These studies were funded by National Medical Research Council Clinician Scientist - Individual Research Grant (CS-IRG) NMRC/CIRG/1372/2013 and NMRC EDG/0022/2008.

1.海藻酸钠溶液制备<ol><li>溶解200毫克藻酸钠（未照射），或者400毫克8兆拉德的在10ml双蒸水照射海藻酸钠振摇1小时的非辐射藻酸盐和辐照藻15分钟。过夜储存在4℃的藻酸盐溶液。滤波器海藻酸钠微珠制作之前无菌0.2微米的注射器过滤的藻酸盐溶液。 </li></ol> 2.海藻酸钠微珠制作<ol><li>消毒静电珠发生器和注射器泵用70％乙醇，并放置在一类二生物安全柜（ 图1）。 </li><li>放置一个玻璃盆珠发生器内磁力搅拌棒。 </li><li>设置珠发生器盆高于9厘米的臂电极。 </li><li>珠发生器的电极电缆连接到手臂电极的滚花螺钉2，倒加入80ml SRCL 2溶液进盆地。 </li><li>负载5毫升0.2微米过滤藻酸盐溶液到注射器和橡胶管。橡胶管连接到臂电极后，以5毫升/小时打开注射泵2分钟以排出管道内的空气，并提供藻酸盐溶液到喷嘴的前端。关掉注射器泵。 </li><li>接下来，切换对封装，然后，将注射泵开始微珠生成。设定为5毫升/小时的藻流速和在5.8千伏的封装上的电压。丢弃前两分钟（或泵出注射器的初始0.5毫升藻酸盐溶液）期间产生的微珠，因为这些微珠趋于不规则尺寸和形状。 </li><li>收集在0.2M的氯化锶溶液随后微珠。同时关闭抽藻酸盐溶液的预先计划体积后注射泵和封装器（按该顺序）。重复此微珠制作后续批次。项目建成后，转F中的注射泵第一，其次是封装。 </li><li>在4℃下储存的微珠在20毫升的0.2M氯化锶溶液过夜以完成交联和稳定的凝胶。 </li></ol> 3.海藻酸钠微球的尺寸测量<ol><li>收集0.5海藻酸钠微珠毫升用塑料移液管，并放置在载玻片上。查看在光学显微镜下微珠在10倍的放大倍率。就拿微珠的十张图片，用显微镜CCD摄像头。在2048点¯x1536的分辨率保存在TIFF格式比例尺（500微米）的图像。 </li><li>在ImageJ的使用长度工具，测量微珠和比例尺（ 图2）的大小。从像素转换微珠长度微米。 <ol><li>点击行工具和绘制穿过藻酸盐珠的中间线。 </li><li>点击菜单栏上的"分析"，然后选择"测量"。会出现一个弹出窗口。 </li><li&gt;重复步骤3.2.1-3.2.2测量图像中的所有藻酸盐珠。测量图像的比例尺。 </li><li>比例尺×500微米的胶珠/长度长度:通过使用公式转换藻酸盐珠的实际长度的直径。例如，1.420（直径ImageJ的测量）/ 2.657（由ImageJ的衡量标尺长度）×500微米= 267微米。 </li></ol></li><li>考虑100微珠的平均尺寸（平均值±标准偏差），为微珠每批代表尺寸。 </li></ol> 4.消毒<ol><li>收集使用100微米的尼龙过滤器微珠并用双蒸水洗涤珠。 </li><li>使用刮刀，传送由0.1mL藻酸盐溶液进行的所有微珠入2ml微量离心管中，用纱布覆盖，以防止干燥。 </li><li>最后，通过使用液体模式高压灭菌消毒的微珠（115℃，15分钟），或按照以manufacturer的规格。加1.5升蒸馏水到室，以防止干燥的珠子。 </li></ol> 5.鱼精蛋白和肝素涂层<ol><li>内的BSL-2罩，孵育1毫升的2毫克/毫升鱼精蛋白溶液在室温下1小时（用0.2μm注射器过滤灭菌的）无菌微珠。 </li><li>孵化微珠1小时（步骤5.1）后，收集150微升的微型二喹啉甲酸（microBCA）测试（第6）鱼精蛋白的解决方案。 </li><li>用双蒸水洗净，鱼精蛋白涂层微珠的两倍。降速使用台式离心机以200×g离心在室温下3分钟。离心后，吸用注射器水中。 </li><li>孵育鱼精蛋白涂覆的微珠用1ml的0.5毫克/毫升肝素的溶液30分钟，以创建聚电解质复合物（PEC）（使用0.2微米的注射器过滤灭菌的）。 </li><li>孵育涂鱼精蛋白微˚F后或30分钟（步骤5.4），收集400微升肝素溶液，以确定肝素含量（第7条）。 </li><li>温育后，通过用双蒸水洗涤两次洗掉从胸肌未结合的肝素。 </li></ol> 6.鱼精蛋白含量<ol><li>根据制造商的说明进行microBCA测试。简要地说，添加150微升的鱼精蛋白溶液（收集前，用微珠孵育后）到96孔板中。添加150微升microBCA工作溶液。 </li><li>使用白蛋白溶液（0,0.5，1，2，5，10，20，40和200微克/毫升）作为校准标准。 </li><li>孵育在60℃下进行60分钟该混合物。测量用在562nm处用分光光度计的吸光度。 </li><li>使用标准曲线来确定根据制造商的说明每个未知样品的鱼精蛋白的浓度。 </li><li>通过在减去鱼精蛋白的总量确定的微珠的鱼精蛋白含量从精蛋白的残留在涂布液（用微珠孵育后）的量涂布液（用微珠孵育之前）。 </li></ol> 7.肝素内容<ol><li>通过将4毫克甲苯胺蓝和在0.01N盐酸20毫克氯化钠制备10毫升工作溶液。 </li><li>添加400微升样品（来自步骤5.5），以工作溶液以2:3的比例为30秒和旋涡。 </li><li>添加600μl的正己烷（等效体积的工作试剂溶液）并涡旋混合物以提取甲苯胺蓝肝素络合物。 </li><li>抽吸将200μl的水相用注射器相分离之后。 </li><li>测量使用分光光度计在631纳米包含在水相中的未提取的甲苯胺蓝的量。 </li><li>准备0-20微克/毫升肝素标准的解决方案。 </li><li>绘制每个标准肝素对肝素浓度的631 nm的读数微克/立方米湖使用标准曲线来确定各样品的肝素浓度。 </li></ol> 8层 - 层结构的共焦成像<ol><li>制造鱼精蛋白，肝素和NELL-1 / BMP-2的荧光类似物的CF-405精蛋白（蓝色），CF 594肝素（红色）和FITC标记NELL-1 / FITC标记根据（绿色），BMP-2 +肝素+精蛋白制造商的技术数据表。 </li><li>外套100微克微珠用300μl荧光类似物（如在5.3-5.6中所述涂覆法）的CF-405精蛋白（蓝色）（2毫克/毫升，1小时温育），CF 594肝素（红色）（0.5毫克/毫升， 30分钟）和FITC标记NELL-1 / FITC标记（绿色）的BMP-2（1.5毫克/毫升，过夜）。用蒸馏水冲洗微珠两次，以消除未结合的荧光鱼精蛋白，肝素和NELL-1 / BMP-2。 </li><li>通过在10倍的放大倍率使用共焦显微镜观察该层的分层结构（ 图3）。7 </li></ol> 9. BMP-2的和NELL-1吸收和释放<ol><li>装载13.3微升1.5毫克/毫升100微克PEC的BMP-2或NELL-1解决方案。在4℃下30 rpm振摇10小时孵育PEC。 </li><li>在37℃下恒定振荡（30rpm下）沉浸在1ml磷酸盐缓冲盐水（PBS）的微珠。 </li><li>收集1毫升上清液和后1,3，6,10和14天用1ml的PBS替换。 </li><li>评估的摄取和使用根据制造商的协议ELISA方法释放的BMP-2的效率。评估的摄取和使用根据制造商的协议的羧基苯甲酰喹啉-2-甲醛（CBQCA）蛋白测定法释放NELL-1的效率。 </li><li>在时间（t）确定累积释放: 在时间（t）=释放累积释放的时间（t）+前一版本在时间（t-1）。 </li><li>绘制BMP-2和NELL-1的累积释放对时间。 </li></ol> 10. 在体外生物活性NELL-1的注:NELL-1 PEC释放的生物活性，通过测定其增加对兔骨髓干细胞（rBMSC）碱性磷酸酶（ALP）的表达能力进行评估。 <ol><li>种子每孔20,000个rBMSCs在24孔板中并允许它们在37℃和5％CO 2的胎牛血清（FBS），1毫升的Dulbecco氏改良Eagle氏培养基（DMEM）+ 10％的增长一天。 </li><li> 24小时后，更换用1ml成骨培养基（DMEM补充有10％胎牛血清，2％青霉素链霉素，50微克/毫升抗坏血酸，10毫摩尔/升的β-甘油磷酸的培养基中，10 -8 mol / L的地塞米松）7天，在37℃和5％的CO 2。 </li><li>将300微克PEC-NELL-1（步骤8.2）和PEC细胞培养插入内（TC插入），以保持胸肌从细胞（这避免了成骨细胞培养基变化期间PEC微珠的冲洗）分开。地方TC插入24以及PL吃了14天。 </li><li>每三天一次，吸将1ml成骨培养基中通过将针TC插入外，并用1ml新鲜成骨培养基的替换。 </li><li>后7和14天的潜伏期，确定与在按照试剂盒生产商的方案的ALP测定试剂盒ALP活性。 <ol><li>裂解细胞用含有0.1％的TritonX-100，在4℃下进行10分钟的测定缓冲液。刮去使用细胞刮粘附细胞​​。在4℃下孵育细胞悬浮液在搅拌下至少60分钟。 </li><li>离心细胞悬浮液以2500 xg离心在4℃下10分钟。收集的ALP测定上清。 </li><li>加入50μl连续稀释碱性磷酸酶标溶液的从200到0纳克/毫升至96孔板的孔中。碱性磷酸酶标准最终量是10，5，2.5，1.2，0.6，0.3，0.15，和0纳克/孔。 </li><li>从步骤10.5.2添加上清液（50μl/孔），并稀释与B类稀释uffer。 </li><li>加入50μl的对硝基苯磷酸酯（物pNPP）底物溶液加入到每个孔中。通过轻轻晃动板30秒混合试剂。孵育在黑暗中30分钟该混合物。测量在通过板读数器405处的吸光度。 </li><li>使用校准曲线计算碱性磷酸酶活性。 </li></ol></li><li>确定根据制造商的说明使用microBCA蛋白测定试剂盒蛋白质含量。由蛋白质含量除以碱性磷酸酶活性规范化碱性磷酸酶活性。 </li></ol> 11.细胞活力<ol><li>在37℃下孵育200毫克PEC-NELL-1用1ml的DMEM + 10％FBS的用于3-（4,5-二甲基吡啶-2-基）24小时-2,5-二苯基溴（MTT）检测。 </li><li>种子2000 rBMSCs每孔（在100μl的DMEM含10％FBS）在96孔板中并孵育1天，在37℃，5％ 的 CO 2。 </li><li>用100μlPEC-NELL-1 / PEC提取物代替DMEM + 10％FBS中孵育在37℃，5％ 的 CO 2。 </li><li>后第1天或培养3天，添加10微升5mg / ml的MTT溶液，在37℃，5％的CO 2进一步孵育在黑暗中4小时。 </li><li>加入100微升的DMSO溶液，以每孔以溶解甲晶体。 </li><li>确定在使用酶标仪570nm处的吸光度。 </li><li>计算相对增长率: <img alt="式（1）" src="/files/ftp_upload/54202/54202eq1.jpg" /></li></ol> 12.包装成支架和BMP-2 NELL-1加载<ol><li>使用BSL-2室内的灭菌抹刀磷酸三钙（MPCL-TCP）脚手架 - 包装胸肌成生物可吸收的医用级聚己内酯的孔中。 </li><li>添加1.5毫克/ BMP-2或NELL-1毫升溶液到填充有PEC的MPCL-TCP支架材料和孵育过夜，在4℃。 </li></ol>

