Concentrating unit</td></tr><tr><td>Amicon Ultra-15 Centrifugal Filter Units</td><td>Merck KGaA</td><td>UFC901024</td><td>Concentrating centrifugal unit</td></tr><tr><td>&amp;Auml;KTA avant FPLC</td><td>&amp;Auml;KTA</td><td>--</td><td>FPLC machine</td></tr><tr><td>Avanti J-26XP</td><td>Beckman Coulter&amp;nbsp;</td><td>393124</td><td>Centrifuge for bacterial culture</td></tr><tr><td>Bacterial Shaking Incubator</td><td>Infors HT</td><td></td><td>Shaking incubator for bacterial culture</td></tr><tr><td>FluorChem Q system</td><td>proteinsimple</td><td>--</td><td>Imaging and analysis system for SDS-PAGE</td></tr><tr><td>Fluorescent miscroscope</td><td>Keyence</td><td>BZ-9000 (BIOREVO)</td><td></td></tr><tr><td>Fractogel&amp;reg; EMD SO&lt;sub&gt;3&lt;/sub&gt;&lt;sup&gt;-&lt;/sup&gt; (M)</td><td>Merck KGaA</td><td>116882</td><td>Ion Exchange Chromatography column material</td></tr><tr><td>Greiner CELLSTAR&amp;reg;&amp;nbsp;96 well plates</td><td>Sigma</td><td>M5811-40EA</td><td>96 well plates for cell culture (ALP Assay)</td></tr><tr><td>Heraeus Multifuge X1R</td><td>ThermoScientific</td><td>--</td><td>Centrifuge</td></tr><tr><td>M-20 Microplate Swinging Bucket Rotor</td><td>ThermoScientific</td><td>75003624</td><td>Rotor for Microcentrifuge for plate during ALP staining</td></tr><tr><td>Microcentrifuge - 5417R</td><td>Eppendorf</td><td>--</td><td>Centrifuge</td></tr><tr><td>OriginPro 9.1 G&amp;nbsp;</td><td>OriginLab</td><td>--</td><td>software for stastic analysis of ALP assay data</td></tr><tr><td>Polysine Slides</td><td>ThermoScientific</td><td>10143265</td><td>microscope slides</td></tr><tr><td>Rotor JA-10</td><td>Beckman Coulter&amp;nbsp;</td><td>--</td><td>rotor for Avanti J-26XP centrifuge</td></tr><tr><td>Rotor JLA 8.1</td><td>Beckman Coulter&amp;nbsp;</td><td>--</td><td>rotor for Avanti J-26XP centrifuge</td></tr><tr><td>Rotor JA 25.50</td><td>Beckman Coulter&amp;nbsp;</td><td>--</td><td>rotor for Avanti J-26XP centrifuge</td></tr><tr><td>Tecan infinite M200 multiplate reader</td><td>Tecan Deutschland GmbH</td><td>--</td><td>Multiplate reader for ALP assay</td></tr><tr><td>Thermocycler - Labcycler Gradient</td><td>SensoQuest GmbH</td><td>--</td><td>PCR</td></tr><tr><td>TxRed - microscope filter</td><td>Keyence</td><td></td><td>Filter for fluorescent microscope&amp;nbsp;</td></tr><tr><td>Ultrafiltration regenerated cellulose discs 3 kDa</td><td>Merck KGaA</td><td>PLBC04310</td><td>used with amicon concentrating cell 400ml</td></tr><tr><td>Ultrafiltration regenerated cellulose discs 10 kDa</td><td>Merck KGaA</td><td>PLGC04310</td><td>used with amicon concentrating cell 400ml</td></tr></tbody></table>

site-directed immobilization of bone morphogenetic protein 2 to solid surfaces by click chemistry

