C.D.M. was supported by the American College of Surgeons (ACS) Resident Research Scholarship. M.S.H. was supported by the California Institute for Regenerative Medicine (CIRM) Clinical Fellow training grant TG2-01159. M.S.H., H.P.L., and M.T.L. were supported by the American Society of Maxillofacial Surgeons (ASMS)/Maxillofacial Surgeons Foundation (MSF) Research Grant Award. H.P.L. was supported by NIH grant R01 GM087609 and a gift from Ingrid Lai and Bill Shu in honor of Anthony Shu. H.P.L. and M.T.L. were supported by the Hagey Laboratory for Pediatric Regenerative Medicine and The Oak Foundation. M.T.L. was supported by NIH grants U01 HL099776, R01 DE021683-01, and RC2 DE020771. D.C.W. was supported by NIH grant 1K08DE024269, the Hagey Laboratory for Pediatric Regenerative Medicine, and the Stanford University Child Health Research Institute Faculty Scholar Award.

注:样品从谁给了知情同意书的患者。所有协议进行了审查，并通过适当的斯坦福大学的机构审查委员会批准。虽然处理人组织和细胞，始终坚持以生物安全2级（BSL2）的预防措施，如您的机构指定。 1.试剂的制备<ol><li>制备FACS缓冲液:添加10毫升胎牛血清，5毫升泊洛沙姆188和5mL青霉素 - 链霉素至500mL无菌磷酸盐缓冲盐水（PBS）。 </li><li>准备消化混合物添加0.375克C. hemolyticum胶原酶II型粉和5毫升泊洛沙姆188到500毫升的无菌199 / EBSS网上平台。 </li><li>制备标准培养基:添加50毫升胎牛血清和5mL青霉素 - 链霉素至500mL Dulbecco改良的Eagle培养基。 </li></ol> 2.采收和携带者的分离注:请确保有足够的机构审批已经到位了使用人体组织和隔离ħUMAN干细胞。获得人的腹部，胁肋，或从健康捐赠者择期吸脂大腿的皮下脂肪。保持脂肪在吸塑罐。 <ol><li> 1的比例的脂肪在原胶罐:在1中添加无菌PBS。 （如果有500毫升脂肪，加入500毫升的PBS）。通过温和搅拌30秒混合。允许将水层沉降至底部（ 图1），然后吸出并弃去水层用附连到抽吸10毫升的塑料吸管。 </li><li>倒出胖成一个大的塑料容器中，加入在1混合消化:1的比例。关闭容器并清洗，用70％的乙醇的外部。密封用石蜡膜盖。 </li><li>以180rpm在轨道摇动器搅拌该容器在37℃下30分钟。 </li><li>比例为1:通过在1中添加标准培养基中和消化。 （如果有1000毫升脂肪混合，加1000毫升标准介质）。 </li><li>分发该混合物同样成偶数的250毫升的塑料锥形离心管中并离心在4°C在300×g离心20分钟将混合物</li><li>吸出上清液，小心留下颗粒完好无损。悬浮颗粒在5毫升每个锥形管标准中。 </li><li>过滤通过100微米过滤器中的悬浮液在一个50毫升锥形离心管中。然后，离心，在300×g离心在4℃下15分钟。 </li><li>吸出上清液并重新悬浮在5毫升室温的RBC裂解缓冲液沉淀。使混合物静置在室温下5分钟，然后在300 xg离心离心在室温（RT）下15分钟。 </li><li>吸出上清液并重新悬浮在15ml标准培养基的沉淀。通过100微米的过滤器再过滤到50mL锥形离心管中。 </li><li>在一个新的50毫升锥形，加入设计用于基于密度分离（见试剂列表）15毫升多聚蔗糖溶液。然后，保持该管在45°角，小心地吸取细胞上的侧管，以使细胞悬浮液轻轻放在多聚蔗糖溶液的顶部层（ 图2）。 注意:这是吸管细胞重要到溶液的​​表面上。不聚蔗糖溶液添加至细胞悬浮液，因为这将产生一个不可逆转的混合物。同样，不要吸管的细胞进入多聚蔗糖溶液的中心。 </li><li>离心该混合物，在300×g离心在RT 10分钟。使用低加速度（2 - 3满分10分）和离心机的制动功能此步骤设置为零。 注:三层将可见:清楚在底部，多云在中间，和鲑色的顶部。中间层含有所关注的细胞。 </li><li>用10毫升吸管小心地取出中间层转移到一个新的50 mL锥形管。 注意:这是更好地完全传送的中间层，并在过程中需要一些比顶层和底层的背后的一些中间层的离开。 </li> &lt;李&gt;计数使用血球细胞，并确定总的活细胞数。 注意:我们通常使用台盼蓝加入到细胞混合物中的0.8％的终浓度，以协助细胞计数。可行的活细胞不会占用的蓝色染料，并会出现白色。 </li></ol> 3.对于FACS BMPR-IB阳性细胞含有细胞的支架材料的制备排序<ol><li>离心细胞悬浮液在300×g离心在4℃下5分钟。重悬在FACS缓冲液将细胞以每100微升（基于在步骤2.13进行的细胞计数）万个单元格的浓度。 </li><li>传送至少10百万细胞与标记的塑料离心管中"BMPR-IB"。另外，将在100微升FACS缓冲区中的一个百万个细胞转移到标记的单独的塑料离心管中"未染色的"。加入1毫升流式细胞仪缓冲的"未染色的"管。保持在FACS缓冲液将细胞的其余部分上的冰，而FACS分拣EXPER的部iment正在做。标签这些细胞"未分类"。使用这些作为对照后在实验中。 </li><li>根据制造商的说明的荧光抗 - 人BMPR-IB抗体适量添加到"BMPR-IB"管。吸取混合物上下轻轻分发抗体。 注意:我们在1:10稀释使用人类BMPR-IB / ALK-6 APC标记的抗体（详见试剂列表）。然而，来自不同制造商的抗体将具有不同的推荐的浓度。 </li><li>覆盖，保持了光的方式冰桶。允许细胞/抗体混合物静置30分钟（或根据抗体制造商的说明书）。 注意:如果防BMPR-IB抗体来作为需要二次抗体的初级未缀合抗体，执行与根据制造商的说明二次抗体在冰上单独染色步骤。 </li><li>无论离心管中300×g离心在4℃下5分钟。吸出上清液，小心不要吸出沉淀。重悬在1mL FACS缓冲液中沉淀的细胞。再次离心管，在300×g离心5分钟。小心吸出上清，重悬在FACS缓冲液将细胞以每1毫升1百万个细胞的浓度。 </li><li>通过40微米的细胞滤网进入新的标记的玻璃FACS管过滤"BMPR-IB"，"未染色的"和"未分类"的细胞。冲洗用500微升FACS缓冲液中的过滤器，以确保细胞中不过滤留下。另外加2毫升标准中到标有"BMPR-IB阳性"和两个独立的塑料离心管"BMPR-IB-负面"。使用这两个收集排序细胞在FACS机器。 </li><li>在FACS机器上，使用未染色细胞以限定一个负栅极为荧光标记物。 </li><li>使用100微米的喷嘴，排序BMPR-IB阳性和阴性细胞进入各管CON泰宁标准网上平台。保持排序在这些管子在4℃下冷藏，如果FACS机器有这个能力。 注:沙龙等请参考JOVE第13条。为FACS分选一个优秀的协议。 </li><li>使用血细胞计数器，计数在每管中的细胞。离心"BMPR-IB阳性"，"BMPR-IB阴性"和"未分类"管300 XG 5分钟，4℃，并吸出上清液，小心不要去打扰细胞沉淀。然后重悬在标准培养基中的细胞，以每250微升万个单元格的浓度。 </li><li>得到预切4毫米PLGA（聚[乳酸 - 共 - 乙醇酸]）涂覆有羟基磷灰石支架。