<ol>
	<li>Yuan, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NELL-1+based+demineralized+bone+graft+promotes+rat+spine+fusion+as+compared+to+commercially+available+BMP-2+product.">NELL-1 based demineralized bone graft promotes rat spine fusion as compared to commercially available BMP-2 product.</a> Orthop Sci. 18, 646-657 (2013).</li><li>Anderson, C. L., Whitaker, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+ossification+associated+with+recombinant+human+bone+morphogenetic+protein-2+(infuse)+in+posterolateral+lumbar+spine+fusion:+a+case+report.">Heterotopic ossification associated with recombinant human bone morphogenetic protein-2 (infuse) in posterolateral lumbar spine fusion: a case report.</a> Spine. 37, 502-506 (2012).</li><li>Glassman, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RhBMP-2+versus+iliac+crest+bone+graft+for+lumbar+spine+fusion:+a+randomized,+controlled+trial+in+patients+over+sixty+years+of+age.">RhBMP-2 versus iliac crest bone graft for lumbar spine fusion: a randomized, controlled trial in patients over sixty years of age.</a> Spine. 33, 2843-2849 (2008).</li><li>Tannoury, C. A., An, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complications+with+the+use+of+bone+morphogenetic+protein+2+(BMP-2)+in+spine+surgery.">Complications with the use of bone morphogenetic protein 2 (BMP-2) in spine surgery.</a> Spine J. 14, 552-559 (2014).</li><li>Carragee, E. J., Hurwitz, E. L., Weiner, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+review+of+recombinant+human+bone+morphogenetic+protein-2+trials+in+spinal+surgery:+emerging+safety+concerns+and+lessons+learned.">A critical review of recombinant human bone morphogenetic protein-2 trials in spinal surgery: emerging safety concerns and lessons learned.</a> Spine J. 11, 471-491 (2011).</li><li>Abbah, S. A., Lam, C. X., Hutmacher, D. W., Goh, J. C., Wong, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+performance+of+a+polycaprolactone-based+scaffold+used+as+fusion+cage+device+in+a+large+animal+model+of+spinal+reconstructive+surgery.">Biological performance of a polycaprolactone-based scaffold used as fusion cage device in a large animal model of spinal reconstructive surgery.</a> Biomaterials. 30, 5086-5093 (2009).</li><li>Abbah, S. A., Liu, J., Lam, R. W., Goh, J. C., Wong, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+bioactivity+of+rhBMP-2+delivered+with+novel+polyelectrolyte+complexation+shells+assembled+on+an+alginate+microbead+core+template.">In vivo bioactivity of rhBMP-2 delivered with novel polyelectrolyte complexation shells assembled on an alginate microbead core template.</a> J. Control. Release. 162, 364-372 (2012).</li><li>Wang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyelectrolyte+Complex+Carrier+Enhances+Therapeutic+Efficiency+and+Safety+Profile+of+Bone+Morphogenetic+Protein-2+in+Porcine+Lumbar+Interbody+Fusion+Model.">Polyelectrolyte Complex Carrier Enhances Therapeutic Efficiency and Safety Profile of Bone Morphogenetic Protein-2 in Porcine Lumbar Interbody Fusion Model.</a> Spine. 40, 964-973 (2015).</li><li>Abbah, S. A., Lam, W. M., Hu, T., Goh, J., Wong, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequestration+of+rhBMP-2+into+self-assembled+polyelectrolyte+complexes+promotes+anatomic+localization+of+new+bone+in+a+porcine+model+of+spinal+reconstructive+surgery.">Sequestration of rhBMP-2 into self-assembled polyelectrolyte complexes promotes anatomic localization of new bone in a porcine model of spinal reconstructive surgery.</a> Tissue Eng. Part A. 20, 1679-1688 (2014).</li><li>Hu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Protamine-Based+Polyelectrolyte+Carrier+Enhances+Low-Dose+rhBMP-2+in+Posterolateral+Spinal+Fusion.">Novel Protamine-Based Polyelectrolyte Carrier Enhances Low-Dose rhBMP-2 in Posterolateral Spinal Fusion.</a> Spine. 40, 613-621 (2015).</li><li>Hu, J., Hou, Y., Park, H., Lee, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-tricalcium+phosphate+particles+as+a+controlled+release+carrier+of+osteogenic+proteins+for+bone+tissue+engineering.">Beta-tricalcium phosphate particles as a controlled release carrier of osteogenic proteins for bone tissue engineering.</a> J Biomed Mater Res A. 100, 1680-1686 (2012).</li><li>Darrabie, M. D., Kendall, W. F., Opara, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+Poly-L-Ornithine-coated+alginate+microcapsules.">Characteristics of Poly-L-Ornithine-coated alginate microcapsules.</a> Biomaterials. 26, 6846-6852 (2005).</li><li>Li, X., Min, S., Zhao, X., Lu, Z., Jin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+entrapping+conditions+to+improve+the+release+of+BMP-2+from+PELA+carriers+by+response+surface+methodology.">Optimization of entrapping conditions to improve the release of BMP-2 from PELA carriers by response surface methodology.</a> Biomed Mater. 10, 015002 (2015).</li></ol>