深入探讨了不同治疗方法对骨缺损不愈合的处理策略。目前使用的治疗存在一些限制, 导致使用生物材料结合成骨生长因子, 如骨形态发生蛋白 (BMPs)。通常使用的吸收或封装方法需要超生理量的 BMP2, 通常导致一种所谓的初始爆裂释放效应, 引发了一些严重的不良副作用。解决这些问题的可能策略是将蛋白质与支架进行共价键。此外, 耦合应以特定于站点的方式进行, 以保证产品的可重现性。因此, 我们创造了一个 BMP2 变种, 其中一个人工氨基酸 (炔丙基赖氨酸) 引入到成熟的部分 BMP2 蛋白由密码子使用扩展 (BMP2-K3Plk)。BMP2-K3Plk 通过铜催化叠氮化物-炔烃 cycloaddition (CuAAC) 耦合到功能化珠。结合 BMP2-K3Plk 的生物活性被证实为体外, BMP2-K3Plk-functionalized 珠的成骨活性在细胞基检测中得到证实。与 C2C12 细胞接触的功能性珠子能够在局部受限的珠子附近诱导碱性磷酸酶 (ALP) 的表达。因此, 通过这种技术, 可以产生功能化的支架, 可以触发细胞分化为成骨的血统。另外, 由于现场定向耦合 BMP2 的控制方向, 降低 BMP2 剂量是足够的。用这种方法, BMPs 总是暴露在细胞表面的适当方向的受体, 这不是情况下, 如果通过非现场定向耦合技术耦合因素。产品的结果是高度可控的, 从而导致材料的均匀性, 提高其适用性, 修复严重大小骨缺损。

在本文中, 我们描述了一种方法, 以共价键夫妇一个新的 BMP2 变体, BMP2-K3Plk, 商业上可利用的叠氮化物功能琼脂糖珠 (图 1)。通过诱导 C2C12 细胞碱性磷酸酶 (ALP) 基因表达, 验证了 BMP2-K3Plk 变异的生物活性。体外测试显示了由野生类型 BMP2 (BMP2-WT) 和 BMP2-K3Plk (图 2) 引起的类似的 ALP 表达式级别。
铜 (II.) 硫酸盐 (丘索4) 和抗坏血酸钠 (NaAsc) 之间的氧化还原反应产生的活性氧种类可能影响结构完整性, 从而影响 BMP2-K3Plk 的生物活性。为了验证蛋白质的结构完整性, 我们进行了一夜孵化与丘索4和 NaAsc, 显示 BMP2-WT 退化以浓度依赖性方式 (由 SDS 页在减少的情况下形象化 (图 3A1)) 和在大约 40 kDa (在不减少的条件下) 可见的多聚体或聚合体的形成(图 3A 2).为避免蛋白质降解, 三 (3-hydroxypropyltriazolyl 甲基) 胺 (THPTA) 被用作保护剂 (图 3B)。使用 THPTA 防止 BMP2 破碎, 而更高分子量结构的形成不能通过添加 THPTA 来防止。反应的进一步改善处理了反应缓冲物的构成, 反应温度和反应时间 (没有显示的数据)。此处描述的协议详细介绍了反应缓冲成分、温度和反应时间等方面的最终成果。
为了证实 BMP2-K3Plk 在偶联后保留生物活性, 我们使用了 I 型受体的荧光标记受体胞外区蛋白 (BMPRIAEC) 来证明固定化蛋白仍能与此受体结合。用染料标记的 BMPREC (图 4) 孵化时, 通过 CuAAC 化学 BMP2-K3Plk 的珠子产生了荧光。与此相反, 与 BMP2-WT 一起孵化的未涂覆的珠子或珠子没有显示出背景水平以上的荧光信号 (未显示数据)14。
在确认固定配体的受体结合能力在体外, 我们还测试了生物反应是否可以触发的功能性珠在细胞的检测。碱性磷酸酶介导的染色仅发生在与 BMP2-K3Plk-functionalized 珠直接接触的细胞中 (图 5)。这证实了蛋白质确实是共价键链接到珠, 而不是仅仅吸收, 因为更广泛的染色, 在更大的距离, 珠子会被观察到。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56616/56616fig1.jpg"> 图 1: BMP2-K3Plk 的描述 (A)BMP2-K3Plk 变异的描述与介绍的氨基酸替换的本地化。(B)耦合方案: BMP2-K3Plk CuAAC 与叠氮化物功能性珠子的反应。转载 (改编), 并允许从 Tabisz et al。(2017): 定点固定 BMP-2: 两种生产创新骨诱导支架的方法。大分子.18 (3), 695-708。(版权所有2017美国化学学会)<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56616/56616fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56616/56616fig2.jpg"> 图 2: BMP2-variants 的生物活性.BMP2-K3Plk 与 wildtype BMP2 (BMP2-WT) 生物活性 (磷酸酶测定) 的比较。x 轴表示测定中使用的 BMP2-WT 或 BMP2-K3Plk 的浓度。EC50 数据代表4个单独实验 (N=4) 的平均值和标准偏差。转载 (改编), 并允许从 Tabisz et al。(2017): 定点固定 BMP-2: 两种生产创新骨诱导支架的方法。大分子.18 (3), 695-708。(版权所有2017美国化学学会)<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56616/56616fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56616/56616fig3.jpg"> 图 3: 还原剂对 BMP2 完整性的影响。(A) BMP2-WT 暴露于抗坏血酸钠 (NaAsc) 与铜 (II) 硫酸盐 (丘索4) 的不同摩尔比率。通过 SDS 页和考马斯亮蓝染色 (图 A1) 和非还原条件 (图 A2) 对样品进行了分析。在减少的条件 (A1) 中, 红色箭头表示 BMP2-WT。在非还原条件 (A2) 中, 红色箭头表示 multimeric BMP2-WT。表示 BMP2 的条纹的带由蓝色箭头指示。图例: NCR -减少了 BMP2-WT 未经处理的情况;数控 BMP2-WT 未经处理;1:1 至 20:1-提高抗坏血酸钠的摩尔比值为丘索4。(B) BMP2-K3Plk 被耦合到 3-叠氮基-7-hydroxycoumarin, 使用 CuAAC 反应条件补充 THPTA。当耦合 3-叠氮基-7-hydroxycoumarin 成为一个荧光染料。图例: NC BMP2-WT 与 3-叠氮基-7-hydroxycoumarin 反应;7:1 至 20:1, THPTA 的摩尔比率增加到丘索4。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56616/56616fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56616/56616fig4.jpg"> 图 4: 固定化 BMP2-K3Plk 的体外生物活性.固定化 BMP2-K3Plk 与德州红标记 BMPREC相互作用的代表性图片。转载 (改编), 并允许从 Tabisz et al。(2017): 定点固定 BMP-2: 两种生产创新骨诱导支架的方法。大分子.18 (3), 695-708。(版权所有2017美国化学学会)<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56616/56616fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56616/56616fig5.jpg"> 图 5: 固定化 BMP2-K3Plk 在基于细胞的检测中的生物活性。(A)碱性磷酸酶 (磷酸) 染色的代表性图片, 用珠子与 BMP2-K3Plk 功能性珠子结合处理。(B)碱性磷酸酶 (ALP) 染色治疗时可溶性 25 nM BMP2-K3Plk. 转载 (改编), 允许从 Tabisz et al。(2017): 定点固定 BMP-2: 两种生产创新骨诱导支架的方法。大分子.18 (3), 695-708。(版权所有2017美国化学学会)<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56616/56616fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry at JoVE.com