将每个支架到24孔板的孔中。覆盖从三个组中的一个与细胞悬液50μL的每个支架:未排序，BMPR-IB-阳性，BMPR-IB-阴性（ 图3）（ⅰ.E，200,000个细胞）。 注意:请是指由罗等人的JOVE第14条。对PLGA支架的施工细节。 </li><li>部分地填充周围空孔与标准介质（防止支架的干燥），覆盖其盖板，并在37℃下在细胞培养培养箱温育30分钟。 </li></ol> 4.创建骨质缺损和含ACS-脚手架中的应用注:请确保有足够的机构审批到位为创造活体小鼠颅骨缺损。该协议已获得了斯坦福大学的机构动物护理和使用委员会。 <ol><li>用麻醉吸入两种异氟醚气体或注入长效麻醉药鸡尾酒CD-1裸鼠。 <ol><li>单独使用气体，将鼠标成麻醉室，并开始在2个氧的流量 - 3L /用异氟烷的2％浓度分钟。一旦鼠标为fully无意识，将其放置容易发生到温水循环毯与吸湿垫和口鼻放入与氧和异氟烷的相同浓度的麻醉鼻锥。仔细监测呼吸频率并根据需要调整异氟醚的浓度。 </li><li>使用一个注射长效麻醉剂鸡尾酒，如上所述麻醉小鼠在麻醉室，然后注入麻醉鸡尾酒。从麻醉室中取出鼠标，并观察它的行为。 注:鼠标可能短暂地从麻醉但是在几分钟内出现应再次充分注入麻醉剂的作用下进行麻醉。鼠标转移到覆盖有吸收剂垫温水循环的毯子。 注:对于麻醉鸡尾酒，我们建议含有氯胺酮10毫克/毫升和赛拉嗪1mg / mL的生理盐水的混合物，腹膜内递送。该混合物的标准剂量是300微升在30 G鼠标，相当于氯胺酮100 mg / kg和赛拉嗪10mg / kg的。请参阅有关注射麻醉剂更多细节讨论部分。 </li><li>用脚趾捏动作，以确定麻醉深度。一个充分麻醉鼠标无法收回它的爪子当它轻轻地由外科医生夹住。 </li><li>应用眼膏，防止角膜干燥的。 </li><li>辖1毫克/公斤丁丙诺啡皮下SR。 注:提供之前，在上级缓解疼痛的手术结果镇痛。 </li><li>在整个过程中，监视小鼠的呼吸频率。 注:鼠标下异氟烷麻醉正常应该有50呼吸频率 - 100次/ min。增加呼吸频率表明，麻醉过浅，同时降低呼吸频率可能表明，麻醉过深。 </li></ol></li><li> PREP颅骨用聚维酮IOD背侧皮肤INE溶液，然后用70％的乙醇三次。用无菌窗帘悬垂性，而使手术部位暴露出来。 注:戴无菌手术手套和手术过程中保持无菌技术。只有触摸无菌的表面和物件，如无菌悬垂性和蒸压仪器。有一个助手调整灯光亮度，打开缝合包等 。 </li><li>使用15刀片的手术刀，使该延伸过多数背侧颅骨的矢状中线切口。用细齿镊，缩回上切口的右侧的皮肤，以暴露右顶骨。 </li><li>使用钻头用蒸压4毫米金刚石涂覆环钻钻头，通过顶骨钻的缺点。不要缺陷过去骨延伸到硬脑膜层。 （ 图4） </li><li>将支架放入缺陷，然后关闭与运行尼龙线将皮肤切口。 </li><li>监视鼠标和提供标准的术后护理一ccording机构指引。 <ol><li>保持一个干净的纸巾鼠标干净的笼子里，而它恢复。笼子的一半应该被放置在温水循环毯和鼠标应首先将存放在此的一半。 </li><li>直到它已经恢复了足够的意识走动不要离开鼠标无人看管。不要返回已动过手术与其他老鼠的笼子，直到它完全恢复了老鼠。 </li></ol></li><li>重复上述步骤与将要测试的每个支架一个新的鼠标。消毒手术器械在使用热珠灭菌或其它合适的方法手术之间。 </li><li>在手术后不久，然后在2，4，6，和8周，使用显微CT扫描分析颅骨缺损闭合的速率。 注:请参阅李维斯等人的文章。 6颅骨缺损的微CT扫描的描述。 </li><li>当实验是完井德，将它们放置在一干净安乐死室和开始以2升/分钟的速率二氧化碳气体的流入腔安乐死的小鼠。呼吸已完全停止后（约10分钟）进行颈椎脱位确认安乐死。 </li><li>任选地，在实验完成后，使用切片和组织学染色来评估缺陷内骨形成。 注:见麦卡德尔等人的文章。 12准备骨标本细节的组织学。对于组织学和染色技术审查，看组织手册外科病理学 15 的手册等。 </li></ol>