我们没有任何利益冲突。

该协议提出了通过层 - 层自组装制备的PEC的方法。的层 - 层结构，使用鱼精蛋白，肝素，BMP-2和NELL-1和共聚焦显微镜的荧光类似物可视化。吸收和释放试验表明，肝素对PEC介导成骨生长因子的吸收和释放。在PEC方法的摄取效率是:NELL-1:86.7±2.7％，BMP-2:70.5±3.1％。在PEC载体具有NELL-1（20％）释放的更好的调制相比，纯表面吸附载体如钙磷灰石颗粒（40-80％）11。 除了 ​​?...

假的发病率已经报道高达10〜45％，在退化性脊柱融合和修订脊柱手术1。以减少脊柱融合和其他重建骨手术，骨生长因子如BMP-2，内尔-1 1和血小板衍生的生长因子（PDGF）已被引入，以促进从头成骨过程中假关节的速度。其中，BMP-2是脊柱融合2的热门选择。虽然BMP-2的诱导和促进新骨形成的效力已被很好地建立3;临床显著并发症，如异位骨形成，血清肿和血肿形成，炎症反应，神经根，椎体骨溶解和逆行射精继续关注的问题，由于使用的4,5超生理金额。 因此，降低的BMP-2的剂量保持在一个相关的策略引诱最小化的副作用。此外，需要有效的载体系统来抑制BMP-2在当代胶原海绵载波系统观察到的突释，并进一步加强这种有效的细胞因子的长期和本地化交货。交替阳离子和阴离子聚电解质的层-层自组装，可以用作建立脚手架基质或植入材料6的表面上的聚电解质复合物的可调谐方法。在这方面，肝素（对于具有所有生物剂的最高负电荷密度已知）已被识别为贪婪地通过静电和肝素结合结构域的多种生长因子结合。的确，肝素已经显示出延长的半衰期，从而使可能的多种生长因子的生物活性。 在此基础上，我们的组适于层 - 层自组装协议来制造基于肝素的聚电解质复合物（PEC），该<html>负载和固定7,8中保留了成骨生长因子的生物活性。藻酸盐微球芯由交联的α-L-古洛糖醛酸盐（G），用二价阳离子钙或锶离子藻酸盐的残基制成。藻酸盐核心是可生物降解的支架基质;该植入后，它是在融合床提供余地骨向内生长吸收。聚-L-赖氨酸（PLL）或鱼精蛋白被用作阳离子层与两个支架矩阵（在这种情况下，藻微珠载体芯）和带负电的肝素隔行;而阴离子肝素层的功能，以稳定和定位加载生长因子。三重层的PEC已经显示出增加的生长因子负载能力在猪模型9。最近，PEC载体已显示出至少20倍的成功减少在大鼠10和脊骨融合8的猪模型的BMP-2的有效剂量。 ntent"&gt;在这里，用BMP-2作为模型骨生长因子，我们报告脊柱融合增强生长因子交付和其他骨重建手术制造佩奇方法。