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

用骨形态发生蛋白 2 (BMP2) 掺杂的生物材料作为治疗非愈合性骨折的新方法。为了克服因无法控制的释放因素而产生的副作用, 我们提出了一个新的策略, 以现场直接固定的因素, 从而创造材料, 改善成骨能力。

点化学法现场定向固定骨形态发生蛋白2到固体表面

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present ...

site-directed-immobilization-bone-morphogenetic-protein-2-to-solid

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

7.5K 次观看.  Institutsteil Würzburg. 用骨形态发生蛋白 2 (BMP2) 掺杂的生物材料作为治疗非愈合性骨折的新方法。为了克服因无法控制的释放因素而产生的副作用, 我们提出了一个新的策略, 以现场直接固定的因素, 从而创造材料, 改善成骨能力。

视频: Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

Methods for rapid surface immobilization of bioactive small molecules with control over orientation and immobilization density are highly desirable for biosensor and microarray applications. In this Study, we use a highly efficient covalent bioorthogonal [4+2] cycloaddition reaction between trans-cyclooctene (TCO) and 1,2,4,5-tetrazine (Tz) to enable the microfluidic immobilization of TCO/Tz-derivatized molecules. We monitor the process in real-time under continuous flow conditions using surface plasmon resonance (SPR). To enable reversible immobilization and extend the experimental range of the sensor surface, we combine a non-covalent antigen-antibody capture component with the cycloaddition reaction. By alternately presenting TCO or Tz moieties to the sensor surface, multiple capture-cycloaddition processes are now possible on one sensor surface for on-chip assembly and interaction studies of a variety of multi-component structures. We illustrate this method with two different immobilization experiments on a biosensor chip; a small molecule, AP1497 that binds FK506-binding protein 12 (FKBP12); and the same small molecule as part of an immobilized and in situ-functionalized nanoparticle.