<ol>
	<li>Silber, J. S., 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=Donor+site+morbidity+after+anterior+iliac+crest+bone+harvest+for+single-level+anterior+cervical+discectomy+and+fusion.">Donor site morbidity after anterior iliac crest bone harvest for single-level anterior cervical discectomy and fusion.</a> Spine (Phila Pa 1976). 28 (2), 134-139 (2003).</li><li>Giannoudis, P. V., Dinopoulos, H., Tsiridis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+substitutes:+an+update.">Bone substitutes: an update.</a> Injury. 36, S20-S27 (2005).</li><li>Laurencin, C., Khan, Y., El-Amin, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+graft+substitutes.">Bone graft substitutes.</a> Expert Rev Med Devices. 3 (1), 49-57 (2006).</li><li>Walmsley, G. G., 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=Nanotechnology+in+bone+tissue+engineering.">Nanotechnology in bone tissue engineering.</a> Nanomedicine. 11 (5), 1253-1263 (2015).</li><li>Chapekar, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering:+challenges+and+opportunities.">Tissue engineering: challenges and opportunities.</a> J Biomed Mater Res. 53 (6), 617-620 (2000).</li><li>Levi, B., 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=Human+adipose+derived+stromal+cells+heal+critical+size+mouse+calvarial+defects.">Human adipose derived stromal cells heal critical size mouse calvarial defects.</a> PLoS One. 5 (6), e11177 (2010).</li><li>Gimble, J. M., Katz, A. J., Bunnell, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cells+for+regenerative+medicine.">Adipose-derived stem cells for regenerative medicine.</a> Circ Res. 100 (9), 1249-1260 (2007).</li><li>Levi, B., 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=CD105+protein+depletion+enhances+human+adipose-derived+stromal+cell+osteogenesis+through+reduction+of+transforming+growth+factor+beta1+(TGF-beta1)+signaling.">CD105 protein depletion enhances human adipose-derived stromal cell osteogenesis through reduction of transforming growth factor beta1 (TGF-beta1) signaling.</a> J Biol Chem. 286 (45), 39497-39509 (2011).</li><li>Chung, M. 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=CD90+(Thy-1)-positive+selection+enhances+osteogenic+capacity+of+human+adipose-derived+stromal+cells.">CD90 (Thy-1)-positive selection enhances osteogenic capacity of human adipose-derived stromal cells.</a> Tissue Eng Part A. 19 (7-8), 989-997 (2013).</li><li>Wozney, J. 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=Novel+regulators+of+bone+formation:+molecular+clones+and+activities.">Novel regulators of bone formation: molecular clones and activities.</a> Science. 242 (4885), 1528-1534 (1988).</li><li>Wan, D. C., 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=Osteogenic+differentiation+of+mouse+adipose-derived+adult+stromal+cells+requires+retinoic+acid+and+bone+morphogenetic+protein+receptor+type+IB+signaling.">Osteogenic differentiation of mouse adipose-derived adult stromal cells requires retinoic acid and bone morphogenetic protein receptor type IB signaling.</a> Proc Natl Acad Sci U S A. 103 (33), 12335-12340 (2006).</li><li>McArdle, A., 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=Positive+selection+for+bone+morphogenetic+protein+receptor+type-IB+promotes+differentiation+and+specification+of+human+adipose-derived+stromal+cells+toward+an+osteogenic+lineage.">Positive selection for bone morphogenetic protein receptor type-IB promotes differentiation and specification of human adipose-derived stromal cells toward an osteogenic lineage.</a> Tissue Eng Part A. 20 (21-22), 3031-3040 (2014).</li><li>Sharon, Y., Alon, L., Glanz, S., Servais, C., Erez, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+normal+and+cancer-associated+fibroblasts+from+fresh+tissues+by+Fluorescence+Activated+Cell+Sorting+(FACS).">Isolation of normal and cancer-associated fibroblasts from fresh tissues by Fluorescence Activated Cell Sorting (FACS).</a> J Vis Exp. (71), e4425 (2013).</li><li>Lo, D. 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=Repair+of+a+critical-sized+calvarial+defect+model+using+adipose-derived+stromal+cells+harvested+from+lipoaspirate.">Repair of a critical-sized calvarial defect model using adipose-derived stromal cells harvested from lipoaspirate.</a> J Vis Exp. (68), (2012).</li><li>Lester, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual of surgical pathology. , (2010).</li><li>Bunnell, B. A., Flaat, M., Gagliardi, C., Patel, B., Ripoll, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cells:+isolation,+expansion+and+differentiation.">Adipose-derived stem cells: isolation, expansion and differentiation.</a> Methods. 45 (2), 115-120 (2008).</li></ol>