<table><tbody><tr><td>Life Science Acrodisc 25&amp;nbsp;mm Syring Filter with 0.2&amp;nbsp;&amp;micro;m Supor Membrane</td><td>PALL&amp;nbsp;</td><td>PN4612</td><td>Sterile protamine, heparin solution by ultrafiltration</td></tr><tr><td>24 well plate</td><td>Cell Star&amp;nbsp;</td><td>662160</td><td></td></tr><tr><td>96 well plate Nuclon Delta Surface</td><td>Thermo Fisher Scientific</td><td>167008</td><td></td></tr><tr><td>(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide), MTT</td><td>Sigma Aldrich</td><td>M5655</td><td>Measure cytotoxicity of PEC-NELL-1</td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>A/0600/17</td><td>Precipitate CF-405&lt;br/&gt; Labeled protamine</td></tr><tr><td>Alamar Blue</td><td>Invitrogen, Life Technologies</td><td>DAL 1025</td><td>Measure cytotoxicity of PEC-BMP-2</td></tr><tr><td>Alkaline Phosphatase Assay (ALP) assay kit</td><td>Anaspec</td><td>AS-72146</td><td></td></tr><tr><td>Ammonium Chloride</td><td>Merck</td><td>Art 1145</td><td>Stop reagent in FITC labeling</td></tr><tr><td>Anhydrous Dimethyl Sulfoxide (DMSO)</td><td>Invitrogen, Life Technologies</td><td>D12345</td><td>Solvent for fluorescent isothiocyanate I</td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma Aldrich</td><td></td><td>Dissolve&amp;nbsp; formazan&amp;nbsp;</td></tr><tr><td>Autoclave</td><td>Hirayama</td><td>HU-110</td><td>Sterilize alginate beads by steam</td></tr><tr><td>Beta-glycerophosphate</td><td>Sigma Aldrich</td><td>G9422</td><td></td></tr><tr><td>BMP-2 (Infuse Bone Graft Large II Kit)&amp;nbsp;</td><td>Medtronic Sofarmor Danek, Memphis TN, USA</td><td>7510800</td><td>Osteogenic Growth Factor, dialysis is needed to remove stabilizer component that interferes with FITC coupling</td></tr><tr><td>Carboxybenzoyl quinoline-2-Carboxaldehyde (CBQCA)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A-6222</td><td>To quantify NELL-1 protein</td></tr><tr><td>Cell Strainer (100&amp;nbsp;&amp;micro;m)</td><td>BD Science</td><td>352360</td><td>Hold PEC for ALP assay</td></tr><tr><td>Cell Scraper 290&amp;nbsp;mm Bladewide 20&amp;nbsp;mm</td><td>SPL Life Science</td><td>90030</td><td>Detach the cell from 24 well plate</td></tr><tr><td>CF 405S, Succinimidyl Ester</td><td>Sigma Aldrich</td><td>SCJ4600013</td><td>Blue fluorescent dye for protamine labeling</td></tr><tr><td>CF 594, Hydrazide</td><td>Sigma Aldrich</td><td>SCJ4600031</td><td>Deep red fluorescent dye for heparin labeling</td></tr><tr><td>Centrifuge</td><td>Beckman Coulter</td><td>Microfuge 22R</td><td></td></tr><tr><td>Confocal Microscope</td><td>Olympus&amp;nbsp;</td><td>FV1000</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma Aldrich</td><td>D4902</td><td>Component of osteogenic growth medium</td></tr><tr><td>Dextran Desalting Columns</td><td>Pierce (Thermo Scientific)&amp;nbsp;</td><td>43230</td><td></td></tr><tr><td>DMEM</td><td>Gibco&amp;nbsp;</td><td>12320</td><td></td></tr><tr><td>BMP-2 Quantikine ELISA Kit</td><td>R&amp;amp;D System</td><td>DBP200</td><td>Determine BMP-2 release</td></tr><tr><td>Fetal Bovine Serum FBS</td><td>Hyclone</td><td>SV30160.03</td><td></td></tr><tr><td>Fluoescein Isothiocyananate, Isomer I</td><td>Sigma Aldrich</td><td>F7250</td><td>Green fluorescent dye for NELL-1 and BMP-2 labeling</td></tr><tr><td>ThinCert Cell Culture Inserts,&lt;br/&gt; For 24 Well plates, Sterile</td><td>Greiner&amp;nbsp;</td><td>662630</td><td>Prevents PEC wash out when changing osteogenic medium</td></tr><tr><td>Havard Appartus Syringe Pump (11 plus)</td><td>Havard Apparatus</td><td>70-2208</td><td></td></tr><tr><td>n-Hexane (&gt;99%)</td><td>Sigma Aldrich</td><td>139386</td><td></td></tr><tr><td>Heparin</td><td>Sigma Aldrich</td><td>H3149</td><td>Binds with osteogenic&lt;br/&gt; growth factor with heparin binding domain</td></tr><tr><td>Hydrochloric acid (37%)</td><td>Merck</td><td>100317</td><td>Highly Corrosive</td></tr><tr><td>Incubator</td><td>Binder</td><td>C8150</td><td></td></tr><tr><td>MicroBCA Protein Assay kit</td><td>Thermoscientific</td><td>23235</td><td></td></tr><tr><td>Microplate Reader</td><td>Tecan</td><td>Infinite M200</td><td>For ALP and microBCA assays</td></tr><tr><td>Nisco cell encapsulator</td><td>Nisco Engineering Inc</td><td>Encapsulation unit VAR V1</td><td></td></tr><tr><td>Fluorescent Microscope</td><td>Olympus</td><td>IX71</td><td></td></tr><tr><td>mPCL-TCP Scaffold (Pore size is 1.3&amp;nbsp;mm)</td><td>Osteopore</td><td>PCL-TCP 0/90</td><td>Hold PEC for &lt;em&gt;in vivo&lt;/em&gt; study</td></tr><tr><td>Penicillin-Streptomycin 10,000 unit/ml, 100&amp;nbsp;ml</td><td>Hyclone Cell Culture</td><td>SV30010</td><td>Antibiotic</td></tr><tr><td>10x Phosphate Buffered Saline (PBS)</td><td>Vivantis</td><td>PB0344-1L</td><td>10x Solution, Ultra Pure Grade</td></tr><tr><td>Poly-L-Lysine MW 15,000-30,000</td><td>Sigma Aldrich</td><td>P2568</td><td>Polycation</td></tr><tr><td>Protamine Sulfate salt, from Salmon</td><td>Sigma Aldrich</td><td>P4020</td><td>Polycation</td></tr><tr><td>Shaker</td><td>Labnet</td><td>S2025</td><td></td></tr><tr><td>Snakeskin Dialysis Tubing 3,500 MWCO 22&amp;nbsp;mm x 35 feet</td><td>Thermo Fisher Scientific</td><td>68035</td><td>Remove unreacted FITC by dialysis</td></tr><tr><td>Sodium Chloride</td><td>Merck</td><td>1.06404.1000</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Qrec</td><td>S5158</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>US Biological</td><td>S4000</td><td>Buffer</td></tr><tr><td>Sodium carbonate</td><td>Sigma Aldrich</td><td>S7795-500G</td><td>Buffer</td></tr><tr><td>Strontium Chloride Hexahydrate</td><td>Sigma Aldrich</td><td>255521</td><td>Crosslinker for alginate</td></tr><tr><td>Spatula</td><td>3dia</td><td></td><td></td></tr><tr><td>5 ml syringe</td><td>Terumo</td><td>140425R</td><td>Diameter of syringe affects the flow rate</td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; Cell Culture Flask Canted Neck</td><td>Corning</td><td>730720</td><td></td></tr><tr><td>Toluidine Blue</td><td>Sigma Aldrich</td><td>52040</td><td>Heparin assay</td></tr><tr><td>Trypsin 1x</td><td>Hyclone Cell Culture</td><td>SH30042.01</td><td></td></tr><tr><td>Sodium alginate</td><td>Novamatrix (FMC Biopolymer, Princeton, NJ)</td><td>Pronova UPMVG</td><td>Core material of microbeads</td></tr></tbody></table>