We present a method for rapid, reversible immobilization of small molecules and functionalized nanoparticle assemblies for Surface Plasmon Resonance (SPR) studies, using sequential on-chip bioorthogonal cycloaddition chemistry and antibody-antigen capture.

微流控芯片上捕获环加成反应可逆地固定小分子或多组分结构的生物传感器应用

Methods  for rapid surface immobilization of bioactive small molecules with control over  orientation and immobilization density are highly desirable for ...

科研

化学

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.

We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell membrane.

BMP-2的共价结合利用表面上的自组装单层膜法

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development.1,2,3 Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction.4,5 Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible.1,2
The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically1 or analytically,2,6 as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness,1,7 (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access.1,2,7
In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N3) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only.
In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein &#x2013; strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.

Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification.

通过Bioorthogonal点击化学化学选择性病毒表面改性

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic ...

免疫与感染

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-&#181;CP) and Sequential Nucleophilic Substitution

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates presenting grafted poly(ethylene glycol) (PEG) brushes can be used to achieve this task by creating microscale, non-fouling and cell adhesion resistant regions as well as regions where cells participate in biospecific interactions with covalently tethered ligands. To engineer complex tissues using such substrates, it will be necessary to sequentially pattern multiple PEG brushes functionalized to confer differential bioactivities and aligned in microscale orientations that mimic in vivo niches. Microcontact printing (&#956;CP) is a versatile technique to pattern such grafted PEG brushes, but manual &#956;CP cannot be performed with microscale precision. Thus, we combined advanced robotics with soft-lithography techniques and emerging surface chemistry reactions to develop a robotic microcontact printing (R-&#956;CP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal &#8216;click&#8217; chemistries. Here, we describe in detail the workflow to manufacture such substrates.

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &#181;CP and Sequential Nucleophilic Substitution

Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.

制造复杂的栽培基质使用机器人微接触印刷（R-μCP）和顺序亲核取代

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates ...

生物工程

Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not occur. With conventional templates, the absence of internal micro-channels results in the lack of cell infiltration, distribution, and inhabitance deep inside the templates. Hence, a highly porous and uniformly interconnected trabecular-bone-like template with micro-channels (biogenic microenvironment template; BMT) has been developed to address these obstacles. The novel BMT was created by innovative concepts (capillary action) and fabricated with a sponge-template coating technique. The BMT consists of several structural components: inter-connected primary-pores (300-400 &#181;m) that mimic pores in trabecular bone, micro-channels (25-70 &#181;m) within each trabecula, and nanopores (100-400 nm) on the surface to allow cells to anchor. Moreover, the BMT has been documented by mechanical test study to have similar mechanical strength properties to those of human trabecular bone (~3.8 MPa)12.
The BMT exhibited high absorption, retention, and habitation of cells throughout the bridge-shaped (&#928;) templates (3 cm height and 4 cm length). The cells that were initially seeded into one end of the templates immediately mobilized to the other end (10 cm distance) by capillary action of the BMT on the cell media. After 4 hr, the cells homogenously occupied the entire BMT and exhibited normal cellular behavior. The capillary action accounted for the infiltration of the cells suspended in the media and the distribution (active migration) throughout the BMT. Having observed these capabilities of the BMT, we project that BMTs will absorb bone marrow cells, growth factors, and nutrients from the periphery under physiological conditions.
The BMT may resolve current limitations via rapid infiltration, homogenous distribution and inhabitance of cells in large, volumetric templates to repair massive skeletal defects.

A step-by-step generic process to create a bone-like template with engineered micro-channels is presented. High absorption and retention capabilities of the template are demonstrated by capillary action via micro-channels.

独特的毛细管作用通过微通道骨般的模板可以增强细胞的招募大骨缺损修复

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not ...