The authors have no disclosures to make.

该议定书中的关键步骤携带者的收获过程中，关键的一步是脂肪用胶原酶消化充分。不足消化将产生的ASC的低产率。在BMPR-IB +细胞的FACS分选，仔细定义大门的积极性是非常重要的。定义门太松可能会导致排序人群是不纯洁的。在创建过程中的颅骨缺损的，关键是通过头骨的骨钻缺陷，但可以不前进到硬脑膜。这将导致大量出血和脑，这将需要的动物实施安乐死的曝光。 修改和故障排除关于麻醉颅骨缺损的手术，我们的实验室倾向于使用吸入异氟醚，但另一选择是腹膜内注射含有氯胺酮10毫克/毫升的在正常山终浓度和赛拉嗪1毫克/毫升的混合物中线。该混合物的标准剂量为300微升，在30 G鼠标。这提供了约30分钟，不需要鼠标手术期间是在异氟醚鼻锥可靠麻醉。使用注射麻醉剂的一个缺点是，它不可能在注射已经给出后降低剂量。与此相反，吸入异氟醚可以方便地或滴定上下实时。 当加入细胞悬浮液的脚手架，研究人员可能会发现，在流体体积太小，在孵育30分钟将部分蒸发。蒸发可引起细胞死亡，这是在骨愈合实验中特别有害的混杂因素。为了解决这个问题，加载支架24孔板的孔中。填充用普通介质直接包围井。这将创建一个湿润的微环境，这有助于保持装支架干燥。 骨组织学染色前dicated上嵌入和切片之前的颅骨正确和全面的脱钙。一般认为，不同年龄段的头骨需要脱钙不同期间，在EDTA溶液做传统该技术的局限性这个协议利用一个4mm的颅骨缺损，以评估细胞群的增强缺陷的封闭件的能力。这种模式可能不是模拟发生在更复杂的环境，例如长骨骨折或肿瘤切除的愈合。出于这个原因，替代动物模型可能需要用来理解的ASC在这些设置的贡献骨愈合。 关于到现有/替代方法的技术意义使用流式细胞术来分离特定的ASC群为在多个组织的背景下和在提高的愈合和再生一个有前途的技术陪审团模型，如血管，脂肪形成，成骨和。亲骨细胞的效用探讨在这项研究中，表现出与BMPR-IB在医治一个颅骨缺损正选择细胞产生深远的影响。 为ASC的隔离现有协议涉及培养细胞对细胞培养板16几天。然而，细胞可能会遇到，而在文化显著表型漂移。因为这个原因，我们使用一个ASC隔离协议，允许对这些细胞的快速分离，随后使用FACS进一步亚群纯化。整个过程在不到一天的时间来完成，最大限度地减少细胞表面标记或表型漂移的影响。 未来应用及路线我们已经表明，携带者高表达BMPR-IB表现出更强的与其他携带者相比，提高再生骨缺损的愈合能力。该机制underlyING这种现象是不知道。例如，它是不明确自己是否将细胞直接分化成骨形成细胞，或他们是否产生鼓励其它细胞这样做的因素。将来的研究将需要执行来回答这些问题。

从损伤，感染，或浸润癌造成重大的骨缺损对患者的康复和生活质量有显著的影响。技术存在从患者自身的身体别处填充健康骨这些缺陷，但这种传输携带它自己的发病率和并发症1，2，3的风险。此外，一些缺陷是如此之大的或复杂的足够的供体骨不可用于填充缺损。假体装置是用于填充骨缺损的潜在的选择，但这些都与若干缺点，包括感染风险，硬件故障，以及异物反应4相关联。 由于这些原因，有在工程使用患者自身的细胞5生物骨替代品的可能性极大的兴趣。脂肪衍生的基质细胞（ASCs）具有潜在的这种应用，因为它们是在患者自身的脂肪组织大量获得，他们已经证明通过产生新的骨组织6,7愈合的骨缺损的能力。携带者的细胞和几个研究的多样化的人口已经表明，选择特定的细胞表面标志物可产生具有增强成骨活性8,9细胞群。最高的成骨能力选择的ASC将增加的可能性，这些细胞接种支架可以再生一个大的骨缺损。 骨形态发生蛋白（BMP）信令是用于调节骨分化和形成10和BMP受体类型IB（BMPR-IB）已知是用于在携带者11成骨重要的关键。最近，我们已经表明BMPR-IB的表达可以b使用E要选择具有增强成骨活性12携带者。在这里，我们证明了的BMPR-IB-表达来自人脂肪的ASCs接着用体内颅骨缺损模型中的他们的成骨活性的测定中隔离的协议。

<table><tbody><tr><td>100 micron cell strainer</td><td>Falcon</td><td>352360</td><td></td></tr><tr><td>15 blade scalpel</td><td>Miltex</td><td>4-515</td><td></td></tr><tr><td>24 well plate</td><td>Corning</td><td>3524</td><td></td></tr><tr><td>40 micron cell strainer</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>50 mL conical centrifuge tubes</td><td>Falcon</td><td>352098</td><td></td></tr><tr><td>6-0 Ethilon nylon suture, 18&quot;, P-3 needle,&amp;nbsp;</td><td>Ethicon</td><td>1698G</td><td></td></tr><tr><td>Anti-BMPR-IB primary antibody</td><td>R&amp;amp;D systems</td><td>FAB5051A</td><td></td></tr><tr><td>BioGel PI surgical gloves</td><td>M&amp;ouml;lnlycke Health Care</td><td>ALA42675Z</td><td></td></tr><tr><td>Buprenorphine SR</td><td>ZooPharm</td><td></td><td></td></tr><tr><td>Castro-Viejo needle driver</td><td>Fine Science Tools</td><td>12565-14</td><td></td></tr><tr><td>CD1 nude mouse</td><td>Charles River</td><td>086</td><td></td></tr><tr><td>Collagenase Type II powder</td><td>Gibco</td><td>17101-015</td><td></td></tr><tr><td>DMEM medium</td><td>Gibco</td><td>10564-011</td><td></td></tr><tr><td>Drill: Circular knife 4.0 mm</td><td>Xemax Surgical</td><td>CK40</td><td></td></tr><tr><td>Drill: Z500 Brushless Micromotor</td><td>NSK</td><td>NSKZ500</td><td></td></tr><tr><td>FBS</td><td>Gicbo</td><td>10437-077</td><td></td></tr><tr><td>Fisherbrand Absorbent Underpads, 20&quot; x 24&quot;</td><td>Fisher Scientific</td><td>14-206-62</td><td></td></tr><tr><td>Fisherbrand Sterile cotton gauze pad, 4&quot; x 4&quot;</td><td>Fisher Scientific</td><td>22-415-469</td><td></td></tr><tr><td>Heating pad</td><td>Kent Scientific</td><td>DCT-20</td><td></td></tr><tr><td>Hyclone 199/EBSS medium</td><td>GE&amp;nbsp; Life Sciences</td><td>SH30253.01</td><td></td></tr><tr><td>Isothesia isoflurane</td><td>Henry Schein&amp;nbsp;</td><td>050033</td><td></td></tr><tr><td>Micro Forceps with teeth</td><td>Roboz</td><td>RS-5150</td><td></td></tr><tr><td>Micro Forceps with teeth</td><td>Roboz</td><td>RS-5150</td><td></td></tr><tr><td>Paraffin film (Parafilm)</td><td>Bemis</td><td>PM996</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>Pen-Strep</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>PLGA scaffolds</td><td>Proprietary Formulation</td><td></td><td></td></tr><tr><td>Poloxamer 188, 10%</td><td>Sigma</td><td>P5556-100ML</td><td></td></tr><tr><td>Polylined Sterile Field, 18&quot; x 24&quot;</td><td>Busse Hospital Disposables</td><td>696</td><td>Cut a rectangular hole of the appropriate size</td></tr><tr><td>Polysucrose Solution: Histopaque 1119</td><td>Sigma</td><td>11191</td><td></td></tr><tr><td>Povidone Iodine Prep Solution</td><td>Medline</td><td>MDS093944H</td><td></td></tr><tr><td>Puralube petrolatum ophthalmic ointment, 1/8 oz. tube</td><td>Dechra Veterinary Products</td><td></td><td></td></tr><tr><td>RBC lysis buffer</td><td>Sigma</td><td>11814389001</td><td></td></tr><tr><td>Webcol alcohol prep swabs</td><td>Covidien</td><td>6818</td><td></td></tr></tbody></table>

rapid isolation of bmpr-ib+ adipose-derived stromal cells for use in a calvarial defect healing model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

微型CT扫描在手术当天做会清楚地显示颅骨缺损。此时不会有长入4毫米的缺陷。后续扫描都随着时间的推移与基线相比随时间量化缺陷的尺寸获得的。当与BMPR-IB-和未分类的细胞（ 图5）相比，用BMPR-IB +细胞接种缺陷应表现出缺陷的更迅速关闭。此外，将含有缺陷的头骨的部分可脱钙并使用标准方法12组织学处理。用Movat的pentachrome染色染色的切片将与BMPR-IB细胞与其他细胞群（ 图6）相比，处理的缺陷揭示更大骨再生。 <img alt="图1" src="/files/ftp_upload/55120/55120fig1.jpg" /> 图1:PBS洗涤后脂肪抽出。 </<html> STRONG&gt;脂肪抽吸的PBS后罐并将混合物静置。顶端组织层是脂肪组织它承载的ASC，而底层是水层，主要由盐水和血的。 <a href="http://ecsource.jove.com/files/ftp_upload/55120/55120fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55120/55120fig2.jpg" /> 图2: 细胞悬浮放置到聚蔗糖溶液。 （一）将细胞悬浮液必须非常轻轻地吸移到管的侧面，允许它在聚蔗糖溶液的顶层。 （二）这是一个细胞悬浮液层上之前，离心多聚蔗糖的顶部正确的外观。"&gt;请点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/55120/55120fig3.jpg" /> 图3: 细胞到脚手架的播种。 （a）在24孔细胞培养板的一个孔的无菌表面，装入约50细胞悬液微升干支架。 （二）孵育在细胞培养孵化器的支架30分钟，以允许细胞粘附。注意:在图中，10厘米的板用于演示目的。 <a href="http://ecsource.jove.com/files/ftp_upload/55120/55120fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55120/55120fig4.jpg" /> 图4:颅骨缺损的创新。颅骨缺损（箭头）距离可见开放皮肤切口。注意完好硬脑膜血管（箭头），这表明在钻井没有违反硬脑膜。比例尺5毫米。 <a href="http://ecsource.jove.com/files/ftp_upload/55120/55120fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55120/55120fig5.jpg" /> 图5: 关键尺寸的骨质缺损不同亚群ASC愈合。 （ - ）的ASC（A）三维CT微重建是为颅骨缺损进行重新与无序，BMPR-IB（+），或BMPR-IB配对。 （B）与未分选的和相比在表现出显著大于骨再生与BMPR-IB（+）的ASC（92％）8周愈合的定量BMPR-IB（ - ）的ASC（分别为58％和46％，** P &lt; 0.01）。在愈合显著差异还观察到在2我们EKS（** P &lt;0.01），第4周（*** P &lt;0.001），和6周（*** P &lt;0.001）。显微CT，微计算机断层扫描。转载来自麦卡德尔等权限。 12。误差棒代表标准偏差。 <a href="http://ecsource.jove.com/files/ftp_upload/55120/55120fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/55120/55120fig6.jpg" /> 图6: 骨重新生成的组织学染色。 （A）Movat的骨pentachrome染色与无序，BMPR-IB（+），或BMPR-IB修复缺损再生（ - ），使用明亮的视野显微镜在5倍放大镜携带者。注意BMPR-IB（+）组与BMPR-IB相比更健壮的骨形成（ - ）组。虚线表示的缺陷区域的程度。黑色矩形内的区域显示在更高的庄重通货膨胀使用（B）10X和（C）40X亮视野缺损面积的显微镜。转载来自麦卡德尔等权限。 12。 <a href="http://ecsource.jove.com/files/ftp_upload/55120/55120fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model at JoVE.com

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

BMPR-IB +快速分离脂肪来源的使用基质细胞在颅骨缺损愈合模型

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

rapid-isolation-bmpr-ib-adipose-derived-stromal-cells-for-use

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

6.6K 次观看.  Stanford University School of Medicine. Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

视频: Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

隔离和人力富集脂肪基质细胞的成骨增强

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

科研

发育生物学

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

人类血管周围干细胞用于骨再生

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

生物工程

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

修复的临界大小的颅骨缺损模型脂肪间充质干细胞的收获Lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. ...

医学

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 ...

Biological Compatibility Profile on Biomaterials for Bone Regeneration

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

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

骨再生生物材料的生物相容性曲线

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

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

兔子骨增增的千叶模型,用于骨代材料骨生长和新血管化的评估

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...

Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous high-quality cells for experimental requirements. mBMSCs could be widely used in therapeutic applications as tissue engineering cells in case of craniofacial diseases and cranio-maxillofacial regeneration in the future due to the excellent self-renewal ability and multi-lineage differentiation potential. Therefore, it is important to obtain mBMSCs in large numbers.
In this study, bone marrow was flushed from the mandible and primary mBMSCs were isolated through whole bone marrow adherent cultivation. Furthermore, CD29+CD90+CD45&#8722; mBMSCs were purified through fluorescent cell sorting. The second generation of purified mBMSCs were used for further study and displayed potential in differentiating into osteoblasts, adipocytes, and chondrocytes. Utilizing this in vitro model, one can obtain a high number of proliferative mBMSCs, which may facilitate the study of the biological characteristics, the subsequent reaction to the microenvironment, and other applications of mBMSCs.

This article presents a method that combines whole bone marrow adherence and flow cytometry sorting for isolating, cultivating, sorting, and identifying bone marrow mesenchymal stem cells from rat mandibles.

大鼠骨髓骨髓的隔离和培养

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous ...

<meta charset="utf8"></head><body>用于再生研究的标准化大鼠颅骨缝合骨缺损模型

The Establishment of Calvarial Suture-Bony Composite Defects in Rats: A Standardized Model for Suture-Regenerative Therapy Investigation

Large-scale calvarial defects often coincide with cranial suture disruption, leading to impairments in calvarial defect restoration and skull development (the latter occurs in the developing cranium). However, the lack of a standardized model hinders progress in investigating suture-regenerative therapies and poses challenges for conducting comparative analyses across distinct studies. To address this issue, the current protocol describes the detailed modeling process of calvarial suture-bony composite defects in rats.
The model was generated by drilling full-thickness rectangular holes measuring 4.5 mm &#215; 2 mm across the coronal sutures. The rats were euthanized, and the cranium samples were harvested postoperatively at day 0, week 2, week 6, and week 12. &#181;CT results from samples collected immediately post-surgery confirmed the successful establishment of the suture-bony composite defect, involving the removal of the coronal suture and the adjacent bone tissues.
Data from the 6th and 12th postoperative weeks demonstrated a natural healing tendency for the defect to close. Histological staining further validated this trend by showing increased mineralized fibers and new bone at the defect center. These findings indicate progressive suture fusion over time following calvarial defects, underscoring the significance of therapeutic interventions for suture regeneration. We anticipate that this protocol will facilitate the development of suture-regenerative therapies, offering fresh insights into the functional restoration of calvarial defects and reducing adverse outcomes associated with suture loss.

<meta charset="utf8"></head><body>作者聚焦:颅骨缝合骨复合缺陷标准化大鼠模型的开发和评估

We describe the detailed surgical procedures of calvarial suture-bony composite defects in rats, alongside the investigations into the short-term and long-term prognoses of the model. We aim to construct a standardized model for developing suture-regenerative therapies.

大鼠颅骨缝合骨复合缺损的建立:缝合再生治疗研究的标准化模型

Large-scale calvarial defects often coincide with cranial suture disruption, leading to impairments in calvarial defect restoration and skull development ...

BMPR-IB +快速分离脂肪来源的使用基质细胞在颅骨缺损愈合模型

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骨再生生物材料的生物相容性曲线

兔子骨增增的千叶模型,用于骨代材料骨生长和新血管化的评估

大鼠骨髓骨髓的隔离和培养

大鼠颅骨缝合骨复合缺损的建立:缝合再生治疗研究的标准化模型

大鼠颅骨缝合骨复合缺损的建立:缝合再生治疗研究的标准化模型

大鼠颅骨缝合骨复合缺损的建立:缝合再生治疗研究的标准化模型