polyelectrolyte complex for heparin binding domain osteogenic growth factor delivery

在骨重建手术，生长因子超生理金额经验装上支架，以促进成功的骨融合。大剂量的高度有效的生物制剂的需要，由于生长因子的不稳定性如快速酶降解的结果，以及在植入物位点定位足够量的生长因子的载体的低效率。因此，该延长的生长因子的稳定性，如BMP-2 / NELL-1，并控制其释放策略实际上​​可以降低其有效剂量，从而减少了需要更大的剂量期间未来骨再生手术。反过来，这将减少副作用和生长因子的成本。自组装胸肌已经制造通过增强的体内稳定性通过肝素结合，并进一步使可能生长因子的生物活性提供的BMP-2 / NELL-1递送的更好的控制。在这里，我们说明了PEC制造这有助于在国内配送简单的过程中重建骨手术的各种生长因子RY。

在我们的载体，鱼精蛋白被选为聚-L-赖氨酸的替代，因为它有类似的化学性质，它是FDA批准作为肝素的解毒剂。光学显微镜结果表明，非照射微珠呈球状的形状，直径为267±14微米。 （0.35毫米喷嘴，流量为5ml /小时和5.8千伏率）。大部分照射微珠是泪滴形状。上照射微珠的轮部测得的直径为212±30微米（0.35毫米喷嘴，流速为4毫升/小时和6千伏率）。 （ 图4）。 ...

Watch this Scientific Journal Video about Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery at JoVE.com

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

聚电解质复合物肝素结合域成骨生长因子交货

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

polyelectrolyte-complex-for-heparin-binding-domain-osteogenic-growth

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

7.5K 次观看.  National University of Singapore. Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

视频: Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

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 ...

科研

化学

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

保利的微波辅助功能化（乙二醇）和On-树脂链聚合反应，并形成水凝胶的肽使用

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

静电生长因子释放到微纤维支架

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

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 ...

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.

Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

界面聚电解质纤维的生物分子在时间控释复合支架

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial ...

Assembly and Characterization of Polyelectrolyte Complex Micelles

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful platform for exploring the physics of polyelectrolyte interactions and also offer a promising solution to the pressing problem of delivering therapeutic oligonucleotides in vivo. Developing predictive structure-property relationships for PCMs has proven difficult, in part due to the presence of strong kinetic traps during nanoparticle self-assembly. This article discusses criteria for choosing polymers for PCM construction and provides protocols based on salt annealing that enable assembly of repeatable, low-polydispersity nanoparticles. We also discuss PCM characterization using light scattering, small-angle X-ray scattering, and electron microscopy.

We provide protocols and representative data for designing, assembling, and characterizing polyelectrolyte complex micelles, core-shell nanoparticles formed by polyelectrolytes and hydrophilic charged-uncharged block copolymers.

聚电解质复合微晶体的组装与特性

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

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

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

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.

This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.

促进FACS纯化的胸腺上皮细胞的3-D聚合，EAK 16-II / EAKIIH6自组装凝胶

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing ...

免疫与感染

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

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

Biomaterials doped with Bone Morphogenetic Protein 2 (BMP2) have been used as a new therapeutic strategy to heal non-union bone fractures. To overcome side effects resulting from an uncontrollable release of the factor, we propose a new strategy to site-directly immobilize the factor, thus creating materials with improved osteogenic capabilities.

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 ...

3D 共培养检测:用于癌症药物筛选的血管化成骨生态位

Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models recapitulating bone- and bone marrow-specific functions, including vascularization, that are compatible with drug screening are highly desirable. Such models can bridge the gap between simplistic, structurally irrelevant two-dimensional (2D) in vitro models and the more expensive, ethically challenging in vivo models. This article describes a controllable three-dimensional (3D) co-culture assay based on engineered poly(ethylene glycol) (PEG) matrices for the generation of vascularized, osteogenic bone-marrow niches. The PEG matrix design allows the development of 3D cell cultures through a simple cell seeding step requiring no encapsulation, thus enabling the development of complex co-culture systems. Furthermore, the matrices are transparent and pre-cast onto glass-bottom 96-well imaging plates, rendering the system suitable for microscopy. For the assay described here, human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) are cultured first until a sufficiently developed 3D cell network is formed. Subsequently, GFP-expressing human umbilical vein endothelial cells (HUVECs) are added. The culture development is followed by bright-field and fluorescence microscopy. The presence of the hBM-MSC network supports the formation of vascular-like structures that otherwise&#160;would not form and that remain stable for at least 7 days. The extent of vascular-like network formation can easily be quantified. This model can be tuned toward an osteogenic bone-marrow niche by supplementing the culture medium with bone morphogenetic protein 2 (BMP-2), which promotes the osteogenic differentiation of the hBM-MSCs, as assessed by increased alkaline phosphatase (ALP) activity at day 4 and day 7 of co-culture. This cellular model can be used as a platform for culturing various cancer cells and studying how they interact with bone- and bone marrow-specific vascular niches. Moreover, it is suitable for automation and high-content analyses, meaning it would enable cancer drug screening under highly reproducible culture conditions.

作者聚焦: 在成像微孔板中使用预制聚乙二醇 （PEG） 水凝胶简单建立血管化成骨骨髓生态位  

An in vitro model of the bone marrow vascular niche is established by seeding mesenchymal and endothelial cells onto pre-cast 3D PEG hydrogels. The endothelial networks, ECM components, and ALP activity of the niches vary depending on the growth factor used. The platform can be used for advanced cancer models.

在成像微孔板中使用预制聚乙二醇（PEG）水凝胶简单建立血管化成骨骨髓壁龛

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models ...