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

表面固定生物分子的单分子荧光检测的自动化系统

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

生物学

Design, Synthesis, and Photochemical Properties of Clickable Caged Compounds

Caged compounds enable the photo-mediated manipulation of the cell physiology with high spatiotemporal resolution. However, the limited structural diversity of currently available caging groups and the difficulties in synthetic modification without sacrificing their photolysis efficiencies are obstacles to expanding the repertoire of caged compounds for live cell applications. As the chemical modification of coumarin-type photo-caging groups is a promising approach for the preparation of caged compounds with diverse physical and chemical properties, we report a method for the synthesis of clickable caged compounds that can be modified easily with various functional units via the copper(I)-catalyzed Huisgen cyclization. The modular platform molecule contains a (6-bromo-7-hydroxycoumarin-4-yl)methyl (Bhc) group as a photo-caging group, which exhibits a high photolysis efficiency compared to those of the conventional 2-nitrobenzyls. General procedures for the preparation of clickable caged compounds containing amines, alcohols, and carboxylates are presented. Additional properties such as the water solubility and cell targeting ability can be readily incorporated into clickable caged compounds. Furthermore, the physical and photochemical properties, including the photolysis quantum yield, were measured and were found to be superior to those of the corresponding Bhc caged compounds. The described protocol could therefore be considered a potential solution for the lack of structural diversity in the available caged compounds.

A protocol for the synthesis and measurement of the photochemical properties of modular caged compounds with clickable moieties is presented.

可点击笼体化合物的设计、合成和光化学特性

Caged compounds enable the photo-mediated manipulation of the cell physiology with high spatiotemporal resolution. However, the limited structural ...

Optimized Methods for the Surface Immobilization of Collagens and Collagen Binding Assays

Fibrosis occurs in various tissues as a reparative response to injury or damage. If excessive, however, fibrosis can lead to tissue scarring and organ failure, which is associated with high morbidity and mortality. Collagen is a key driver of fibrosis, with type I and type III collagen being the primary types involved in many fibrotic diseases. Unlike conventional protocols used to immobilize other proteins (e.g., elastin, albumin, fibronectin, etc.), comprehensive protocols to reproducibly immobilize different types of collagens in order to produce stable coatings are not readily available. Immobilizing collagen is surprisingly challenging because multiple experimental conditions may affect the efficiency of immobilization, including the type of collagen, the pH, the temperature, and the type of microplate used. Here, a detailed protocol to reproducibly immobilize and quantify type I and III collagens resulting in&#160;stable and reproducible gels/films is provided. Furthermore, this work demonstrates how to perform, analyze, and interpret in vitro time-resolved fluorescence binding studies to investigate the interactions between collagens and candidate collagen-binding compounds (e.g., a peptide conjugated to a metal chelate carrying, for example, europium [Eu(III)]). Such an approach can be universally applied to various biomedical applications, including&#160;the field of molecular imaging to develop targeted imaging probes, drug development, cell toxicity studies, cell proliferation studies, and immunoassays.

This work presents an optimized protocol to reproducibly immobilize and quantify type I and III collagen onto microplates, followed by an improved in vitro binding assay protocol to study collagen-compound interactions using a time-resolved fluorescence method. The subsequent step-by-step data analysis and data interpretation are provided.

Fibrosis occurs in various tissues as a reparative response to injury or damage. If excessive, however, fibrosis can lead to tissue scarring and organ ...

生物化学

Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects on bone repair in a unicortical defect model. Treatment of mesenchymal cells produced an increase in alkaline phosphatase activity, showed nodule formation by the cells, and increased the expression of genes for runx2, osterix, bone sialoprotein, and osteocalcin. A collagen sponge soaked with peptide promoted repair of bone defects, whereas the control was less effective. The results from this study demonstrated that mesenchymal cells treated with peptide in vitro differentiate towards osteogenesis, and, that peptides delivered in vivo using a collagen sponge promote the repair of unicortical defects.

A phage display library was used to identify peptide sequences that target bone. The objective was to investigate the effect of these peptides on mesenchymal cell differentiation and to determine their effect on bone regeneration.

从噬菌体抗体库的肽调节间质细胞的基因表达和使可能在Unicortical骨缺损的成骨

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects ...

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

基于多步法链位移光刻的基因功能表面固定化研究

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic ...